MANUAL ON QUALITY STANDARDS FOR HIV TESTING LABORATORIES

March 2007

National AIDS Control Organisation Ministry of Health and Family Welfare

2007@ National AIDS Control Organisation. All rights reserved.

TABLE OF CONTENTS
S.NO. CHAPTERS Table of contents Foreword Acknowledgements List of abbreviations 1. Basics of HIV Structure Transmission Immunopathogenesis Susceptibility Laboratory diagnosis of HIV / AIDS and National HIV testing strategies Introduction Laboratory investigation for detection of HIV infection Causes of false positive and false negative results Strategies/algorithms of HIV testing Detection of p24 antigen Polymerase chain reaction (PCR) Virus culture Viral load assay HIV Testing at counseling and testing sites (ICTCs/VCTCs and PPTCTC's), etc. Introduction Types and Technologies of rapid tests - Immunoconcentration - Agglutination - Immunocomb assay - Immunochromatography - Possible outcomes of these rapid tests Accuracy of rapid tests Strategies/algorithms for rapid tests Uses of rapid tests Quality assurance practices Total quality management Management requirements Technical requirements Laboratory management Laboratory configuration Stock/inventory management of reagents and consumables Data management 1---5

2.

6--15

3.

16--20

4.

21--25

5.

26--27

6.

Quality control Introduction Scientific principles involved Definition of terms Essential components Categories of controls Dispensation and storage of quality control samples Determination of acceptable range of quality control to validate each test run Variations in the quality control validation guidelines Importance of 'E' ratios over OD values of external controls to check validity of runs Interpretation of aberrant result of QC samples: shift and trend Collection, transport & storage of specimens for HIV testing Performing venipuncture Transport and storage of samples\ Standard operative procedures (SOP) The structure of SOP An example of SOP Documentation and document control Selection of HIV test kits ELISA Rapid tests Details of some of the commercially available kits Evaluation of HIV test kits Introduction Preparation of serum panel Composition of serum panel Reference test Testing conditions Parameters for performance characteristics of a kit Selection of HIV test kits Equipment maintenance and calibration Introduction Checklist before deciding on purchase of instrument Maintenance and calibration of different equipment Calendar of activities for maintenance & calibration of equipment Function check Documentation Preventive maintenance Operation of ELISA Reader (Multiskan plus)

28--46

7.

47--50

8.

51--52

9. 10.

53--54 55--59

11.

60--66

12.

67--75

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13.

Troubleshooting Introduction Specific assay problems: some examples and their possible solutions Variations in test results Common errors in HIV testing and resolution of the same Vigilance / supervision Record keeping Implementation of external quality assessment programme at different levels of the HIV/AIDS testing network and job definitions of each level Equipment, supplies and reagents for practicing quality control in HIV testing Biosafety practices in HIV testing laboratory Introduction Modes of exposure to blood borne pathogens in the laboratory Essential steps of biosafety Biosafety guidelines for laboratory workers Chemical disinfection commonly used in HIV laboratory Transport of specimens Disposal of laboratory waste Processing of syringes, needles and gloves for reuse Packing, storage and transport of treated (disinfected) waste Final disposal of laboratory waste Container and color coding for disposal of bio medical waste Spills and accidents Post exposure prophylaxis against HBV and HIV Selected list of references consulted Annexures: Annexure I: Consent form for HIV testing Annexure II: Proforma for requisitioning HIV test Annexure III: Worksheet for ELISA Annexure IV: HIV test report form Annexure V A & B: Formats for reporting to SACS and NACO Annexure VI: Proforma for External quality assessment programme for HIV testing Annexure VII: Sample EQA Questionnaire for lab supervisors Annexure VIII: Sample EQA Questionnaire for lab staff Annexure IX: States allotted to the national reference centers Annexure X: Pipetting techniques List of Experts For Revised Manual List of Experts involved in Field Testing of the Original Manual

76--80

14.

81--84

15.

85--86

16.

87--98

17. 18.

99--100

101 102 103 104 105--106 107--111 112--115 116-118 119 120-122 123 124-125

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K. Sujatha Rao
Additional Secretary & Director General National AIDS Control Organisation, Ministry of Health and Family Welfare, Government of India

FOREWORD
In order to ensure that the result produced by a HIV Testing Laboratory (ICTC/VCTC, PPTCT, and blood bank laboratory) is reliable, accurate and reproducible, it is essential that laboratory personnel have the knowledge about all aspects of HIV testing and undertake stringent quality control measures every day. There is a vast network of HIV testing laboratories in the country; however there is a need to institute uniform testing and quality control protocols in these laboratories across the Nation especially in view of the wide variety of tests kits used by them. The generation of false positive and false negative results is associated with immense social, ethical and legal implications. It is indeed critical to assure quality in HIV testing so that accurate results are produced every time the test is performed. Manual on "Quality Standards for HIV Testing Laboratories" will provide guidelines for practicing quality assurance required for HIV testing on a day to day basis; training, implementation and establishment of quality assurance programs in all the HIV testing facilities across the country (blood banks, ICTCs/VCTCs, PPTCTs and Blood Bank labs); evaluation and selection of HIV test kits and standard formats for requisitioning, performing and reporting the test results. The guidelines in this manual conform to the recommended quality standards for HIV testing laboratories as well as the needs of the country based on available infrastructure. We appreciate the efforts of all the expert who prepared the document; those who field tested the manual and those who updated the manual.

Director General & Addl. Secy, National AIDS Control Organization

9th Floor, Chandralok Building, 36 Janpath, New Delhi - 110001 Phone : 011-23325331 Fax : 011-23731746 Email : asdg@nacoindia.org

viuh ,pvkbZoh voLFkk tkusa; fudVre ljdkjh vLirky esa eqr lykg o tkp ik,A
Know your HIV status; go to the nearest Government Hospital for free Voluntary Counselling and Testing.

ACKNOWLEDGEMENTS
The manual entitled Quality Standards in HIV Testing Laboratories is intended for use by laboratory specialists, laboratory supervisors/in charges and technical staff engaged in HIV testing across the country .The manual provides background knowledge of HIV; details of laboratory diagnosis of HIV; step by step practices of quality assurance; HIV testing procedure; evaluation and selection of HIV kits; troubleshooting and biosafety practices. The document also includes samples of standard formats used for HIV testing and reporting. The manual can be referred to for preparing Standard Operative Procedures for any HIV test, practicing quality assurance and producing accurate results. We thank Ms. Sujatha Rao, Additional Secretary and Director General, NACO for giving us the opportunity to revise and update the existing manual. We are extremely grateful to Dr. Jotna Sokhey, Addl. Project Director NACO, Dr M. Shaukat, Joint Director, NACO, Dr. D. Bachani, Joint Director, NACO and to all the contributors and experts and the team at Clinton Foundation who contributed, field tested, and updated this manual to bring the manual to its final form.

The Contributors

LIST OF EXPERTS FOR REVISED MANUAL

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Dr. Jotna Sokhey, Addl. Project Director, NACO Dr. M. Shaukat, Joint Director, NACO Dr. D. Bachani, Joint Director, NACO Dr. Sandhya Kabra, Deputy Director, NACO Dr. D. Kasana, former Deputy Director ,R & D , NACO Dr. Preeti Mehta, KEM College, Mumbai Dr. Madhuri Thakar, NARI, Pune Dr. R. K. Lodha, AIIMS, Delhi Dr. Mario Tamburini, WHO, SEARO Dr. Usha K. Baveja, former Lab Specialist, Clinton Foundation Dr. Namita Singh , Lab Specialist, Clinton Foundation Dr. Sanjay Sarin, former Lab Specialist, Clinton Foundation Dr. S. N. Misra, Clinton Foundation

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vi vii

vi vii

123

LIST OF EXPERTS INVOLVED IN FIELD TESTING OF THE ORIGINAL MANUAL

The manual on Quality Assurance practices in HIV Testing Laboratory was field tested on two different occasions at NIB ,Noida. The following experts contributed: 1. Dr. (Mrs.) Usha K. Baveja Head, HIV/AIDS Division, NICD 22, Shamnath Marg, New Delhi 2. Dr. PL Joshi Ex-Additional Project Director (Technical) National AIDS Control Organization Chandralok Building, 9th Floor 36- Janpath, New Delhi- 110001 3. Dr. Rajesh Bhatia Ex-Director, National Institute of Biologicals, A-32, Sector- 62, Institutional Area Phase II, Noida- 201301 4. Dr. Pradeep Seth Ex-Professor & Head Department Of Microbiology, All India Institute of Medical Sciences New Delhi: 110029 5. Dr. S.N Misra Consultant, NACO 9th Floor, 36 Janpath, Chandralok Complex, New Delhi-110001 6. Dr. Shobha Broor Professor Department Of Microbiology All India Institute of Medical Sciences New Delhi: 110029 7. Dr. Shashi Khare Ex-Deputy Director (Quality Control), National Institute of Biologicals, A-32, Sector- 62, Institutional Area Phase II, Noida- 201301

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8.

Dr. V.Ravi

Professor & Head Department Of Neurobiology, NIMHANS, Bangalore.

9.

Dr D.K. Neogi

Professor & Head Department of Virology School of Tropical Medicine, Calcutta.

10.

Dr. N.M Samuel

Professor Department of Experimental Medicine MGR University, Chennai.

11.

Dr. Brajachand Singh

Professor Department of Microbiology, Regional Institute of Medical science, Imphal 795004

12.

Dr. Charanjeet Sokhey

Senior Scientist Department of Blood products & Microbiology, National Institute of Biologicals NOIDA 201301

13.

Dr.Ramesh Paranjape

Office-in-charge National AIDS Research Institute, Plot No. 73, G Block, MIDC Bhosari, Pune 411026

14.

Dr. Arun Risbud

Microbiologist National AIDS Research Institute, Plot No. 73, G Block, MIDC Bhosari, Pune 411026

15.

Dr. Shameem Banu

Professor of Microbiology Madras Medical College, Chennai - 600006

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LIST OF ABBREVIATIONS
BTC CCR-2, 3, 5, CXCR4 cDNA CMV CD4 CSF CV DNA EIA ELISA EQA E ratio E/R gp HBIg HBV HCW HCV HIV HIV 1 HIV 2 HIV -I M HIV -I O : : : : : : : : : : : : : : : : : : : : : : : Blood testing centre Co-receptors on T cells/ macrophages for HIV complementary DNA Cytomegalovirus Cluster determinant 4 Cerebrospinal fluid Coefficient of variation Deoxyribonucleic acid Enzyme Immuno Assay Enzyme Linked Immunosorbent Assay External Quality Assessment ELISA ratio ELISA / Rapid Glycoprotein Hepatitis B Immunoglobulin Hepatitis B virus Health care worker Hepatitis C virus Human Immunodeficiency Virus Human Immunodeficiency Virus Type -1 Human Immunodeficiency Virus Type -2 HIV -1 Major group HIV -1 Outlier group

vi

HIV I N HTLV IB NPV PPV NRL SRL OD PCR PEP PHA PI PPTCTC QA QAP RIA RNA SD SOP TQM UV VCTC ICTC WB

: : : : : : : : : : : : : : : : : : : : : : : :

HIV -1 New group Human T lymphotropic virus Immunoblot Negative Predictive Value Positive Predictive Value National Reference Laboratory State Reference Laboratory Optical density Polymerase Chain Reaction Post Exposure Prophylaxis Passive haemagglutination Protease inhibitors Prevention of parent to child transmission centre Quality Assurance Quality Assurance Programme Radio Immunoassay Ribonucleic acid Standard deviation Standard Operative Procedure Total Quality Management Ultra violet Voluntary Counseling & Testing Centre Integrated Counselling and Testing Centers Western blot

vii

CHAPTER 1 BASICS OF HIV

Introduction
Human immunodeficiency viruses (HIV) belong to the family Retroviridae and subfamily Lentivirinae and Genus Lentivirus . Two types of HIV are recognized; HIV-1 and HIV-2. Both differ in geographical distribution, biological and molecular characteristics and extent of transmissibility. These viruses store their genetic information as ribonucleic acid (RNA). RNA must be converted to DNA by a special enzyme, reverse transcriptase. HIV-1 has 3 groups, HIV-1 major group (HIV 1-M), outlier (HIV 1-O) and new (HIV 1-N) group. The strains of HIV-1 isolated from people in USA and Europe are genetically diverse from strains isolated in Africa and Asia. HIV-1 major group can be further classified into subtypes or clades designated A through K excluding I. Such subtypes have envelope gene sequences that vary by 20% or more between subtypes. The subtypes also differ in geographical distribution, biological characteristics and major mode of transmission, etc. HIV-1 subtypes O and N are more distant to all other HIV-1 subtypes but less so compared to HIV-2. So these are classified under HIV-1 only and have limited distribution in West Africa. HIV-2 has also been reported from other countries and this also comprises of heterogeneous group of viruses.

Structure
HIV is a 90-120 nm icosahedral, enveloped, RNA virus. HIV comprises of an outer envelop consisting of a lipid bilayer with uniformly arranged 72 spikes or knobs of glycoprotein (gp) 120 and gp 41 in HIV-1 and gp140 and gp 36 in HIV- 2, respectively. Glycoprotein 120/140 protrudes out on the surface of the virus and gp 41/36 is embedded in the lipid matrix. Inside is the p24 protein core surrounding two copies of RNA. This core also contains viral enzymes: reverse transcriptase, integrase and protease, all essential for viral replication and maturation. Proteins p7 and p9 are bound to the RNA and are believed to be involved in regulation of gene expression.

Genetic structure
The genetic structure of virus contains both highly conserved and highly variable regions. The high variability of the virus accounts for drug resistance and evasion from immune response. This also poses problems for development of a successful vaccine. In an infected individual, quasi-species of a particular viral subtype may be found on account of constant variability. As a result many recombinant strains like AE, AG and AB may be present in infected individuals. HIV has structural and regulatory genes coding for structural and regulatory products, respectively. Structural genes direct the synthesis of physical components of the virus and are also responsible for viral size, shape, structural integrity and its compartmentalization in host cell. The regulatory genes direct synthesis of proteins that affect the synthesis of viral components and viral replication. Structural genes are gag, pol and env. HIV also has some accessory genes vpu, vpr and vif, etc. which increase infectivity and budding.

Replication
Glycoprotein 120/140 of HIV binds to a receptor / receptors on HIV permissive host cell. Predominant receptor is the CD4 molecule present on T lymphocytes and macrophages, though others such as galactosyl-ceramide (gal C) have 1

also been proposed. Receptors are molecules (proteins and / or glycoproteins) present on the surface of host cells which facilitate the attachment and entry of viruses into the cell. Entry of virus into the host cell requires certain cellular co-receptors / factors e.g. CCR-5, CXCR-4, CCR-2 and CCR-3, etc. designated collectively as cell infectivity factors (CIF). CIF may be a co-receptor or enzyme helping in virus interaction with host cell. Most convincing candidate is the chemokine receptor related protein, fusin (CXCR-4). Once the gp41/36 of the virus fuses with the host cell membrane, the capsid is uncoated and a ribonucleoprotein complex capable of reverse transcription is formed. During the process of reverse transcription cDNA is formed under the effect of viral enzyme, the reverse transcriptase. Reverse transcription is inefficient in quiescent cells suggesting the involvement of host components in the process. The nucleoprotein complex formed after transcription comprises of linear double stranded DNA, the gag matrix (MA) protein, the accessory vpr protein and the viral integrase (IN). This is called pre-integration complex and is transported into the host cell nucleus. It mediates a complex series of enzymatic steps and integration occurs at cellular loci with open chromatin structure. Integration probably is an essential step for viral replication. The integrated virus is called provirus. The virus may not be expressed in many cells and is considered latent. Virus expression can be stimulated by many viral, cellular and exogenous factors. Other co-existent viral infections e.g. cytomegalovirus, herpes virus etc. can make the non permissive cells permissive. Maturation of virus also takes place after virus assembly and budding.

Immunopathogenesis
HIV enters the circulation and targets onto cells expressing CD4 on the surface in various organs including brain and lymphoid tissues. Infected CD4 lymphocytes migrate to lymph nodes, become activated, proliferate and start releasing HIV. The antigen driven migration and accumulation of CD4 cells within the lymphoid tissues may account for dramatic decrease in CD4 cells and generalized lymphadenopathy characteristic of acute retroviral syndrome. Cellular and humoral responses are established within one week to three months (window period) and host enters the phase of clinical latency characterized by lack of symptoms and almost initial normal levels of CD4cells. There is gradual deterioration of the immune system due to depletion of CD4 cells. CD4 cells are destroyed by HIV mediated cell lysis; formation of syncytia and multinucleated giant cells (infected as well as uninfected cells are destroyed); virus specific immune responses; superantigen mediated activation of T cells making them permissible to HIV and programmed cell death (apoptosis). The ability of HIV to replicate in T cells depends on the state of activation of cells. So, a vicious cycle is established leading to depletion of CD4 cells, destruction of lymphnode architecture and progressive increase in the viral load. The progression of HIV disease also depends upon the biological properties of the infecting HIV strain (high vs. low replicating, syncytium vs. nonsyncytium forming virus). The HIV disease progresses fast in infants infected in utero because of the immature immune system and rapid spread of HIV in the body. Most cases vertically infected have a shorter incubation period and a more rapid, fulminant disease process.

Transmission
Risk factors for transmission of HIV include: unsafe sexual practices with multiple sexual partners, recipient of multiple 2

blood transfusions, use of infected/unsterilised needles or syringes (whether therapeutically or for recreational drug use), transmission from an infected mother to the foetus/infant before, during, or shortly after delivery and during breastfeeding by an untreated mother who is infected (perinatal factors that increase transmission include prematurity, maternal STI, episiotomy, and prolonged rupture of membranes and others). The efficiency of transmission of HIV is determined by the amount of virus in a body fluid and the extent of contact. High concentration of free infectious virus and virus infected cells have been reported in blood, genital fluids and cerebrospinal fluid. Breast milk and saliva yield varying numbers, whereas, other body fluids have a low viral content. Saliva in adults contains some non-specific inhibitory substances like fibronectins and glycoproteins which could prevent cell to cell transfer of virus. Thus, saliva is not a likely vehicle of transmission. Urine, sweat, broncho-alveolar lavage fluid, amniotic fluid, synovial fluid, faeces and tears have been reported to yield zero or few HIV particles. Hence, these vehicles also do not appear to be important in virus transmission, though standard work precautions should be undertaken while handling all the body fluids Breast milk at the time of primary infection in a feeding mother has a high content of virus and may transmit the infection to the baby. In contrast, cerebrospinal fluid (CSF) also has a high content of virus particularly in individuals with neurological disease; CSF is not a natural source of virus transmission. Table1 Efficiency of different routes of HIV transmission and their contribution to total number of cases reported in India. Exposure route Percent efficiency * Percentage of total Worldwide Blood transfusion Perinatal Sexual intercourse Vaginal Anal Oral Injecting drugs use Needle stick exposure Exposure unspecified
* see references Percentage of total in India from NACO website

India 2.5

90-95 20-40 0.1 to 10 0.05-0.1 0.065-0.5 0.005-0.01 0.67 0.3

5 10 75 (60) (15) Case reports only 10 0.1

86 85 (heterosexual) 0.548 (homosexual)

7.3

4.2

The most efficient vehicle of HIV transmission is blood (Table 1). However, the risk of infection via blood transfusion is now extremely low due to strict HIV screening of donated blood. The most common route of transmission is unprotected, penetrative sexual contact. Different forms of sexual practices carry a variable risk gradient of acquiring HIV. Cell associated rather than free virus is responsible for disease transmission. Anal intercourse carries a high risk of transmission because of bowel mucosa which acts as a portal of entry for virus, and also because of a greater chance of injury to the mucosa. Risk to insertive partner is through infection of lymphocytes and macrophages in the foreskin or along the urethral canal. In females, HIV transmission occurs when infected cells in the semen gain entry 3

into the female genital tract, and infect the resident lymphocytes, macrophages and probably the uterine epithelial cells. The transmission from infected mother to child appears to occur in 20-40% children born to HIV positive mothers without PPTCT intervention. The source of virus in the new-born is controversial. HIV infection can occur via amniotic fluid, genital secretions, maternal blood and through the breast milk. Transmission to the baby can occur inutero, and during or after delivery. Transmission of HIV infection to Health Care Professionals (HCP) is extremely uncommon. Pooled data from 20 prospective studies suggests that risk associated with needle stick injury from HIV infected blood is approximately 0.3 percent. Further, the risk associated with mucocutaneous contact is too low to be reliably estimated. The risk from mucosal or non-intact skin is also minimal. Transmission of HIV through casual contact, sharing utensils, lavatories and through insect bites has not been documented. Prospective studies offer a conclusive evidence that family members and close household contacts of HIV infected individuals are not at risk of acquiring HIV infection through casual human contact (shaking hands, kissing and by sharing of utensils, toilet, bed linen etc.) or by providing routine nursing care. Mosquito bite does not transmit HIV.

Prevention of HIV Transmission

Transmission of HIV through sexual route can be prevented by creating awareness; promoting mutually faithful sexual practices, safer sexual behaviors, or celibacy; treating existing sexually transmitted infections. All individuals should be counseled to take personal responsibility for their sexual health and behavior. Transmission of HIV through blood and blood products can be prevented by mandatory screening of donated blood and rational use of blood. It should be emphasized that it is safer to use blood components rather than whole blood. Whole blood should only be used when so indicated. Transmission of HIV infection through contaminated needles can be prevented by using sterile needles and syringes; by reducing the number of injections i.e. prescribing oral drugs in place of injections wherever possible and practice of standard work precautions at all times while rendering medical services. Post exposure prophylaxis with anti retroviral drugs as per guidelines should be taken within two hours in case of accidental exposure to HIV-infected blood. Transmission of HIV infection from infected mother to the new born is prevented currently through intervention with anti retroviral drug nevirapine under the PPTCT program of NACO.

Susceptibility of HIV
Sterilization Autoclaving at 121C at 15 lb pressure for 20 minutes Dry heat 170 C for 1 hr. (holding time) Boiling for 20 minutes (this kills HBV and HCV also ).

Disinfection minimum 30 minutes contact time Sodium hypochlorite 0.5% to 1% 4

Ethanol 70% Povidone iodine (PVI) Formalin 3-4% Glutaraldehyde (activated) 2%

References for Table 1:

1. 2. 3. 4. Donegan E, Stuart M, Niland JC, et al. Infection with human immunodeficiency virus type 1 (HIV-1) among recipients of anti- bodypositive blood donations. Ann Intern Med 1990; 113:7339. Kaplan EH, Heimer R. HIV incidence among New Haven needle exchange participants: updated estimates from syringe tracking and testing data. J Acquir Immune Defic Syndr 1995; 10:1756. European Study Group on Heterosexual Transmission of HIV. Comparison of female to male and male to female transmission of HIV in 563 stable couples. BMJ 1992; 304:80913. Varghese B, Maher JE, Peterman TA, Branson BM, Steketee RW. Reducing the risk of sexual HIV transmission: quantifying the per- act risk for HIV on the basis of choice of partner, sex act, and 5. 6. condom use. Sex Transm Dis 2002; 29:3843. Bell DM. Occupational risk of human immunodeficiency virus infection in healthcare workers: an overview. Am J Med 1997; 102:915. Leynaert B, Downs AM, De Vincenzi I; European Study Group on Heterosexual Transmission of HIV. Heterosexual transmission of HIV: variability of infectivity throughout the course of infection. Am J Epidemiol 1998; 148:8896.

CHAPTER 2 LABORATORY DIAGNOSIS OF HIV/AIDS AND NATIONAL HIV TESTING STRATEGIES

Introduction
Two distinct human immunodeficiency viruses, HIV-1 and HIV-2 are the aetiologic agents of AIDS. Phylogenetically, HIV-1 is divided into Group M [10 subtypes A through K excluding I]; Group O (9 subtypes) & Group N (new virus). HIV-2 has been categorized into 5 subtypes (A to E). In Thailand, India and sub-Saharan Africa, ~ 90% of HIV-1 infections are acquired through heterosexual transmission in contrast to 10% in the U.S. and Western Europe. Subtypes A, C and D predominate in Africa, subtypes E and B are commonly found in Thailand and C is the main subtype in India; whereas, subtype B predominates in the U.S. and Western Europe. In 50-93% of cases primary HIV infection is symptomatic with a variety of symptoms ranging from influenza-like or mononucleosis-like illness to more severe neurological symptoms which can persist from a few days to as long as two months. Longer acute clinical illness is associated with rapid progression to AIDS. Laboratory diagnosis is the only method for determining HIV status of such an individual. A number of tests and diagnostic kits are available to assess the HIV status of individuals. Serological tests are most commonly performed. The choice of test protocol depends upon: Objectives of HIV testing Sensitivity and specificity of the test used The prevalence of HIV infection among the population Resources available Appropriateness to the strategy of testing Infrastructural facilities available

Objectives of HIV testing

Blood and blood products safety. This is determined by mandatory screening of all donated blood Donors of sperms, organs and tissues are tested for HIV to prevent HIV transmission to the recipient Diagnosis of HIV infection in clinically suspected individuals Prevention of parent to child transmission after counseling and informed consent Voluntary testing: high risk groups after counseling and informed consent Sentinel surveillance to monitor epidemiological trends Research, surveys, etc.

