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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

Laboratory Guidelines
General Anatomic Pathology Chemistry Hematology Transfusion Medicine

2013 Edition

Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

LABORATORY GUIDELINES - 2013


SUMMARY OF CHANGES The following GUIDELINES have been revised, added, or deleted in this edition of the document. REVISED: Reference Textbook List for Laboratories Urinalysis Retention Guideline eGFR Differential Quality Control Procedure for WBC Estimate

ADDED: International Sensitivity Index (ISI) Verification Verifying or Establishing a Normal Reference Range for Routine Coagulation Testing

DELETED: Performance of Whole Blood Glucose Testing D-dimer Semen/Sperm Analysis

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

Table of Contents
General
Retention Guideline Recommended Reference Textbook List for Laboratories 4 7 10 12 13 15 16 17 19 20 21 21 22 23 26 27 28 31 32 33 35 37 39 42 43 44 45 46 47 49 50 51 53 56 58 60 62 64 66 67 71 72

Anatomic Pathology
Specimens Exempt from all Gross &/or Microscopic Pathology Laboratories Surgical Pathology Reports Removal of Tissue, Blocks or Slides from the Original Hospital Site Performance of Non-Gynecological Cytology Follow-up Reports for Gynecological Cytology Follow-up Program for Cytology

Chemistry
Estimated Glomerular Filtration Rate eGFR Cholesterol/Triglyceride/Lipid Testing Urinalysis Reporting Sperm in Urine Quality Control Diagnosis and Monitoring of Thyroid Disease Presence of Small Amounts of Albumin Crosscheck/Validation Guideline for Those Facilities with Multiple Chemistry Instruments Procedure/Method Statistical Work-up/Validation Study Guidelines

Hematology
Principles for Hematology Practice Hematology Films/Labelling of Slides Morphology of Lymphocytes Differential Performance and Referral Practice Guideline Red Blood Cell Morphology Reporting Guideline Differential Quality Control Smudge Cells Flow Cytometry for the Diagnosis of Lymphoma/Leukemia Malaria Erythrocyte Sedimentation Rate (ESR) 3.2% Na Citrate Anticoagulant Recommended vs. 3.8% for Coagulation Studies Vitamin B12 & Folate Bleeding Time Crosscheck Validation Guideline for Facilities with Multiple Hematology Instruments Procedure/Method Statistical Work-up/Validation Study Guidelines Protocol for Validation of Linearity on Automated Hematology Analyzers Procedure for WBC Estimate Establishing Conversion Factor for WBC Estimation Procedure for Platelet Estimates Establishing Conversion Factor for Platelet Estimation Indirect Platelet Count International Sensitivity Index (ISI) Verification Verifying or Establishing a Normal Reference Range for Routine Coagulation Testing

Transfusion Medicine
Retention of Transfusion Medicine Records Procedure/Method Statistical Validation/Work-up Guidelines Trans. Med.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

GENERAL

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RETENTION GUIDELINE

Laboratories vary in size, facility and extent of services provided. Clinical laboratories must maintain thorough, accessible records that can demonstrate an acceptable standard of care and compliance with the accreditation requirements. The Laboratory Quality Assurance Program of the College of Physicians and Surgeons urges laboratories to retain records, materials, or both for a longer period of time than specified for educational and quality improvement needs. Laboratories must establish policies that meet or exceed the following minimum requirements for retention of documents and specimens as established by professional and/or regulatory organizations.
References include: CSTM, CSCC, CAP, CSA, ISO, CPSS

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

Record Retention
Accession record Worksheets Instrument print-outs Paper copy of patient reports Quality control/PT documents Maintenance records Service records Method /instrument evaluation Procedure Manual Technologist ID & initials log/computer Telephone logs Requisition Laboratory Information Systems Records - Validation records, including transmission of results and calculations - Database changes - Hardware and software modifications - LIS downtime and corrective action Biomedical Waste

Storage Time
Hematology Biochemistry Microbiology Cytopathology Surgical Pathology

1 year 1 year 1 year 3 months 2 years 2 years

1 year 1 year 1 year 3 months 2 years 2 years

1 year 1 year 1 year 3 months 2 years 2 years

2 years 6 months n/a indefinite

2 years 6 months n/a indefinite

1 year 3 months 3 months 2 years

2 years 2 years Life of the instrument, plus 2 years Life of the instrument, plus 2 years 2 years after the method has been discontinued 2 years after procedure has been discontinued 1 year 1 year 1 year 1 year 3 months 3 months 2 years 3 months 1 year 2 years 3 months 5 years 2 years 3 months 5 years 2 years

Manifests must be retained for a minimum of 1 year

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

Specimen Retention

Hematology
Peripheral Blood Smear -7days Peripheral Blood Smear reviewed by a pathologist -1 yr Semen smears - 3 months Bone Marrow Slides/Reports - 20 yrs (adults) - 50 yrs (children) Whole blood/Plasma - 24 hrs after report has been finalized Body fluids - 48 hrs after report has been finalized Urine - 24 hrs after report has been finalized

Chemistry
Whole blood, serum & plasma - 48 hrs after report has been finalized Body fluids - 48 hrs after report has been finalized Urine - routine - 24 hrs after report has been finalized 24hr Urines - samples discarded 48 hrs after report has been finalized

Microbiology
Swabs or specimens - 24 hrs after report has been finalized Positive Blood Culture - 5 days after reporting Gram Stain - one week or until final report is sent Ova & Parasite slides - one month

Cytopathology
Slides
neg/unsatisfactory

Surgical Pathology
Blocks & slides - 20 yrs (adults) - 50 yrs (children) Autopsy - 20 yrs Gross specimen - min. 8 weeks after issue of report Wet Autopsy Tissue - 8 weeks after issue of report Bone Marrow Slides/ Reports - 20 yrs (adults) - 50 yrs (children) Photographic Transparencies indexed and kept indefinitely

- 5 years Slides suspicious/pos - 20 years Fine-needle aspiration slides - 20 years Cytology Consultation/ Requisition - Indefinitely Male fertility slides - 1 year Cytology paraffin blocks - 20 years

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RECOMMENDED REFERENCE TEXTBOOK LIST FOR LABORATORIES

Microbiology 1) Manual of Clinical Microbiology 10th Edition; American Society for Microbiology; Murray, Patrick R. 2) Bailey & Scott's Diagnostic Microbiology 13th Edition, Mosby, Inc.; Forbes, Betty A., et al. 3) Clinical Microbiology Procedures Handbook 3rd Edition; Issenburg

Transfusion Medicine 1) Circular of Information, Canadian Blood Services (most recent version) 2) Canadian Standards Association Blood and Blood Component Z902 (most recent version) 3) Canadian Society for Transfusion Medicine (most recent version) 4) Modern Blood Banking and Transfusion Practices 6th Edition; Harmening, Denise M. 5) Canadian Medical Association Journal Guidelines for Red Blood Cell and Plasma Transfusion for Adults and Children; supplement to CAN MED ASSOC J 1997; 156 (11) 6) American Association of Blood Banks Technical Manual - most recent edition 7) Bloody Easy 3: Blood Transfusions, Blood Alternatives and Transfusion Reactions 3rd Edition; Ontario Regional Blood Coordinating Network

Hematology 1) Color Atlas of Hematology, Hematology and Clinical Microscopy Resource Committee, CAP; Glassy, Eric F. 2) Clinical Hematology: principles, procedures, correlations 2nd edition; Stiene-Martin, Lotspeich-Steininger, Koepke 3) Hematology: Clinical Principles and Applications 4th Edition; Rodak, Fritsma, Doig 4) Clinical Hematology Atlas 4th Edition; Carr/Rodak

Chemistry 1) Tietz Fundamentals of Clinical Chemistry 6th Edition, W. B. Saunders Company; Burtis, Ashwood, Bruns

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2) Clinical Chemistry Principles, Procedures, Correlations 6th Edition; Bishop

Urinalysis 1) Graffs Textbook of Urinalysis and Body Fluids 2nd Edition; Mundt, Shanahan

Anatomic Pathology 1) Histotechnology: A Self Instructional Text 3rd Edition; Carson & Hladik 2) Principles of Anatomy & Physiology 13th Edition; Tortora & Grabowski

Safety 1) Transportation of Dangerous Goods Act and Regulations Supplement Canada Gazette, Part II [www.tc.gc.ca/eng/tdg/clear-tofc-211.htm] 2) CSMLS Guidelines Laboratory Safety, 7th Edition

Competency Evaluation 1) Canadian Society of Medical Laboratory Science PO Box 2830 LCD 1 Hamilton, ON L8N 3N8 website www.csmls.org Certification - Competency Profiles

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ANATOMIC PATHOLOGY

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SPECIMENS EXEMPT FROM ALL GROSS &/OR MICROSCOPIC PATHOLOGY LABORATORIES

Irrespective of the exemptions listed below, gross &/or microscopic examinations will be performed whenever the attending physician requests it, or at the discretion of the pathologist when indicated by gross findings. SPECIMEN TYPE Abdominal pannus Accessory Digits Amputation Bone or cartilage removed from the arthritic joints during joint replacement surgery Bone segments removed as part of corrective orthopedic procedures (for example: rotator cuff, synostosis repair, spinal fusion) Bunions and hammer toes Calculi (renal bladder etc.), are sent for chemical analysis and description Disc Materials Extraocular muscle from corrective surgical procedures (strabismus repair) Femoral head removed for prosthesis (if straight forward) Foreign bodies, such as bullets or medicoloegal evidence that is given directly to law enforcement personnel Gangrenous and traumatized limbs Intrauterine contraceptive devices without attached soft tissue Loose bodies (joint) Medical devices such as catheters, gastrostomy tubes, myringotomy tubes, stents and sutures that have not contributed to patient illness, injury or death Middle ear ossicles Nasal bone and cartilage from rhinoplasty or septoplasty Orthopedic hardware and other radioopaque mechanical devices provided there is an alternative policy for documentation DISCRETIONARY (By Chemistry Dept.) GROSS &/OR MICROSCOPIC

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

SPECIMEN TYPE Placentas which do not meet the criteria for examination Prosthetic breast implants Prosthetic cardiac without attached tissue Rib segments or other tissue removed only for the purpose of gaining surgical access from patients who do no have a history of malignancy Saphenous vein segments harvested for coronary artery bypass Skin or other normal tissue removed during cosmetic or reconstructive procedures (blepharoplasty, cleft palate repair, abdominoplasty, rhinectomy or syndactyly repair) that is not contiguous with a lesion and that is taken from a patient who does not have a history of malignancy Teeth without attached soft tissue Therapeutic radioactive sources Tonsils and adenoids if clinically not suspicious Torn menicus Varicose veins

DISCRETIONARY

GROSS &/OR MICROSCOPIC

(under 10 yrs old)

(over 10 yrs)

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SURGICAL PATHOLOGY REPORTS

Timeliness of reports is critical to providing quality of care. Guidelines for surgical pathology reporting include: (i) Routine Surgical Pathology reports Complete within 2 working days. Where additional procedures are being performed, an extension of 24 hours is appropriate. Autopsy Reports Written initial reports of gross pathological findings within 72 hours. Final Report 30 days for routine cases, 90 days for complicated cases

(ii)

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

REMOVAL OF TISSUE, BLOCKS OR SLIDES FROM THE ORIGINAL HOSPITAL SITE Anatomic Pathologists are charged with the responsibility of keeping and guarding the integrity of the ever-growing number of tissue containing paraffin blocks and slides derived from surgical, cytological and autopsy diagnostic services. Documentation and maintenance (tracking) of the continuous care shall ensure quality practice. These specimens must be maintained in orderly files to ensure ready access. There are inevitably increasing demands for slides, blocks or tissues to be retrieved from the original site. *A release form must be provided and retained on file at the original institution for permanent release. Summary Follow HIPA guidelines Ensure there is sufficient material for further work-up Indicate reason for request Return all material as soon as possible The lab is the custodian of tissue, blocks or slides collected. The source of material remains the property of the patient. These are the various suggested categories to be considered: In-Province Consultation Request may be initiated by the primary physician, surgeon or oncologist for review by local or out-of-district pathologist. Request may be initiated by the original signing out pathologist who is responsible for maintaining records and assuring return of the material. Note: The return of materials to the original site must be documented and a consult report sent to the original pathologist as well as the requesting pathologist. Out-of-Province (recommended slides only) Request from an originating pathologist to seek out-of-province consultation for diagnostic purpose. Research or national study groups request specimen be referred to another institution for treatment. If blocks are requested, cut a set and send the cut set. The originals should be maintained at the processing site. Educational Consultation Requests for educational rounds should be restricted to slides; to ensure integrity of patient property. Request should indicate for rounds and materials returned promptly to ensure ongoing patient care.

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Research Regulations require pathologists to obtain patient authorization and/or an Institutional Review Board (Ethics Committee) waiver of informed consent when using any identifiable patient health information for research purposes. Requests must ensure the integrity of the patient material. All materials that have critical diagnostic, prognostic or medical-legal implication may be retained at the discretion of the releasing institution. Return all materials as soon as possible.

References: Guardians of the Waxand the Patient. Editorial.; American Journal of Clinical Pathology 1995 104 p 356-7 Use of Human Tissue Blocks for research. Association of Directors of Anatomic and Surgical Pathology. Human Pathology 1996.27 p 519-520

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PERFORMANCE OF NON-GYNECOLOGICAL CYTOLOGY Non-gynecological cytology comprises of fine needle aspiration biopsies (FNAB) of organs/tissues such as lungs and other visceral lesions, effusion cytology of pleural, peritoneal, pericardial fluids, cytology of urine, CSF, sputum, broncho-alveolar lavage (BAL) and brush biopsies of endoscopic procedures, (gastrointestinal tract, etc). Scrapings of open lesions and nipple discharges may also be included along with cytology of transplant organs to test for rejection (kidneys), or for cyclosporin toxicity. BAL and transplant cytology is usually done in specialized centers as it requires specific interpretation and often special tests. Most of the other samples can be handled and processed in a routine surgical pathology/cytology lab equipped with basic facilities including a biological safety cabinet (fume hood), cyto-centrifuge and staining capability for H & E and PAP stains. In contrast to gynecological cytology, non-gynecological cytology (NGC) does not necessarily require a screening step. If adequate diagnostic material is present the focus is on diagnosis of and interpretive correlation with the clinical setting. If cell block or cytospin samples are available, further testing with special procedures could be performed. Some aspects of NGC require a rapid turnaround time such as FNAB performed under CT-scan or ultrasound guidance and intra-operative cytology requests. It is important for institutions with CT scanner facilities to be able to provide cytology service in house. However, it is acceptable, if the lab does not have a cytology department, the technologists in the histology lab are trained in processing the specimens. Such training is easily obtained and can be provided by short courses provided on site and documented. Most NGC procedures are performed on patients who are in-patient residents in a hospital/health care institution or are required to come in for a day procedure/ambulatory care. Due to the time factor involved in patients institution stay; a rapid turnaround time becomes a key factor in availability of the service. On the other hand, the patient in an acute care setting may have an infectious process or malignancy requiring rapid diagnosis and treatment. The final interpretation and reporting of non-gynecological cytology shall be made by a pathologist.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

FOLLOW-UP REPORTS FOR GYNECOLOGICAL CYTOLOGY

To ensure a quality cytology service, a follow-up mechanism must be in place to provide reports to the primary and/or consulting physician. In an attempt to eliminate the potential for LOST TO FOLLOW reporting situations, the following require follow-up letters to the primary and/or consulting physician: 1. A repeat smear was requested at the time of reporting and 3 months have lapsed since the date of request. A diagnosis is rendered requiring follow-up and none has occurred. For example: HSIL required follow-up a.s.a.p. and if this has not occurred within 3 months, a letter is required. LSIL (ASCUS, AGUS) within 6 months requires a letter in 9 months. A malignant diagnosis with no apparent followup. A follow-up letter has been previously issued with no reply. These letters should be automatically generated by the computer system and then replies must be recorded and reviewed quarterly. A minimum laboratory requirement of a computer system used to report gynecological cytology shall have the ability to generate automatic follow-up letters that are linked to diagnostic codes.

2.

3.

4.

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FOLLOW-UP PROGRAM FOR CYTOLOGY

A follow-up mechanism shall be in place to ensure that actions appropriate for abnormal findings are implemented.

a)

All patients who are reported to have a significant abnormality should be followed up by the laboratory or other agency to which this task may be delegated, to obtain final clinical or preferably tissue confirmation of the diagnosis. Statistical data should be maintained which would include the number of cases screened annually in each category, and all correlative follow-up data available. Discrepancies, if any, should be included with this information.

b)

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CHEMISTRY

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

ESTIMATED GLOMERULAR FILTRATION RATE - eGFR

The glomerular filtration rate is the estimated volume of glomerular filtrate that moves from renal glomerular capillaries into the Bowmans capsule per unit time. Evidence-based clinical practice guidelines suggest that an estimate of GFR (eGFR) provides the best clinical tool to gauge kidney function. The Canadian Society of Nephrology has recommended that laboratories report eGFR routinely for adult patients, in order to detect chronic kidney disease. Serum creatinine results can vary significantly and tend to be ineffective in general practice as an early marker. 24 hr. collection for creatinine clearance is impractical and prone to error. eGFR should be reported on outpatients over the age of 18 years. eGFR has not been validated for use in hospitalized patients and therefore, is not recommended for reporting on inpatients. eGFR is less reliable in,
patients with near normal eGFR unstable serum creatinine acute illness extremes of body composition (eg. obesity, cachexia) unusual muscle mass (e.g. marked muscularity, muscle disease, amputation) pregnancy age under 18 years or over 70 years drugs with significant renal toxicity or clearance drugs affecting creatinine metabolism or clearance unusual dietary intake (e.g. vegetarians) other serious comorbid conditions

Summary: Reporting of eGFR is becoming the standard of care in helping identify, stage and monitor patients with chronic kidney disease.
eGFR > 60 eGFR 30-59 eGFR 15-29 eGFR < 15

Normal or slightly decreased kidney function (stages 1 or 2) Moderately decreased kidney function (stage 3) Seriously decreased kidney function (stage 4) Kidney failure (stage 5)

eGFR is frequently used for DRUG DOSING using the Cockroft-Gault equation. eGFR-MDRD has not been validated for this purpose. eGFR-MDRD assumes steady state. For rapidly changing kidney function, monitor serum creatinine. (MDRD: Modification of Diet in Renal Disease) The reported eGFR shall be multiplied by 1.21 for patients of African descent.

NOTE: This information is intended for clinicians, patients and allied health professionals.
References: www.jasn.org, www.csnscn.ca, www.renal.org http://www.kidney.org/professionals/kdoqi/gfr_calculator.cfm

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CHOLESTEROL / TRIGLYCERIDE / LIPID TESTING

Cholesterol results are based on optimal performance of testing. Considerations to be included: a) b) c) d) treatment/preparation of the patient the emergent or non-emergent nature of the test appropriate technical equipment adequate quality control

The accomplishment of treatment goals also demands accurate cholesterol measurements. This requires standardization of all cholesterol measurement for accuracy to minimize the methodspecific biases. This can be achieved ONLY by standardizing the cholesterol measurements and ensuring accuracy that is traceable to the National Reference System for Cholesterol (NRS/CHOL), National Cholesterol Education Program. Clinical protocols are well established and should be followed by all testing sites. Laboratories testing for lipids shall be capable of performing the entire profile, to include: Cholesterol, Triglycerides, HDL and LDL, for diagnosis and assessment. All lipid measurements should be performed by the same methodology. Only instrumentation capable of maintaining intralaboratory precision that is less than or equal to 3 % (C.V.); and can demonstrate an accuracy bias of less than 3 % from the true value may be used for cholesterol analysis.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

URINALYSIS

Complete urinalysis shall include microscopic examination if the following are present: leukocyte esterase, blood, protein, turbidity and nitrites. The microscopic examination should be completed within four hours of collection. (Note: specimens not tested within 2 hours should be refrigerated.) Report semi-quantitative results in SI units.

REPORTING SPERM IN URINE

Sperm are not normally present in urine and if presence is detected, it is usually considered a contaminant. When sperm appear in a microscopic exam, they shall be reported as present. However, prior to reporting, results should be discussed with the attending physician, in order to avoid errors (ie. mislabelling, etc.).

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

QUALITY CONTROL (QC)

The purpose of QC is to detect the problems early enough to prevent their consequences. QC emphasizes statistical control procedures, but may also include non-statistical check procedures, such as linearity checks, reagent and calibration checks, etc. Two or three different materials should be selected to provide concentrations that monitor performance at different levels of medical decision-making. For quantitative tests, the use of two levels of control material shall be run each day of use, as a minimum. For qualitative tests that include built-in controls, a positive and negative control shall be performed a minimum of once per month and upon initiation of a new lot number and shipment. For those that do not include a built-in control, known positive and negative external controls shall be tested each day of use.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

DIAGNOSIS AND MONITORING OF THYROID DISEASE

Thyroid function tests are among the most commonly ordered laboratory tests in the province. In the past, investigations of thyroid disease required more than one test. Sensitive thyroid stimulating hormone (TSH) is the initial test in the diagnosis of hypothyroidism (TSH elevated or above normal) and hyperthyroidism (TSH is suppressed or below normal). Free T4 (FT4) is preferred over total T4 (TT4) measurement to confirm the diagnosis of hypothyroidism or hyperthyroidism. FT4 is 0.02 0.04% of total T4. FT4 is the metabolically active form of TT4 and is a better indicator of thyroid status than TT4 because it is unaffected by protein binding abnormalities such as pregnancy and oral contraceptives. Free T3 (FT3) is mainly of value in diagnosing T3 toxicosis, in determining the T3 response to therapy, and clarifying protein binding abnormalities. It can also be of use in the early progression of subclinical hyperthyroidism to overt thyrotoxicosis when FT4 is normal and TSH is suppressed. FT3 is often the first to be increased. FT3 is approximately 0.2 0.5% of total T3. American Association of Clinical Endocrinologists (AACE) recommends that the TSH reference range run from 0.3 - 3.0, versus the old range of 0.5 - 5.5. Keep in mind that there is disagreement among practitioners. Limitations: 1. These guidelines do not apply to neonates. 2. TSH is not reliable in the investigation of hypothalamic or pituitary disease. 3. TSH may be an unreliable indicator of thyroid status in patients with acute severe nonthyroidal illness (e.g. CCU and ICU patients) and the test is only recommended when there are clinical indicators of possible pre-existing thyroid disease. 4. Medications such as lithium, amiodarone, glucocorticoids, and dopamine affect TSH and may also affect the individuals thyroid status. Clinical Aspects of Testing: 1. Screening asymptomatic, apparently healthy patients for thyroid disease is not considered indicated at this time. 2. Testing is indicated in the presence of symptoms or signs that are suggestive of thyroid disease especially in high risk populations. 3. High-risk groups include women over 50, the ambulatory elderly, postpartum, individuals with a strong family history of thyroid disease and other autoimmune diseases such as Type I diabetes. Symptoms and Signs of Hypothyroidism: Cold intolerance, lethargy, depression, constipation, menstrual disorders, dry skin, weight gain. There will be slow growth in children. Symptoms and Signs of Hyperthyroidism: Palpitations, fatigue, weakness, increased appetite, heat intolerance, usually enlarged thyroid, weight loss, warm moist skin, tremor and tachycardia. Restlessness, sleep disturbances, difficulty maintaining attention and concentration occur in children.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