Sensitivity
It is the accuracy with which a test can confirm the presence of an infection in an infected individual. Tests with high sensitivity show few false negative results and are meant to be used to screen blood prior to transfusion and ensure blood safety. It is recommended that 3rd or 4th generation HIV test kits which are 100% sensitive should be preferred for use at blood banks for screening donated blood. 6

Specificity
It is the accuracy with which a test can confirm the absence of an infection in an uninfected individual. Tests with high specificity show few false positives and are to be preferred for the diagnosis of HIV infection in an individual.

Prevalence of HIV infection

The probability that a test will accurately determine the true infection status of a person being tested varies with the prevalence of HIV infection in the population from which the person comes. The higher the prevalence, greater is the probability that a person testing positive is truly infected i.e. greater is the positive predictive value (PPV) of the test. The likelihood that a person showing a negative result is truly uninfected i.e. the negative predictive value (NPV) decreases as the prevalence of HIV infection among the general population increases.

Laboratory investigations for HIV infection

HIV infection can be detected in the laboratory either by detection of antibodies to HIV, or by detection of the virus, its antigen and its DNA or RNA. Detection of specific antigens, viral nucleic acid & isolation / culture of virus are all tests in which the presence of the virus is detected. But most of these tests are very laborious and difficult to perform, require expertise and hence are to be done only in specified laboratories The indirect predictors of HIV infection (CD4 cell count, 2 microglobulin, etc.) are monitors of immune status of patients. CD4 cell enumeration is to be done at routine intervals as per the guidelines, to monitor the progression of disease and response to ART, etc. The specimens which can be utilized to detect various markers of HIV infection are given below. (i) Antibody detection Blood / serum / plasma 3-5 ml. of blood is collected in clean, screw capped plain vial for ELISA and for the supplemental tests. Saliva and urine have been used to detect antibodies to HIV but the assays have not been validated in India. (ii) Antigen detection Blood/serum / plasma (most commonly used sample) Cerebrospinal fluid (used sometimes) Cell culture supernatant (i.e. the tissue culture fluid-for research only) Virus isolation: HIV can be isolated from HIV infected tissues. It can be successfully isolated from blood (PBMN cells), semen, vaginal / cervical specimen, tissue, CSF and plasma. It is less successful on other body fluids like saliva, urine, breast milk, tears and amniotic fluid. Virus isolation is done for research purposes only.

(iii)

Detection of specific antibodies

This is done by performing initial screening test, which if positive, is followed up by supplemental tests to confirm the diagnosis. 7

Screening tests
ELISA/EIA: (Enzyme Linked Immunosorbent Assay/Enzyme Immunoassay) It is the most commonly performed test at blood banks and tertiary labs to detect HIV antibodies. There are various kinds of ELISA based on the principle of test: Indirect ELISA Competitive ELISA Sandwich ELISA Immuno Capture ELISA ELISA is also classified on the basis of the antigens utilized, into: 1st generation: Infected cell lysate is used as the antigen. 2nd generation: Glycopeptides (recombinant antigens) are used as the antigen. 3rd generation: Synthetic peptides are used as the antigen. 4th generation: Antigen and antibodies are detected simultaneously. The assays may use a combination of recombinant and synthetic peptides as antigens. When a serum sample tests reactive once by a system of ELISA / Rapid (E/R) test, the test is to be repeated immediately by a different system in order to confirm the diagnosis at VCTCs and PPTCTCs. If it tests reactive a second time, the sample is to be taken up further for supplemental tests to confirm the diagnosis. Supplemental tests are E/R based on different principles of test or different antigen systems. Sometimes, WB, etc. may have to be used to resolve the sero-discordant results. ELISA takes up to three hours to yield results. It has a major advantage of being economical. Although rapid tests give results within minutes, these are relatively expensive per test. Commercial kits are available for both ELISA and rapid tests. Rapid tests include: Dot blot assays (immunoconcentration based) Particle agglutination (gelatin, RBC, latex, microbeads) Dip stick and comb tests, etc (EL ISA technology based). Immunochromatography based tests (lateral flow of reagents) Tests which detect antibody to HIV 1 and 2 and all the subtypes of HIV1 and HIV2, are to be employed

Supplement tests
Second and third ELISA/Rapid Western blot (WB)

WB is expensive, time consuming and requires expertise to perform. This is mostly done to confirm the diagnosis on samples which given discordant results in E/R and in legal cases.

Causes of false positive and false negative results

There are many conditions which may give rise to false positive or false negative test results. False positive results have been reported in cases of haematologic malignant disorders, DNA viral infections, autoimmune disorders, 8

multiple myeloma, primary biliary cirrhosis, alcoholic hepatitis, chronic renal failure, positive RPR (rapid plasma regain) test and false negative results have been reported in cases of window period prior to seroconversion, immunosuppressive therapy, malignant disorders, B-cell dysfunction and bone marrow transplantation, etc.

Strategies / Algorithms of HIV testing

Because of the enormous risk involved in transmission of HIV through blood, safety of blood products is of paramount importance. Since the PPV is low in populations with low HIV prevalence, WHO/GOI have evolved strategies/algorithms to detect HIV infection in different population groups and to fulfil different objectives. The various strategies/algorithms, so designed, involve the use of categories of tests in various permutations and combinations. 1. 2. Screening ELISA/ Rapid tests (E/R) Used in strategy I, II & III Supplemental test: E/R and Western Blot(WB) WB is used in cases where discordant serological results are obtained; if WB is not available, then client should be retested all over again after 2-4 weeks.

Strategy I (algorithm I)
Blood/plasma/serum is subjected once to E/R for HIV. If negative, the sample is considered free of HIV and if positive, the sample is taken as HIV infected for all practical purposes. This strategy is used for ensuring donation safety (blood, organ, tissues, sperms, etc.). Units of blood testing positive by the screening E/R are destroyed as per guidelines. A donor who gives consent to know the result of HIV test is informed about the result. In case the test is positive, the donor is informed about the possible reactive nature of the result and is advised that the same requires confirmation. The individual is accordingly referred to VCTC for counseling, testing and confirmation of the test result.

Strategy II A (algorithm II A)
A serum sample is considered negative for HIV if the first ELISA or rapid (screening) test is negative, but if reactive, the same sample is subjected to a second ELISA or rapid test which utilizes a system different from the first one. i.e. either the principle of test and/or the antigen used is different. It is reported reactive only if the second ELISA/ rapid test confirms the positive report of the first test. In case the second E/R is non reactive, then the result is taken as negative for surveillance purposes (II A). Strategy II A is used for sentinel surveillance (anonymous, unlinked testing).

Strategy II B (algorithm II B)
This strategy is used for detection of HIV infection in symtomatic individuals with symptoms of AIDS clinically. The sample is processed as in Strategy IIA, but a sample reactive with first assay and non-reactive with second assays subjected to the third tie breaker E/R. If the third E/R is reactive, the samples reported as indeterminate and patient is called back for repeat testing after 2-4 weeks. If the third test is negative, it is reported as negative. Two to three different test kits with different antigen system or different principle of the test are required to follow this Strategy IIB of testing. In this strategy if the first two consecutive tests are positive for presence of HIV antibodies, a positive report can be given to the patient.

Strategy III (algorithm III)

Testing is done as in strategy IIB. However, added confirmation of a third reactive E/R test is required for a sample to be reported as HIV positive. If the sample gives reactive result with two E/R and non reactive with the third assay, it is reported as indeterminate and patient is called back for repeat testing after 2-4 weeks. The test used as the screening test is one with the highest sensitivity (may give high number of false positives) and the supplementary second and third tests used are with the highest specificity (to minimize false positive reactions). This strategy is used for diagnosis of HIV infection in asymptomatic individuals. Counseling and informed consent are a must in these cases. Three different kits with different antigen system and / or different principles of tests are required to follow this strategy. Strategy IIB & III are to be used for diagnosis of HIV infection in symptomatic and asymptomatic individuals, respectively. A sample reactive in first assay and non-reactive in second assay is subjected to a third tie breaker assay. However, in case the third assay also gives non- reactive result, sample is reported as negative for HIV infection. In such cases when a negative report has been issued but the client history is suggestive of high risk factors/behaviour, he may asked to come for follow up testing after 2-4 weeks. Antibodies to HIV-1 are most commonly and reliably detected by E/R tests and confirmed by different E/R tests. Antibody testing by ELISA remains the standard method for screening potential blood donors; simultaneous testing for p24 antigenemia is superfluous because of low sensitivity and expense. Use of improved, third and fourth generation serological assays demonstrates that seroconversion typically occurs within 3-12 weeks post-infection, although significant delay can occur in some individuals. In diagnosing HIV-1 infection, the specificity of ELISA is >99% when properly performed and the sensitivity is >98%. In low-risk populations, the false-positive rate of combined EIA and WB testing is estimated to be <1 in 100,000. Highly sensitive and specific agglutination and EIA methods for detection of type-specific antibodies to HIV-2 are also available. Strategy I (algorithm I)

(For Transfusion/ transplantation safety) One test kit required A1

A1 + Consider Positive2 (Destroy the unit of blood as per guidelines refer to VCTC for confirmation of status after consent) 10

A1 Consider Negative

Strategy/Algorithm II A

(For surveillance) 2 Test kits required A11

A1 +

A1 Report Negative

A21

A1 + A2 + Report positive

A1 + A2 Report negative Strategy/Algorithm II B

(Diagnosis of an individual with AIDS indicator disease symptoms.) 3 Test kits required. A11

A1 +

A1 Report Negative

A21

A1 + A2 + Report positive with post test counselling

A1 + A2 -

A31

A1 + A2 - A3 + Indeterminate
3

A1 + A2 - A3 Report Negative 11

Strategy/Algorithm III [To detect HIV infection in asymptomatic individuals (VCTCs, PPTCTs) ] 3 Test kits required
A11

A1 +

A1 Report Negative

A2

A1 + A2 +

A1 + A2 -

A31

A1 + A2

A3 +

A1 + A2 + A3 Indeterminate3 A31

Report positive with post test counseling

A1 + A2 - A3 + Indeterminate3

A1 + A2 - A3 Report Negative

The screening test selected preferably should be fourth generation and the sensitivity of the test should be 100%. The supplemental/confirmatory tests selected should be >99.8% sensitive and >98.5% specific. Highly sensitive tests reduce the occurrence of false negative result and fourth generation HIV tests reduce the window period to 2-3 weeks, thus playing an important part in ensuring blood safety. Highly specific tests reduce the occurrence of false positive result and thus ensure that a person testing positive as per the strategy II/ III is actually HIV infected.
1 2

Assays A1, A2, A3 represent 3 different assays Such a result is not adequate for diagnostic purposes: use strategies IIB or III. Whatever the final diagnosis,

donations which were initially reactive should not be used for transfusions or transplants. Refer to VCTC after
3

informed consent for confirmation of HIV status Testing should be repeated on a second sample taken after 14-28 days. In case the serological results continue to be

indeterminate, then the sample is to be subjected to a Western blot /PCR if facilities are available or refer to the National Reference Laboratory for further testing. 12

All diagnostic results to be reported after post-test counseling.

Detection of p24 antigen

Detection of p24 viral antigen is expensive and not very reliable. The sensitivity of the test is also limited. Though a positive test may indicate HIV infection in adults, a negative test does not rule out HIV infection. It is undertaken in the following situations: To detect infection in the newborn (not reliable) To resolve equivocal Western Blot results To detect infection during early window phase To diagnose CNS (central nervous system) disease Late stage of disease (immune collapse) For research To monitor response to anti-retroviral therapy ( quantitative assay)

EIA for HIV-1 antigen detects primarily uncomplexed/dissociated p24 antigen, in serum, plasma, CSF or cell culture. It indicates active infection and allows diagnosis before seroconversion. Quantitative test can predict prognosis and may be useful for monitoring response to therapy. Disadvantages of antigen detection assays include: poor sensitivity (only 69% in patients with AIDS and low in neonates < 1 month old); detection is not possible in patients with high titers of p24 antibody (which complexes with the antigen); and failure to detect HIV-2 antigen.

Polymerase chain reaction (PCR)

PCR can detect proviral DNA during window period, can differentiate latent HIV infection from active viral transcription and can quantitate the copy number of HIV RNA when used with external standards (e.g. viral load assays). PCR can successfully differentiate between HIV-1 and HIV-2 infections. Proviral DNA can be detected in peripheral blood mononuclear cells before seroconversion. Limitations to the diagnostic use of PCR are rare false-negatives, some of which can be avoided by the use of multiple primer pairs and primers from conserved regions of the genome, and false-positives due to cross-contamination of the PCR reaction mixture. PCR is not to be used as a screening test. HIV-1 can be detected by PCR in the CSF of HIV-infected patients independently of disease stage; spread of HIV-1 to the brain represents an early event during infection which occurs in most asymptomatic individuals. PCR can also be used to detect HIV infection in neonates born to HIV infected mothers.

Virus culture
Virus culture is another method for identifying HIV infection. Positive culture rates of up to 98% are reported in confirmed seropositive individuals. The culture method is, however, expensive, labour-intensive, can take weeks for complete results and potentially exposes laboratory workers to high concentrations of HIV. Virus culture is used for research (drug sensitivity, vaccine studies, etc.) only.

Viral load assay

Quantitation of HIV RNA in plasma is useful for determining free viral load, assessing the efficacy of anti retroviral 13

therapy and predicting progression and clinical outcome. Because baseline HIV viral load is predictive of survival at 10 years in patients with nearly identical CD4 counts, assessment of baseline viraemia prior to initiation of therapy may be useful in patient management.

Indirect predictors of HIV infection

Decreased CD4 cells Increased 2 microglobulin Increased serum neopterin Increased IL-2 receptors AIDS defining illnesses.

Details about enumeration of CD4 cells and the uses of this marker in management of HIV infected individuals are given in Guidelines on enumeration of CD4 cells using single platform technology.

Unlinked anonymous testing

Such type of screening or testing is not directed to the individual, but has as its objective, the public health surveillance of HIV infection. It is a method for measuring HIV prevalence in a selected population with the minimum of participation bias. Unlinked anonymous screening offers a distinct advantage over mandatory or voluntary testing. Unlinked anonymous testing involves use of blood already collected for other purposes; therefore, the effect of selection bias will remain, though minimal, and will depend upon time, location and other details of blood collection.

Voluntary confidential testing

Testing is often done for diagnostic purposes. Here it is important that the issues related to confidentiality receive great attention. Since this method is based on voluntary HIV testing or testing for diagnosis of HIV/AIDS cases, it is imperative to respect the individual's need to maintain confidentiality. By maintaining confidentiality, it will not only instill faith in the individual about the health care system in the community but also encourage more and more people practicing risk behavior to come forward for an HIV test. This testing is done after counseling and informed consent of the client.

Mandatory testing
When testing is done without the consent of the patient and when the data could be linked to identify the person, it is called mandatory testing. Mandatory testing is recommended only for screening donors of semen, organs or tissues (to ensure transplantation safety) in order to prevent transmission of HIV to the recipient of the biological products.

Choice of HIV tests

The choice of tests is also based on the different objectives of HIV testing. The tests that are adopted are the ELISA or Rapid Test, clubbed together as 'E/R'. One E/R denotes a test done on one single antigen preparation; two is when all positive samples on first antigen test are repeated on a second antigen preparation / principle and three is when this test is repeated on the same sample for a third time using a different antigen /principle system. For transfusion safety purposes, one E/R is used; for surveillance two E/Rs are used; for diagnosis of AIDS cases two or three E/Rs are used, and for asymptomatic individuals three E/Rs are used. 14

General principles of HIV testing

Testing policy in general should consider the following points: It should be part of the overall comprehensive preventive programme. Testing should be technically sound and appropriate to the objective of testing. Test procedure must be appropriate to the field situation. Testing procedure must be cost effective. Laboratory procedures must be monitored for ensuring quality.

HIV testing in health care settings

The fear and apprehension that exists among health care workers in managing HIV infected individuals and AIDS patients is largely due to the risk of HIV transmission due to a needle stick or other sharp injury. Thus the demand for mandatory HIV testing of patients admitted in hospitals or undergoing surgery, etc is not rational or appropriate. A mandatory HIV test is no substitute for Standard Work Precautions that need to be adopted for every patient in a hospital or any other health care setting. On the other hand testing without explicit consent of the patient has been proven to be counterproductive in the long run. In the control of the HIV epidemic, such testing can drive the target people underground and make it more difficult for launching interventions. The national testing policy reiterates the following: No individual should be made to undergo mandatory testing for HIV. Mandatory HIV testing should not be imposed as a precondition for employment or for providing health care services and facilities. Any HIV testing except that undertaken for blood safety and transplantation safety must be accompanied by pretest and post test counseling services and informed consent. Confidentiality of the result should be maintained.

15

CHAPTER 3 HIV TESTING AT COUNSELLING AND TESTING SITES ( VCTCs AND PPTCTCs) USING RAPID TESTS
Introduction:
Rapid tests are in vitro qualitative tests for the detection of antibodies to Human Immunodeficiency Viruses (HIV) types 1 and 2 in human serum, plasma, whole blood saliva and urine. Currently HIV testing in India is performed on serum/whole blood (fingerprick), and plasma. This is because the HIV testing on urine and saliva samples has not been evaluated and validated in India. In recent times, a large number and wide range of rapid tests of high quality have become available and are being currently used in laboratories under the following conditions: Facilities to perform ELISA test are absent, In emergency cases Remote blood banks where the collection volume is low and the facilities for ELISA are not available (ELISA is the test of choice for HIV testing at blood banks) . Point of care settings like VCTCs, PPTCTCs, and also intervention healthcare sites, etc.

Types and technologies of rapid tests:

Different types of rapid tests are available. The various technologies on which rapid tests are based include: Immunoconcentration ( flow through) ( dot blot assays) Immunochromatography ( lateral flow assays) Particle agglutination (latex, gelatin, RBCs, etc) Immunocomb (Dip stick/ comb tests) ( mostly ELISA based)

Immunoconcentration:
This technology is available in the form of dot blot assays. These are multi step tests, very rapid to perform and results are obtained within a few minutes. The antigens (recombinant / synthetic peptides) are passively blotted on the nitro cellulose membrane matrix which is bound to the solid support and contains the adsorbent pads to collect the serum and reagents after their addition. Inbuilt control (internal control) in the form of a spot or line to indicate the validity of the test is available with each test.

Agglutination:
Agglutination assays are easy to perform and require no wash procedures. The antigens are adsorbed passively on the carriers (red blood cells, latex particles, gelatin particles and or micro beads). The antibody present in the serum sample reacts with the antigen adsorbed carriers, resulting in clumping of agglutinated particles. However, if in the reaction mixture the concentration of antigen and antibody is not optimum, phenomenon of prozone reaction (antibody excess) occurs which may lead to a false negative reaction. To overcome this initial dilution of the test sample is to be done as recommended by the manufacturer. Reactions are read visually. The control (uncoated or un sensitized particles) is run to detect non specific agglutination. If agglutination is detected in both the control and the test samples the assay needs to be repeated. Examples of these tests are Capillus and Serodia. 16

Capillus agglutination test

Reactive
Latex Aggregation White Clumping

Non-reactive
No Latex Aggregation no white clumping

Immunocomb assays:
The immunocomb is a rapid test intended for the qualitative and differential detection of antibodies to HIV-1 and HIV-2 in human serum or plasma. The immunocomb is an indirect solid phase enzyme immunoassay (EIA). The solid phase is a comb with 12 projections. Each tooth has 3 spots: Upper spot: Goat antibodies to human immunoglobulin (control to validate the test) Middle spot: HIV-1 synthetic peptides Lower spot: HIV-2 synthetic peptides The developing plate has 6 rows (12 wells each) with each row containing a reagent solution ready for use at different steps in the assay. Immunoglobulin present in the testing samples is captured by the anti human immunoglobulin antibody on the upper spot (internal control). Unbound components are washed away. IgG from the sample is captured on the teeth and reacts with antihuman IgG antibody labeled with alkaline phosphatase which react with chromogenic components and the results are seen as gray blue spots on the surface of the teeth of the comb Interpretation of controls: Appearance of upper spot- Negative control Appearance of all 3 spots- Positive control Upper spot does not appear - Invalid result Interpretation of results: Appearance of upper spot - Non reactive sample Appearance of upper and middle spot- Reactive for HIV-1 Appearance of upper and lower spot- Reactive for HIV-2 Appearance of all 3 spots - Reactive for HIV-1 and HIV-2 Post-assay activities: After the assay, discard the test devices in the box containing 1% sodium hypochlorite Discard the used filter paper into the bio hazard bags Swab the workbench and all equipments after use with 1% sodium hypochlorite followed by 70% alcohol 17

Diagramatic representation immunocomb test

Human Ig HIV-1 HIV-2

Positive control Reactive for both HIV-1 and HIV-2

Negative for HIV Control dot seen

Reactive for HIV-2

Reactive for HIV-1

Test not valid (Human Ig spot control dot not seen)

Immunochromatography:
These are lateral flow one step tests. These tests are usually temperature stable and can be stored at the room temperature. For HIV testing the rapid tests are used to detect the HIV antibodies. Examples of kits based on this technology are Determine, Unigold and Hemastrip.

Reactive
2 lines of any intensity appear in both the control and patient areas.

Non-reactive
1 line appears in the control area and no line in the patient area.

Invalid
No line appears in the control area. Do not report invalid results. Repeat test with a new test device even if a line appears in the patient area.

Possible outcomes of these rapid tests( Immunoconcentration, Immunofiltration, Dipsticks and Immunocombs) : Reactive or positive : Appearance of test band and control band Non reactive or negative : Appearance of control band only Invalid : Absence of control band. Test has failed. Repeat the Test with a new device

Advantages:
No equipment is required to perform the test. Micropipettes may be needed to dilute the sample for some assays eg. agglutination assays Limited infrastructure is needed. Some of the rapid tests can be stored at room temperature. These tests have wide temperature range stability. Rapid assays can be used in remote peripheral labs and for circumstances where same day results are required e g VCTCs and PPTCTs 18

The rapid assays are versatile as far as sample stability is concerned. These can be used on whole blood, serum, plasma, saliva or urine. Different kits are available for use on these different samples Most of the rapid tests are one to five step procedures Reading is subjective and visual with naked eye examination. However now rapid test readers are available for some tests, allowing us to perform semiquantitative analysis. Rapid immunoassay results can now be read objectively using CCD chips (as in digital cameras). Rapid tests are now more robust Management of waste generated by performing rapid tests is easier to manage as compared to that generated by performing ELISA Rapid tests work out more cost effective overall particularly in set ups like that in VCT and PPTCTCs as they are more robust, there is lesser cost of the requisite lab infrastructure and lesser cost for sample collection

Disadvantages
More expensive because of single test packaging and performance. Test performance may vary by the product. Refrigeration required for some of the products. Each test card cannot be quality controlled with an external quality control sample. There may be issues of sensitivity, specificity, negative predictive value and positive predictive value in relation to the reference gold standard. These should be carefully considered while procuring the product.

Accuracy of rapid tests

Many rapid tests available in the market are as accurate as the classical assays, e.g., ELISA. Some of the rapid tests may be less accurate. This happens when manufacturers do not follow total quality management and good manufacturing practices and cut the costs. In order to ensure quality of rapid tests, the WHO carries out and publishes evaluations as part of its Bulk Procurement Scheme. In addition, rapid tests can be validated vis--vis the classical assays in the specific locations before use. This was done by NACO before the rapid test kits for use at VCTC's were recommended i.e., the available rapid test kits were evaluated in a blinded multicentric study vis--vis available ELISA's by NICD on behest of NACO and the rapid test kits were recommended for use at VCTS's and later at PPTCTC on the basis of evaluation results obtained. HIV rapid tests were found to perform as reliably as ELISA's in the field.

Strategies for rapid test use

Rapid tests are used following the algorithm based on serial testing following the national strategies of testing appropriate to the objective of testing. The rapid test selected for screening should be 100% sensitive. Highly specific (> 98.5%) rapid tests should be selected for supplementary and confirmatory testing.

Uses of rapid tests

These tests can be performed on patients at point of care facilities like peripheral health care centers, pharmacies, emergency vehicles, out patient clinics, VCTC's, blood banks, PPTCT centers, hospital wards, homes, in the field and hospital wards.

19

Quality assurance practices

Most of the rapid test kits have inbuilt IgG / sample / reagent control spot/line which monitors the quality of kit as well as the quality of performance of the test. The IgG spot/line always shows up as positive provided the kit reagents are functioning optimally and the test has been performed as per the manufacturer's instructions. However these are the kit controls. In order to ensure the validity of the test result, and quality performance of test, a known positive external control must be tested along with the test samples using the same kit (same lot number and expiry, etc.) on day of performance of the assay. This will ensure quality of the kit as well as the procedure.

20

CHAPTER 4 TOTAL QUALITY MANAGEMENT

Introduction
Medical science is advancing rapidly and everyday new technologies, which are more sensitive and specific, are being developed. The more sensitive the technology, the more precise and stringent the processes have to be, to produce accurate results. This can only be achieved by practicing total quality management. A variety of highly sensitive tests have been developed and are being used to detect HIV infection in different healthcare settings. Minor error in performance of HIV tests can lead to repercussions of far reaching significance. A false positive result may have catastrophic implications for an individual and a false negative result may have disastrous implications for transfusion safety. Efficient performance of tests can only be ensured by rigorously conforming to the norms, elements and practices of total quality management.

Elements of total quality management

Management requirements Organization and management Quality system Document control Procurement of equipment, reagents and supplies Maintenance contracts Internal audit Complaints Preventive action Corrective action

Technical requirements
Personnel Civil Infrastructure Environment Equipment Sampling Handling of test and calibration items Assuring quality of test and the results Reporting of results Biosafety and safe waste disposal

Management requirements Organization

The organization under which the laboratory functions, is legally responsible for the activities undertaken in the laboratory. These activities (HIV testing, etc.) should meet the requirements of the clients and regulatory authorities. 21

The organization provides the managerial and technical personnel who have the resources and authority needed to carry out the assigned functions i.e. HIV testing in this case. The organization ensures that policies and procedures are in place right from the collection of the sample to the reporting of results. The quality system practiced is able to identify the occurrence of departure from standard procedures and errors and takes corrective actions as and when required. The organization is responsible for assigning responsibilities, ensuring smooth inter relationship of personnel who manage, perform and verify the laboratory activities. Organization also provides supervision and has overall responsibility for technical operations and provision of resources (equipment, space and supplies) and manpower to carry out quality HIV testing.

Quality system
Quality System also dictates that there will be a quality manual which gives details of all processes and procedures to be followed for HIV testing; a biosafety manual which will give details of standard work precautions to carry out the procedures and tests in the lab and guidelines on safe disposal of waste.

Document control
The documents form a part of the quality system. There are a number of documents generated in the laboratory ranging from the policy documents, procedure documents, worksheets, instruction sheets, equipment documents, reports and records, etc. A system has to be in place to control all these documents. The documents should be numbered and available with the organization. All the documents are available and accessible to the laboratory personnel. Documents with controlled access should be clearly listed. Authorized, current documents should be available at all locations where operations are performed. The old documents should be archived as per the laboratory norms for historical reference. Documents on procedures, equipment, etc. should be revised from time to time as required. Details on documentation are given in the relevant chapters .

Procurement of equipment, reagents and supplies

Procurement of equipment and supplies is an integral part of the quality system. Mostly it is done through the standard tendering process and contracts. The laboratory should ensure that the equipment, kits, reagents, disposables and other supplies meet the quality standards, are inspected and checked for quality before actual use. The procedure to procure equipment and kits is discussed in detail in the relevant chapters.

Maintenance contracts
All equipment in the laboratory must be under the annual maintenance contract (AMC) so that there are no interruptions in the functionality due to sudden breakdowns. AMC should be inbuilt in the procurement process.

Internal audit
Internal audits should be conducted periodically and in accordance with a predetermined schedule and procedure to verify and ensure that the laboratory procedures fulfill the requirements of the quality system. Internal audit serves to identify any lapses/errors in the system which can be corrected by corrective actions. The internal audit report should be tabled and discussed with the lab administration/ management so as to ensure adequate addressal of the nonconformances raised during the audit. 22

Complaints
There should be a procedure and policy in place to resolve the complaints received from a client. These may include delay in reporting the result, rude behavior of personnel, break in confidentiality and wrong result, etc. There should be an investigation following the complaints, the responsibility should be fixed and corrective action taken to resolve the complaint. A note on corrective action taken should be made in the records.

Preventive action
Preventive Action is a pro-active process to identify opportunities for improvement to prevent breakdown in equipment, work and quality system.

Corrective action
A problem with the quality system or with technical operations of the laboratory may be identified during internal/ external audit, management reviews, feedback from clients or staff observations. The corrective action has to be taken to resolve the problem. For this, an investigation to identify the cause of problem should be carried out by analysis of all potential sources of the problem. Many a times this may be the most difficult task. However once the root cause/ causes of the problem are identified corrective action should be taken (such as repair of an equipment, training of staff), documented and effect of corrective action must be monitored

Technical requirements Personnel

It is the responsibility of the management to ensure that staff posted in the laboratory has the requisite competence to carry out testing. The personnel working in the HIV testing laboratory should know how to operate and manage the equipment; how to interpret, record and report test results and how to implement all components of quality management system .The staff must be adequately trained and continue to undergo refresher trainings to maintain competence. All materials and supplies required to practice standard work precautions must be provided by the management. Immunization against HBV must be given to the staff working in the laboratory.

Civil infrastructure
The laboratory should have adequate space to carry out the procedures, house the equipment and space for personnel. There should be enough lighting and all power requirements should be met. Surfaces should be smooth and disinfectable. There should be a pest control system in place. Working surfaces preferably should be acid resistant.

Environment
A clean environment should be provided for the laboratory. The temperature and humidity should be maintained as per the requirements of the testing procedure and the equipment maintenance etc .The environmental conditions should not adversely affect the required quality of test or invalidate the test. Smoking, eating and drinking should be prohibited in the laboratory and signs to this effect must be displayed. 23

Equipment
It must be ensured that the equipment needed for the required testing is available and functional. The list of equipment required for the HIV testing is given in the relevant chapter . The equipment should be maintained as per the instructions of the manufacturer and calibrated as recommended. The equipment should be operated by authorized staff and the documents on equipment should be a part of document control. The details to be documented about equipment include name of equipment, name of manufacturer, serial number or any other unique identification, record of checks as per specifications, manufacturer's instruction manual, and the contract for maintenance, malfunction modification/ updating, etc. The procedure for calibrating the measuring equipment should be available. Other details on equipment procurement, maintenance and use are given in the chapter on equipment maintenance and calibration.

Sampling
Sampling is a defined procedure whereby part of a substance, material or product is taken to provide for testing or calibration of a representative sample of the whole. The sampling procedure should describe the selection, sampling plan, withdrawal and preparation of a sample from a substance, material or product to yield the required information. For example before the HIV test kits are imported in the country ,the Drug Controller General of India lifts a sample of kits for evaluation by the statutary institute. .

Handling of test and calibration items

The procedures for identification, transportation, receipt, handling, protection, storage, retention and disposal of test / or calibration items , including all provisions necessary to protect the integrity of the test or calibration items should be established and made available to the staff. There should be no deterioration of test and of calibration items during storage, handling and preparation. The instructions for transportation, etc. if any accompanying the test or calibration items should be provided and followed.

Assuring the quality of test results

Quality control procedures for monitoring the validity of the test should be undertaken each time the test is performed. Appropriate statistical techniques should be employed to review the validity of test results. All such techniques should be listed in the quality manual/SOP for the benefit of the lab staff. The standards can be achieved by: Regular use of certified reference material. Participation in external quality assessment/proficiency testing program. Replicate the test using same or different methods. Retesting of sample if required

Reporting the results

The results of the test carried out should be reported accurately, clearly, unambiguously and to the right person in the right manner i.e. after post test counseling. The result should be written clearly for interpretation by the clinician. 24

The report should include patients ID, name and address of the laboratory, mention of the sample, name of the test carried out, name of the test performed, name of the person who performed the test and the name of the person who reviewed the test result. All results should be reported with appropriate units and with applicable reference range.

Biosafety
It is essential to have the guidelines on standard work precautions in the laboratory. The bio safety practices are extremely important to prevent transmission of blood borne viruses to the technologist. These precautions should be practiced diligently at all times while working in the laboratory and/or providing other services in the health care facility. The bio safety practices are detailed in a separate chapter.

Safe disposal of waste

Much of the waste generated in the laboratory may be infectious. The procedure for disinfection and disposal of waste must be laid down and practiced strictly. Waste should be disposed as per the guidelines given elsewhere in this manual.

25

CHAPTER 5 Laboratory Management

Introduction
There is always pressure on the laboratory to produce quality results, using current technology, while keeping up with increasing demand to aid clinicians and program managers. This can be ensured by instituting a proper lab management system that looks after all the various aspects of HIV testing. A well developed lab management system should work to increase staff efficiency and reduce reagent wastage. It should therefore cover pre-analytical, analytical as well as post-analytical stages of laboratory testing. A well designed lab management system should consist of the following:

Laboratory Configuration
The key to a reliable HIV testing lab is for it to be optimally configured. This should involve the following: Equipment layout: The lay out of the equipment should be in line with the testing requirements of the lab. This may vary for a lab with high or a low testing throughput. This would include layouts for refrigerator, water bath, incubator, centrifuge, ELISA reader, etc. Workflow staging and direction: Work areas should be arranged to allow uni-directional sample flow and defined space for each test step. Defined areas are needed for: Specimen collection Specimen receiving and storage Specimen preparation Specimen testing-instrument Result production, validation and release Reagent and consumable storage Electrical requirements: These should be in line with the testing requirements of the laboratory. Staffing requirements: Decisions on staffing should give due consideration to the required level of expertise in terms of the type of laboratory and the tests to be performed. It should also take into account the number of trained staff required on the basis of the operational volumes.

Stock / inventory management of reagents and consumables

The laboratory should have a well defined inventory management system. The inventory or the stock management system could be manual or electronic depending upon the available resources. The system should be designed to ensure the following: Uninterrupted reagent supply to prevent reagent stock-outs. Clearly define the buffer stocks as well as the re-order levels Availability of other consumables, (e.g. pipette tips, gloves, needles, syringes, vacutainers, and other tubes). Stocks are in line with the testing demand/needs of the lab to prevent a situation where there is an excess stock of reagents / consumables. Any such situation would lead to wastage of precious resources on account of expiry of these goods.

26

Data management
Proper record keeping of patient results is vital for providing optimal patient care and gaining knowledge from patient data collected. A well defined data management system should: Ensure reliable and rapid delivery of results to clients / clinic sites Ensure clinics have systems for receiving and processing result data Ensure the laboratory maintains records of result data for defined periods Use standard reporting formats Ensure that dedicated human and other resources for data management are assigned

In addition, the data management system should work to ensure that: Laboratories examine all specimens accepted to ascertain that they meet the proper criteria for data entry and processing Laboratories verify that the results are for the intended patient Laboratories enter and process data correctly, to ensure that the results are given out in a correct and efficient manner. This can be achieved in the following manner : Results should be entered on both the worksheet and patient result form Worksheets should be filed by date in the laboratory for easy result retrieval Patient report form results should be entered and processed. All patient reports should be signed by the concerned technologist / technician and the same should be verified by the lab head. The release of results in the correct form is as important as conducting the test. The results should only be handed over to the concerned individual after confirming all the antecedents / particulars of the concerned individual & posttest counseling. Confidentiality should be maintained in all respects. Another important aspect of data management system is the archiving of results. Archives can be be electronic or paper-based. It should use a consistent system for data storage based on one or more variables (collection date, clinic site, patient ID) that allows easy reference and retrieval of records.

27

CHAPTER 6 QUALITY CONTROL

Introduction
Quality control practice aims to assess the measurement error. It is defined as the set of systematic procedures required to be implemented in the laboratory to evaluate and monitor the accuracy and precision of any analytical process. Quality control indicates that the assay is valid, all test conditions for that run have been met, and all test results for that run are reliable. Quality control however does not ensure that the results are accurate since the accuracy of the result may be influenced by factors like characteristics and limitations of the test employed. Moreover a run that passes the quality control does not ensure that the results have been reported properly and/or reported to the right individual in the right manner. In order to assure that the results produced by a HIV testing laboratory (BTC, surveillance centre, VCTC and PPTCTC) are accurate, it is essential that laboratory personnel have the knowledge about each and every aspect of HIV testing. There is a vast network of HIV testing laboratories in the country. Though the monitoring of the proficiency program has been started (EQAP) there is still a lot to be done to make the program a success. The laboratories are not practicing internal quality control on a day-to-day basis. The problem is further compounded by a wide variety of test kits used by these laboratories. As the generation of false positive and false negative results is associated with social, ethical and legal implications, it is extremely important to practice quality assurance for HIV testing to avoid the occurrence of inaccurate results. The standard operative procedures for practicing quality assurance are available with few HIV testing laboratories across the country. Moreover, the simple measures that could be undertaken to ensure quality of testing in the labs is often viewed as a complicated task due to lack of background knowledge. This chapter has been designed as an attempt to help the laboratory personnel at HIV testing laboratories to understand, adopt and practice quality control for HIV testing in their respective facilities. The chapter describes the procedures for quality control in HIV testing presuming that infrastructure, manpower and equipments required for the purpose are in place. This can also be used as a model to prepare standard operating procedures for practicing quality assurance for other transfusion transmitted infections.

Scientific principles involved

Routine procedures for separation, handling and storage of serum / plasma. Preparation of serial dilutions of serum / plasma. Running Enzyme Immunoassay (EIA) for HIV, employing HIV specific antigen coated plates. Calculation of statistical values like mean, standard deviation and coefficient of variation. Plotting of quality control graphs. Calculation of performance characteristics of an EIA kit in terms of sensitivity, specificity, predictive values and delta value. Ensuring quality assurance of rapid HIV testing procedures

28

Definition of terms Total quality management

Total quality management (TQM) refers to a comprehensive organizational approach that is focused on continually improving the quality and efficiency with which the laboratory operates. TQM is a component of quality assurance (QA) that implements and increases overall quality of the programme.

Quality assurance
Quality assurance is defined as the means by which the laboratory ensures that the final results reported are correct and are achieved in a standard, reproducible and traceable manner. Quality assurance helps ensure avoidance of mistakes; consistency of performance; data integrity; efficiency and cost effectiveness; customer satisfaction and laboratory esteem and credibility. Quality assurance practices are important to acquire standards to national and international accreditation.

Quality assessment
Quality assessment also known as proficiency testing and external quality assessment (EQA) is a means to determine the quality of results generated by the laboratory, which involves incorporation of proficiency panels prepared by an external agency into routine testing and analysis of the results produced after the testing of this panel. The process is known as the External Quality Assessment Programme. These results are then assessed and compared with the standard results, a performance score is developed and feed back is given to the participating laboratories. The aim is to assess the performance of the laboratory for specific tests, identify the factors responsible for incorrect results (material, processes, performance) and try to correct these through corrective actions and training. Specimens of EQAP (external quality assessment programme) must be handled in the same way as a specimen is handled for diagnostic purposes in the laboratory. The EQA process involves the following elements:Gathering information from the participant laboratories regarding the kits/assays being used. Preparation of serum panel. Distribution of panel Collection of results from the participating laboratories Collation and analysis of results Sending the analysis report to the participating laboratories Taking corrective actions for the participating laboratory performing poorly on EQA panel through locating the problem and resolving the same. Issuing participation certificate to all the participating laboratories.

Quality control (QC)

Quality control refers to the continual measures which must be undertaken for each assay to ensure that the test is working accurately as per the limits of the test so as to produce valid and acceptable results. 29

Essential components
The following essential components of quality control must be performed during every assay: Each test run must include one full set of controls that should yield results within the limits of the standard for acceptability and validity of the run. The values of controls obtained for the first run of the day cannot be used for subsequent runs. The controls have to be included in each run. Any run not having at least the minimum recommended number of controls falling within the acceptable range is invalid and should be repeated. All test kits should be used before the expiry date. Physical parameters of the test such as incubation time and temperature must be followed as per manufacturer's instructions.

Categories of controls Internal controls/kit controls

Refers to the negative and positive controls provided in the kits. These controls are useful but have the following limitations: They can be used only in those batches of kits from which they originate. Most of the controls in commercial kits are established artificially in a manner that minor deterioration of the kit may not be detected by the readings of kit controls. For the above reasons, the quality control programme relies more on the use of external controls for internal quality control.

External controls
These are a set of controls included from outside i.e. not belonging to the kit. They are applied for each run/assay to continually monitor assay performance (validity). A model Quality Control Programme that incorporates the external controls has the following main components: Making an external control with desirable anti HIV titer available Determination of acceptable ranges Inclusion of external controls in each run Data collection Analysis Reporting

Sources of external controls:

National Reference Laboratories. Commercial control panels (e.g. BBI panel from Boston Biomedical Inc.). However, these controls are expensive and thus may not be affordable for routine use by most laboratories. Producing in-house external controls from the sera subjected routinely for testing. Samples collected from another laboratory. However, the samples should be recharacterized and well standardized before being employed as external QC sample. Pooled test kit controls - ideal economically. 30

Use of freeze-dried controls

Freeze dried control serum samples can be stored at 2-8C for a very long time. In addition, the shipment of freeze dried samples is very simple and straight forward. However, during the process of freeze drying, antibody activity, especially of the borderline samples may be lost. Borderline control samples should not be freeze dried.

Preparation of external controls with desired titer

The categories of external controls to be used with each run comprise of a positive, a borderline reactive and a negative control. However, the ideal external control (or if one has to employ only one category) is the borderline reactive sample since this control is capable of detecting any minor error in assay performance. This is important in order not to generate false results among test samples having an OD near the cut off value.

The steps for preparing a borderline reactive external quality control sample are:
Selection of a high titer HIV positive plasma/serum. Serial dilution of the high titer HIV positive sample using normal HIV negative human serum. Selection of a suitable sample dilution to achieve the desired titer for use as external control with borderline reactivity. Batch production: preparation of bulk external (low positive) control sample. Batch validation. Dispensation and storage. Validation after storage.

Selection of a high titer HIV positive sample

Sources could be (i) either an HIV infected individual or (ii) a unit of blood found to be positive for HIV. In the former case, adequate quantity of serum could be obtained from blood collected from the individual and allowed to clot while in the latter case, the plasma is separated and recalcified to yield serum. Both plasma and serum are heat inactivated at 56C for 30 minutes. The source is subjected to an appropriate EIA (preferably more than one variety) to confirm high titer of anti HIV antibody before collecting bulk specimen.

Procedure for obtaining serum from plasma

Ideal material for preparation of external positive controls is serum. This is because plasma tends to be unstable on long storage and may clot spontaneously during transport. Under many circumstances blood collected from a blood bank may be the only source for HIV positive and HIV negative samples and thus plasma must be recalcified to produce serum. However, recalcification should be done with care because excessive recalcification may affect some assays like gelatin particle agglutination, adversely. Only citrated blood should be recalcified.

Recalcification of plasma to obtain serum

The procedure for obtaining serum from plasma is given below: A 10X recalcification solution (0.25M CaCI2. 6H2O, 55g and 0.08M MgCl2. 6H2O, 16g dissolved in 100 ml distilled water) is prepared and autoclaved at 121C for 20 min at 15 lbs pressure. 1.5 ml of recalcification solution is added to 1 unit of blood or 250 ml of plasma which has been brought to room temperature. Incubation is carried out at 37C for 30-60 min (until clot formation) followed by overnight storage at 4C. 31

Centrifugation is carried out at 1500 rpm for 20 min. The serum is separated aseptically. A small volume of serum is tested for HIV antibodies by screening and confirmatory tests (as well as for HBV and HCV). The bulk (i.e. remaining quantity of serum) can be stored in smaller and

convenient volumes at -20C, after labeling. If plasma is more than 7 days old, thrombin should be added to the plasma before re-calcification.

Serial dilution of the high titer HIV positive sample

This procedure is intended to prepare a sample with low reactivity to be included as external QC sample in each run. Many commercial organizations supply low positive QC panel sera which do not require this exercise unless there is a reason to recheck the titer.

Selection of diluents
The recommended diluent is normal human serum rather than normal saline or other sample diluents. This is to keep the antibodies in natural serum protein environment. Adequate quantity of serum for use as diluent can be obtained from a pool of normal healthy individuals (HIV, HBV and HCV negative) or from the unit of blood negative for HIV, HBV and HCV. In the later instance, plasma has to be recalcified to obtain serum as per the procedure mentioned earlier.

Protocol for serial dilution

Two fold serial dilutions(doubling dilutions) are prepared where a fixed volume of HIV positive serum is mixed and transferred into successive tubes containing an identical volume of diluent (i.e. normal human serum). When one unit volume of positive serum is mixed with an equal volume of normal human serum (diluent) the final dilution of the positive control becomes two fold or 1 in 2 (i.e. tube No. 2 in the figure 1). Accordingly when unit volume of material from tube number 2 (already diluted to 1 in 2) is mixed with unit volume of diluent in the next tube (tube no 3 in the figure) the dilution of positive serum will be again two fold of that of tube No 2 i.e. 1 in 4 of original and so on. This exercise of doubling dilutions will be helpful in subsequent steps (vide below) when ultimately the requisite dilution of positive serum, that would represent the low positive serum as external control, is to be worked out. For example, if it is found that dilution 1 in 256 of the positive control serum gives value acceptable to be used as external control, then 100l (or 0.1 ml) of this positive serum can be added to 25.50 ml of normal human serum to get 1: 256 dilution.

Fig. 1 Scheme for preparation of doubling dilution of HIV positive serum

Discard 1 Tube No 11 Positive serum Diluent Final dilution (reciprocal) Log2 dilution 2 3 4 5 6 7 8 9 10

200ml N 0

100ml 2 1

100ml 4 2

100ml 8 3

100ml 16 4

100ml 32 5

100ml 64 6

100ml 128 7

100ml 256 8

100ml 512 9

100ml 1024 10

32

Performance of EIA with serially diluted HIV positive sample

Each dilution is considered as a separate sample. An EIA kit is selected that is quality control approved and is well within the valid expiry date. Each dilution made above is charged in triplicate in the EIA plate (triplicates are shown in figure 2 as A1, A2 and A3 for each dilution). The triplicates of any particular dilution should not be charged in adjacent wells, but should be spaced apart randomly all over the plate to minimize well to well variation (intra run variation) as shown in Figure - 2. The EIA is performed as per the manufacturer's protocol.

Fig. 2 Conducting EIA with the dilutions of (in triplicates) HIV positive serum.
1 A B C D E F G H
CP = positive control (kit controls); CN = negative control (kit controls) 1, 2, 3...12 = Indicate sample (pre-diluted) numbers Note: Controls in the plate map are displayed as per the protocol of Innotest ELISA.

2 1

5 2

10

11 1

12

CP CP CP CN CN CN

3 1 2 3 3 2

Recording the results

The OD values of the samples (i.e. triplicates of each dilution as well as the controls supplied with the kit) are recorded as per the validity of the run. Cut off value is calculated on the basis of the reading of the controls supplied with the kit (internal controls) as per the kit protocol. The mean of the triplicate OD values of each dilution is calculated. The dilutions are plotted in X axis while the E ratios are plotted on the Y axis (vide below for the significance of E ratio).

Expression of antibody level in terms of 'E' ratio

Cut off values in EIA may differ depending on the principle of test, manufacturer and the recommended protocol for its calculation. Following are the few examples of acceptable ranges of cut off values recommended by different manufacturers. (Table 1). Even with the day to day use of kits from same manufacturer with identical batch numbers, some degree of variations in the internal controls (supplied with the kits) are encountered, that result in turn in the variation of the calculated cut off value, that is calculated on the basis of OD values of the internal controls. This is due to variation in factors like incubation conditions, preparation of the reagents, plate to plate as well as well to well variation in the amount of 33

coated antigens, etc. However, such factors influencing the OD values of the controls would also expectedly influence the OD values of the test samples in similar direction. Thus the relative reactivity of a given test sample in relation to cut off value would not vary much. This relative reactivity of a test sample in relation to cut off value in a particular run is expressed and termed as E ratio. This is the ratio between the sample OD and cut off OD. Hence it is more appropriate to express the degree of ELISA reactivity in terms of E ratio rather than individual OD values as explained. Following are the examples of two unknown samples, designated X and Y, run on different dates using kits from same manufacturer and same batch (Table 1 and 2).