Recommended Testing Algorithm


TSH

Increased

Normal

Decreased

FT4 low

Primary hypothyroidism

If clinical suspicion is high for secondary hypothyroidsm order FT4 as initial test

FT4 high

FT4 normal

hyperthyroidism

FT3 if clinical suspicion is high

If TSH screen is abnormal, a FT4 will be done on the same sample reducing the need to call back the patient for subsequent testing. If the TSH is less than 0.3mU/L a FT4 and FT3 will be done on the same sample. Free T3 is a better indicator of the degree of thyrotoxicosis in most patients. Further Testing Recommendations: 1. Follow-up for Primary hypothyroidism or replacement therapy: TSH should be performed 6 8 weeks after start of therapy or dosage adjustment. After normal results have been achieved, TSH should be done annually, unless the clinical condition changes or unless the clinical condition warrants re-testing. In children under one year of age the TSH and FT4 should be measured every 3 months and every 6 months for children under six years of age. Also rapidly growing adolescents should have a TSH checked once every 6 months. 2. After Radioactive iodine treatment: A FT4 should be done at 4-6 weeks interval for the first 6 months or until normal. At 6 months and then annually a TSH should be done to detect hypothyroidism. In most children and young people following thyroid ablation the FT3 is a better indicator of control. 3. Antithyroid drugs for Hyperthyroidism: Patients should be monitored by means of FT4 monthly until controlled and then at least every 3 months while on medication. If clinical signs and symptoms are present a FT3 may be indicated. In cases of T3 toxicosis a FT3 should be ordered. In patients with thyrotoxicosis the TSH may not recover for quite some time after euthyroidism has been achieved and sometimes requires a period of hypothyroidism before recovery. 4. Suppressive doses of thyroxine: Designed to support a neoplasm or goiter. TSH and FT4 every 2 months until TSH has reached a level of suppression acceptable to the clinician. 5. Subclinical hypothyroidism: Borderline results are fairly common in elderly patients and individuals with an autoimmune mechanism are more likely to progress to a hypothyroid state. Observation and monitoring by TSH at 6-12 month intervals is recommended.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

6. Subclinical hyperthyroidism: Patients with low or suppressed TSH but thyroid hormones in the normal levels with minimal or no symptoms are followed by FT4 and/or FT3 at 6 12 months to gauge the progression of their condition.

References: 1. 2.

HSURC guidelines. Nov 1992 Ontario Association of Medical Laboratories. Guidelines for the use of serum tests to detect thyroid dysfunction. May 1987 3. Ontario Association of Medical Laboratories. Guidelines for the use of serum testing in the management of primary hypothyroidism. June 1998 4. Alberta Medical Association. Laboratory Testing Guidelines for Investigation of Thyroid Dysfunction. May 1999 5. Protocol Steering Committee B.C. for the use of Thyroid function tests in the diagnosis and monitoring of patients with thyroid disease. Aug 1997 6. The College of Physicians and Surgeons of Manitoba. Investigation of Thyroid Disease. February 1995 7. National Academy of Clinical Biochemistry. Laboratory Support for the Diagnosis and Monitoring of Thyroid Disease. November 2000 8. Vanderpump MPJ, Ahlquist JAO, Franklyn JA et al 1996. Consensus statement of good practice and audit measures in the management of hypothyroidism and hyperthyroidism. BMJ 313: 539-44. 9. Singer PA, Cooper DS, Lewy EG et al 1995. Treatment guidelines for patients with hyperthyroidism and hypothyroidism. JAMA 273: 808-12. 10. Ladenson PW, Singer PA, Ain KB et al 2000. American Thyroid Association Guidelines for detection of thyroid dysfunction. Arch Intern Med 160: 1573-5.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

PRESENCE OF SMALL AMOUNTS OF ALBUMBIN

Presence of small amounts of albumin in urine is considered an early predictor of the development of glomerular damage in the absence of overt nephropathy. Patients with diabetes and hypertension are the primary risk groups. The Canadian Diabetes Association recommends testing for small amounts of albumin once/year after the onset of diabetes. The presence of small amounts of albumin in urine is detectable by dipstick methodologies, and is approved as a screen for renal damage in the known diabetes patient. All positive results must be confirmed by quantitative analysis.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

CROSSCHECK/VALIDATION GUIDELINE FOR THOSE FACILITIES WITH MULTIPLE CHEMISTRY/HEMATOLOGY INSTRUMENTS PERFORMING THE SAME TEST PROCEDURE Proficiency testing registration is mandatory for each analytical test. Rotating proficiency testing result submission between analyzers is not a requirement, but is suggested where appropriate (e.g. Blood Gases). Where there are multiple analyzers performing the same test procedure in larger facilities, it may be more appropriate for proficiency testing submission to be consistently submitted from the same analyzer for tracking purposes. An internal cross-check/validation protocol is required to ensure that there is correlation between all analyzers providing the same test result in the same facility. If this protocol is not followed, then each analyzer must be registered in the external proficiency testing program as mandated by LQAP. This procedure is recommended every six months. Chemistry/Hematology High Volume Analyzers (e.g. Electrolytes/CBC) Validation/Crosscheck Element:
Patient correlation

Requirement:
Regularly scheduled intervals Whenever criteria for recalibration/validation is met: - change of manufacturer for reagents or equivalent - after maintenance or service as per manufacturers recommendations - as required for purposes of troubleshooting /validation of reagent lot # changes or as indicated by quality control data

Frequency/Data Points:
Minimum of 20 patient specimens/2 times per year or equivalent (i.e. 10 patient specimens/4 times per year or on-going data collection as appropriate) As necessary per recalibration/validation event

Chemistry/Hematology Low Volume Analyzers (e.g. Fibrinogen) Validation/Crosscheck Element:


Patient correlation

Requirement:
Regularly scheduled intervals Whenever criteria for recalibration/validation is met: change of manufacturer for reagents or equivalent after maintenance or service as per manufacturers recommendations as required for purposes of troubleshooting /validation of reagent lot # changes or as indicated by quality control data

Frequency/Data Points:
Minimum of 5-10 patient specimens/2 times per year or equivalent As necessary per recalibration/validation event

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

PROCEDURE/METHOD STATISTICAL WORK-UP/VALIDATION STUDY GUIDELINES: CHEMISTRY/HEMATOLOGY

Work-up guidelines and definitions (change in method/instrument):


Work-up Element
1. Imprecision within run between run

Definition: CLIS or International Federation of Clinical Chemistry (IFCC)


The variation in analytical results demonstrated when a particular specimen of aliquot is analyzed multiple times or on multiple days. Imprecision is expressed quantitatively by a statistic such as standard deviation or coefficient of variations.

Minimum Data Requirements (where appropriate):


Within run use preferably a patient sample or pool close to the decision levels with a minimum of 10 data points. Between run 20 results from 20 separate runs on 2 levels over a 10-day minimum time period using appropriate QC material. 40 data points are recommended with a minimum of 20 having 50% of the data points outside the reference intervals, if possible. Correlations should involve comparison with an acceptable reference method or laboratory. 4 data points each in duplicate as a minimum requirement, but 5 data points are preferred (over reportable range). Linearity studies are expected on an initial method work-up and further studies as defined by the College guidelines (i.e. troubleshooting). The minimum requirement is 20 data points for confirmation of an established reference range and 120 for the establishment of a new reference range. 3 data points using acceptable reference material (i.e. CEQAL or CAP) 1 data point may be acceptable for haematology accuracy studies if related to sample stability. Sensitive studies are only required for those methods which have clinical relevance at values close to 0 (i.e. TSH)

Applicability:

Qualitative

Quantitative

2. Patient Correlation

The correlation coefficient is a means to look for a relationship, not agreement, between pairs. Two methods may have a perfect correlation throughout the measuring range but may not agree in value (i.e. one may be double the value of the other).

n=20

n=40

3. Linearity

(IFCC) The range of concentration or other quantity in the specimen over which the method is applicable without modification (CLIS) when analytical results are plotted against expected concentrations; the degree to which the plot curve conforms to a straight line is a measure of the system linearity.

4. Reference range validation 5. Accuracy

It is common convention to define the reference range or interval of a laboratory test as the central 95% interval bounded by the 2.5 and 97.5 percentiles of the selected patient population. Validation of an established reference range requires a minimum of 20 samples. Closeness of the agreement between the result of a measurement and the accepted reference value (true value of the analyte)

6. Sensitivity

Measure of the ability of an analytical method to detect small quantities of the measured component. When concern is performance at a very low concentration it is useful to determine the detection limit as influenced by imprecision.

When clinically relevant

When clinically relevant


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7. Specimen Stability

The conditions of handling and storage, which permits the measurement and reporting of a clinically relevant result.

8. Interference

The effect of any component of the sample on the accuracy of the measurement of the desired analyte.

9. Recovery

A recovery procedure involves the addition of a known amount of analyte to an aliquot of sample. Recovery is defined as the ratio of the amount of the analyte recovered to amount added and is given as percentage.

No data generally required. As per manufacturers guidelines. If manufacturers stability window is to be extended a stability study is expected. Document the manufacturers interference information. The method should include a disclaimer or a process for dealing with a lipemic, icteric or hemolyzed sample. Recovery studies should only be necessary for those methods or analytes where organic extractions or equivalent are required as part of the methodology (i.e. Toxicology)

Storage and transportation dependent Methods with known interferences

Storage and transportation dependent Methods with known interferences Method specific/organic extraction

Work-up requirements when an instrument is moved from site A to site B: (It is assumed that the instrument has been in recent use with acceptable performance). Work-up Element 1. Imprecision studies, QC only 2. Patient correlation Minimum data requirements: As above 10 data points, where feasible.

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HEMATOLOGY

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PRINCIPLES FOR HEMATOLOGY PRACTICE

Hematology incorporates leading edge technology to help decipher and treat troubling diseases. As hematology has evolved, there are some generic principles that must be simply stated for quality patient care. Quality management practices are essential to ensuring quality care. Some of the core principles include: 1) Hemoglobin, the single most common complex organic molecule (Hb) shall be determined by spectrophotometric methodology. 2) WBC and platelet counts shall be tested by automated methodology as part of a CBC on whole blood specimens. If necessary, WBC and platelet counts may be estimated on the peripheral smear. 3) Manual PT (INR)/APTT testing shall be discontinued. Please refer to CLSI H21-A4 for reference on collection and storage. 4) Reticulocyte Count shall be performed by automated methodology. Manual counts may used as a QC method for automated analyzers. 5) All differential leukocyte counts shall be reported in absolute values. Reporting percentages is optional, and would be in addition to absolute values.

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HEMATOLOGY FILMS/LABELLING OF SLIDES

Unequivocal patient identification is the first step to ensuring a quality hematology slide. With no formal standards in place for labelling slides, the basic principles require: Unique identification of the patient (at least two identifiers) Written instructions for labelling Labels shall be clear and legible Date of collection CLSI Document H-20-A states: Label the samples uniquely. Positive Patient ID

_______________________________
Last Name

_______________________________
First Name

PHN______________________________________________________________
Date of Birth Unique ID #

Date of Slide___________________________________

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MORPHOLOGY OF LYMPHOCYTES

Benign versus Malignant A variety of diseases and disorders may produce changes from the normal in numbers and/or morphology and functions of one or more of the leukocytes. The most important feature of variant lymphocyte morphology is the recognition of its benign nature. The pertinent fact is that these lymphocytes are normal cells that have been altered as the result of a normal response to stimulus. When changes in WBCs are produced by non-malignant disorders (e.g. infections), the cells formerly called atypical, are now referred to as reactive lymphocytes. When changes are suspicious of being produced by malignant disorders (leukemias, lymphomas, gammopathies) and additional investigations may be required, the cells are often referred to as atypical and/or abnormal. In non-malignant disorders, the variant lymphocytes, reactive lymphocytes, atypical lymphocytes, virocytes, stress lymphocytes, Downey cells, transformed lymphocytes, transitional lymphocytes, and glandular fever cells, among others, are normal cells reacting to a stimulus, whether it be viral or other. The designation of reactive lymphocytes is preferred. In Chronic Lymphocytic Leukemia, the lymphocytes are somewhat larger than normal, have nuclei with clumped or condensed chromatin, and may have prominent nucleoli. The cytoplasm may be abundant, nongranular and moderately basophilic, or it may be relatively scant. In Prolymphocytic Leukemia, the prolymphocyte is a relatively large mononuclear lymphoid cell with an oval to round nucleus, coarse-appearing chromatin strands and one or two large vesicular nucleoli with perinuclear condensations of chromatin. The cytoplasm is abundant and usually granular and is basophilic with Romanowsky stains. In Waldenstrms Macroglobulinemia, the abnormal B-lymphocytes involved are transitional cells. They have the ability to differentiate into large plasmacytoid lymphocytes and plasma cells. These malignant cells circulate in the peripheral blood only in the terminal stages. In Lymphomas, peripheral blood involvement (i.e., abnormal circulating cells) is seen late in the disease. Lymphoma cells can exhibit a variety of appearances and the cellular morphology is variable and depends on the underlying type of lymphoma. These cells can exhibit variable size, shape, nuclear, and cytoplasmic characteristics. Lymphoma cells are usually round to oval, and can be irregular. Cell size ranges from 8 to 30 m and the N-C ratio varies from 7:1 to 3:1. In diffuse small lymphocytic lymphoma (the tissue equivalent of chronic lymphocytic leukemia), the cells are generally small with round to oval nuclei, compact and coarse chromatin, and have a scant amount of basophilic cytoplasm. They may be the same size as normal lymphocytes or may be slightly larger. Occasionally, the nuclei exhibit an angulated appearance with slightly more open chromatin. A small nuclear indentation may be present. Nucleoli are not seen. Scattered prolymphocytes, which are larger cells with a centrally placed nucleus, a prominent single nucleolus, and moderate basophilic cytoplasm, often are seen. In the small-cleaved cell lymphomas, the cells are slightly larger than normal lymphocytes and have an angulated

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appearance. The majority of nuclei have clefts, indentations, folds, convolutions, and may even be lobulated. The chromatin is moderately coarse and one or more nucleoli may be prominent. Their cytoplasm is scant to moderate and basophilic. The cells in small noncleaved lymphomas (Burkitts lymphoma) appear similar to L3 lymphoblasts. These cells are generally moderate i n size (10 to 25 m) and have a round to oval nucleus with moderately coarse chromatin, and one or more prominent nucleoli. The cytoplasm is moderate, stains dark blue, and may contain numerous small vacuoles. Large cell lymphomas and immunoblastic lymphomas may exhibit some of the most blast-like and abnormal morphology. These cells are large (20 to 30 m) and have scant to moderate amounts of deeply basophilic cytoplasm. The nuclei are generally round to oval, but may be angulated, folded, indented, or convoluted. Nucleoli are prominent and may be single or multiple. Vacuoles can occasionally be seen in the cytoplasm. These cells can be easily confused with blasts. T cell lymphomas can exhibit similar morphology to any of the above types of lymphomas. The typical appearance is a moderate-size cell with a markedly convoluted nucleus giving a cerebriform or grooved pattern. Their chromatin is moderately coarse and nucleoli are not apparent. The cytoplasm is generally scant and blue. In Hairy Cell Leukemia, the abnormal lymphocytes (Hairy Cells) have scant to abundant, agranular, light grayish-blue cytoplasm. The plasma membrane appears irregular with hair-like or ruffled projections, which are seen more easily with phase microscopy. These cells often have a round or oval nucleus; sometimes, the nucleus appears folded or bilobed. The chromatin is loose and lacy, and one or two nucleoli are commonly seen. In Szary Syndrome, the abnormal lymphocyte is larger than normal with scanty cytoplasm, and the nucleus is large with clefting. Nuclear folding can be so extensive as to suggest an image of the brain, and these nuclei are thus described as cerebriform. The nuclear chromatin is fine with little condensation. There may or may not be visible nucleoli. Note: Performance of manual differentials is required when abnormalities/unexpected results are found in the WBC.

References: McTaggart Bill, SAIT Hematology Updating Correspondence Course, 5th Edition, 1993. pp. 10. Stiene-Martin, 1998, pp. 355-356, 484-485, 490, 507-508. College of American Pathologists, Surveys, Hematology Glossary, 2001

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DIFFERENTIAL PERFORMANCE AND REFERRAL PRACTICE GUIDELINE


Blood Film WBC Count adults Lower referral range Upper referral range children (2-14 years) children (90 days 2 yrs) newborn (0 90 days) Absolute Neutrophils Absolute Granulocytes Absolute Eosinophils Absolute Basophils Absolute Lymphs adults children (0-14 years) Absolute Monocytes Hemoglobin Lower referral range adult female adult male Upper referral range adult female adult male Pediatric ranges children (1 mo 14 yrs) newborn (0 1 month) HCT MCV Lower referral range > 3 months (adult) 0 3 months MCHC Upper referral range MCH RDW RBC COUNT adult female adult male Reference Range 4.0 - 11.0 x 109/L <1.5 x 109/L (all age groups) >25.0 x 109/L 5.0 15.0 x 109/L 5.0 20.0 x 109/L 7.0 20.0 x 109/L 1.5 7.5 x 109/L 1.5 7.5 x 109/L 0.0 0.6 x 109/L 0.0 0.2 x 109/L 1.1 4.4 x 109/L 0.2 0.8 x 109/L 120 160 g/L 135 180 g/L 120 160 g/L 135 180 g/L 105 145 g/L 135 195 g/L 0.37 0.50 L/L 79 97 fL 98 114 fL 310 360 g/L 27 32pg 11.5 14.5 % 3.2 5.4 x 1012/L 4.6 6.2 x 1012/L >360 g/L None None >6.5 x 1012/L >365 g/L none none >6.5 x 1012/L <1.5 x 109/L (all age groups) <1.5 x 109/L (all age groups) >1.0 x 109/L (all age groups) >0.3 x 109/L (all age groups) >5.0 x 109/L >7.0 x 109/L >1.0 x 109/L (all age groups) <1.0 x 109/L (all age groups) <1.0 x 109/L (all age groups) >2.0 x 109/L (all age groups) >0.5 x 109/L (all age groups) >7.0 x 109/L >10.0 x 109/L >1.5 x 109/L (all age groups) <1.0 x 109/L (all age groups) >25.0 x 109/L Perform a Differential or Scan on First Occurrence or Significant Change Referral Unexpected or Unexplained

<100 g/L (Combined with MCV <70 fL) <100 g/L (Combined with MCV <70 fL) >165 g/L >185 g/L <100 g/L (Combined with MCV <70 fL) <160 g/L or >210 g/L None

<100 g/L (Combined with MCV <70 fL) <100 g/L (Combined with MCV <70 fL) >165 g/L >185 g/L <100 g/L (Combined with MCV <70fL) <135 g/L or >210 g/L >0.65 L/L

<70 fL or >100 fL (Combined with HGB <100 g/L) <97 fL (Combined with HGB <135 g/L)

<70 fL (Combined with HGB <100 g/L) <90 fL (Combined with HGB <135 g/L)

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Blood Film

Reference Range 7.4 10.4 fL 150 400 x 109/L

MPV Lower referral range Upper referral range Platelet Count Lower referral range Upper referral range WBC Morphology

Perform a Differential or Scan on First Occurrence or Significant Change None

Referral Unexpected or Unexplained

<6.0 fL >14.0 fL <100 x 109/L >600 x 109/L <100 x 109/L >600 x 109/L > 10% Reactive Lymphs Pelger-Huet anomaly Hypogranulated neutrophils Hairy Cells Blasts/Immature Cells Hypersegmented neutrophils >5 NRBC/100 WBC RBC inclusions: Pappenheimer, Howell-Jolly or Heinz Body, basophilic stippling Presence of schistocytes, echinocytes, bite cells, sickle cells, rouleaux, autoagglutination, significant polychromasia, oval or round macrocytes, target cells, tear drops, spherocytes, elliptocytes, acanthocytes, stomatocytes Dimorphic picture Parasites Malaria none

NUCLEATED RBCS RBC Morphology

PLATELET MORPHOLOGY OTHER CRITERIA Specified Instrument Flags Ordered by Physician Technologist Discretion

When indicated Physician request Technologist initiated Physician request Technologist initiated if suspicious cells are present, refer to a pathologist

The Color Atlas of Hematology, Hematology and Clinical Microscopy Resource Committee, CAP; Glassy, Eric F. is recommended as a resource.

NOTE: This table is provided as a guideline only. Consult a larger centre for more specific ranges.

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RED BLOOD CELL MORPHOLOGY REPORTING GUIDELINE

Red Blood Cells/100x oif (200 RBC field) x 10 fields Any present

Abnormality to be Reported Schistocytes/Helmet Cells

Implications for Diagnosis Thrombotic thrombocytopenic purpura (TTP), RBC fragmentation syndromes such as hemolytic uremic syndrome, DIC, microangiopathic hemolysis, malignant hypertension, eclampsia, Cardiac valve hemolysis, some renal vascular diseases Kidney Disease Drug or chemical induced oxidative damage, unstable hemoglobins Sickle Cell Anemia, Hemoglobin SC/SD Disease Lead Poisoning, Thalassemia, Sideroblastic & Megaloblastic Anemia, Sickle Cell Anemia Megaloblastic Anemia, Postsplenectomy state Hemorrhage, response to treatment, Sideroplastic anemia, post-transfusion Paraproteinemia, increased fibrinogen, inflammatory disorders Autoimmune Hemolytic Anemia (Cold Agglutinin Disease) Identify specific forms Sever hemolysis, part of Leukoerythroblastic picture, Bone marrow stress Response to treatment, blood loss, Hemolysis Megaloblastic state, Aplastic Anemia, Myelodysplastic Syndrome

Echinocytes/Burr Cells Bite Cells

Sickle Cells Basophilic Stippling

Howell Jolly Bodies Dimorphic

RBC Rouleaux (5cells stacked) RBC Aggutination Parasites Nucleated RBC

>5

Polychromasia Macrocytes (oval)

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

Target Cells

Tear Drops Spherocytes Pappenheimer Bodies

Liver Disease, Postsplenectomy/hyposplenism, Hemoglobinopathy, Thalassemia Myelofibrosis, Pernicious Anemia Hereditary Spherocytosis, Autoimmune Hemolytic Anemia Sideroblastic Anemia, Chronic Hemolysis, Liver Disease Liver Disease, Alcoholism Hereditary Elliptocytosis Post-splenectomy state, Liver Disease, Abetalipoproteinemia Liver Disease Iron Deficiency, Thalassemias, Treated Polycythemia

>10

Macrocytes (round) Elliptocytes Acanthocytes/Spur Cells Stomatocytes Microcytes (hypochromic cells)

Avoid using the terms anisocytosis and/or poikilocytosis since they convey no specific meaning.
The numeric value is meant for internal use to indicate a significant abnormality presence. No numeric value is reported, just the abnormality.