Table 1 Ranges of cut off values recommended by different manufactures

Name of the kit Principle of Manufacturer Range of cut off OD values (in 10 consecutive runs employing same batch of kit ) 0.176 - 0.313

1.

Innotest HIV 1+2

Indirect EIA

Innogenetics NV, Belgium Biochem Immuno System Inc, Canada United Biomedical Inc., USA Cambridge Biotech Ltd., Ireland

2.

Detect HIV

Indirect EIA

0.160 - 0.240

3.

UBI HIV 1/2 EIA

Indirect EIA

0.125 - 0.205

4.

Recombigen HIV - 2 EIA

HIV-1

Indirect EIA

0.393 - 0.518

Table - 2 Variations in the EIA values of two samples X and Y run in different EIA kits
Sample number X 1 Y X 2 Y X 3 Y 0.037 0.032 1.778 0.518 0.071 0.029 1.512 0.417 0.076 3.432 0.363 0.066 3.625 Run day Sample OD 1.262 Cut off OD E ratio 3.476

As evident from above example, the variation of OD values expressed as 'E' ratio is much less compared to OD value above.

Selection of suitable sample dilution from the dilution curve

Results usually show a sigmoidal curve. As mentioned earlier, dilutions suitable for using as low positive controls are generally selected at 'E' ratio of 1.5-2.0 and around 0.70 (i.e. 1.5-2.0 times and about 0.70 times the cut off OD value) for an indirect and competitive EIA respectively (Figure 3.). 34

Fig. 3 EIA reactivity (E ratio) of HIV-1 positive serum by indirect EIA Innotest HIV-1/HIV-2, (Innogenetics kit) in relation to serial dilutions in normal serum.
10 9 8 7 6 o 5 4 3 2 1 0 1 2 3 4 5 6 7 8 9 10 11

Log2 dilution of HIV positive serum

Solid line indicates the cut-off limit for interpretation of result Arrow indicates suitable dilutions for preparation of external control

Batch production: Preparation of bulk external control Volume requirement

The requisite volume of the low positive control to be prepared for use as external control will depend on: How long the external control of the current batch needs to be used (e.g. for one year or 6 months). The sample volume required for the assay (e.g. 10 - 20l for most indirect EIAs). How often the assay is performed (i.e. number of specimens tested per day). This will indicate the number of times the external control would be needed over a specified period. Number of laboratories to which QC sample is to be supplied for use as external control.

Procedure
HIV positive sample and diluent are filtered to remove bacteria and fungi. However, filtration of recalcified serum is often difficult as filters tend to clog by fibrin clots. Hence the positive sample should be centrifuged, prefiltered with a 0.8m biological filter to remove aggregated proteins or debris and then filtered through a 0.22 m biological filter to remove contaminating bacteria, although there will be inevitable reduction in volume of the valuable specimens selected as controls. The positive sample is centrifuged and filtered (0.22 m) followed by heat inactivation at 56C for 20 minutes. Positive sample and volume of diluent i.e. normal human serum containing preservative (e.g. Bronidox) is mixed in the volumes required to yield the required titer (as described above) and kept for 16-20 hours at 4C on a magnetic stirrer. The required titer is ensured by running EIA for 10 runs for 10 days. The batch thus produced is aliquoted into large polypropylene storage containers (e.g. 250 ml). Polyethylene containers are not recommended as these absorb antibodies and thus may affect the titer of the QC sample. 35

An I.D number is assigned to the lot with date of production mentioned. All containers to be used to produce the batch should be autoclaved.

Batch validation: Determination of degree of inter-aliquot variation Principle

This process is to check that the batch has been sufficiently mixed and is homogenous so as to minimize the interaliquot as well as inter run variation. Higher the dilution required to produce QC sample of required titer, more is the importance of such validation. Aliquots (25 shown in figure) are dispensed from each storage bottle. This means 125 aliquots from 5 storage bottles. These aliquots are tested in the assay or assays in which the sample is to be used as a QC sample subsequently. The batch should be accepted if (i) the reconstituted batch after aliquoting yields the targeted titer (ii) the samples show minimal variation following aliquoting (i.e. inter aliquot variation). The aliquots of samples as selected above are subjected to EIA by the EIA kit of the brand that is going to be employed in daily use. The internal controls supplied with the kit are included to check validity of the run. The kit selected for this purpose should be licensed and quality checked. Preferably, an external control from the previous lot should also be included in the run. The mean (X), standard deviation (SD) and coefficient of variation (CV) of the 'E' ratios of a total of 25 aliquots are calculated as per example given below in Table - 3.

Table - 3 An example of values obtained while testing batch variation of 25 batches of external control

36

Mean
The mean (expressed as X) is calculated by adding individual observations (X1+X2+X 3------Xn or X) and then dividing by the number of observations (expressed as n). Thus mean is calculated as follows:

In the data shown in table, the mean 'E' ratio calculated as the sum of individual 'E' ratios divided by number of aliquots is found to be as follows:

Standard deviation
Standard deviation (SD) is a measure of the variation or dispersion of observations about the mean and defines the expected range of a control in relation to the mean value.

Standard deviation is calculated as below: _

Each of the individual values ('E' ratios) are compared with the mean (X) to find out the deviation from the mean. Thus for sample with 'E' ratio as X1 the deviation will be X1 ~ X, for the next sample with 'E' ratio as X2, the deviation will be X2 ~ X and so on. The deviation is also expressed as 'd' conventionally.

37

Coefficient of variation
The coefficient of variation (CV) is expressed as percentage and the following formula is used.

Fig. 4 Batch validation (inter-aliquot variation) of 25 aliquots of external control sample; coefficient of variation (CV) is calculated to be 6.82%.

A Levy-Jennings chart in which E ratios are plotted against the Y axis and the individual samples (aliquots) plotted against the X axis are shown in Figure - 5. 38

Fig. 5 Levy-Jennings chart to plot a QC graph on the basis of values ('E' ratios) obtained in 20 consecutive days run of external control.

Coefficient of variation less than 10% is considered to be an indication of little inter aliquot (batch) variation and thus consistency can be expected in the performance of various aliquots as external quality control in daily run.

Dispensation and storage of quality control samples

Even though, one may start with sterile serum samples, subsequent use and manipulation in the laboratory may easily contaminate these. Some of the preservatives that could be used are (i) Bronidox (5-bromo-5-nitro-1, 3-dioxane) in propylene glycol to a final concentration of 0.05% in serum, may be used conveniently. (ii)Thiomersal (mercuric chloride) can be used in a final concentration of 0.01%, but is effective only for a few weeks as it loses its activity when exposed to light. (iii) Sodium azide is not recommended as it inactivates peroxidase conjugate. Following validation, all the batches of reconstructed low positive controls are stored at -20C or below in non self defrosting freezer upto 1 year. These batches (already validated) are to be utilised one at a time for preparation of large number of aliquots, with volume per aliquot sufficient to last for one week in routine testing (e.g. 200l/ aliquot). It is recommended that the aliquots are stored divided in two freezers in different locations keeping in mind the possibility of electrical failure. One aliquot is thawed at the beginning of the week for use for that week only, following which it is discarded. It is stored at 2 to 8C in between, during the week. Use of quality control samples is to validate each test run.

Determination of acceptable ranges of quality control to validate each test run

Range for accepting individual runs of the external control as valid is an important parameter that needs to be established. This is carried out by testing the external controls for a sufficient time to establish the limits before the actual use of external controls to validate the runs. This is done by employing statistical parameters like mean, standard deviation and coefficient of variation. Subsequently, the external controls, in conjunction with the internal kit controls, can be used to validate all test runs.

Procedure
The external QC sample is tested in at least 20 runs (e.g. in 20 consecutive days) to make statistically significant observations. 39

The mean and standard deviation of 'E' ratio of the external controls are calculated (as per method mentioned above) in each of the 20 runs separately. The coefficient of variation (CV) of the external controls is calculated on the basis of mean and standard deviation of the 'E' ratios.

It is important that coefficient of variation (CV) of the 'E' ratios observed on different dates is minimal (i.e. <20%). An example of values obtained in 20 consecutive runs of external controls is shown in Table - 4.

Table -4 Examples for calculations of acceptable range of external control

Data analysis
As per the standard methods of calculations the following observations are made about the above mentioned runs of EIA: Mean ( X) Standard deviation (SD) Limit of Mean 1SD Limit of Mean 2SD Coefficient of variation (%) = = = = = 1.90 0.19 1.71-2.09 1.52-2.28 11.6% 40

Having assured the minimal batch variation (inter aliquot variation) i.e. CV < 20%, as tested in at least 20 consecutive runs, the external control is considered to be suitable for inclusion as external control in subsequent daily runs of EIA.

Preparation of a QC graph (Quality Control Chart)

Following validation of minimal variation of the external control values (i.e. CV < 20%) the 'E' ratios are plotted on the Y axis in the Levy-Jennings chart against consecutive dates of runs plotted on the X axis. On the basis of standard deviation (SD), calculated as above, a limit of 2 SD is drawn on either side of the mean This limit of mean 2 SD is accepted as the limit for assessing the function of external controls in day to day monitoring of the run of EIA to check validity Figure 5.

Incorporation of external control in daily test run

The external controls are treated as any other serum sample being tested in the EIA run. EIA is performed as per manufacturer's guidelines with the conditions identical for the external control as well as test samples. The observed OD values of the internal (kit) controls are checked and matched with the limits prescribed by manufacturer as acceptable to ensure that they are within acceptable limits. The OD value of the external control is recorded. The 'E' ratio for the external control is calculated on the basis of OD value of the external control and cut off value obtained in the same EIA run. OD value of external control Cut off OD value

'E' ratio =

The observed 'E' ratio is matched with the Levy-Jennings chart prepared (as described above).

Interpretation
If the 'E' ratio of the external control falls beyond 2 SD limit of mean in Levy-Jennings chart, the run of EIA is treated as quality control failed or invalid even though the internal kit control values are within acceptable limits. On the other hand, if it falls within the limit mentioned above, the run is considered as valid.

Variations in the quality control validation guidelines

Some authorities recommend inclusion of the borderline external control in duplicate in each run. A run is considered as invalid if (i) both external control values are beyond the Mean 2SD limit or (ii) one of external control values is beyond 3 SD limit or (iii) the external control value exceeds the standard deviations in two consecutive runs. While adding the duplicate external controls, it is recommended that one is charged into the well in the first vertical row immediately after dispensing the internal kit controls, while the other is charged in the last vertical row randomly. Care is taken to add the duplicate controls as per the normal sequence of charging the specimens in the wells i.e. the first of the duplicate control is charged in the beginning while the second one is charged at the end along with the test specimens which are being charged in the last row. This procedure is likely to take care of the variation of values due to time delay of operation of various steps between the Ist row and last row.

41

According to some authors, setting up the acceptability limit as Mean 2 SD is likely to lead to perceptible degree of rejection due to random error. Hence, they recommend the extension of the limit of SD to + 2.5 instead of 2 with the assumption that 5% or less of any set of control values will fall outside the range due to chance.

A relatively more stringent measure of quality control recommends incorporation of external controls (or even internal controls) in triplicate at 3 random wells in the ELISA plate in each run to check intrarun variation.

Importance of 'E' ratio over OD value of external controls to check validity of runs
There are two methods by which the reference QC graph may be represented. The first one is to plot the OD values of the external control on the Y axis and the consecutive run members (i.e. run dates in case one plate is run per day) on the X axis. The second option is to plot the 'E' ratios i.e. ratio between the OD value of external control and the cut off value on the Y axis instead of the OD value of the external control alone, the determinants on the X and Y axis remaining as in the first method. The second method is considered to be more accurate alternative since any general change in the conditions during the run of EIA on a specific day/run will affect the OD value of the external control as well as kit controls towards a similar direction (explained in details previously in section 2). This is exemplified in the following example of six EIA runs on consecutive dates using permissible limit of (i.e. mean 2SD) in the values of 20 runs mentioned in example for setting the limit of acceptability of external controls for validity of runs. The acceptability limits of external controls for validation of test results calculated as below (based on OD values of 20 runs above). Mean (X) of OD SD of OD Limit of Mean 1SD Limit of Mean 2SD = = = = 0.428 0.055 0.373 - 0.483 0.318 - 0.538

On the other hand, as shown in Table -4, the following values are obtained if the 'E' ratios in the same runs of EIA are taken into consideration to validate the run. Mean (X) of 'E' ratio SD of 'E' ratio Limit of Mean 1SD Limit of Mean 2SD = = = = 1.90 0.19 1.71 - 2.09 1.52 - 2.28

Now, let it be presumed that the following six consecutive runs of EIA are evaluated for their validity based on the performance of external control Table 5.

42

Table - 5 Recording of OD values in six consecutive days of test runs.

It can be observed from Figure - 6 and 7 that when the OD values of the external controls on different days of test run are matched against the acceptability limit (mean 2SD) drawn on the basis of OD values of the external control alone, the run on day 2 appears to be invalid since the observed OD value on that particular day (i.e. day 2) lies beyond acceptability limit i.e. more than mean 2 SD limit or 0.538). On the other hand, if the corresponding 'E' ratios are matched against the acceptability limit (i.e. mean 2SD ) drawn on the basis of 'E' ratios of the external control, the run on day 2 appears to be within the acceptability limit, while the run on day 4 appears to be invalid (Figures - 6 and 7.).

Fig. 6 Validity of runs evaluated on the basis of OD values of the external control alone. Cut off OD values are shown at the bottom as broken line

Fig. 7 Validity of runs evaluated on the basis of ELISA ratios (E ratio i.e. external control OD/ cut off OD) of the external control.

If the OD values are further examined for the runs in question, it is clear that, on day 2 although the OD value of the external control went up, there was some degree of rise in cut off OD value as well. Thus the net change in the ratio between the external control sample OD and cut off OD (i.e. 'E ratio) did not fall outside the acceptability range when 43

matched in terms of 'E' ratio. On the other hand, on day 4 of run, there was some degree of elevation of external control OD value while cut off value did not show any such change rather it showed change on reverse side which is not as per expectation (as explained in earlier section). Thus there is a rational ground to consider the run on day 4 as invalid Table - 3 & 4, Figure - 6 and 7

Fig. 8 Interpretation of aberrant results of QC: shift and trend Illustrations of shift and trends

Interpretation of aberrant results of QC: shift and trends (Figure - 8)

Control values of six consecutive runs fall on one side of the mean line. Can be due to the following: Switching to a new lot of kits New reagents Changes in incubation temperature New technician

Trend
Six successive points distributed in one general direction (towards either higher result or lower result) within the acceptable range. Can be due to the following: Deteriorations of reagents Slowly faltering equipments e.g. pipette

Actions taken for aberrant QC readings include the following checks:

The date the aliquot of external QC sample in use was opened is checked. If aberrant results are enumerated in 3 consequent runs the aliquot in use is discarded and a new vial of QC sample is opened. Both aliquots (new and discarded aliquot) are tested on the next run to verify the QC sample's deterioration. The storage conditions of the QC sample: The aliquot in use can be stored at 4 C for 1 week, all other aliquots must be stored at -20C. No QC sample should be freeze/thawed and refrozen. 44

The date of expiry of kit/reagents and/or any indications of deterioration/or contamination. The performance of the operator of the test to ensure the assay is being performed correctly. Whether or not the aberrant results were produced with a particular batch of kit.

Further actions to be taken may include

Checking equipment (e.g. washer, incubator, reader). Checking the kit and reagents more thoroughly for deterioration or contamination.

Flow CHART FOR PERFORMING HIV TESTING USING QUALITY CONTROL ALIQUOT Performance of EIA with external control (HIV)

45

FLOW CHART FOR PERFORMING RAPID HIV TESTING USING QUALITY CONTROL ALIQUOT

Selection of Rapid test

* *

Licensed Quality checked As per manufacturers guidelines Internal controls are checked Include one positive external control daily Examine for either coloured dot/dots, or coloured line/lines and/or agglutination Ig spot should always give positive result. Positive control should give positive result (colored dot, line and/or agglutination

Performance of Rapid HIV test

* *

Inclusion of external control in daily run Examine cartridge, comb, etc. for result

* *

Validation of rapid test result

Test result invalid

-Ig spot not seen -Positive control result does not show as positive

46

CHAPTER 7 COLLECTION, TRANSPORT AND STORAGE OF SPECIMENS FOR HIV TESTING

Introduction
Almost all laboratory procedures for HIV testing are performed on patients' blood; serum or plasma; hence the collection of blood is described below.

Performing venipuncture:
Gloves should be worn and sterilised /disposable syringes and needles should be used. For avoiding soiling, a piece of linen with a layer of dressing pad ( a sheet of absorbent cotton between two layers of gauze piece) or simply a big piece of absorbent cotton may be placed below the forearm before commencing veni-puncture. After collecting 3- 5 ml of blood aseptically, it should be carefully transferred from the syringe without squirting into a sterile plastic leak proof specimen container preferably screw capped. The containers should be labelled before commencement of venipuncture. The cap may be tightly screwed after the blood has been transferred to the vial. After blood is collected, the tourniquet is removed and the needle is withdrawn. The patient is given a dry sterile cotton swab to press over the site of venipuncture. Elbow may be flexed to keep the cotton swab in place till the blood stops. Any blood spill is carefully wiped with 70% ethanol/10% bleach solution. All the swabs and cotton pieces are placed in plastic bags for disposal. If the outside of the vial is visibly contaminated with blood, it should be cleaned with 10% freshly prepared sodium hypochlorite solution. The blood is allowed to clot for 30 minutes (not more than 2 hours) at room temperature. The clot may be gently broken if necessary using sterile pasteur pipettes. Alternatively vacuum based blood collection systems (vacutainers) can also be used. These vacuum based collections are relatively safe for usage and harbour minimum risk of unwanted exposure to infected blood.

Separation of sera samples:

The vial / vacutainer should be centrifuged at 3000 rpm for 10 minutes to separate serum to avoid haemolysis. If no centrifuge is available, the blood with clot may be left overnight in the refrigerator at 4C.The clot will retract and get separated from serum.

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The specimen vial is un-stoppered, the serum is drawn off by sterile Pasteur pipette and transferred to a sterile plastic screw capped leak proof tube.

Addition of preservative:
The usual preservative should not be added since it inactivates conjugates and gives rise to false serological results. If necessary, 5 bromo, 5 nitro, 1-3 dioxane in propylene glycol at a final concentration of 0.05% is recommended as preservative. Thiomersal at a final concentration of 0.01% is effective only for a few weeks as it loses activity when exposed to light.

Storage of serum specimens:

The sera samples are placed in leak proof plastic containers in the refrigerator at 2-8C, for upto 48 hours for storage. The storage of samples should be as per test requirements. The outside of the container is checked for visible contamination with blood which should be cleaned. All the specimen vials must be adequately labelled with patient details. Then the specimen vials are packed in a second tightly capped unbreakable container surrounded by adequate packing material (see figure 1 ahead). For storage for a longer time, specimens must be frozen at - 200C or deep-freezing at - 70C is advised, in labs where deep freezers are available.

Sample transportation
These instructions are recommended for specimen transportation: The shipment of infectious agents is regulated by the Transportation of Dangerous Goods Act and the International Air Transport Association (IATA) dangerous goods regulations. HIV infected specimens are classified as infectious class 6.2 substances under the United Nations (UN) no. 2814. The packaging must adhere to UN class 6.2 specifications. Packaging requires a 3 layer system as described below (see Fig. 1 for a diagrammatic representation): The specimen tube, in which serum is to be transported, should not have cracks / leakage. It should preferably be made of plastic and be screw capped. The outside of the container should be checked for any visible contamination with blood which should be disinfected. Place the tube containing the specimen in a leak-proof container (e.g. a sealed plastic bag with a zip lock or alternatively the bag may be stapled and taped) and pack this container inside a cardboard canister / box containing sufficient material (cotton gauze) to absorb all the blood should the tube break or leak. Cap the canister/box tightly. Fasten the request slip securely to the outside of this canister. This request slip should have all details i.e. name, age , sex , risk factors, history of previous testing, etc. and should accompany the specimen. The request slip should be placed in a plastic ziplock bag to prevent smudging on accountsof spillage. 48

For mailing, this canister/box should be placed inside another box containing the mailing label and biohazard sign.

The diagram ( fig 1) depicts the method of sample transport for a single/ few ( 2-3) samples that could fit into the secondary container shown in the diagram. The size of the primary sample container will vary with the number of samples being transported. For a larger number of samples ,a tube rack (or some such container ) may be used wherein the samples can be transported in the upright position and at appropriate temperature. The packaging instructions for the transport of a larger number of samples are given below: The specimen should be carefully packaged to protect it from breakage and insulated from extreme temperature Label appropriately and mention the test/s being requested for that sample. The collection site should make use of a unique identification number as sample identity. Names of the patients should be avoided to prevent confusion on account of duplication of names as well as to maintain confidentiality. Secure the vacutainer cap carefully and seal it further with sticking tape ( placed so that it covers the lower part of the cap and some part of the tube stem. During packaging, the tubes containing specimens should be placed in a tube rack and packed inside a cool box (plastic or thermocol) with cool/ refrigerated / frozen gel packs ( as appropriate to keep the sample at the recommended temperature for the test ) placed below and on the sides of the tube rack. Place some cotton or other packaging material between the tubes to ensure that they do not move or rattle while in transit.. Cool box required for transportation could be a plastic bread box or a vaccine carrier. Seal/secure the lid of the cool box . This cool box should then be placed in a secure transport bag for purposes of shipping to the testing facility. The request slips should be placed in a plastic zip lock bag and fastened securely to the outside of the cool box with a rubber band and sticking tape. A biohazard label should be pasted on the visible outer surface of the package containing the samples. The package must be marked with arrows indicating the 'up' and 'down'side of the package Samples should be transported to the receiving laboratory by commercial courier or be hand delivered by a trained delivery person. The collection site must have prior knowledge of the designated testing days of the laboratory to which the samples are being sent. No transport should be done during weekends and holidays or non-testing days of the testing laboratory unless prior arrangement has been made with the receiving laboratory.

Note: Use overnight carriers with an established record of consistent overnight delivery to ensure arrival of specimen within the specified time.

Figure 1 Packaging of specimen for transport to the laboratory.

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Safe Handling and disposal of sharps:

Extreme care should be used to avoid auto-inoculation. All chipped or cracked glassware should be discarded in appropriate containers. Broken glass should be picked up with a brush and pan. Bare hands must never be used. The disposable needles should never be manipulated, bent, broken, recapped or removed from syringes. The used sharps should never be passed directly from one person to another. One should always dispose of his/her own sharps. Used needles should be discarded in puncture-proof rigid containers (plastic or cardboard boxes) after disinfection in 0.5-1% freshly prepared sodium hypochlorite solution (common bleach) and never in other waste containers. If a needle shredder/destroyer is available, only the needles or the needles along with syringe nozzle may be shredded depending upon the type of the shredder Sharp disposable containers should be located close to the point of use. Sharp disposal containers should be sent for disposal when three-fourth full. In case suitable means of disposal of syringe / needles are not available, these disposable syringes should be heated in dry ovens and be allowed to mutilate to prevent recycling of plastic syringes. Needles can be incinerated.

IN CASE OF DAMAGE OR LEAKAGE IMMEDIATELY NOTIFY PUBLIC HEALTH AUTHORITY

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CHAPTER 8 STANDARD OPERATIVE PROCEDURES

Standard operative procedure (SOP) must be prepared for each activity and all tests being conducted in the laboratory. The SOP should give detailed instructions on the following: Name and scope of the test Name of the kit being used Name of the procedure (ELISA, Rapid Test) Collection, transport and management of the specimen. Storage and inventory (logging in the specimen and storing for reference or retesting). Test requisition: the information required to be collected on the sample which may be relevant and have implications for interpretation of the test. Details of environmental requirements (temperature, humidity etc.) Details of performance of test-each step of sample processing to arrive at the result. Instructions for quality control practices, types of internal (kit) and external controls required to be processed for validation of the test run. Instructions regarding the interpretation of the test. Instructions on use of the algorithm as per the policy. Instructions on recording the test result. Instructions on reporting the test result, whom to report and how to report.

SOP should be available at testing site and should be followed every time the test is performed. The SOP must be simple to follow and should be in the form of flow diagram / chart, one step leading into the next and easy to interpret. SOP must also include all the pre-analytic, analytic and post-analytic requirements for performing the test keeping the quality issue in view.

A sample SOP
Name of the test procedure. Intended use: detection of HIV-1 and HIV-2 antibodies. Principle of the test procedure: details as per the manufacturer's kit insert have to be given. Details of the kit contents like conjugates, negative and positive controls, plate / cartridge / comb etc. List the materials which are required for performance of the test but are not provided in the kit. Storage instructions.

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The precautions to be taken while performing the test, standard work precautions and any other precautions specific to the test.

Specimen collection and storage of whole blood, serum or plasma; How the serum / plasma should be separated; How the specimen is to be used and how it should be stored.

Quality controls required to be put to validate the tests. Details of the test procedure as per the instructions in the kit insert. Details about how to validate and interpret the results as per instructions of the kit insert. Limitations of the test in detecting infection. Standard work precautions. Details about disposal of left over samples and other wastes generated. Details about recording the result and reporting.

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CHAPTER 9 DOCUMENTATION AND DOCUMENT CONTROL

Introduction
Quality management system documents are extremely important to ensure quality practices in the laboratory. All instructions, processes, procedures must be documented; the staff must be aware about the documents and understand how the documents work. Documents must be available to the staff whenever they are needed. The staff must follow the instructions provided in the documents. Since, the functions, processes and procedures of laboratories are constantly changing, so the documents should be revised constantly as required. Verbal instructions are very unreliable as a means of communication as these may not be heard, may be misunderstood, may be ignored or may be quickly forgotten particularly so if it is complex technical information. The documents required for smooth functioning and accreditation of a laboratory include: Quality manual: This documents and gives details of the quality policy, the quality system structure and the key areas of activity. It gives an overview of all the functions carried out by the laboratory and provides link to all other documentation. Procedure documents: gives details of processes and each procedure being performed in the laboratory documented in the form of standard operative procedures (SOP). SOP must be easily accessible and available to the staff. It also gives details of assignment of various procedures to the staff. Work instructions: These are to be specified for each staff member and should be clear and understandable by the staff - what to do and how to do it? It also describes how specific tasks referenced in procedures are to be carried out. The jobs for each staff are defined and documented for optimal and quality functioning and to avoid conflicts. Forms: This should include all types of forms being used by an organization / laboratory to perform the required jobs, collect the relevant information important for performing a test / job, analysis and interpretation of results, etc. Sample collection forms, report forms, indent forms, are some of the examples of the forms. Records: A number of records are generated in the course of activities of the organization / laboratory. These records must be maintained for future reference, audit and legal purposes, etc. These records include details of personnel, training, results, etc. Documents need to be numbered and filed. Documents need to be revised/changed as and when required e.g. when a procedure is changed, the documents i.e. work instructions and work-sheets need to be changed accordingly. The documents should be designed in such a way that they are easy to follow, legible and serve the required purpose. The aim is that the document should work well for communicating information. The current documents should be available & accessible for use. The old documents should be archived and not destroyed. This is important to provide an accurate historical record. For example, if a modification is made to a laboratory method, it is important to know what was the modification, who made the modification and when was the modification made. The revised/changed document must be shared/ communicated to the staff. 53

Types of documents
Customer complaint document. Corrective and preventive action document. Equipment maintenance, calibration, and monitoring document (details in chapter on equipment maintenance and calibration). Equipment inventory document. Reagent inventory document. Safety manual. List of suppliers of equipment, reagents and other materials used in the laboratory. Documents detailing the contracts to perform special activities (e.g. research projects, etc). Samples of contracts to be stored. Documents on standards being used by the organization.

For example, corrective and prevention action document will detail what preventive action needs to be taken to avoid breakdown of an equipment. Preventive actions are defined as the proactive processes to identify the potential sources of non-conformance. This is discussed in details in the chapter on equipment maintenance and calibration. The corrective actions taken to repair equipment, improve a procedure, improve performance, etc. must also be documented.

Example Inventory on equipment

Record the unique identification facts of each equipment (Manufacturers serial no. or lab number). Maintain record of name, number, date received and condition upon installation, maintenance history, future maintenance policy - all are documented in the equipment inventory document. Maintain records of service of equipment and calibration of equipment. There must be a document giving the instructions for proper use, maintenance, and frequency of servicing required. Maintain details of calibration of equipment like name of equipment, date of calibration, name of person who calibrated.

Document control
Certain documents need to be controlled i.e. such documents may not be accessible to everyone in the lab. All such documents which need to be controlled should be clearly specified and the accessibility must also be defined. These may include financial records of the lab and all those documents which the management feels are essential to be controlled. At the same time, control of documents should be exercised in a manner that it does not interfere with the quality management system of the lab and should not affect the quality of the result.

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CHAPTER 10 SELECTION OF HIV TESTING KITS

Introduction
Selection of the appropriate HIV test kits is an essential prerequisite for accuracy and reproducibility of test results and for quality management. A large number of assays are available commercially, offering essential as well as attractive performance characteristics namely sensitivity, specificity, efficiency and predictive values. However, the performance of the test will depend upon the conditions in the laboratory, technical expertise, population being tested, etc. An endeavour should be made to selects kits which are evaluated and approved by WHO and DCGI. The characteristics of the selected kits should fulfil the objectives of testing. The kits as far as possible should be user friendly.

The performance characteristics to be considered for the selection of kits are as under:
Sensitivity Specificity Efficiency Positive predictive value Negative predictive value Stability Ease of performance of test.

The parameter's values should be within those recommended by the technical expert committee, G.O.I. on this subject. The final approval of the test kit is given by DCGI on the basis of the evaluation reports submitted by the identified evaluation centres Sensitivity: Specificity: Efficiency: Predictive values: Sensitivity of a test is defined as its ability to detect truly infected individuals as also its ability to detect very small amounts of analyte. This is the ability of a test to correctly identify all the uninfected individuals i.e. there should be no false positives. It is the overall ability of a test to correctly identify all positives as positive and all negatives as negative. This is the measure of value of a test in relation to the prevalence of the disease in the population. The value of a test in addition to the parameters described above depends on the population being tested. Positive predictive value (PPV) is the ability of the test to identify actually infected individuals among all persons giving a positive result with the kit being evaluated. Negative predictive value (NPV) is the ability of the test to correctly identify the really non infected individuals from among all the persons giving negative result. 55

If the prevalence is high, the PPV of a test will be high i.e. an individual testing positive will mean actual infection. Whereas, in low prevalence area the chance that an individual testing positive is actually infected is lower.

RECOMMENDATIONS FOR SELECTION OF TEST KITS

The testing centre should procure and have available at all times, HIV test kits based on three different principles and /or antigen systems to enable them to perform HIV testing as per NACOs HIV testing strategies at VCTCs and PPTCTCs and one highly sensitive test kit (100%) to screen blood at blood banks to ensure blood safety.

HIV (ELISA) test kits - general specifications

1. 2. 3. 4. 5. 6. 7. 8. 9. Should be solid phase microplate ELISA using HIV I & II recombinant and/or synthetic peptide antigens. The assay should detect HIV I and II antibodies. The assay should detect antibodies to all sub-types of HIV I. The assay should be able to detect antibodies of HIV I and II during early seroconversion period. The assay should have reactive and non-reactive controls with each kit. The kit should have a shelf life of minimal 12 months at the port of discharge or consignees end which ever is applicable. Adequate literature detailing the components, methodologies, validity criteria, performance characteristics, storage conditions, manufacturing and expiry dates should be provided with each kit. The assay should have sensitivity level at 99.8% and above and specificity level at 98% and above. The manufacturer/ authorized agent should ensure maintenance of cold chain during storage and transport at 20C - 80C.

HIV test kits (sandwich ELISA)

1. Should be solid phase microplate sandwich ELISA using HIV I & II recombinant and/or synthetic peptide antigens

The selection criteria 2-9 are identical to those applicable as above for HIV (ELISA) test its general specifications

HIV (ELISA) capture/competitive test kits

1. 2. Solid phase microplate capture ELISA in which solid phase is coated with anti-immunoglobin (anti IgG, IgM, IgA). If assay is competitive ELISA, the solid phase should be coated with recombinant and/or synthetic peptide antigen.

The selection criteria 3-9 are identical to those applicable as above for General Specifications

HIV Rapid test kits-general specifications

1. 2. 3. 4. 5. Should be solid phase/particle coated with synthetic/recombinant HIV I & HIV-II antigens. The assay should detect antibodies to HIV I & HIV II by Enzyme Immuno Assay / Agglutination/ Immunochromatography/any other principle. The product should be able to detect antibodies to HIV I and II during early sero-conversion period. The product should have positive and negative controls. The kit should have a shelf life of minimal 12 (twelve) months at the port of discharge or consignees end which ever is applicable. 56

6. 7. 8. 9. 10. 11.

Adequate literature dealing the components, methodologies, validity criteria and performance characteristics of the product should be provided with each kit. Should have sensitivity level at 99.8% and above and specificity level at 98% and above. The supplier/local agent should have facility for storage of kits at 2C - 8C The total procedure time should not be more than 30 minutes. Provision shall be made for conducting single test at a time. The tests should be easy to perform and the kits should preferably be stable at room temperature (22 25C.

HIV Rapid test kits- principle of Enzyme Immuno Assay

1. 2. Should be a Solid phase/particle coated with synthetic/recombinant HIV I & HIV II antigens. The assay should detect HIV I and II antibodies by principle of Enzyme Immuno Assay.

The selection criteria 3-11 are identical to those applicable as above for HIV Rapid test kits (general specifications)

HIV Rapid test kits -principle of agglutination

1. 2. Should be solid phase/particle coated with synthetic/recombinant HIV I & HIV II antigens. The assay should detect HIV I and II antibodies by agglutination.

The selection criteria 3-11 are identical to those applicable as above for HIV Rapid test kits (general considerations)

HIV Rapid test kits - any other principle excluding Enzyme Immuno Assay and Agglutination
1. Should be solid phase/particle coated with synthetic/recombinant HIV I & HIV-II antigens. The assay should detect antibodies to HIV I & HIV II by any other principle excluding Enzyme Immuno Assay and Agglutination. The selection criteria 3-10 are identical to those applicable as above for HIV Rapid test kits-general specifications

2.

DETAILS OF SOME OF THE COMMERCIALLY AVAILABLE HIV RAPID TEST KITS

Immunofiltration*

Immunoassay

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Agglutination

Different manufacturers use different raw materials as matrix for adsorbing the HIV peptides. The immunofiltration involves the vertical flow of the reagents. As the reagents pass through the matrix the reactants get adsorbed and concentrated (immunoconcentration) on the viral peptides on the matrix in case of HIV positive sample to produce a colored spot /line

ELISA TESTS

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Combinations as per principle

I. II. III. IV. Immunofiltration + Immunoassay + Agglutination Immunofiltration + Immunoassay + Lateral Flow/ Immuno chromatography Immunofiltration + Agglutination + Lateral Flow/ Immuno chromatography Immunoassay + Agglutination+ Lateral Flow/ Immuno chromatography

Examples
I. II. III. IV. Tridot/Pareekshak/ HIV Spot + Immunocomb HIV 1& 2Bispot/HIV EIA Comb/CombAIDS RS + Capillus/NEVA Tridot/Pareekshak/ HIV Spot + Immunocomb HIV 1& 2Bispot/HIV EIA Comb/CombAIDS RS + SD Bioline HIV 3.0/ Retrocheck HIV/ Precise Tridot/Pareekshak/ HIV Spot + Capillus/NEVA + SD Bioline HIV 3.0/ Retrocheck HIV/ Precise Immunocomb HIV 1& 2Bispot/HIV EIA Comb/CombAIDS RS + Capillus/NEVA + SD Bioline HIV 3.0/ Retrocheck HIV/ Precise Care has to be taken that atleast two kits selected should be able to differentiate between HIV 1 & 2 For example: Do not select Pareekshak + Comb AIDS RS + Capillus as none of these kits can differentiate between HIV 1 & 2, instead Tridot + HIV EIA Comb + Capillus can be selected

Combinations as per antigen

All the kits have recombinant antigens except Immunocomb & HIV 1& 2 Bispot, which have synthetic antigen. CombAIDS RS has a combination of recombinant & synthetic peptides. To avoid confusion it will be better to use kits with different principles rather than different antigens. But in dire situations, a combination of two immunoassays having different antigens can be used along with one more test of other principle.

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CHAPTER 11 EVALUATION OF HIV KITS

Introduction
Periodic evaluation of HIV test kits is a prerequisite of good quality management. A large number of assays are available commercially each offering essential as well as attractive performance characteristics namely sensitivity, specificity, efficiency and predictive values. However, the performance of the test will depend upon the conditions in the laboratory, technical expertise, population being tested, etc. So, wherever possible, evaluation of kits should be done under the same conditions and in the same locale using the local panel of sera to assess the performance characteristics and efficiency of a test kit. New and improved kits are also being developed continuously and these also need evaluation in the same way as the established kits. Reports of evaluation of kits provide a feed back to the manufacturers to improve their products so that the kits may be fit for use under different situations and in different geographical areas. To have an idea about the performance characteristics of these HIV kits, prior evaluation by some notified agency should be made and is mandatory. Situations under which these kits may have to be evaluated can be as follows: Kits under import; evaluation with local serum panel is very important. New kits developed/manufactured indigenously; to assess efficacy. To resolve controversies regarding discordant results or deterioration of components and reagents. This may be due to break in cold chain during transit and/or storage of in-use kits

As far as the first category is concerned DCGI asks the importer to get the representative sample of kits evaluated, using a locally made and well characterized panel by the Institutes identified for this purpose. Currently NIB is the statutory institute which perform evaluations. If found suitable, only then the kits should be allowed to be imported for distribution, both in Government as well as private sector. In second case, when the kits are manufactured indigenously, the performance characteristics of the kits should be ascertained by evaluating different batches of kits (the final product) with the standard serum panel for evaluation of kits at least at two to three different geographical sites and consistent results should be obtained. Only the kits performing to the standards (sensitivity and specificity, etc.) as laid down should be licensed for marketing. In the third case, the licensed, and/or approved kit's performance may be questioned particularly when different centers report inconsistent results. This can happen due to break in cold chain during transport and storage of HIV kits. To resolve this problem the kits have to be evaluated by the identified centers to ensure optimal performance. This chapter gives the basic guidelines to be followed to ensure proper evaluation of HIV test kits.

Kit evaluation process

A process must be followed to perform evaluation of kits. The process comprises of: Gathering information on the kit to be evaluated from the manufacturer Examining the available data on the kit. Writing an evaluation protocol. Selecting an evaluation panel. 60

Performing the testing Collating and analyzing the data.

Information about the performance characteristics (sensitivity, specificity, predictive values, etc.) of the kit is ascertained from the manufacturer. The information helps to select the right type of evaluation panel. The evaluations can be done by a single site or multiple sites. In case of multi-site evaluations, a QC sample should be included to assess inter-laboratory variations.

Evaluation panel
A serum panel is defined as a collection of well characterized serum samples for a specific reference/purpose. Serum panel can be prepared and used for different purposes. These are: Panel for evaluation of kits i.e. evaluation panel including seroconversion panel Panel for proficiency testing (external quality assurance) Panel for quality control in the laboratory (internal quality control)

Volume of panel samples

The volume of serum being processed to prepare panel should be sufficient to meet the requirements for at least 2-3 years for the purpose for which the panel is prepared. For example 6 ml. of serum will be sufficient to evaluate approximately 100 kits.

Collection of specimen for panel

Ideally material used for panel should be serum although plasma can also be used. However, plasma tends to be unsuitable as it clots spontaneously on storage. Therefore plasma should be defibrinised with thrombin or recalcified and then processed for making the panel.

Aliquoting of serum
Repeat freezing and thawing of specimens affects the quality of the panel. To avoid this, samples should be aliquoted in volumes that may meet the particular panel requirement at one time. Suitable anti-bacterial agents e.g. Bronidox (0.05%) should be added to panel samples to prevent contamination.

Characterization of serum panel

Serum samples should be well characterized so that there should not be any doubt about the final outcome of the test results. Specimens should be tested using high quality HIV antibody ELISA or any other screening assay followed by confirmatory Western Blot assay to determine their true status and should also distinguish between HIV-1 and HIV - 2 types. As characterization of each unit in the panel is quite expensive, use of large volume of each specimen will be more cost effective.

Storage
Aliquots must be stored at or below -70C. Glass vials should not be used as they may eventually crack with long term storage in frozen condition. 61

Transport of panel
To provide safety for laboratory staff, couriers and community, specimens should be transported under International Air Transport Association (IATA) regulation, for this purpose. Specimens should be packed, labeled and documented as classed under 6.2 UN 2814 to minimize the risk of infection to anyone during transit.

Composition of serum panel

These are well characterized serum samples, those which have given consistent results with all the available HIV test kits. These sera should be from individuals in whom the true HIV status (infected or not infected) is known with high degree of certainity. Such samples are called pedigreed sera. Panel must include reactive, nonreactive, weakly reactive (grey zone) and indeterminate classified sera. Both HIV-1 and HIV-2 reactive sera should be included in the panel. The panel should be representative of the population/area where the kits are going to be used. To prepare a local panel, sera should be tested with all the available kits. At least 50 specimens should be included in each panel of reactive, weakly reactive, indeterminate seroconversion and non reactive. The panel size can be increased as more the number of sera in each panel, more statistically valid is the comparison. Non reactive panel should also include representative sera from individuals suffering from local endemic diseases (e.g. malaria, tuberculosis, hypergammaglobulinaemia, etc.) and sera with high IgM levels. Panel specimens should be appropriately processed/characterized and stored as aliquots in frozen condition. All specimens should be in the same condition as far as storage is concerned when test for evaluation of kits is performed. Specimens should not be haemolysed, lipaemic and turbid etc.

Reference test
A well established, reference gold standard test must be available to the laboratory for comparison, the results of which should correlate with the actual status of the patient. Currently, such a test in serologic testing for HIV infection is considered to be Western blot (WB) test. However, sometimes WB may give indeterminate results in which case more sophisticated tests like synthetic peptide based line immunoassay, polymerase chain reaction (PCR) and virus culture can be undertaken. The tests are expensive, labor intensive and require higher level of technical expertise. Thus for the moment WB usually is regarded as the Reference/Gold standard test. When a number of kits are being evaluated the tie breaker for false positive/false negative is usually a WB test, sometimes supported by more sophisticated tests as mentioned above.

Testing condition
All evaluations must be performed under identical conditions. The tests must be conducted in the same laboratory using same equipment (pipettes, instruments, etc.), by the same technician, using kits which have not expired and have been stored properly and using the exact protocols provided in the package insert.

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The technicians must not know the results obtained on the panel with other kits i.e. the evaluation testing is done blindly.

Parameters for performance characteristics of a kit

The parameters to be calculated to assess the performance characteristics of the kits include: Sensitivity Specificity Efficiency Delta values Positive predictive value Negative predictive value

Sensitivity
Sensitivity of a test is defined as its ability to detect truly infected individuals as also its ability to detect very small amounts of analyte. Sensitivity can be calculated by the following formula: True positives Sensitivity = ----------------------------------------------------- X 100 True positives + false negatives Or Number of samples detected as positives by the kit under evaluation ----------------------------------------------------------------------------------------------X 100 Total number of positives by the reference test employed

Sensitivity =

Example Total sera tested= 100 HIV infected = 10 HIV noninfected = 90 Results with the reference test

Results with kit under evaluation: 9 out 10 HIV infected detected as positive 9 Sensitivity of the kit under evaluation will equal to --------- X 100 = 90% 9+1

Specificity
This is the ability of an assay to correctly identify all the uninfected individuals i.e. there should be no false positives (reactive result in the absence of disease). The specificity can be calculated by the following formula: True negatives ------------------------------------------------ X 100 True negatives + False positives

Specificity =

63

Specificity =

Or Number detected as negatives by the kit under evaluation -------------------------------------------------------------------------------------- X 100 Total number of negatives detected by reference test employed

Example Total sera tested= 100 HIV infected = 10 HIV noninfected = 90 Results with the reference test

The results obtained with kit under evaluation, 9 positive out of 10 HIV infected and 1 positive out of 90 HIV noninfected. 89 Specificity of the kit under evaluation will equal to -------------X 100 = 98.9 % (approximately) 89+1

Efficiency
It is the overall ability of a test to correctly identify all positives as positive and all negatives as negative. Efficiency is determined as below: True positives + True negatives Efficiency = ----------------------------------------------------------------------X 100 True positives + False negatives + True negatives + False positive Or Total number detected as positives + negatives by test kit ---------------------------------------------------------------------------------------------------- X 100 Total number of positives + negatives detected by reference test employed

Efficiency =

Example Total sera tested= 100 HIV infected = 10 HIV noninfected = 90 Results with the reference test

The kit being evaluated detected 9 out of 10 HIV infected as positive and one out 90 non-infected also as positive. 9+ 89 So efficiency of the kit will equal to ------------------- X 100 = 98% 9+1+89+1

Delta value
This is a statistical measure of sensitivity and specificity and is calculated from the actual OD values generated by the test. This also helps to determine how close the O.D. value of false positive and or false negative is to the real positive and or real negative value, respectively. If these values are too close the test is flawed. Delta values are defined as the distance of the mean O.D. ratio of the sample population from the cut off (C.O.) measured in standard deviation units.

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Delta values =

Mean O.D. / Cut off ratio (log 10) -------------------------------------------------------- X 100 Standard deviation

Positive delta value is calculated on mean of ODs of positive samples and negative delta value is calculated on mean ODs of negative serum samples. When two tests are being compared, the test that has a higher positive delta value would characterize positive samples more accurately; similarly the test with higher negative delta value will better classify the negative samples.

Example

Positive predictive values This is the measure of value of a test in relation to the prevalence of the disease in the population. The value of a test in addition to the parameters described above depends on the population being tested. Positive predictive value (PPV) is the ability of the test to identify actually infected individuals among all persons giving a positive result with the kit being evaluated. True positives -------------------------------------------------- X 100 True positives + False positives

PPV =

Negative predictive value (NPV) is the ability of the test to correctly identify the actually non infected individuals from among all the persons giving negative result. True negatives NPV = -------------------------------------------------- X 100 True negatives + False negatives If the prevalence is high the PPV of a test will be high i.e. an individual testing positive will mean actual infection. Whereas, in low prevalence area the chance that an individual testing positive is actually infected is lower. Example Low prevalence area 1% Total sera tested = HIV infected = HIV noninfected =

1000 10 990 65

with the reference test

Results obtained with kit under evaluation: Positive results obtained in 8 individuals, two from HIV infected group and 6 false positives from HIV non infected group. Negative results obtained in 992 individuals, 984 from the non infected group and 6 false negatives from HIV infected group. 2 ------- X 100 = 25% 8 984 ------------- X 100 = 99.3% 992

The PPV =

The NPV = Example

High Prevalence area 10% Total sera tested = 1000 HIV infected = 100 HIV noninfected = 900

with the reference test

Results obtained with HIV kit under evaluation; 105 tested positive, 95 from HIV infected group and 10 from HIV non infected group i.e. gave false positive result. Negative result was obtained in a total of 900 cases. 892 of 900 were from HIV non infected group and 8 were from HIV infected group. 95 --------------- X 100 = 90.5% (approx.) 105 892 --------------- X 100 = 99.12% 900

PPV =

NPV =

All the above parameters are ascertained by the laboratory carrying out the evaluation of kits. The parameter values should be within those recommended by the Technical Expert Committee, G.O.I. on this subject. The final approval of the test kit is given on the basis of the evaluation reports submitted by the identified evaluation centers. For further information on test kit evaluation, see also: Guidelines for Assuring the Accuracy and Reliability of HIV Rapid Testing: Applying a Quality System Approach. 2005. Centers for Disease Control/WHO.

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CHAPTER 12 OPERATION, MAINTENANCE AND CALIBRATION OF EQUIPMENT

Introduction
Preventive maintenance is the best measure to ensure that the equipment will function properly. Preventive maintenance schedules should be followed regularly for optimal performance of equipment. Ordinarily, maintenance schedules are set up as tasks to perform daily, weekly, monthly, every six months, and yearly. Depending on such factors as work load, types of instrumentation and number of employees, a record or check-sheet of such activities can be adopted for any given laboratory to help the laboratory manager keep track of necessary tasks.

Checklist before deciding on the purchase of an instrument

The instrument specifications should fit the purpose. The specifications should conform to local conditions such as power supply, humidity and climate. The advice of other laboratories using a similar instrument should be sought. The prices of different brands with similar specifications should be compared, not forgetting prices of spare parts. Running costs should be compared. Availability of after-sale services, including maintenance, should be assured. An operations and maintenance manual must be supplied. On delivery, an extra supply of commonly needed replacement parts (e.g. carbon brushes, fuses, bulbs, electrodes, heating elements) should be obtained. The equipment must be assessed for technical safety. The need for an operator from the laboratory to receive on-the-job training in operation, maintenance and minor repairs must be recognized by the supplier or a competent national agency.