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DIFFERENTIAL QUALITY CONTROL

The following is a list of measures undertaken by each laboratory to ensure the quality of differential results reported on patients. Good Laboratory Practice: 1. Develop a protocol for determining if a manual differential is required based on instrument capabilities. (i.e. Hematology GuidelinesDifferential Reporting Guidelines) 2. Develop a list of abnormalities, which must be reviewed by the supervisor or pathologist before results are reported. Refer to Blood Film Guideline. 3. Differentials must be repeated if each cell does not meet the limits set out in the table below. If the repeat is still not within the established limits, a second technologist should repeat the differential. 4. Whenever the tech1 has concerns with a differential the rest of the CBC can be released with a notation Differential to follow. The requesting physician can be invited to review the smear if they so choose. The smear should be evaluated as soon as possible. 5. Leukocyte abnormalities seen during a smear review require a manual differential completed regardless of the protocol for when to perform a manual differential. 6. Perform the manual differential and compare it to the automated differential using the 95% confidence limits table. Each cell should compare within the range set. 7. Differentials between technologists should also fall within the established limits (95% confidence limits). 8. If the manual differential performed by two technologists agrees within the established limits, but is not in agreement with the automated differential, then the manual differential should be reported out instead of the automated differential. 9. It is good laboratory practice to circulate unknown QA slides quarterly. The results will be compared with peers and should be within the 95% confidence limits as set by the following table. Any problem areas will be covered between the technologist and the supervisor. Procedure: How to use the following 95% confidence limits table: 1. Look up each cell number you want to compare to in column a. E.g. 25% neutrophils

Tech refers to the medical laboratory technologist or combined laboratory and x-ray technologist.

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2. If a 100 cell differential was performed go to the column n=100 to determine the acceptable range. If a 200 cell differential was done refer to column n=200. E.g. you counted 32 neutrophils in a 100-cell differential. 3. Determine acceptability of the differential by checking if the number you counted for a certain cell falls within the stated range. E.g. Stated range is 16-35%; you are within this range. 4. Repeat for each cell type. 5. Determine acceptability of each cell line by comparing automated to manual differential. For the neutrophils/granulocytes, segmented neutrophils, band neutrophils and other neutrophil precursors must be added together and for lymphocytes, reactive lymphocytes and lymphocytes must be added together. E.g. 77 segs and 15 bands = 92% 6. You must be within this range, or the differential must be repeated. Example: The automated or technologist differential indicated the following differential and the acceptable range for each number was looked up in n=100 column: Neu: 70 acceptable range 60-79 Lymph: 15 8-24 Mono: 7 1-12 Eos: 3 0-9 A 100 cell differential is completed by another technologist and based on the acceptable range the results are as follows: Neu: 52 not within limits Lymph: 27 not within limits Mono: 12 within limits Eos: 7 within limits Baso: 2 within limits This manual differential would have to be repeated.

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The 95% confidence limits for various percentages of leukocytes of a given type as determined by differential counts on stained blood smears. n 0 1 2 3 4 5 6 7 8 9 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 91 92 93 94 95 96 97 98 99 100 n=100 0-4 0-6 0-8 0-9 1-10 1-12 2-13 2-14 3-16 4-17 4-18 8-24 12-30 16-35 21-40 25-46 30-51 35-56 39-61 44-65 49-70 54-75 60-79 65-84 70-88 76-92 82-96 83-96 84-97 86-98 87-98 88-99 90-99 91-100 92-100 94-100 96-100 n=200 0-2 0-4 0-6 1-7 1-8 2-10 3-11 3-12 4-13 5-14 6-16 10-21 14-27 19-32 23-37 28-43 33-48 37-53 42-58 47-63 52-67 57-72 63-77 68-81 73-86 79-90 84-94 86-95 87-96 88-97 89-97 90-98 92-99 93-98 94-100 96-100 98-100 n=500 0-1 0-3 0-4 1-5 2-7 3-8 4-9 4-10 5-11 6-12 7-13 11-19 16-24 21-30 26-35 30-40 35-45 40-50 45-55 50-60 55-65 60-70 65-74 70-79 76-84 81-89 87-93 88-94 89-95 90-96 91-96 92-97 93-98 95-99 96-100 97-100 99-100 n=1,000 0-1 0-2 1-4 2-5 2-6 3-7 4-8 5-9 6-10 7-11 8-13 12-18 17-23 22-28 27-33 32-39 36-44 41-49 46-54 51-59 56-64 61-68 67-73 72-78 77-83 82-88 87-92 89-93 90-94 91-95 92-96 93-97 94-98 95-98 96-99 98-100 99-100

References: 1. Abbott training seminarthe following table was provided. 2. Clinical Hematology Principles, Procedures, Correlations, Cheryl A Lotspeich-Steininger et al, 1992. 3. FHHR protocol

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SMUDGE CELLS

Distinguished by their naked amorphous nuclear chromatin material, smudge cells were initially described as white blood cells with broken-down nuclei in patients with chronic lymphocytic leukemia. Subsequently, these nuclear shadows have most often been referred to as smudge cells, but the term basket cells is used synonymously. The mechanism is often associated primarily with traumatic disruption of cells during blood film preparation. In the process, the cell membrane ruptures and when viewed under a microscope, what remains looks like a smudge, hence the term, smudge cells. To ensure reliability of results, it is important to understand the effects of variables associated with smudge cell formation, particularly the blood film preparation. Thus, the angle and the degree of incline of the slide spreader, the type of slide spreader (sharp or smooth), the cleanliness of the slides, and the overall quality of the blood films cannot be overemphasized. For minimal morphologic alterations, blood films should be made within three hours and not more than twelve hours after collection. It is recommended to include smudge cells in the differential as an absolute count, especially when the smudge cell numbers are noticeably increased. This identifies a more appropriate count because smudge cells are actually lymphocyte artifacts. It also avoids the need for repeating or verifying abnormal counts by the time - consuming albumin treated method. Education is needed (for the ordering physicians especially) to eliminate the risk of misinterpreting this smudge cell count as a new cell type. Criteria for Reporting Smudge Cells Absolute lymphocyte count should be greater than 5.0 x109/L. Patient age should be more than 30 years*. Smudge cells should be reported if greater than 10 per 100 leukocytes. Report smudge cells in absolute numbers.

*Although CLL is not often diagnosed in patients under the age of 40, patients over 30 years of age should be considered potentially at risk. CLL is rare in patients under 30 years of age. *In children (18 & under), the smudge cells are not counted as part of the differential. However, the presence of smudge cells may be noted on the report. *Smudge cells are present in those candidates for which definitive diagnostic criteria are well established. Examples include: CLL & Acute Leukemia.

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FLOW CYTOMETRY FOR THE DIAGNOSIS OF LYMPHOMA/LEUKEMIA

Immunophenotypic analysis of hematological malignancies is crucial for accurate diagnosis and classification of these complex malignancies. Flow cytometric data shall be interpreted in the context of additional information and correlation with other clinical findings, obtained through genetic studies, and through conventional morphologic and cytochemical methods. Flow cytometry by itself does not provide enough information for diagnosis. Flow cytometric analysis has become an acceptable medical practice in the diagnosis and characterization of hematologic neoplasia and its role in the management of patients with these diseases is well recognized. Despite its extraordinary power, there is great concern regarding the inconsistent practices and wide variation in styles among laboratories involved in the flow cytometric analysis of leukemias and lymphomas. Of particular importance are the deficiencies in standardization and validation of procedures used in the analysis, the manner by which the information is generated and reported to pathologists or treating physicians, and the appropriate utililzation of this technology in patient care. (US-Cdn Consensus Recommendations on the Immunophenotypic Analysis of Hematologic Neoplasia by Flow Cytometry)

a)

A MLT with Hematology experience, and appropriate Immunology background, and training in Flow Cytometry is required for the performance of Flow Cytometry clinical testing of Lymphoma and Leukemia. A qualified Pathologist/Hematologist who has training in both Hematopathology and Flow Cytometry must perform interpretation of Flow Cytometry results. Flow cytometry in diagnostic purposes for Lymphoma/Leukemia shall only be performed in pathologist directed laboratories.

b)

c)

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MALARIA

Malaria can be a life threatening infection and a rapid diagnosis is necessary to institute appropriate management in a timely manner. The diagnostic information required for patient management is best obtained by microscopy; however, a rapid test (immunologically-based) is a very useful screening test. The Rapid Diagnostic Tests (RDT) results shall be confirmed by microscopy. Rapid Diagnostic Tests (RDTs): 1. Should be available in all acute care sites in Saskatchewan 2. A single RDT should be recommended for use across the province. 3. Standardized reporting terminology should be used. 4. The test should be handled as a STAT procedure. 5. Results should be phoned to the requesting physician. 6. All requests should automatically trigger microscopic examination. 7. Results in a patient suspected of falciparum infection should include prompt referral (of the patient or transport of sample) to a centre where microscopy is available on an urgent basis. 8. Reports must indicate that the RDT is a screening test, with confirmation by microscopy to follow. Microscopic Diagnosis: 1. The test should be available 24/7. 2. Upon receipt of specimens at the testing laboratory, TAT is 4-6 hours. 3. The testing should be based on the protracted (20 min.) examination of four thick and four thin blood smears. 4. Positive slides must be confirmed by a laboratory physician and/or pathologist. 5. The information in the report should include the following; a) positive or negative, b) speciation, c) quantification of parasitemia. 6. All positive results should indicate the requirement to report Malaria cases to Public Health and a recommendation to consult with an Infectious Disease specialist as soon as practically possible. 7. Results must be called to the physician by the testing laboratory 8. The laboratories must participate in an external/internal quality assurance program.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

ERYTHROCYTE SEDIMENTATION RATE (ESR)

The ESR is a commonly ordered test for the assessment of inflammation and is not meant to be used to screen an asymptomatic person for disease. Although relatively easy to perform it is not easily monitored by conventional QC method. Therefore, the following practice is recommended. Perform at least one ESR at two separate times. Perform within the 4 hr timeframe from collection; Results must be comparative i.e. If the first results fall within the reference range, the second result should be similar. Significant variations indicate inaccuracy and testing should be discontinued until resolved. Perform this practice every six months.

ESR is indicated in the diagnosis and therapeutic monitoring of temperal arteritis and polymyalgia rheumatica. The ESR may be helpful in resolving conflicting clinical evidence in patients with rheumatoid arthritis and with the evaluation and management of patients with specific autoimmune inflammatory or infectious disorders (e.g. pelvic inflammatory disease, bacterial endocarditis, septic arthritis and osteomyelitis).

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3.2% Na CITRATE ANTICOAGULANT RECOMMENDED VERSUS 3.8% FOR COAGULATION STUDIES

The International Standards Committee on Thrombosis and Hemostasis, and the CLSI have recommended guidelines to standardize whole blood collection to 3.2% sodium citrate anticoagulant. Several publications have indicated that the clotting times for PT and APTT are significantly shortened with the 3.2% NaCitrate. For this reason, exchange between the two concentrations is not acceptable. When selecting an anticoagulant for collection, it is important to note that 3.2% NaCitrate is used for ISI assignments for thromboplastin. For these reasons, it is important for laboratories to standardize the choice of anticoagulant for sample collection. The anticoagulant used for coagulation assays should be 3.2% trisodium citrate. The proportion of blood to anticoagulant volume is 9:1. Inadequate filling of the collection device will decrease this ratio, and may lead to inaccurate results. The manufacturer recommendations should always be followed. If the hematocrit is very high and the usual relative volumes of blood and citrate solution are mixed, a prolonged prothrombin time results from overcitration of the reduced proportion of plasma in the blood sample. This means the final citrate concentration in the blood should be adjusted in patients who have hematocrit (PCV) values above 0.55 (55%). Adjust the volume of NaCitrate in the draw tube by applying the calculation outlined in the CLSI H21-A3. Note: To maintain the vacuum in the NaCitrate collection tube, use a tuberculin syringe to draw out the anticoagulant. The anticoagulant volume may be calculated from the expression: X = (100 PCV) x draw volume of tube (mLs) (595 PCV) X = the volume of anticoagulant required to prepare volume of anticoagulated blood PCV = packed cell volume (Hct) in % For example: To determine the volume required for 5 mLs anticoagulated blood, calculate x 5 mLs
References: H21-A3 CLSI, BD Vacutainer Systems, Azko Nobel Assessment of the influence of citrate concentration on the international Normalized Ratio (INR) determined with twelve reagent-instrument combinations. Chantarangkul, Tripodi, Clerici, Negri, Mannucci Effect of concentration of trisodium Citrate Anticoagulant on Calculation of the international Normalized Ratio and the International Sensitivity Index of Thromboplastin, Duncan, Casey, Duncan, and Lloyd The Prothrombin Time Test: Effect of Varying Citrate Concentration, Ingram, Hills. Comparison of 3.2% vs. 3.8% Sodium Citrate Anitcoagulant Collection Tubes for Coumadin, Heparin, Abnormal and Normal Specimens. Phillips, Sunnybrook ON

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

VITAMIN B12 & FOLATE

Background: The normal proliferation of cells depends on adequate folate and vitamin B12. Folate is necessary for efficient thymidylate synthesis and production of DNA. B12 is needed to successfully incorporate circulating folic acid into developing RBCs retaining folate in the RBC. MEASURING BOTH B12 AND FOLATE LEVELS IS NOT NECESSARY IN ALL PATIENTS. Serum B12: The clinical indications for ordering serum B12 include: 1) Evaluation of patients with MACROCYTIC (high MCV in the CBC) anemia and the clinical information suggesting possible B12 deficiency; 2) Evaluation of patients with psychiatric and neurologic impairment (symptoms of subacute combined degeneration of spinal cord). Red Cell Folate: Red cell folate is ordered as it is an indication of the folate status over a longer period of time (several months) as opposed to serum folate that reflects levels over the last few days. Population studies have shown that dietary supplements have increased average folate levels and therefore folate deficiency is much rarer. The clinical indications for ordering red cell folate include the evaluation of MACROCYTIC (high MCV in the CBC) anemia and the clinical information suggesting possible folate deficiency. Practice Tips: 1. Persons on B12/folate supplements or other multivitamins do not require testing. 2. Lab tests are used to confirm a specific diagnosis and not as a fishing expedition. 3. Marginal B12 deficiency in any elderly patient with dementia, peripheral neurological symptoms or impaired immunity should be taken seriously. 4. B12 should not be done on patients on oral contraceptives.
Prepared by Dr. A. Saxena, Department of Laboratory Medicine, Saskatoon Health Region

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Current Indications for B12 and Folate Investigation


Measuring both B12 and Folate levels is not necessary in all patients.

CBC

Normal

MCV

Neurological or Clinical symptoms suggestive of B12 &/or Folate deficiency

No further testing

YES

Notes: *B12 tests should not be done if the person is on B12 *False low B12 in pregnant women and women on oral contraceptives Serum folates are not a useful screening tool Folic Acid Deficiency is rare due to folate fortification in food RBC FOLATE IS MORE INDICATIVE OF TISSUE FOLATE LEVELS Folate tests will not be done if the person is on folate B12 and RBC folate

Abnormal

Normal

Requires further patient evaluation

No further testing

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BLEEDING TIME

Bleeding Time (BT) is defined as the time between the making of a small incision and the moment the bleeding stops. It indicates how well platelets interact with blood vessel walls to form blood clots (a test of platelet and vascular function). Purpose: Bleeding time is used most often to detect qualitative defects of platelets, e.g. Von Willebrands disease (vWD). However, it is prolonged in many situations including vascular disorders, e.g. Ehler-Danlos syndrome, pseudoxanthoma elasticum. Issue: The use of BT is declining and at many institutions this test has been eliminated. This is because of three main reasons: It is neither a sensitive nor a specific test. A specific diagnosis of vWD can be made based on history and other tests. More reliable information about platelet function can be obtained by other tests. Notes: Use of bleeding time is not recommended (not by itself). Current practice varies and if BT is required, the recommended method is the template device. BT is generally not recommended for children under the age of 5. Reference Interval: Results are reported to the nearest half-minute. The reference range varies and is usually 1.5-9.5 minutes. The test should be discontinued if the patient hasnt stopped bleeding by 15-20 minutes.

References: Andrew M, Paes B, Bowker J, et al, "Evaluation of an Automated Bleeding Time Device in the Newborn,"Am J Hematol, 1990, 35(4):275-7. Basili S, Ferro D, Leo R, et al, "Bleeding Time Does Not Predict Gastrointestinal Bleeding in Patients With Cirrhosis,"J Hepatol, 1996, 24(5):574-80. Brown BA, Hematology: Principles and Procedures, 6th ed, Philadelphia, PA: Lea and Febiger, 1993, 267-70. de Rossi SS and Glick MG, "Bleeding Time: An Unreliable Predictor of Clinical Hemostasis,"J Oral Maxillofac Surg, 1996, 54(9):1119-20. George JN, Shattil SJ: The Clinical Importance of Acquired Abnormalities of Platelet Function. NEJM 324:27-29, 1991. Gewirtz AS, Miller ML, and Keys TF, "The Clinical Usefulness of the Preoperative Bleeding Time,"Arch Pathol Lab Med, 1996, 120(4):353-6. Henry, J. B. Clinical Diagnosis and Management by Laboratory Methods. Philadelphia: W. B. Saunders Co., 1996. Munro J, Booth A, and Nicholl J, "Routine Preoperative Testing: A Systematic Review of the Evidence,"Health Technol Assess, 1997, 1(12):1-62. Lewis SM, Ban BJ, Bates I. Dacie and Lewis Practical Haematology. 2006. Churchill Livingstone Elsevier, Philadelphia (PA). Lind SE: Prolonged Bleeding Time. Am J Med 77:305-312, 1984. Peterson P, Hayes TE, Arkin CF, et al, "The Preoperative Bleeding Time Test Lacks Clinical Benefit,"Arch Surg, 1998, 133(2):134-9. Rodgers RP and Levin J, "A Critical Reappraisal of the Bleeding Time, "Semin Thromb Hemost, 1990, 16(1):1-20. Triplett DA. Laboratory Evaluation of Coagulation, 1982. American Society of Clinical Pathologists Press, Chicago (IL).

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CROSSCHECK/VALIDATION GUIDELINE FOR THOSE FACILITIES WITH MULTIPLE CHEMISTRY/HEMATOLOGY INSTRUMENTS


PERFORMING THE SAME TEST PROCEDURE

Proficiency testing registration is mandatory for each analytical test. Rotating proficiency testing result submission between analyzers is not a requirement, but is suggested where appropriate (e.g. Blood Gases). Where there are multiple analyzers performing the same test procedure in larger facilities, it may be more appropriate for proficiency testing submission to be consistently submitted from the same analyzer for tracking purposes. An internal cross-check/validation protocol is required to ensure that there is correlation between all analyzers providing the same test result in the same facility. If this protocol is not followed, then each analyzer must be registered in the external proficiency testing program as mandated by LQAP. This procedure is recommended every six months. Chemistry/Hematology High Volume Analyzers (e.g. Electrolytes/CBC)

Validation/Crosscheck Element:
Patient correlation

Requirement:
Regularly scheduled intervals Whenever criteria for recalibration/validation is met: - change of manufacturer for reagents or equivalent - after maintenance or service as per manufacturers recommendations - as required for purposes of troubleshooting /validation of reagent lot # changes or as indicated by quality control data

Frequency/Data Points:
Minimum of 20 patient specimens/2 times per year or equivalent (i.e. 10 patient specimens/4 times per year or on-going data collection as appropriate) As necessary per recalibration/validation event

Chemistry/Hematology Low Volume Analyzers (e.g. Fibrinogen) Validation/Crosscheck Element:


Patient correlation

Requirement:
Regularly scheduled intervals Whenever criteria for recalibration/validation is met: change of manufacturer for reagents or equivalent after maintenance or service as per manufacturers recommendations as required for purposes of troubleshooting /validation of reagent lot # changes or as indicated by quality control data

Frequency/Data Points:
Minimum of 5-10 patient specimens/2 times per year or equivalent As necessary per recalibration/validation event

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PROCEDURE/METHOD STATISTICAL WORK-UP/VALIDATION STUDY GUIDELINES: CHEMISTRY/HEMATOLOGY Work-up guidelines and definitions (change in method/instrument):
Work-up Element
1. Imprecision within run between run

Definition: CLIS or International Federation of Clinical Chemistry (IFCC)


The variation in analytical results demonstrated when a particular specimen of aliquot is analyzed multiple times or on multiple days. Imprecision is expressed quantitatively by a statistic such as standard deviation or coefficient of variations.

Minimum Data Requirements (where appropriate):


Within run use preferably a patient sample or pool close to the decision levels with a minimum of 10 data points. Between run 20 results from 20 separate runs on 2 levels over a 10-day minimum time period using appropriate QC material. 40 data points are recommended with a minimum of 20 having 50% of the data points outside the reference intervals, if possible. Correlations should involve comparison with an acceptable reference method or laboratory. 4 data points each in duplicate as a minimum requirement, but 5 data points are preferred (over reportable range). Linearity studies are expected on an initial method work-up and further studies as defined by the College guidelines (i.e. troubleshooting). The minimum requirement is 20 data points for confirmation of an established reference range and 120 for the establishment of a new reference range. 3 data points using acceptable reference material (i.e. CEQAL or CAP) 1 data point may be acceptable for haematology accuracy studies if related to sample stability. Sensitive studies are only required for those methods which have clinical relevance at values close to 0 (i.e. TSH)

Applicability:

Qualitative

Quantitative

2. Patient Correlation

The correlation coefficient is a means to look for a relationship, not agreement, between pairs. Two methods may have a perfect correlation throughout the measuring range but may not agree in value (i.e. one may be double the value of the other).

n=20

n=40

3. Linearity

(IFCC) The range of concentration or other quantity in the specimen over which the method is applicable without modification (CLIS) when analytical results are plotted against expected concentrations; the degree to which the plot curve conforms to a straight line is a measure of the system linearity.

4. Reference range validation 5. Accuracy

It is common convention to define the reference range or interval of a laboratory test as the central 95% interval bounded by the 2.5 and 97.5 percentiles of the selected patient population. Validation of an established reference range requires a minimum of 20 samples. Closeness of the agreement between the result of a measurement and the accepted reference value (true value of the analyte)

6. Sensitivity

Measure of the ability of an analytical method to detect small quantities of the measured component. When concern is performance at a very low concentration it is useful to determine the detection limit as influenced by imprecision.