Operation, Maintenance and calibration of different equipment Centrifuges

Two types of centrifuges are currently used, mechanical and electrical. The major aspects in maintenance of different types of centrifuges used in laboratory routine are as follows: The centrifuges must be positioned exactly horizontally to prevent the instrument moving away from its place when out of balance during centrifugation. Check if the rubber buffers are in the buckets. It is critically important that the centrifuge load is balanced at all times. Therefore buckets loaded in matched pans and tubes should be arranged so that balanced tubes oppose each other in the centrifuge head. This is necessary to maintain the same forces of gravity in the opposite positions of the tubes. This arrangement involves placing a dummy, i.e., a tube filled with the appropriate volume of water corresponding to the weight of the volume, in the oppositely positioned test tube, when an odd number of specimens must be centrifuged. Haematocrit centrifuges need not be balanced before use, as the capillary tube samples are small. Capillaries should be plugged at one end to avoid spilling and loss of blood. The plugged end should always be placed against the sealing gasket. 67

Turn the speed control slowly up and down. Stop the centrifuge immediately if it makes an abnormal noise. After use, the buckets should be inverted to drain dry. After any sample spillage, wipe and disinfect immediately. Clean the centrifuge at short intervals (preferably daily) because it is one of the most frequently used instruments. Check mounting and replace if necessary. Check brushes and bearings every 3 months. Replace if necessary. Check for corrosion and clean and repaint if necessary. Calibration: use a pre-calibrated tachometer to check centrifuge speed.

Refrigerators
The following general advice may be helpful for maintenance: Refrigerators must be so placed that sufficient air can pass the condenser (at the back of the Daily Checks Check temperature daily. It should not exceed 12C. Application of battery driven mobile or stationary thermometers is recommended, preferably those including continuous printing or plotting of temperature measured when heatsensitive reagents are stored for long periods. Monthly Checks Clean cool chamber and defrost the evaporator monthly. Clean refrigerator from outside. Clean condenser of dust. Clean door gasket. refrigerator) for exchange of heat and also to facilitate cleaning of the condenser. The refrigerator door must seal perfectly to prevent warm outside air from entering the cool chamber. Calibration: Use a pre-calibrated thermometer to ensure accuracy of temperature check.

Hot air oven

Hot air ovens are mainly used for drying laboratory equipment and medical devices in dry air. Some hot ovens are also used for sterilization. Sterilization in dry air is only effective when the material is exposed for 60 minutes to 160C or for 40 minutes to 180C. It is important to remember that the timing of sterilization is sufficient when the holding period begins after the air in the oven has reached its expected temperature. Use of hot air ovens Set up the thermostat at the required temperature prior to sterilization. If there is a fan, check if it is working. Allow to continue heating for an additional 60 minutes after the temperature reaches the pre-set degree. Switch off the heat when the time is up. Wait until the temperature falls to 40C before opening the door. Calibration: use a pre-calibrated thermometer to ensure accuracy of temperature checks. 68

Autoclave
Bacteria cannot survive in an autoclave environment; however, sterilization does not necessarily kill viruses. It should be borne in mind that autoclaves need careful handling and must be regularly inspected. They can be hazardous and can seriously injure a person with hot steam accidentally escaping from the instrument. The main factors influencing perfect steam sterilization are saturated steam temperature time Use of Autoclaves Prepare the material for autoclaving with indicator paper. Fill the bottom of the autoclave with demineralized water up to the support. Before placing the material in the autoclave, ensure that there is an adequate amount of water. Close the lid and make sure that the rubber washer is in its groove. Screw down the clamps firmly. Open the air outlet valve. Turn on the heating (electric element). Do not leave the autoclave unattended. Close the outlet valve when a constant jet of steam starts to escape. Reduce the heat as not to heat too quickly. Once the expected temperature is reached, reduce the heat to maintain the temperature. Do not touch drainage tap, outlet or safety valve while heating under pressure. When the time required for sterilization has passed, turn off the heat completely. When the temperature falls below 1000C, open the outlet valve slowly. Do not leave the outlet valve unopened for too long. Never unscrew the lid clamps or open the lid except after the whistling sound stops. Leave the sterilized material to cool before its removal from the autoclave. Check the autoclave tape (used in the preparation of the material to be sterilized) which must have turned black and the covering paper brown (not yellow or black). Calibration: use a pre-calibrated pressure gauge to check accuracy of pressure in the autoclave.

Adequate sterilization by autoclaves and dry ovens should be monitored by the weekly use of a biological (spore suspension) or chemical indicator.

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Incubators
Incubators should be subjected to continuous recording of temperature. However, if it is not possible, the temperature must be recorded every day and before opening of the incubator. The incubator should have a fan to maintain uniform temperature. Incubators are used for bacterial culture by laboratories working in microbiology. The incubator must maintain a constant temperature of 37 1 C. Temperature should be daily recorded in incubators. Like all laboratory instruments, incubators must be cleaned and disinfected routinely at short intervals (at least every fortnight) and after spilling of infectious material. The actual temperature must correspond to the thermometer control when the instrument is used. Calibration: use a pre-calibrated thermometer to ensure accuracy of temperature checks.

Water bath
It is important that the water bath maintains a constant temperature within a narrow range ( 0.5C) when used for kinetic measurements such as determination of enzyme activities or clotting assays. Inadequate adjustment and insufficient stabilization of the temperature will strongly affect the results of kinetic measurements. Use of water baths The level of water in the water bath must be above the level of the solution to be incubated. Open containers, vials or tubes must not be incubated in a water bath with an open lid to avoid contamination and dilution of the incubated material by condensed water. The water bath must be refilled regularly to prevent growth of algae and bacteria.

Pipettes
Each pipette, whether manual, semi-automated or automated, must be tested periodically to determine if it is delivering the correct volume. Pipettes are used for volumetric measurements of liquids. They may be made of glass or plastic and may have different volumes, ranging from 2 mL to 100 mL. They may be calibrated or non-calibrated. Glass pipettes are either calibrated to contain (In) or to deliver (Ex). The variety of designs for these devices is so numerous that it is difficult to discuss the subject systematically. It must be noted, however, that the replacement of broken conventional glass pipettes can be very costly, and that there may be other economic advantages in using mechanical pipettes. Mechanical pipettes are preferably used to pipette small volumes (4L to 1000L); they have a higher precision and are less subject to errors in pipetting than glass pipettes. Volumes less than 500L should be pipetted with mechanical pipettes. However, mechanical micropipettes can only be recommended where a reliable supply of standardized disposable tips can be guaranteed. They are usually of air displacement (indirect), or direct displacement design. To avoid contamination during pipetting samples, most designs use a disposable tip which is discarded after each delivery. The practice of washing and reusing disposable tips is to be discouraged, as any cleaning procedure will change the wettability of the plastic. In addition, drying even to only slightly elevated temperatures may distort the tip. This will prevent a good pneumatic seal with the pipette body and change the volume of liquid to be pipetted. 70

After each use, the mechanical pipettes must be kept in an upright position and thoroughly cleaned at periodic intervals. Pipettes are precise and important basic instruments of the laboratory, and as such, need to be calibrated at least every 3-6 months. Several methods of pipette calibration are available (see annexure X)

pH meters
A pH meter needs to be standardized before each run with a standard buffer of pH 7.0. However, in instances when the work is related to a pH range of less then 6.0, it is advisable to use a standard buffer of pH 4.0. The buffer solution should be checked monthly with another pH meter and discarded if the pH deviates by more than + 0.4 or if the buffer is contaminated with microorganisms. Glass electrodes must be stored in buffer solutions at pH 4 to pH 8. The buffer solution must be regularly changed at short intervals. Glass electrodes which have been stored for longer periods must be soaked in 0.1 molar HCI for at least four hours. Thereafter they must be carefully washed with distilled water. The same procedure must be applied for dried-up electrodes. The life-span of properly maintained glass electrodes is about two years. Thereafter they should be replaced. Aging of an electrode is indicated when the constant electrode potential does not develop 20 seconds after insertion of the electrodes into the ion solution. Glass electrodes are sensitive to mechanical damage. Calibration of pH Meters To obtain a precise measurement of pH, the pH meter must be calibrated with two different buffers at pH 4 and pH 7 every day (two-point calibration). For the calibration of a pH meter special buffer solutions must be used, the pH of which should be near the pH of the solution to be measured. Phosphate buffers and acetate buffers are preferable. Calibration measurements should be done using plastic containers. Use of the pH Meter Switch off the measuring circuit Wash the electrode with deionized water Standardize the electrode at the start of the day with two standard buffers (pH 7.0, pH 4.0) Transfer the electrode to a small beaker containing the standard buffer, pH 7.0 Measure the pH of the standard buffer and adjust the buffer control knob to reading Wash the electrode and gently wipe with fibre-free material. Transfer the electrode to another beaker containing the standard buffer, pH 4.0 Read the pH of the standard buffer. It should be very close to expected pH value (pH 4.0) Use fresh standard buffer every day. Discard standard buffer if it is contaminated or cloudy

Measuring the pH of the test solution The following steps must be taken: Wash the electrode with deionized water and transfer it to the test solution. With proper electrodes the potential establishes 5 to 20 seconds after insertion of the electrodes into the sample solution. Air bubbles at the electrodes must be avoided since they cause a drift of the electrode potential. Compensate for the temperature at each run. Measure the temperature and adjust the temperature dial to the reading. 71

Do not read the pH before the reading is stable. Record the pH reading of the pH solution. Wash the electrode and store it in a container with storage solutions.

Balances
Balances are used to measure weight and mass. The principle of weighing is based on attracting forces between two separate masses. In daily life this is the attracting force between mass and the earth. In laboratories, weighing is an essential step in preparing defined quantities of reagents and reaction mixtures. There are two main categories of balance Mechanical balances Electromagnetic balances (battery driven or connected to the mains)

The following factors influence weighing and can cause errors in measurement Temperature Moisture (atmospheric humidity) Electrostatic effects Magnetism Gravitational forces Air

Maintenance of balances The following guidelines are worth remembering: The balance should be placed on a solid vibration-free surface, free from dust and at even temperature, away from sunlight. The instrument must be placed in an exactly horizontal position. The balance should be zeroed prior to each use. Use the smallest possible vessel for weighing. Avoid weighing in vessels made of plastic, because they can become electrostatically charged. Use instead glass vessels or weighing paper, as applicable. The weighing vessel and the sample to be weighed should be at the ambient temperature. Never put your hand into the weighing chamber so as not to warm it up. Place the sample to be weighed in a weighing vessel in the middle of the weighing pan, to avoid corner-load error. Liquids or powders should never be directly weighed on the pan. The weight of the weighing vessel needs to be determined prior to placing the substance to be weighed. A tweezer is useful as a substitute for hands in the weighing chamber. Use clean weighing vessels. Keep the working place, weighing chamber and weighing pan clean. To avoid any possible corroding effect of chemicals, any spillage must be cleaned immediately. Biological materials may be a source of infection. Disinfection can be done with 70% alcohol. After completing weighing return the balance to zero weight. Keep the working place at the balance as clean as possible. Weight of the material to be weighed should be within the range of the balance.

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Photometer instruments (ELISA reader etc.)

Most photometric instruments require calibration to ensure accuracy and linearity of their readings. This is usually accomplished using special calibration plates, available from the manufacturer. The plates consist of different wells, each capable of producing a different O.D. reading. Observed readings are compared to theoretical values and evaluated using confidence limits. Likewise, automated diluters must be calibrated. This is usually accomplished by diluting a standard colour solution and reading O.D. spectrophotometrically. Results must be within 10% of the expected limits. The manufacturer of any automated instrument can be contacted for details of these procedures. The annual maintenance contract should include the above mentioned check-ups and laboratory incharges should ensure the implementation.

Calendar of activities for maintenance and calibration of equipment

Daily Weekly Monthly Electronic or optical checks are performed on all components. Many automated and semiautomated instruments have built-in programs for calibration. Every 6 months Filters are cleaned or changed, fluid lines and tubing for signs of deterioration are checked and replaced as needed. All pipettes are regressed as needed. Yearly All fluid lines and tubing of major instruments are changed. A service call for factory representative if possible is done. Pipettes are recalibrated. Optical components etc. are kept free from dust. Surfaces of instruments are cleaned. Fresh batches of reagents are prepared as needed. Controls or calibrators added to each run. Recalibration of instruments if necessary; For ELISA readers, the proper filter is put in place. Waste containers, rinse sample ports, etc. are emptied with distilled H2O. All spills cleaned up. Reagent levels are checked. ELISA washers flushed. Biohazardous waste disposed.

Function checks
It is essential that laboratory personnel know and document that all equipment is in good condition each day of use. This can be accomplished by undertaking function checks, often referred to as calibration and validation. 73

Calibration That process which is applied to quantitative measuring or metering of equipment to assure its accurate operation throughout its measuring limits. Validation The steps taken to confirm and record the proper operation of equipment at a given point of time in the range in which tests are performed. Documentation The assurance that a piece of equipment is operating properly can best be judged by examining its performance over time. Records of performance parameters, therefore, are a vital element in the proper operation of laboratory equipment. Some suggested information is provided below: Name and serial number of instrument Elements to be checked and kind of data to be collected Frequency of checking Record of data Changes made to restore accuracy and precision, if any Signature with date of the person performing these tasks.

Preventive maintenance
Maintenance of equipment is an extremely important function in the microbiology laboratory. Unfortunately, this is often grossly neglected because of indifference on the part of laboratory workers and on the erroneous belief that it is too costly. The expense of such maintenance policies as inspection, lubrication and adjustment of instruments is insignificant when compared with the cost of emergency repairs, rebuilding or overhauling equipment, and the additional personnel time and materials involved in producing test results when equipment is down. Preventive maintenance is defined as a programme of scheduled inspections of equipment and instruments resulting in minor adjustments or repairs for the purpose of delaying or avoiding major repairs and emergency or premature replacements. It provides the following advantages over breakdown maintenance. Better quality results Identification of components showing excessive wear Greater safety Fewer interruptions in services Lower repair costs Less stand by equipment requirements

ELISA Reader
Regular maintenance of the ELISA machine should be carried out according to the manufacturers' instructions and may vary between brands. The machine itself should not be opened.

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Note: The filters must be protected from moisture and fungal growth. Keep Silica gel packet in the filter box.

Plate washer
Plate washers are critical in ELISA assay performance. The washer works on a simple principle and comprises of wash fluid, waste fluid reservoirs, pressure and vaccum pumps, a dispense manifold and a plate carrier. The wash fluid is pressurized and a valve opens and allows the fluid through a manifold and into assay microwells. The waste fluid under vaccum is aspirated back through the manifold to the waste container. A number of cycles of dispense and aspiration comprises the washing of a plate. At the end of the wash procedure the wells are empty of the fluid. After use care Fill the rinse bottle with about 500 ml of distilled water. Dispose off the unused wash buffer. Rinse with distilled water, a couple of times and leave about 500 ml in the wash bottle. Fix the cap tightly. After using the washer switch off power.

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CHAPTER 13 TROUBLESHOOTING
Introduction
In spite of being vigilant and following the SOP, many a times a test run fails. The reason may be apparent in some cases but not in others. Troubleshooting refers to measures undertaken to determine why a run has failed.

General approach to trouble shooting Factors responsible for some common problems / errors
Specimen: lysed blood, lipaemia, deterioration, volume not sufficient. Clerical/transcription: wrong name/ID no/wrong result transcribed, labeling, etc. Kit related: conjugate gone bad (deterioration of reagents), storage not proper and inter lot variations, etc. Laboratory variations: due to use of different kit, different testing procedure (SOP), different technique. This kind of variation may be inter-laboratory as well as intra-laboratory. So, results are non-reproducible. Environmental conditions: higher temperature, humidity. Equipment problems: ELISA reader, washer, calibration of pipettes, water bath and refrigerator, etc. Technical errors: carelessness, not following SOP, pipetting errors, worksheets not maintained. Borderline reactor/grey zone reactors. Laboratory does not practice quality control procedures.

Methods for identifications of some common problems

Review protocol with the technician who ran the test, carefully checking for procedural omissions or changes. Double check component expiration dates. Verify that all physical parameters of the assay were followed and met (e.g. times and temperatures). Confirm that the support equipment such as pipettes, plate washing and reading systems, etc. are working properly. Verify that the preventive maintenance and servicing procedures have been performed. Check the wavelength of the ELISA plate reader selected for the test. Verify the calibration of the pipettes. If the assay calls for 10L of specimen and the pipette delivers 9L, this constitutes a 10% error and may be sufficient to cause the controls to fall outside acceptable limits. Proper calibration of pipettes is essential for accurate results. Check the quality of the distilled water (pH). Check that reagents are not contaminated and were prepared and stored properly. There are several situations in which characteristic appearances in a test system may indicate problems. Examples 1) include : Change in the colour of the substrate e.g. if tablets appear orange when they should appear yellow to white - indicating that the tablets have been contaminated or have deteriorated, and thus cannot be used. 76

2)

If the reagent indicator cells or carrier particles in an agglutination assay appear clumped or otherwise heterogeneous - this reagent may have been contaminated or may have deteriorated. Most package inserts describe the appearance of the reagents contained within the kit, and thus ensure comparison of the actual appearance with what is expected.

Specific assay problems: some examples and their possible solutions

All wells in an ELISA are of the same colour in each row. This could be either due to conjugate going bad, not working or substrate going bad. Try another kit from the same lot to pinpoint the cause of error. The plate visually reads O.K. i.e. different wells have different colours but the result on spectrophotometer shows almost identical ELISA values. This is due to error in detection and/or reference wavelength of the spectrophotometer/ELISA reader. Correct the wavelength and then re read the results. Negative control strip has an unexpected band. Due to contamination between wells and/or reaction has been allowed to develop too long. Repeat the entire procedure with same controls plus negative control from another lot number to identify error.

Variations in test results

The HIV test results produced by the same laboratory (reproducibility) and or by a different laboratory on the same specimen may vary. This can be due to error at any step right from collection of specimen to testing and final reporting of the result. The reasons for inaccurate results may be on account of: Specimen problems Clerical errors Kit dependent problems Technologists dependent errors Equipment problems Environmental problems and their influence on the test result. Non repeatable (reproducible) results.

Specimen problems
1. 2. Error Insufficient volume for repeating the test in case of reactive result. Haemolysis can cause false positive or false negative result in ELISA and background noise on spot test. Lipaemia causes pipetting error, otherwise it causes no error in the test result. Bacterial contamination degrades antibody and will affect the borderline reactive result. Resolution Collect appropriate volume (3-5 ml) of blood depending upon the objective of testing and strategy of testing. Repeat sample. In case it is not possible record "sample haemolysed" in the result. Buy widebore pipettes and pipette tips. Record "sample lipaemic" in the result. Refrigerate the samples for short storage (one week) and store at -20C for long storage(>one week). 77

3. 4.

5. 6.

Freeze/thaw cycle may affect the borderline positive samples. Aliquoting errors

Avoid freeze-thawing as far as possible till the test are performed. All aliquots should be labeled appropriately. A uniform method of storing samples/aliquots should be followed in the laboratory. Organise the specimen in racks and leave a tube space empty between aliquots made from different specimens.

Clinical/clerical errors
Error 1. 2. Logging in specimen : there may be mixing of names, wrong history, wrong number, etc. Result print out : the well number on worksheet and the print out not matched properly (transcription error). Errors during translating of results from the worksheet to the report form. Reporting to the wrong person or result communicated without post test counselling and confidentiality. Resolution Set up the log books/records properly ; collect second sample from the same patient and perform testing. Transcribe the result from the print out sheet to the worksheet carefully. Repeat the test for resolution of the result. Supervisory review/vigilance of results by a second person can correct the error. Report result to the correct person after post test counselling and maintain the confidentiality. Reporting should be as per the "National HIV Testing Policy".

3. 4.

Kit dependent errors

Error 1. Used after expiration date. Resolution Expiry dates should be displayed in bold letters on the kit packing. Regular checks should be made to ensure that no kits remain in the lab after the expiry dates. Stocks should be rotated to ensure that kits with lesser expiry are used before the kits with longer expiry dates. Use reagents for the kits for which they are made. Do not mix reagents from different kits. Do not use unsatisfactory kits i. e. those which do not meet the quality standards. Do not use contaminated/deteriorated (control values different from those mentioned in package insert) kits. Store at the optimal temperature and maintain cold chain during transport of kits. Random monitoring of performance characteristics of the kits to ensure the quality.

2. 3. 4.

Mixing of reagents from different kits/lot numbers. Performance characteristics (sensitivity, specificity and delta values) not satisfactory. Deterioration/contamination of one or more component or reagents of the kit. (faulty transport/storage). Intra-lot and inter-lot variation in kit performance due to faulty manufacturing practices.

5.

78

Common errors in HIV testing and resolution of the same

Procedural error / technologist dependent errors 1. Dilution errors. Resolution Carefully calculate the volumes of all components required for the test and perform dilutions of reconstituted components accordingly to get the desired volumes. Do not allow pipette tip to touch the plate well while adding the sample. Test each sample as well as the quality control sample in exactly the same way. Use reagents for the kits for which they are prepared . Do not mix reagents. The worksheets should be meticulously prepared indicating which specimen goes into which well. The internal quality control samples should be randomly placed for each run. Each step of SOP must be diligently followed. Supervision vigilance and training for accurate pipetting.

2. 3. 4. 5.

Scratching off the coated antigen from the well. Inconsistent technique for test and controls particularly quality control samples. Mixing reagents from different lots of kits. Mixing of samples.

6. 7.

SOP not followed to perform the test. Wrong pipetting due to carelessness.

Equipment based errors

Error 1. 2. Use of inappropriate pipette tips. Equipment not maintained as per requirement. Resolution Use appropriate volume pipette tips to deliver accurate volumes. Maintain and calibrate the equipments as per the requirement (Refer to chapter on equipment maintenance and calibration). The washer should be placed under annual maintenance contract for optimal working. Use the correct U.V. filters for reading results (as per the directions in package insert). ELISA reader must be placed under maintenance for accurate results. Calibrate refrigerator to ensure that right temperatures are maintained in refrigerator/deep freeze.

3. 4. 5. 6.

ELISA washer not working satisfactory. Use of incorrect U.V. filters. Improper reader Refrigerator/deep freeze not working to optimum level.

Environment dependent errors

Error 1. Improper temperature can affect the enzyme reaction and the result. 2. Drying up of ELISA plates Resolution Calibration of incubators to maintain the right temperatures is very important. Maintain humidity so that the plates do not dry. 79

Non repeatable/non-reproducible results

Causes 1. 2. 3. 4. 5. Mislabelling of specimen. Specimen deterioration. Borderline reactors. Carelessness of the technologist. SOP not changed with the change in kit. Resolution Repeat testing on 2nd sample collected from the same patient. Parallel testing of the sample with stored sample. Repeat testing. Follow up and repeat testing. Vigilance in the laboratory. Training of personnel. Review SOP and change if required particularly if new types of kits are purchased.

Vigilance / Supervision
Periodic monitoring of the performance of laboratory workers is essential for ensuring quality of testing. This can be done by officer incharge of laboratory by resubmitting an already tested specimen as a test sample without the knowledge of the laboratory worker. If the results of the resubmitted sample are consistent with the previous results that means quality is being maintained. This should be done in a healthy way and under no circumstances the laboratory worker should be intimidated/reprimanded/ticked. Vigilance means scrutiny of laboratory and every step of the test to ensure accurate results every time. Scrutiny has to be random and of each step of the test right from collection, transport of specimen to reporting of result to the right person in the right manner. Every step of quality control has to be practised every day to ensure that the procedures and results are accurate and no false positive or false negative results are generated by the laboratory. This may be done by regular internal audit of the laboratory.

Record keeping
Monitor the records from collection and logging in of the specimen in the laboratory to reporting of the results. This will include the following details: Name of individual or specimen identification number. Date of collection of specimen and date received in laboratory. Name of requesting physician. The test requested. Worksheet. Result of the test.

The worksheet records the date of test, type of kit (Lot no. etc.) used, the test samples (nos.), quality control samples (nos.), name of laboratory worker, O.D. values obtained and cut-off values, etc. The worksheets are an important document for quality performance of tests. A model of worksheet is given in annexure III.

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CHAPTER 14 IMPLEMENTATION OF EXTERNAL QUALITY ASSEMENT PROGRAMME AT DIFFERENT LEVELS OF THE HIV TESTING NETWORK AND JOB DEFINITIONS OF EACH LEVEL
A wide network of HIV testing laboratories operating at different levels exist in the country. The network includes National and State Reference Laboratories (NRLs / SRLs,), Voluntary Counseling and Testing Centers (VCTCs), PPTCTCs and blood bank HIV testing laboratories. These centers and laboratories are uniformly distributed across the country. Each and every facility / laboratory engaged in HIV testing has to practice quality assurance to ensure that every time the testing is performed the results produced are accurate, within the limits of the procedure and the reagents / kits used. The network of laboratories all over India is huge and scattered. So, the Quality Assurance Programme (QAP) is being implemented in three phases.

Phase I
All the National Reference Laboratories participate in this phase of QAP. Apex centre i.e. NIB distributes the characterized serum panel (10) to the National Reference Lab along with the procedural proforma. The participating labs process the External Quality Assessment serum panel exactly the same way as the routine samples and send the results to NIB within 15 days for analysis and feedback. This exercise is repeated once every 6 months. NIB also conducts EQA training program for the NRLs. Those National Reference Laboratories which are already participating in any of the External Quality Assessment Programmes being conducted by WHO Reference / collaborative laboratories may continue to do so.

Phase II
All the NRLs conduct training programme on EQAP and Internal Quality control as well as aspects of HIV Testing for the identified State Reference Laboratories (SRL) and distribute the EQA panel. Composition of serum panel is the same as that from apex reference laboratory. The SRLs test the EQA panel in the same way, under same conditions and using same kits which are used for testing client / patient sample within two to four weeks. The reports are sent back to the respective NRLs for analysis and feedback.

Phase III
Each National Reference Laboratory has been allotted states for implementation of EQA for all the HIV testing facilities existing in the respective states. For this purpose the National Reference Labs and or SRLs prepare the External Quality Assessment (EQA) serum panel of ten well characterized sera as per details already given. This panel comprising of 10 sera is sent to each and every HIV testing facility namely VCTC, PPTCTC & blood banks by the NRL/SRL in the respective allotted states. National AIDS Control Organization, Government of India facilitates the conduction of EQAP at various levels by 81

providing funds to NRLs/SRLs and issuing directives to SACS, NRL and participating VCTC, PPTCT and blood bank labs, etc for compliance, emphasizing the importance of EQAP. The participating laboratories in the states test the EQA panel sent by NRL/SRL as routine test samples and send the results to the assigned NRL/SRL within 2 weeks. NRL/SRL analyse the data and provide feed back to the participating labs. In case of discordance of result the experts from NRL visit the defaulting centre / laboratory for trouble shooting. NRL/SRL also conduct trainings for VCTCs and other HIV testing labs in the state. The National Reference Labs/SRL liase with the Project Director, State AIDS Control Society to train the staff from VCTC, PPTCTC and Blood Banks to participate in the External Quality Assessment Programme. The training programme is funded by respective Project Directors. It is a five days hands on training programme. The modus operandi for implementation of EQAP in states at grass route level can be decided by the Officer In charges, NRLs/SRLs corresponding to the size of states, etc. The panel is sent to the participating laboratories maintaining the cold chain (2-80C). The serum panel comprises of a total of 10 samples (4 positive, 4 negative and 2 borderline positive). The NRL sends all the relevant papers, proformas (Annexure VI) for performing the test and reporting back of the results obtained by the participating laboratories to the SRL/ NRL. The networking of HIV testing facilities for EQAP is given in annexure IX. Each National Reference Laboratory has been allotted SRLs/HIV testing cnentres in various states as mentioned in the annexure. The NRLs/SRLs cater to the ever expanding HIV testing facilities in their respective regions for EQAP.

Job definitions for the network of labs engaged in implementing EQAP

The levels of labs: Apex Reference Laboratory National Reference Laboratories (NRLs) and State Reference Laboratories (SRLs). Blood bank laboratory, VCTC, PPTCTC Apex Laboratory Job definition Preparation of serum panels for conducting external quality assurance. Evaluation of panels prepared by the National Reference Laboratories Participation in the EQA program conducted by the WHO collaboration Centre. Trouble shooting the problems faced by the National Reference Laboratories. National Reference Laboratories and State Reference Laboratories Job definition To send panel to apex centre for quality control and evaluation of the panel Distribution of panels to SRLs, VCTCs, PPTCTCs and blood banks. Analysis of data and feedback to participating SRLs, VCTCs, PPTCTCs and blood banks. Solving problems faced by SRLs and other HIV testing centers with support from apex laboratory as and when required. Testing representative samples received from SRLs and other HIV testing labs (20% of all positives and borderline positive sera and 5% of all negative sera). 82

Training of SRLs and HIV testing laboratories. Visiting SRLs and HIV testing labs for trouble shooting

VCTCs, PPTCTCs and blood bank laboratories, Job definition Testing of external quality control panel sent by NRL/SRL. Generation of data from blood bank labs, VCTCs and PPTCTCs. Referring lab related problems and problem sera (sero-status not resolved) to respective SRL Sending representative samples to SRL for quality assurance (20% of all positives and borderline positive sera and 5% of all negative sera). Should possess an independent HIV testing laboratory with adequate floor space, personnel for carrying out quality assurance work.

Recommendation on development of serum panels and evaluation of kits

Development of panels Panels will be prepared by the NRLs and SRLs. The serum panel should comprise of 40% positive, 40% negative and 20% border line reactive sera.

Flow of sample panel from Apex Laboratory /NRL/SRL

Apex Reference Laboratory National Reference Laboratories State Reference Laboratories VCTCs, PPTCTs, Blood Banks

Transportation of panel
All the panels are to be transported from one laboratory to another in the temperature range of 2-8C. The panel should consist of 10 serum samples and each individual sample should have a volume of at least 100 microlitres.

Turn around time for reporting

The participating laboratory will send back the result of the panel within four weeks of the receipt of the panel to the next level of laboratory linked with the centre and the higher laboratory will provide feed back to the respective laboratories within 8 weeks.

Frequency of external assessment exercise

It will be conducted at least once a year to begin with and preferably twice a year, during months of February/March and August/ September preferably.

Panel evaluation
Apex laboratory as well as the National Reference labs will carry out the evaluation of panel and analysis of the results. NACO will inform the apex laboratory and NRLs about the details of HIV test kits that are likely to be purchased and distributed to the blood banks and blood testing centers. 83

Kit evaluation
The apex laboratory and the National Reference Laboratories will have the responsibility of evaluation of kits as per requirement of NACO and other national bodies. It is also suggested that each fresh batch of kits purchased by NACO should be evaluated for sensitivity, specificity and delta values before it is distributed to the blood banks and other laboratories so that the quality of the kits supplied is assured. The apex laboratory and the NRL should put together efforts to prepare panel of sera to be used for evaluation of kits. This panel should include (i) sera from different geographical locations in the country (ii) sera against prevalent types and subtypes (iii) sera from patients with chronic diseases such as tuberculosis, leprosy, kala azar, auto immune diseases, malaria, etc. and (iv) sera from HIV sero-negative individual with risk behaviour for acquiring HIV infection. The panel established by the Apex Laboratory and National Reference laboratories will be tested for thermostability and reproducibility in the Apex Laboratory and NRLs. It is also recommended that regular surveillance should be maintained at each level. Five percent of negative samples, 20% of positive samples and all border line reactive samples may be monitored at the respective higher levels; grey zone samples should be repeated.

The networking of HIV testing facilities for EQAP:

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CHAPTER 15 EQUIPMENT, SUPPLIES AND REAGENTS FOR PRACTICING QUALITY CONTROL IN HIV TESTING*
Biosafety " " " " " " " " " " " Collection of serum samples from individual/plasma from blood bags (from blood banks). " " " " " " " " " Preparation of external control " " " " " " " " " " " " " " " " Laboratory coats/gowns (front closed). Chappals, slippers (top covered). Rubber gloves. Sodium hypochlorite / ethanol / glutaraldehyde / any other appropriate disinfectant. Discarding jar. Disposable paper sheets. Disposable bags. Autoclave, preferably incinerator. Lysol/labolene (for disinfecting floor) . Formalin (for disinfecting rooms). Needle destroyer Disposable syringes and needles. Screwcapped plastic tubes. Centrifuge for separation of serum / plasma. Vacutainers (vacuum based blood collection systems) CaCl 2. 6H2O; MgCl2. 6H2O. Centrifuge bottles (250 ml, presterilised, screwcapped). Water bath. Vacuum pump, seitz filter with filter pads, biological filters ( 0.8m, 0.6m). Magnetic stirrer. Water bath (35-44C). Incubator BOD ( 35-44C). Balance (physical / electrical). Rocker (40 - 60 rockings / minute for running Western Blot). ELISA reader filters (405nm, 492nm, 540nm and paper role). ELISA washing system. ELISA / rapid kits for HIV testing. Glass pipettes - 2ml, 5 ml, 10 ml. Microtips ( capacity 25l, 50l, 100l). Multichannel pipettes ( 50-200l). Micro pipettes ( 5-50l, 50-200l, 200-1000l). Microtip container. Pasteur pipette, rubber teats. Plastic forceps, plastic troughs. Tube racks. Graph paper. 85

Recalcification of plasma to serum.

" Sterilisation " " " " " " " " " "

Calculator (scientific). Hot air oven. Autoclave. Refrigerator (domestic with freezer compartment). Deep freeze (-30C, -70C). Polypropylene storage bottles ( 250 ml.). Plastic storage vials . Syringes for filtration through biological filters (50 ml.). Racks for holding storage vials. Biological filters ( 0.8m, 0.2 m). Bronidox (5 - bromo - 5 - nitro - 1, 3dioxane) and Propylene glycol (solvent for Bronidox)/ Thiomersal. Sample carrier ( e.g. Thermocol box). Water distillation plant. Glass ware (e.g. Beakers, flasks, measuring cylinders, etc). Markers, parafilm (for sealing storage vials). Air conditioner. UPS (uninterrupted power supply). Biological safety cabinet. ELISA washer (automated / semi-automated).

Storage / preservations

Referral Expected existing back up

" " " " " " " "

*Uses of these components have been included in relevant sections.

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CHAPTER 16 BIOSAFETY FOR HEALTH CARE WORKERS WITH SPECIAL REFERENCE TO HIV
Introduction
Ever since AIDS has been recognized in 1981, the concern about bio safety in medical science has attained a new dimension. However, any measure regarding safety, needs assessment of the extent of risk, failing which, there is possibility of undue panic or carelessness due to over-estimation or under-estimation of the risk respectively. Fortunately, the threat of HIV infection to health care workers (HCW) has never assumed so much of problem. This has been mainly due to the fact that HIV is known for its fragile nature, having very low power of surviving in natural conditions like drying, temperature, etc. as well as due to its relatively lower concentration in blood and body fluids. Blood is the single most important source of HIV, HBV, HCV and other blood borne infections for HCWs. The risk of infection varies with the following factors: Type of exposure e.g. exposure of intact skin, nonintact skin, mucous membrane or needle stick injury. The amount of blood involved in the exposure; hollow bore needles, cannula, etc. carry more blood than IM (intramuscular) needles. The amount of virus in patient's blood at the time of exposure (maximum during acute viraemia). Prevalence of infection in the population. Number of exposures in case of needlestick injuries from individuals with unknown serostatus. Timely availability of post exposure prophylaxis (PEP).

Table1.

Modes of exposure to blood borne pathogens in the laboratory

87

Table2.

Essential steps of biosafety

The essential biosafety measures in relation to various steps of activities in a health care setting are as follows.

Bio safety guidelines for laboratory workers General precautions for laboratory workers
Wear gloves for all manipulations of infectious materials or where there is possibility of exposure to blood or body fluids containing blood. Ensure generous supply of good-quality gloves. Discard gloves whenever they are thought to have become contaminated; wash your hands, and put on new gloves. Do not touch your eyes, nose, or other exposed membranes or skin with gloved hands. Do not leave the workplace or walk around the laboratory wearing gloves. Wash your hands with soap and water immediately after any contamination and after work is completed. If gloves are torn, wash your hands with soap and water after removing the gloves. Wear a laboratory gown, smock, or uniform when in the laboratory, wrap-around gowns are preferable. Remove this protective clothing and leave it in the laboratory when leaving the laboratory. When work with material potentially infected with HIV is in progress, close the laboratory door and restrict access to the laboratory. The door should have a biohazard symbol and No Admittance sign posted. Keep the laboratory clean, neat, and free of extraneous materials and equipment. Disinfect work surfaces when procedures are completed at the end of each working day. An effective all-purpose disinfectant is a 1% hypochlorite solution. Place used needles, syringes, and other sharp instruments and objects in a puncture-resistant container. Do not recap used needles and do not remove needles from syringes. Never use mouth pipetting.

88

Perform all technical procedures in a way that minimizes the creation of aerosols, droplets, splashes, or spills. Do not eat, drink, smoke, store food or personal items, or apply cosmetics in the laboratory. Have an effective insect and rodent control programme as per standard recommendation.

Guidelines for those who collect blood samples Major hazards

Blood contamination of the hands while drawing blood. Penetrating injuries caused by needles and other sharp instruments or objects.

Guidelines
Inspect your hands for cuts, scratches, or other breaks in the skin. If the skin is broken, cover with occlusive bandage and wear gloves. Take care to avoid contaminating your hands during the collection of blood. If blood gets on gloves, these should be discarded. Place used needles and syringes in a puncture-resistant container. Do not recap used needles. Do not remove needles from syringes. Do not use needle clippers. Seal specimen containers securely. Wipe the exterior of the container free of any blood contamination with a disinfectant. Wash your hands with soap and water immediately after any blood contamination and after work is completed. Wear the laboratory gown. In the event of needlestick or other skin puncture or wound, wash the wound thoroughly with soap and water. Encourage bleeding. Report any contamination of the hands or body with blood, any puncture wound, or cut to the supervisor and the health service.

Table 3. Safety guidelines for laboratories engaged in serological testing of HIV

Chemical disinfectants commonly used in HIV laboratory

The common disinfectants that are used for bio safety against HIV are mentioned below:

89

Chlorine - sodium hypochlorite For wiping work benches and specimen containers, cleaning gloves, etc. 1% freshly prepared sodium hypochlorite solution is used. For heavily soiled equipment, blood spills, etc. 10% sodium hypochlorite solution is needed. Sodium hypochlorite solutions gradually lose their strength, requiring daily preparation of fresh solutions for use. The effectiveness of hypochlorite solutions is neutralized by blood, serum, and other proteinaceous material. Daily replacement is required. Chlorine - calcium hypochlorite Available in powder, granule, or tablet form. Decomposes at a slower rate than sodium hypochlorite. Availability of chlorine - (70%) 0.1% solution is obtained by dissolving 1.4 g dry chemical in 1 litre of water. 1.0% solution is obtained by dissolving 14 g dry. chemical in 1 litre of water. Chlorine - sodium dichloroisocyanurate (NaDCC ) Available as tablet. 60% available chlorine. 0.1% available chlorine solution is obtained by a dilution of 1.7 g stock in 1 litre of water. 1.0% solution is obtained by a dilution of 17 g in 1 litre of water. More stable than sodium and calcium hypochlorite. Chloramine

Available in powder or tablet form 25% available chlorine Releases chlorine at a slower rate than other chlorine compounds. Higher concentrations are required for effective disinfection. General purpose - 20 g stock in 1 litre of water. 40 g stock in 1 litre of water for disinfecting heavily soiled equipment, blood spills, etc. Chloramine is more stable than sodium and calcium hypochlorite.

Alcohol-ethanol (ethyl alcohol) and 2-propanol (isopropyl alcohol) Ethanol and 2-propanol are effective disinfectants, particularly for cleaning surfaces such as the exterior of specimen containers and bench tops. For maximum effectiveness they should be used in a 70% concentration (70% alcohol, 30% water). Iodophore - povidone iodine (PVI) Disinfectant activity is similar to that of hypochlorite solutions. More stable and less corrosive (however, do not use on aluminium or copper). Formaldehyde -formalin Excellent disinfectant Formalin containing 35%-40% formaldehyde, 10% methanol and water. Should be diluted 1:10 (a solution containing 3.5%-4% formaldehyde) for disinfection. Rinse the equipment before reuse.

90

Glutaraldehyde High-level disinfectant. Extremely useful for the disinfection of non-disposable heat-sensitive equipment and instruments. Usually available as a 2% aqueous solution. Immersion in the solution destroys vegetative bacteria, fungi, and viruses in less than 30 minutes. Ten hours' immersion is required for the destruction of spores. Treated equipment must be thoroughly rinsed. Prepared solutions should not be kept more than 2 weeks, or according to the manufacturer's instructions.

The selection of an appropriate disinfectant will also be weighed in terms of contact time, physical characteristics like irritant and toxic properties etc. In a developing country like India cost is another important consideration.

Transport of specimens
Transport of specimens (e.g. serum, blood etc.) is a frequently performed activity for a laboratory engaged in HIV diagnosis. The broad guidelines are as follows: General guidelines Specimen containers should be screwcapped, leak-proof resistant plastic or glass. After the container is closed and sealed it should be wiped with a disinfectant - e.g. 1% sodium hypochlorite solution. On receipt of a specimen, before the container is opened it should be wiped with a disinfectant as above. Within the health care facility and laboratory, the specimen containers should be placed in racks to maintain them in an upright position. The racks are to be transported in leak-proof plastic, metal or rigid thermocol containers. From field collection sites or between laboratories, leak-proof plastic or metal boxes with secure, tight-fitting covers should be sought.

Disposal of laboratory waste Waste should be segregated into different categories at the site of generation i.e. in the laboratory. Non infectious waste is collected in black bags and disposed as general waste. Infectious waste should be decontaminated prior to its storage, transport and disposal. Solid infectious waste is disposed as follows Sharps needles and syringe nozzles are shredded in needle-destroyer (if available); if not available decontaminated as in below. Scalpel blades/ lancets / broken glass should be put in an impermeable container with bleach that should be filled to three fourth only, transferred to plastic/ cardboard boxes, sealed to prevent spillage and transported to incinerator. Glass ware should be disinfected and sterilised by autoclaving followed by cleaning; never attempt cleaning before autoclaving. Swabs are chemically disinfected followed by incineration.

Disposable items include single use products (syringes, gloves, sharps, etc.). As these items are often recycled and have the risk of being reused illegally, these should be disinfected by dipping in freshly prepared 1% sodium 91

hypochlorite for 30 minutes to 1 hour. Bins which can be used for this purpose are a set of twin bins, one inside the other with the inner one being perforated and easily extractable. This minimizes contact when the contents are being removed. Disposable items like gloves, syringes, etc. should be shredded, cut or mutilated before disposal followed by deep burial or properly accounted before disposal. Extreme care should be taken while handling the needles. Liquid wastes generated by the laboratory are either pathological or chemical in nature and are disposed of as follows: Non infectious chemical waste should first be neutralised with reagents and then flushed into conventional sewer system. The liquid infectious waste should be treated with a chemical disinfectant for decontamination then neutralized and flushed into the sewer. Solid wastes are collected in leak-resistant single heavy duty bags or double bags may be used. Bags having different colour with red labels mentioning date and details of waste are recommended. The bags are tied tightly after they are three-fourths full. Processing of glass syringes, needles and gloves for reuse These are the items most frequently handled in any laboratory handling blood or other infectious fluids/materials. Hence procedures for their reuse are mentioned below. Reuse of needles and glass syringes Gloves must be worn. Extreme care must be exercised to prevent needle stick injuries and/or cuts. Leave the needle attached to the syringe. Aspirate hypochlorite or another suitable disinfectant into the syringe. Immerse the syringe and the attached needle in disinfectant solution, horizontally in a flat tray. Leave them immersed in disinfectant solution for 20 minutes. Discharge the disinfectant solution from the syringe and needle. Rinse the syringe and needle, filling and emptying several times. Examine needles and syringes for needle barbs, the rubber ring, the needle hub fit to the syringe, readable syringe markings, etc. Disinfect or sterilize the syringe and needle by autoclaving (in a steam steriliser) or boiling in water prior to reuse. Reuse of gloves Generally recommended for laboratory workers who handle blood specimens or other suspect fluids potentially infected with HIV. Gloves reduce the risk of contamination of the hands with blood, but will not prevent the occurrence of penetrating injuries or cuts caused by needles, other sharp instruments, or broken glass or plastic. It is important to remember that gloves are meant to supplement, not replace good infection control practices, including the hygienic practice of proper hand washing. Test the gloves for peel, cracks, punctures, etc. Thoroughly rinse your gloved hands in a hypochlorite disinfectant solution. Rinse your gloved hands in clear water. Wash your gloved hands with soap and water and rinse. Remove the gloves and hang them up to dry. Wash your hands. 92

Packing, storage and transport of treated (disinfected) wastes All segregated and disinfected waste should be packed in proper containers and colour-coded bags with red label biohazard signs (Table - 4). All containers used for storage of such waste shall be provided with a properly covered lid to avoid spillage during handling and transit of such waste. Such containers should be inaccessible to scavengers and protected against insects, birds, animals and rain. The waste should be transported in vehicles authorised for this purpose only. No such waste should be stored in the place where it is generated for a period of more than two days.

Disposal of laboratory wastes

Waste which cannot be incinerated (e.g. plastics) should be pre-treated by disinfection and disposed of in an environmentally sound manner. Waste should not be dumped, discharged or disposed in any place other than a site identified for the purpose. All treatment and disposal facilities including burial pits shall be located at a specified area away from the general service area of the hospital, public places and residential areas. All precautions and personal safety measures should be taken (including provision of protective clothing, masks, gloves, gumboots, goggles, etc. as may be necessary). Hepatitis B vaccine is recommended for affording protection to all personnel engaged in handling biomedical waste, or being exposed to such wastes against infection from handling or exposure. All liquid waste should be disinfected and flushed in the sinks at the point of generation.

Table 4.

Container and colour coding for disposal of bio-medical wastes

ks

93

ks

Spills and accidents

Spills of blood /infectious material can be quite frequent in a busy laboratory. The essential steps and requirements in case of spills are as follows: Essential steps Wear gloves Cover with paper towel/absorbent material Pour hypochlorite 10% Leave for 30 minutes Clean the area Finally, wipe the surface with disinfectant again & mop up to leave a clean area. Essential requirements Gloves to be worn, avoid direct contact of gloved hand with spill. Sweep broken glass/fractured plastic with dustpan and brush. Needle stick or other puncture wounds, cuts and skin contaminated with spill should be washed with soap and water; encourage bleeding. Report immediately. Maintain record.

Occupational exposure to blood/body fluids and contaminated sharps, etc.

An "exposure" that may place a Health Care Provider (HCP) at risk of bloodborne infection is defined as a percutaneous injury (e.g. needle-stick or cut with a sharp instrument), contact with the mucous membranes of the eye or mouth, contact with non-intact skin (particularly when the exposed skin is chapped, abraded, or afflicted with dermatitis), or contact with intact skin when the duration of contact is prolonged (e.g. several minutes or more) with blood or other potentially infectious body fluids. Body fluids that are potentially infectious include - blood, semen, vaginal secretions, cerebrospinal fluid, synovial, pleural, peritoneal, pericardial and amniotic fluids or other body fluids contaminated with visible blood. Exposure to tears, sweat, urine, faeces, saliva of an infected person is normally not considered as an "exposure" unless these secretions contain visible blood. 94

The exposure must be reported to the appropriate authority and condition must be treated as an emergency. Prompt reporting is absolutely essential.

Post exposure prophylaxis against HBV, HCV & HIV

HBV In recent years immunization of HCWs against HBV has been successfully employed widely. However some of the potential limitations are lack of consensus regarding the ideal regime of vaccination, the required level of protective antibody and poor understanding about the variables that might hamper expected response.

Table 5. Recommendations for Hepatitis B prophylaxis following percutaneous or permucosal exposure

* HBIG dose 0.06 ml/kg IM @ Adequate anti-HBs Ag is > 10 SRU by RIA or positive by EIA.

Hepatitis C (HCV)
Hepatitis C is most commonly transmitted by parenteral route, i.e., through infected blood transfusion or sharing of needles and syringes in IV drug users. It is a common agent of co-infection with HIV. Like the other bloodborne infections, the mainstay of preventing occupational infection is through prevention of exposure. Risk of transmission after needlestick injury is 2-3%. There is no vaccination at this time, and no recommended chemoprophylaxis. For HCW exposed to Hepatitis C, follow up care is recommended.