When clinically relevant

When clinically relevant

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7. Specimen Stability

The conditions of handling and storage, which permits the measurement and reporting of a clinically relevant result.

8. Interference

The effect of any component of the sample on the accuracy of the measurement of the desired analyte.

9. Recovery

A recovery procedure involves the addition of a known amount of analyte to an aliquot of sample. Recovery is defined as the ratio of the amount of the analyte recovered to amount added and is given as percentage.

No data generally required. As per manufacturers guidelines. If manufacturers stability window is to be extended a stability study is expected. Document the manufacturers interference information. The method should include a disclaimer or a process for dealing with a lipemic, icteric or hemolyzed sample. Recovery studies should only be necessary for those methods or analytes where organic extractions or equivalent are required as part of the methodology (i.e. Toxicology)

Storage and transportation dependent Methods with known interferences

Storage and transportation dependent Methods with known interferences Method specific/organic extraction

Work-up requirements when an instrument is moved from site A to site B: (It is assumed that the instrument has been in recent use with acceptable performance). Work-up Element 1. Imprecision studies, QC only 2. Patient correlation Minimum data requirements: As above 10 data points, where feasible.

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PROTOCOL FOR VALIDATION OF LINEARITY ON AUTOMATED HEMATOLOGY ANALYZERS

Purpose: To validate the entire range through which patient values can be reported or to verify the manufacturers stated linearity from the analysis of undiluted or diluted specimens for all measured parameters. Specimen Selection: For each parameter requiring linearity validation, choose a specimen with results at/near or above the high end of the linear range published by the manufacturer of the instrument. Ensure that there were no interfering substances noted during analysis of the specimen. This value is equivalent to 100% while patients plasma or instrument diluent is equivalent to 0%. For RBC and HGB use a polycythemic or concentrated sample. A concentrated specimen may be prepared by centrifuging the specimen and removing plasma to produce a value near or above the high end of the linear range published by the manufacturer. Linearity of MCV, MCH and MCHC can also be performed by concentrating the specimen. For RBC and HGB, do not exceed an HCT of 0.60-0.65. For WBC use a leukocytosis sample diluted in patients own plasma or instrument diluent. For PLT count use a thrombocythemic sample diluted in patients own plasma or instrument diluent. Ensure that sufficient specimen is collected for dilution preparations and instrument aspiration.

Procedure: 1. Verify that instrument reagents are not expired and that sufficient volume of reagents is loaded on instrument to cycle all the prepared dilutions. 2. Check that background counts, instrument precision and calibration of the instrument are acceptable before starting procedure. 3. Determine the volume per dilution by calculating the volume required for aspiration on the instrument. One specimen may not provide sufficient volume. If more than one tube is drawn, pool the tubes into one aliquot. 4. Label five clean plastic tubes 80%, 60%, 40%, 20% and 0%. 5. Prepare the dilutions using the original specimen as the 100% as shown in table provided below. Make sure the 100% sample remains well mixed throughout the dilution preparation process.

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Dilution (%) 100 80 60 40 20 0

Specimen (Parts) 10 8 6 4 2 0

Diluent (Parts) 0 2 4 6 8 10

6. Analyze each well-mixed dilution in triplicate on the instrument. Results with suspect parameter flags should not be used. 7. Record all results for each parameter from all three runs on table provided. 8. Calculate Obtained Mean value for each parameter. 9. Calculate Expected value for each parameter by multiplying the Reference Mean (100% Obtained Mean) by the multiplication factor. 10. Calculate the Difference between Obtained Mean and Expected Mean. 11. To graphically illustrate linearity, plot each parameter on linear graph paper with the obtained mean value for the parameter on the X axis and the expected value for the parameter on the Y axis. Draw a line through all the points on the graph. When the obtained mean is plotted against the corresponding expected value, the plotted curve will approximate a straight line for a linear method. Excel spreadsheet can also be used to show linearity. Note: Linearity is performed initially and when calibration fails to meet the laboratorys acceptable limits.
References: 1. Package insert from R&D Systems Inc. which sells a CBC-LINE Full Range Hematology Linearity Kit and CBC-LINE Low Range Hematology Linearity Kit 1994 2. Shinton NK, England JM, Kennedy DA, Guidelines for the evaluation of instruments used in Haematology laboratories J Clin Path 1982;35; 1095-1102 3. Package insert from STRECK Calibration Verification Assessment 2007 4. Quam EF, Method Validation The Linearity of Reportable Range Experiment ASCP

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WBC Linearity
WBC Results Run #1 Run #2 Run #3 Obtained Mean Reference Mean X Multiplication Factor Expected Value Difference Allowable Evaluation Limits 0% 20% 40% 60% 80% 100% (Reference)

X 0.0

0.2

0.4

0.6

X 0.8

1.0

0.4
0%

0.4
20%

0.4
40%

0.4
60%

0.4
80%

0.4
100% (Reference)

RBC Linearity
RBC Results Run #1 Run #2 Run #3 Obtained Mean Reference Mean X Multiplication Factor Expected Value Difference Allowable Evaluation Limits

X 0.0

0.2

0.4

0.6

X 0.8

1.0

0.1
0%

0.1
20%

0.1
40%

0.1
60%

0.1
80%

0.1
100% (Reference)

HGB Linearity
HGB Results Run #1 Run #2 Run #3 Obtained Mean Reference Mean X Multiplication Factor Expected Value Difference Allowable Evaluation Limits

X 0.0

0.2

0.4

0.6

X 0.8

1.0

3
0%

3
20%

3
40%

3
60%

3
80%

3
100% (Reference)

PLT Linearity
PLT Results Run #1 Run #2 Run #3 Obtained Mean Reference Mean X Multiplication Factor Expected Value Difference Allowable Evaluation Limits

X 0.0

0.2

0.4

0.6

X 0.8

1.0

10

10

10

10

10

10

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PROCEDURE FOR WBC ESTIMATE

WBC Estimation is to be used for cases in which the instrument flags invalid data or suspect WBC populations and for which you cannot obtain a valid WBC in any mode. 1. Examine the specimen for presence of a clot, strands of fibrin, or gross hemolysis before you proceed. 2. Next examine the histograms for likely causes of interference. Histogram review may help you decide which version to use if it is necessary to issue a partial CBC report. (If the HGB or PLT results are critical or STAT, these should not be held back.) Most interferences are visible in the lower region of your first WBC histogram, e.g. resistant RBC, NRBC, megathrombocytes, clumped platelets, fibrin, etc. In some cases, the analyzer will incorrectly include these interferences in the lymphocytes. This results in both a falsely elevated WBC and lymphocyte count. Rule of thumb: the lowest WBC from the analyzer is usually the most correct. 3. Make and stain two blood smears. 4. Two techs shall do an estimate, each using a separate slide (if a second tech is not available, one tech can do two estimates, one on each slide) a. Examine the smears under 10x objective for even distribution of WBC before proceeding. If there is a ridge or layer of WBC in the tails, the estimate will be invalid. Re-make the slide. b. Choose an area from the tails, where the RBC are evenly spread, or just beginning to overlap (where 50% of the red cells are overlapping doubles or triplets). This should be the same area where you would do RBC morphology or platelet estimation. Be aware that if the RBC/PCV are low, you must avoid going in too deep. Then count in ten consecutive fields. c. Switch to hpf. Count the total number of WBC seen in 20 fields. Include disintegrated cells. Do not include NRBC. d. Divide your total by 20 to obtain the average number of WBC/hpf, to two decimal places. Multiply the average by the conversion factor for the microscope. Refer to conversion factor procedure for hpf. Average your result with that of the other tech. e. If your estimate is 0.40 or lower, just call it less than 0.5 f. If your estimate is over 0.40 calculate the estimate as a range of +/- 40% from this number. Round off the results of 2.0 or lower to one decimal place; round off results over 2.0 to the nearest whole number.

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The examples below are using a microscope conversion factor of 2.5. Example 1: Avg. number of WBC/hpf on #1 slide = 1.55 x conversion factor of 2.5 = 3.875 Avg. number of WBC/hpf on #2 slide = 2.00 x conversion factor of 2.5 = 5.00 Average estimate from two slides (3.875+5.00) 2=4.4375 40% Range=4.4375x4.0=1.775 Estimate =4.4375 +/- 1.775, or 2.6625 to 6.2125. Round off to whole numbers. WBC Estimate is 3 to 6 x 10^9/L Example 2: Estimate is calculated as 0.30. Estimate is 0.40 or lower, so interpret range as less than 0.5 Example 3: Estimate is calculated as 0.45, range of 0.27 to 0.72. Round off as 0.3 to 0.7. Example 4: Estimate is calculated to be 2.5, range of 1.5 to 3.5. Round off as 1.5 to 4. 5. If the estimate range agrees with the analyzer WBC, report the best results from the analyzer. Example: Analyzer count = 5.5 Estimate from films is 4.4 +/- 40%, or 3 to 6. Report the analyzer WBC of 5.5. 6. If the WBC estimate does not agree with the analyzer count, report the estimate as a range. Examples: WBC estimate on film appears to be less than 0.5 x 10^9/L. WBC estimate on film appears to be 1.5 to 4 x 10^9/L. WBC estimate on film appears to be 3 to 6 x 10^9/L. 7. If it is absolutely necessary to report a differential with a WBC estimate, it must be a manual differential in relative units (%).
References: Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al: J.B. Lippincott Company, 1998

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ESTABLISHING CONVERSION FACTOR FOR WBC ESTIMATION

Purpose: To provide for instruction for establishing a conversion factor for the hematology microscope by calculating the ratio of electronic count to the number of WBC on either hpf or per oil immersion field. Frequency: The conversion factor must be established once for every microscope used in hematology or when there are major repairs or changes in the analyzer for electronic counts or a new microscope is used for estimates. Procedure: Note: Total leukocyte counts can be estimated roughly either by hpf or 100x oil immersion field. Therefore 100x oil immersion field can be substituted in procedure that states hpf. Step: 1. Action: Perform electronic WBC count on 30 consecutive fresh patient blood samples. Alternatively if unable to perform 30 consecutive samples it is acceptable to perform 10 samples per day on 3 consecutive days. Prepare and stain one peripheral blood film for each sample. For each film, under hpf microscopy, find an area where 50% of the red cells are overlapping doubles or triplets. Then count WBC in 10 consecutive fields. Divide by 10 the total number of WBC found to obtain the average number per hpf. Divide the electronic WBC count by the average number of WBC per hpf. Add the numbers obtained in step 5 and divide by 30 (number of observations in this analysis) hpf. Round the number calculated in step 6 to the nearest whole number to obtain an estimation factor.

2. 3. 4. 5. 6. 7.

Reference: Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al: J.B. Lippincott Company, 1998.

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WBC ESTIMATION CONVERSION FACTOR LOG SHEET Microscope Model: _______________________ ID: ______________________________ Column 1 Column 2 Column 3 Column 4 DIVIDE SPECIMEN ID AUTOMATED WBC WBC ESTIMATION AUTOMATED WBC COUNT COUNT BY WBC ESTIMATION 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. Average Estimation Factor __________________ Calculation: To determine Average, total the values in Column 4, divide by 30. Round off to nearest whole number to obtain the estimated factor.