95

HIV
Management of Exposure : Steps to be taken on accidental exposure to blood (or body fluid containing blood) are: Wash wound immediately with running water and soap Inform the lab /hospital management and document occupational accident Consult with nearest ART centre/ resource for Post-exposure prophylaxis, evaluation, and follow-up (as per the National guidelines on PEP) Counselling and collection of blood for testing from the exposed HCW with written informed consent must be done. Whenever possible confidential counselling and testing of source for Hepatitis, HIV etc must be done. A history should be taken as well to ascertain likely risk of the source. (PEP should be provided to the exposed HCW until report of source is available and confirmed negative). Risk of infection and transmission must be evaluated Never delay start of therapy due to debate over regimen. Begin with basic 2-drug regimen, and change if warranted, once expert advice is obtained Reevaluation of the exposed person should be considered within 72 hours post exposure, especially as additional information about the exposure or source person becomes available. The exposed person is advised to seek medical evaluation for any febrile illness that occurs within 12 weeks of exposure. Administer PEP for 4 weeks .PEP should be provided until result of the source's test is available and confirmed negative or until course completed ,if source positive or unknown A repeat HIV test of the exposed individual should be performed at 6 weeks, 12 weeks and 6 months postexposure, regardless of whether or not PEP was taken

Ideally, prophylaxis should be begun within 2 hours of exposure. Late PEP (within 72 hours) may still be useful as early treatment of HIV infection, in case infection has occurred. Donts: Do not panic! Dos: Do not reflexively place pricked finger into mouth Do not squeeze blood from wound, this causes trauma and inflammation, increasing risk of transmission Do not use bleach, alcohol, betadine, or iodine, which may be caustic, also causing trauma

Remove gloves, if appropriate Wash site thoroughly with running water. Irrigate thoroughly with water or saline if splashes have gone into the eye or mouth.

Seroconversion after occupational exposure If transmission of a bloodborne infection occurs after occupational exposure which has been documented, the HCP has a right to receive treatment and care for this illness. It is also the right of all persons infected thusly, to freedom from discrimination regarding their working conditions. Such persons are entitled to have all of their human rights respected, beginning firstly with the right to confidentiality regarding their health care.

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Table 6. HIV Postexposure prophylaxis evaluation

Table7. Post-exposure chemoprophylaxis when source patient is drug nave

of

Nelfinavir 750 mg three times daily or any other boosted protease inhibitor. (for higher risk categories - consult expert).

Drugs not recommended for PEP:

l l

ddI and d4t; together Nevirapine

In case the source patient is on ART and considered to be having drug resistance (clinically) then the ART 97 124 125 124 125

physician should be consulted and appropriate ARVs should be given for PEP. Schedule of necessary measures regarding therapy and follow up in case of PEP The person should be provided with pre test counseling and PEP be started as discussed above. Before starting PEP, 3-5 ml of person's reference blood sample is to be taken and sent to the laboratory for testing and storage. It is important that a serum sample is collected from the HCW as soon as possible (zero hour) after exposure for HIV testing, failing which it may be difficult to attribute the acquired infection due to exposure in occupational setting. This may have bearing on the claims of compensation from health authorities. The first sample for HIV testing is collected immediately after exposure and 2nd at 6 weeks, 3rd at 12 weeks and last at 6 months after the exposure. During the follow-up period, especially the first 6-12 weeks when most infected persons are expected to show signs of infection, the recommendations for preventing transmission of HIV are to be followed by the HCW. These include refraining from blood, semen, organ donation and abstaining from sexual intercourse. In case sexual intercourse is undertaken a latex condom must be used correctly and consistently. This reduces the risk of HIV transmission. In addition, women should not breast-feed their infants during the follow-up period after exposure to prevent exposing their infants to HIV in breast milk. The antiretroviral drugs for PEP are to be given for four weeks. Government of India has already made the resources available with various State AIDS Societies to meet with the expense of PEP for HCWs. The drugs for PEP must be available round the clock. The report of exposure and PEP has to be sent to Addl. Director (Technical) National AIDS Control Organisation, GOI. Pregnancy and PEP If the HCW is pregnant at the time of occupational exposure to HIV, the designated authority/physician must be consulted about the use of anti-viral drugs for post exposure treatment. Facts known about the safety and side effects of these drugs Most of the information known about the safety and side effects of these drugs is based on studies of their use in HIVinfected individuals. For these individuals, ZDV and 3TC have usually been tolerated well except for nausea, vomiting, diarrhoea, tiredness, or headache for people taking ZDV. Steps to be undertaken by the infection control officer on receiving information about occupational exposure All the needle stick injuries should be reported to the State AIDS Society giving the details of the type of exposure. and the measures taken to manage the same.The State AIDS Societies should in turn inform NACO about the cases periodically. A registry is available with NACO for follow-up of all such cases. Infection control officers in all hospital have been directed to ensure that anti retroviral drugs for PEP are available in casualty at all the times.

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CHAPTER 17 SELECTED LIST OF REFERENCES CONSULTED

1. 2. WHO publication Safe blood and blood products introductory Modules No. 1 to 3, 1995. WHO publication, Eastern Mediterranean series No. 14, 1995. Quality System for Medical Laboratories-guidelines for implementation and monitoring. Retroviral testing - Essentials for quality control and laboratory diagnosis. Constantine. NT. Callahan JD and Watts DM, (Eds) CRC Press, London, 1992. 4. Gradwohl's Clinical Laboratory Methods & Diagnosis. Sonnenwinth CA & Jarett. C.V. (Eds) Mosby Company, St. Louis, Missouri, 1980. 5. Quality Control in Microbiology, Prier JE, Bartoia J and Friedman H. (Eds) University Park Press. Baltimore, 1975. 6. 7. WHO publication, Blood Transfusion - A guide to the formulation and operation of a transfusion service. Talib VH and Dutta AB ; A text book of Blood Banking and Transfusion Medicine, CBS publishers and distributions, New Delhi, 1st Edition, 1995. 8. 9. 10. Pavri K. Standard bio safety guidelines for blood banks; Transfusion bulletin, 1991, 4: 6 - 11. Saran RK and Makroo RN. Transfusion Medicine Technical Manual, DGHS, Govt. of India, 1991. Ministry of Health & Family Welfare, Government of India publication. Standards for Blood Banks and Transfusion Services, DGHS, New Delhi 1987. 11. American Association of Blood Banks. Standards for Blood Banks and Transfusion Services, 12 th edition, 1987. 12. WHO/UNAIDS publication Guidelines for Organising National External Quality Assessment Schemes for HIV serological testing; 1996. 13. Report of Quality Assurance Workshop - South East Asian & Western Pacific Region. Bangkok. Thailand, 23 rd to 31 st January, 1997. 14. Workshop Document : SEAWP Region Laboratory Quality Assurance Workshop 7-11 February, 2000, National Serological Reference Laboratory, Australia in collaboration with Bureau of Laboratory Quality Standards, Department of Medical Sciences, Ministry of Public Health, Thailand. 15. 16. HIV testing policies and guidelines WHO 1994. AIDS HIV: Reference Guide for Medical Professional 4th Ed. Fahey, John L, and Flamming D.S., CIRID, UCLA Williams and Wilkins, London, 1996. 99

3.

17.

AIDS Etiology Diagnosis Treatment and Prevention 4th Ed. Curran J., Esses M., and Fauci A.S, Pippincott Raven, 1997. HIV Testing Manual: Laboratory Diagnosis, Biosafety and Quality Control. NACO document. NABL documents on accreditation. ISO 17025 and other ISO documents.

18. 19 20

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Annexure I

Consent form for HIV Testing

This is to state that I have been counselled about the HIV test to be conducted on me and have been explained about the implications of the test result- positive, negative or indeterminate. All the details pertaining to HIV, its transmission, and testing procedure have been explained to me. Its limitations and interpretation of results have been explained to me in a manner that I can understand. I, hereby, give my consent for the test to be conducted on me in order to ascertain my HIV sero-status.

Signature: Date:

Note:
1. It may be noted that general consent obtained for carrying out procedures in hospital does not include HIV consent. 2. 3. In case of minor, the consent should be obtained from the parents. In case of unconscious patients, where there is a need for diagnosis of HIV for management of the patient, consent should be obtained from the parents, spouse/closest relative available at that time. 4. In case no attendant is available, the test, if necessary for management of the patient, may be carried out on recommendations of two attending doctors.

101

Annexure II

102

Annexure III

103

Annexure IV

104

105

Additional Project Director (Technical) N.A.C.O., Ministry of Health & Family Welfare

106

Additional Project Director

Annexure VI

107

Annexure VI contd.

HIV ANTIBODY TEST RESULTS ELISA RESULTS (SRL/BLOOD BANKS/OTHER LABS PERFORMING ELISA)
Please record the reading for the sample (column A) and the appropriate cutoff value/rate (column B) and calculate the Sample Ratio/Cut off Ratio (A-B) for each panel sample. Please record the test interpretation given to the sample after the testing.

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Annexure VI contd.

HIV ANTIBODY TEST RESULTS RAPID (e.g. particle agglutination results)

Please record if a reaction is observed with unsensitised particles for each sample (see control column). If your laboratory titrates positive samples please record the titre (note that this is not essential, but optional). Please list under 'Remark' if you performed an absorption step with any samples showing agglutination activity with unsensitised particles.

109

Annexure VI contd.

HIV ANTIBODY TEST RESULTS EXTRA TEST RESULT FORM

110

Annexure VI contd.

HIV ANTIBODY TEST RESULTS HIV ANTIBODY STATUS REPORT

Please give a final anti-HIV status for each of the panel samples. Example: Report the status of each panel sample as Negative, Indeterminate / Equivocal or Positive and place a tick in the right hand column of this form if you consider any panel sample for further testing.

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Annexure VII Name of the NRL / SRL Conducting the Training Programme (Sample Questionnaire) Workshop on External Quality Assessment Programme for HIV Testing Participant Group: Laboratory supportive personnel Pre-Test / Post-Test Evaluation Time :30 min. Name of the Participant: Name of the Institute:

TICK (
1.

) THE CORRECT ANSWER

The choice of protocol for serological testing does not depend on the following: (i) Objective of HIV testing (ii) Sensitivity and specificity of the test (iii) Prevalence of HIV infection in (iv) Time limit for performance of test the population (v) Serum or plasma as specimen In multiple testing strategy for HIV infection, the strategy II B is employed for (i) Blood bank (ii) Diagnosis of individual with AIDS defining symptoms (iii) Detection of HIV status in VCTC The most important limitation of p24 antigen for diagnosis of HIV infection is: (i) Poor specificity (ii) Poor sensitivity (iii) Poor correlation with therapy (iv) Inability to diagnose HIV-2 infection External quality assessment scheme is usually organized by (i) Local officer-in-charge of the (ii) Local supportive laboratory personnel laboratory (iii) External agency / National / Regional reference laboratory Recommended method for conversion of plasma to serum is (i) Calcification (ii) Thrombinisation (iii) Heating the plasma at 56C in (iv) Incubating at 37C water bath Recommended diluent for making serial dilution of strong reactive serum for preparing level control is following expert (i) Normal saline (iii) Serum negative for infectious markers (like HIV, HBV, HCV etc.) (ii) Phosphate buffer saline at pH 7.2 (iv) Distilled water 112

2.

3.

4.

5.

6.

Annexure VII.....contd. 7. In order to find out the extent of variation of reactivity of a sample over 7 consecutive days in ELISA kits of same batch number, it is preferable to draw inference from (i) OD values of the samples (ii) Positive control values (iii) Negative control values (iv) 'E' ratio of the sample 8. Any given HIV positive serum when subjected to 4 kits of different brands following things are likely to be observed (i) 'E' ratio differs (ii) 'E' ratio does not differ Recommended range of 'E' ratios of an external control in indirect ELISA is (i) 10-20 (ii) 1.5-2 (iii) 0.01-0.1 (iv) 0.1-1 Levy Jenning's chart for validation of test result usually employees the range of (i) Mean 1SD (ii) Mean 2SD (iii) Mean 3 SD (iv) Mean 4 SD Six consecutive points of external control values gradually moving in one direction up or below the mean line in the Levy Jenning's chart possibly indicate: (i) Sudden change in the batch of kit (ii) Change in the technician Performing the test (iii) Change of incubator with (iv) Gradually deteriorating reagents different temperature set point 12. For storage of the aliquot of external control sample for daily use over 7 days Temperature required is (i) 0C (ii) 2-8C (iii) <-30C (iv) <-70C The recommended preservative for long term preservation of external control serum is (i) Sodium azide (ii) Thiomersal (iii) Bronidox Standard quality control practice requires (i) Inclusion of external control in (ii) (iii) Changing the working hand (performing the test frequently)

9.

10.

11.

13.

14.

Quality check of every run equipments

15.

Two populations X and Y were subjected to a screening test (p) for HIV infection with sensitivity and specificity of 99.5% and 99% respectively. Prevalence of HIV infection in population X is 20 per 1000 which the same in population Y is 5 / 1000. The positive predictive value of the screening test (p) in population X will be: (i) Lower than that in population Y (iii) Identical compare to that in population Y (ii) Higher than that in population Y

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Annexure VII.....contd. 16. While choosing a kit for diagnosis of HIV infection, three kits were evaluated with a panel of 200 sera comprising of 100 true positive and 100 true negatives the following characteristics were obtained. Mark the one with maximum positive Delta value.

17.

The most important limitation of a rapid test for diagnosis of HIV infection is (i) Poor sensitivity (ii) Poor specificity You want to buy one micropipette for your laboratory for HIV testing. Placed below is a checklist of major (basic) specifications that you need to consider before the purchase. Specify (mark ) if any of these is considered unnecessary by you. Name sticker to avoid mixing among personnel (iii) Colour of the micropipette black or grey (v) Slim design to negotiate through narrow neck containers upto a volume of 50 ml. (vii) Simple for cleaning and servicing matching stickers for (i) Devise for comfortable resting on hand (iv) Easy tip purging (vi) Autoclavable (ii)

18.

(viii) Colour coded caps, \ selection of appropriate tips.

19.

Some common methods for calibration of micropipette are the following with the exception of (Mark ). (i) Pipetting of coloured solutions (ii) Weighing distilled water or followed by spectrophotometric mercury analysis (iii) Pipetting volumes of radioisotopes (iv) Commercially available spectrophotometric calibration kit (i) Weighing the micropipette at weekly interval for 4 week The 99% Sensitivity of an HIV test means that: The test will correctly identify 99 to 100 infected individuals (iii) The technician must be careful in running the test (v) The test will rarely give a false positive results (i) The test will be negative in 99% of uninfected persons (iv) The accuracy of the test divided by its cost (ii)

20. (A)

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Annexure VII.....contd. (B) The 99% Specificity of an HIV test means that: (i) The test will correctly identify (ii) 99 to 100 infected individuals (iii) The technician must be careful in (iv) running the test (v) The test will rarely give a false positive results The test will be negative in 99% of uninfected persons The accuracy of the test divided by its cost

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Annexure VIII Name of the NRL / SRL Conducting the Training Programme (Sample Questionnaire) Workshop on External Quality Assessment Programme for HIV Testing Participant Group: Laboratory supportive personnel Pre-Test / Post-Test Evaluation Time :30 min. Name of the Participant: Name of the Institute:

TICK (
1.

) THE CORRECT ANSWER

3rd generation ELISA uses the following as antigen of HIV (i) Infected cell lysate (ii) Recombinant glycopeptides (iii) Synthetic peptides Internal kit controls (serum) refers to following: (i) Controls supplied with the kit (ii) Controls prepared out of own Laboratory samples (iii) Controls procured from commercial (iv) Controls procured from the agency reference laboratory External control (serum) refers to (i) Controls supplied with the kit (iii) Control procured from commercial agents

2.

3.

Controls prepared out of own laboratory samples (iv) Controls procured from the reference laboratory

(ii)

4.

ELISA ratio or E ratio refers to (i) Ratio of sample OD/ cut off OD (iii) Ratio of sample OD upon positive control OD

(ii)

Ratio of sample OD upon negative control OD

5.

Calculation of standard deviation of external control values over 20 observations requires. (i) Mean value of the 20 observations (ii) Square of the individual values

(iii) Square of the mean values of the 20 observations. 116

Annexure VIII.....contd. 6. The recommended storage temperature of most of the HIV diagnostic kits is (i) 0C (iii) <-30C 7. (ii) 2-8C (iv) <-70C

For general purpose use the recommended concentration of sodium hypochlorite is (i) 0.01% (ii) 0.1% (iii) 1% (iv) 10% To mop up accidental spillage of blood, the recommended concentration of Sodium hypochlorite is (i) 0.01% (ii) 0.1% (iii) 1% (iv) 10% In the multiple test strategy for diagnosis of HIV infection in an individual (i) (ii) The specimen is tested thrice by a particular rapid test with high sensitivity. The specimen is tested by indirect EIA from three different companies that employ identical antigens for

8.

9.

coating the EIA plate. (iii) The specimen is tested by three tests (EIA/rapid) based on different principles / different antigen preparations 10. Ideal choice of a kit for use in a blood bank is the one with (i) Maximum sensitivity(100%) (ii) Maximum specificity Third generation/fourth generation Discrimination between HIV-1 and HIV-2 infections is possible by (i) All EIA kits (ii) All rapid / visual kits (iii) Some rapid / visual kits On a particular day while running routine EIA for detection of HIV reactive units among donated blood samples it was observed that all the wells in the plate including the control well did not show any colour at the end of the run. The possibilities considered are: (Mark ) the one you think valid. (i) The plate is not coated with antigen Washing has not been proper or omitted at the stage following addition of conjugate (iv) Plate has been incubated more than the recommended time (vi) The plate has been exposed to light for long time after addition of substrate (ii)

11.

12.

(iii) Substrate has been broken down before addition (v) Stop solution has been added late in the final stage 13.

On a day of EIA run, the whole plate, including the positive control wells show colour. The possibilities considered are as follows: (Mark ) the one you consider valid: (i) Washing has not been done (ii) Substrate has lost its potency properly (iii) Conjugate has been deteriorated (iv) Incubation has been carried out for less than recommended period 117

Annexure VIII.....contd. 14. Most commonly employed laboratory tests for diagnosis of HIV infection. (i) Serological diagnosis by (ii) HIV isolation antibody detection (iii) Serological diagnosis by antigen (iv) Haematological studies detection (iv) Skin test Collection of blood for diagnosis of HIV infection by ELISA (i) Clotted blood (ii) Heparinised blood (iii) Blood in tissue culture media Mention the appropriate storage temperature for 3 days for serum for serological test of HIV infection: (i) 2-8C (ii) 0C (iii) Less than 20C Mention the storage temperature of ELISA kits for HIV (i) 2-8C (ii) 0C (iii) Less than 20C The most commonly used disinfectant in a blood bank / HIV testing laboratory is (i) Absolute alcohol (ii) Formaldehyde (iii) Dettol (iv) Sodium hypochlorite The recommended working concentration of sodium hypochlorite is (put tick against the appropriate concentration) (i) ELISA plate / tips used in routine serology (ii) Blood spillage .001% 100% .001% 100% .01% 0.1% .01% 0.1% 1% 1% 5-10% 5-10% 1

15.

16.

17.

18.

19.

20.

ELISA positivity in an HIV infected means that person has AIDS: (i) True (ii) False

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Annexure IX

STATES ALLOTED TO THE NATIONAL REFERENCE CENTERS

Sr.No. National Reference Centers 1. 2. 3. 4. 5. Christian Medical College, Vellore Madras Medical College, Chennai National AIDS Research Institute, Pune Institute of Immunohaematology, Mumbai All India Institute of Medical Sciences, New Delhi National Institute of Communicable Diseases, Delhi National Institute of Cholera & Enteric Diseases, Calcutta School of Tropical Medicine, Calcutta Regional Institute of Medical Sciences, Imphal National Institute of Biologicals, Noida MGR Medical University, Chennai National Institute of Mental Health & Neurosciences (NIMHANS), Bangalore State Kerala, Lakshadweep Tamil Nadu, Pondicherry Maharashtra, Goa, Gujarat Mumbai, Madhya Pradesh Himachal Pradesh, Chandigarh and Punjab Rajasthan, Haryana, Delhi, Jammu & Kashmir Assam, Orissa, Andaman & Nicobar Islands West Bengal, Bihar, Sikkim Manipur, Tripura, Arunachal Pradesh, Mizoram, Nagaland Uttar Pradesh Andhra Pradesh Karnataka

6.

7.

8. 9.

10. 11. 12.

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ANNEXURE X

PIPETTING TECHNIQUE
The following information is consolidated from operational instruction manuals from several pipette manufacturers. Complete information and more detailed instructions may be seen in the specific pipette instruction manuals. Read the manufacturer's manual carefully before beginning the pipetting procedure. Choose the appropriate disposable pipette tips for your application. Hold the pipette in one hand and use a slight twisting movement to seat the tip firmly on the shaft of the pipette to ensure an air-tight seal Select the desired volume (with manual pipettes, higher volumes should be set first; if adjusting from a lower to a higher volume, first surpass the desired volume and then slowly decrease the volume until the required setting is reached). If applicable, select the desired mode (e.g., reverse pipette). This is recommended for optimal precision and reproducibility. Reverse pipetting can be done with a manual pipette by pressing the control button slightly past the first stop when aspirating, taking up more liquid than will be dispensed, then pressing the control button only to the first stop when dispensing. A small volume will remain in the tip after dispensing. Select an appropriate tip.

Pre-rinsing
The following procedures will help ensure optimal precision and accuracy. Volumes >10 L: Prerinse pipette 23 times for each new tip (this involves aspirating and dispensing liquid several times). Reasons for prerinsing include the following: to compensate for system pressure, for slight differences in temperature between pipette and liquid, and for properties of the liquid; to clear the thin film formed by the liquid on the inside of the pipette. Without prerinsing, retention of a thin film on the inside wall of the tip would cause the first volume to be too small. The thickness and nature of this film, and therefore the potential source of error, will vary depending on the nature of the liquid being pipetted. Volumes <10 L: Do not prerinse pipette, but rinse tip after dispensing to ensure that the whole volume was dispensed. For smaller volumes, prerinsing is not recommended because the dispensed volume may be higher than the requisite volume.

Filling
Make sure tip is securely attached. Hold pipette upright. When aspirating, try to keep the tip at a constant depth below the surface of the liquid. Glide control button slowly and smoothly (electronic pipettes perform this step automatically). When pipetting viscous liquids (e.g., whole blood), leave the tip in the liquid for 12 seconds after aspirating before withdrawing it. After liquid is in the tip, never lay the pipette on its side.

120

Dispensing
Hold the tip at an angle, against the inside wall of the vessel/ tube if possible. Glide control button slowly and smoothly (electronic pipettes perform this step automatically).

Testing pipetting precision

The precision of pipetting should be evaluated periodically (e.g., monthly or the lab can establish its own frequency) to ensure the accuracy of results. All records of this evaluation procedure must be retained for quality assurance purposes. Using the reverse pipetting technique, pipette 10 replicates of blood and record the weights. Select a volume normally used in the performance of the assay. Using the reverse pipetting technique, pipette 10 replicates of bead suspension and record the weights (this applies to methods in which the beads must be pipetted into the tubes). Calculate the mean, standard deviation, and coefficient of variation (CV). The CV for replicates should be <2%.

Testing pipetting accuracy

The following procedure can be used to test the pipette and how accurately it measures volume. Water is used because the weight of 1 L of water is 1 g. Using the reverse pipetting technique, pipette 10 replicates of distilled water and record the weight. (100 L of water should weigh 0.1000 grams.) (Table 1) Calculate the mean, standard deviation, and CV. The CV must be <2% (range: 0.0980.102).

Table 1. EVALUATION OF PRECISION AND ACCURACY

TO EVALUATE : Replicate # (grams) 1 2 3 4 5 6 7 8 9 10 Mean S.D. % CV ACCURACY 100 l of water (grams) 0.1036 0.1018 0.1020 0.1026 0.1008 0.1002 0.0989 0.1019 0.1009 0.1027 0.1015 0.0014 1.35 PRECISION 100 l of blood (grams) 0.1072 0.1071 0.1067 0.1069 0.1067 0.1060 0.1072 0.1090 0.1070 0.1066 0.1070 0.0008 0.73 PRECISION 100l of microbeads 0.1056 0.1056 0.1055 0.1056 0.1052 0.1055 0.1056 0.1047 0.1050 0.1050 0.1053 0.0003 0.31

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Other Recommendations :
To ensure optimal performance, the temperature of the pipetted solution and the pipette and tips should be the same (volume errors may occur because of changes in air displacement and viscosity of the liquid). Do not pipette liquids with temperatures >70C. Volume errors may also occur with liquids that have a high vapor pressure or a density/viscosity that differs greatly from water. Water is most commonly used to calibrate pipettes and to check inaccuracy and imprecision. A pipette could possibly be recalibrated for liquids with densities that vary greatly from that of water. Pipettes should be checked regularly for precision and accuracy. Regular maintenance (e.g., cleaning) should be performed either by the user or a service technician according to manufacturer's instructions. All calibration and maintenance records must be maintained.

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