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PROCEDURE FOR PLATELET ESTIMATES

1. Platelet estimation is to be used for cases in which instrument flags identify invalid platelet data, platelet clumps and for verifying platelet counts below 100x109/L. Scan the slide for clumps under hpf. If platelet clumps are present, they cause a false decrease in platelet count. Edit out the platelet count by replacing it with the message Platelets clumped, results invalid. Do not report the MPV. NOTE: For this purpose, platelet clumping is defined as at least 5 clumps, with at least 5 platelets per clump. Rare or occasional smaller clumps should be ignored. 2. Switch to 100x objective. Total the number of platelets in at least 10 fields. Determine the average number per field. Multiply by the conversion factor established for your microscope. The estimate should agree with the analyzer count +/- 40% Example: Average of 6.3 cells per field x conversion factor of 11 = estimate of 69 x 10^9/L. Analyzer count of 88. Multiply by 0.6 or 1.4 for +/-40% range of 53 to 123. Platelet estimate agrees with analyzer. 3. If your estimate disagrees with the instrument platelet count by more than 40% in the absence of clumps: a. Have another technologist do an estimate, when possible, to confirm the discrepency. b. If the patient is anemic or polycythemic (RBC below 4.0 or over 6.0), the estimate may be inaccurate. A second blood film should be prepared, (see Indirect Platelet Count Procedure) and performed by two technologists, this may require referral. c. Examine the platelet histograms and the blood smear for the following sources of error: i) RBC fragments: may be small enough to be counted as platelets. ii) Megathrombocytes: if many of the platelets on the film appear abnormally large, i.e. approximately the size of RBC, the analyzer may not be including them in the count. If your estimate varies from the analyzer by more than 40%, add the message Platelet estimate appears higher on film; megathrombocytes may be excluded from count. Do not comment anything if occasional large platelets are present, and your estimate agrees within 40%. iii) Leukocyte cytoplasmic fragments, if present in large numbers, may falsely elevate the instrument platelet count. They appear as particles similar in size to platelets, but with a homogeneous, rather than granular, structure. 4. If you are reporting the platelet estimate instead of the analyzer count, append an appropriate comment:

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Platelet estimate 20 x 10^9/L Platelet estimate > 20 x 10^9/L Platelet estimate > 50 x 10^9/L Platelet estimate >100 x 10^9/L Note: You should not use this method if RBC is abnormal. 5. Send to Senior Technologist/Pathologist for review on first occurrence for each patient for whom the instrument count is determined to be invalid due to RBC fragments, leukocyte fragments, or megathrombocytes.

Reference: Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al: J.B. Lippincott Company, 1998

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ESTABLISHING CONVERSION FACTOR FOR PLATELET ESTIMATION

Purpose: To provide instructions for establishing a conversion factor for the hematology microscope by calculating the ratio of electronic count to the number of platelets per oil immersion field. Frequency: The conversion factor must be established once for every microscope used in hematology or when there are major repairs or changes in the analyzer for electronic counts or a new microscope is used for estimates. Procedure: Step: Action: 1. Perform electronic platelet count on 30 consecutive fresh patient blood samples. Alternatively if unable to perform 30 consecutive samples it is acceptable to perform 10 samples per day on 3 consecutive days. 2. Prepare and stain one peripheral blood film for each sample. 3. For each film, under oil immersion (100x) microscopy, find an area where 50% of the red cells are overlapping in doubles or triplets. Then count platelets in 10 consecutive fields. 4. Divide by 10 the total number of platelets found to obtain the average number per oil immersion field. 5. Divide the electronic platelet count by the average number of platelets per oil immersion field. 6. Add the numbers obtained in step 5 and divide by 30 (number of observations in this analysis) to obtain the average ratio of the platelet count to platelets per oil immersion field. 7. Round the number calculated in step 6 to the nearest whole number to obtain an estimation factor, the number of peripheral blood platelets represented by one platelet in an oil immersion field.

Reference: Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al; J.B. Lippincott Company, 1998

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Platelet Estimation Conversion Factor Log Sheet

Microscope Model: __________________________ ID: __________________________ Column 1 Column 2 Column 3 Column 4


PATIENT ID AUTOMATED PLATELET COUNT PLATELET ESTIMATION DIVIDE AUTOMATED PLATELET COUNT BY PLATELET ESTIMATION

1. 2. 3. 4. 5. 6. 7. 8. 9. 10 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Average Estimation Factor

__________________ Calculation: To determine Average, total the values in Column 4, divide by 30. Round off to the nearest whole number to obtain the estimated factor.

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INDIRECT PLATELET COUNT

Purpose: An indirect platelet count may be obtained by determining the ratio of platelets on a blood film to RBC. The ratio is then used to calculate the number of platelets in comparison to the number of RBC x 1012/L. This method is useful for situations in which an accurate platelet count cannot be obtained from the CBC analyzer e.g. samples with marked megathrombocytes, RBC fragments, or leukocyte cytoplasmic fragments. It is particularly more accurate than conventional estimation when the RBC is abnormal. Procedure: 1. Make two good quality blood smears. Air dry and stain as for differentials. The peripheral smears must be well made, not touching edges of slide, with rounded end and no visible tails. 2. Obtain the RBC count from the automated CBC analyzer. 3. Using a 100x oil immersion objective, count the number of platelets per 500 RBC. Two technologists should count one slide each and the platelets counts should check within 10 platelets of each other. If the counts do not check, a third technologist counts in the same manner. 4. Add the two agreeable counts together and multiply this result by the RBC count to obtain the indirect platelet count in 109/L. Next calculate +/- 10 to report the result as a range rather than an exact number. Example: RBC = 4.10 1st count = 11 platelets per 500 RBC 2nd count = 15 platelets per 500 RBC 11+15 = 26 26 x 4.10 = 107 Add and subtract 10 to establish the reportable range. The indirect platelet count is reported as 97 to 117 x 109/L. 5. Compare the indirect platelet count to the analyzer count. If the indirect count is within the analyzer count +/- 30%, report the analyzer count. 6. If the indirect count is not within the analyzer count +/- 30%, report the indirect platelet count.

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Procedure Notes: The Indirect Platelet Count is preferable to platelet estimation when an accurate platelet count cannot be obtained on the CBC analyzer. It is particularly more accurate than conventional estimation when the RBC is abnormal. Limitations of the Procedure: The indirect method is not recommended as the method of choice for enumerating platelets. However, it is useful when an accurate platelet count cannot be obtained by the CBC analyzer.

References: 1. Raphael SS. Lynchs Medical Laboratory Technology, Third Edition, Philadelphia PA: WB Saunders Company, 1976, page 1084. 2. Hematology Laboratory Safety Manual, Royal University Hospital, Saskatoon, SK 1998 3. Constantino BT. Leukocyte Cytoplasmic Fragmentation: Its Causes and Laboratory Evaluation, Canadian Journal or Medical Laboratory Science -60, 1998, page 195

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

INTERNATIONAL SENSITIVITY INDEX (ISI) VERIFICATION Purpose: Verification of ISI is mandated for all laboratories using assigned instrument-specific ISIs. Verification must be performed in each laboratory at least annually, to ensure the accuracy of the International Normalized Ratio (INR) result. Verification is done using a set of certified plasmas. NOTE: This guideline does not apply to point-of-care/rapid cartridge-based technology, at this time. Frequency: Verification of ISI is required: when a new instrument is put into place; with any change in reagent type; with any change in reagent lot number; with any change in instrument; following instrument repair, if QC is outside acceptable limits; at a minimum of once per year.

Procedure: 1. Determine Geometric Mean Normal Prothrombin Time (MNPT) for current lot. [*contact LQAP office for Geometric Mean Calculator] 2. Obtain a set of certified plasmas from the manufacturer. A minimum of three certified plasmas with an INR range of 1.5 - 4.5 are recommended. The certified plasma must be appropriate for the instrument in which they will be used. 3. Perform INR test on certified plasma in duplicate over three sessions. 4. Determine mean INR from the three sessions of testing on all certified plasmas. a) If the mean INR is within 15% of assigned INR on all certified plasmas then the ISI is valid and verification is complete. b) If the mean INR is not within 15% of assigned INR revalidate the MNPT. i. If MNPT is valid, then a local system calibration must occur; go to step 5. ii. If MNPT is not valid, then it must be re-established, go back to step 1. 5. Contact the manufacturer for further instructions (ie. calibration). Return to step 3.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

VERIFYING OR ESTABLISHING A NORMAL REFERENCE RANGE FOR ROUTINE COAGULATION TESTING

Purpose: To provide instructions for establishing or verifying a Normal Reference Range for routine coagulation testing. 1. Verifying a Normal Reference Range: To verify a normal reference range, 20 representative donor samples should be used. 2. Establishing a Normal Reference Range: For an assay that has never previously had a reference range study, 120 donor samples should be used. To provide instructions for lot number changes of reagent or quality control (QC) material. Frequency: Verify a Normal Reference Range:

With any change in reagent lot number Following instrument repair if QC is outside acceptable limits At a minimum of once per year

Establish a Normal Reference Range:


When a new instrument-reagent system is put into place With any change in instrument With any change in reagent type

Sample Information: Specimens must be the same type and collected in the same container required for a given test (usually 3.2% citrated plasma; see specific procedure). Accumulate the appropriate number of normal donors that meet the following guidelines: 1. Healthy with no known pathological conditions (in-patients should be avoided they are hospitalized for a medical reason). 2. On no medications affecting coagulation, including anticoagulants/blood thinners. 3. Span the adult age range (unless a pediatric range is required). 4. Equally divided between males and females. If unable to maintain equal male/female ratio, a larger center may be able to assist with donor specimens. Donor samples can be collected in advance to speed up the evaluation process. The samples should be double spun, aliquoted and stored for up to 14 days at -20 C or up to 6 months at -80 C. Spun plasma should have <10x109/L platelet counts.

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

Procedure for Verifying a Normal Reference Range Prompt Run the applicable tests(s) as per procedure manual. Print all results. Run applicable QC for each assay. Additional Information Examine all results for outliers. Repeat outliers to rule out an analyzer error. When determining new reference ranges, it is acceptable to exclude outlier values particularly any values that fall outside the current normal range - providing a minimal number of data points are involved. An assay with more than 5% unexplained outliers should be discussed with the laboratory supervisor. Calculate the geometric mean Compare reference range with previous range and submit and SD from the data (Refer to to the lab supervisor for review before implementing. the Geometric Mean Calculation). Establish a reference range based on the mean 2 SD.

Procedure for Establishing a Normal Reference Range Consultation with Laboratory Manager/Director (or a coagulation specialist) is recommended when establishing a normal reference range. Sequestering Reagents/Quality Control Materials: 1. All routine coagulation reagents (Prothrombin Time, APTT and Fibrinogen) and related QC materials should be sequestered from the supplier in appropriate volumes for long term use (usually up to 1 year). 2. When sequestering new lot items, be sure to ask for an expiry date longer than the time sequestered for. 3. When sequestering a new lot of PT reagent, always check the ISI assigned to your specific instrument. An ISI close to 1.0 is preferable and improves correlation between instruments and peer laboratories. 4. Be sure to allow adequate time for shipping and preliminary testing prior to depleting your current stock.

Lot Number Changes: Note: It is not recommended to change reagent and QC lots for the same assay, at the same time. 1. Reagent Lot Number Changes

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

a. Establish a new mean and 2 SD normal range for the applicable assay as described in the procedure above. b. Compare the new reference range with the previous. Variation should be minimal. If there is a significant fluctuation (> 5%), consult with supervisor to obtain new lot number of reagent. c. Establish new mean and SD for all QC levels associated with the assay using the new lot number of reagent (see below - Quality Control Lot Number Changes). Note: If possible, run the new lot of reagent in parallel with your current lot whenever QC is run. When performing parallel testing with new lots of reagent, it is not recommended to load any new patient samples on the analyzer this will prevent possible release of erroneous results. 2. Quality Control Lot Number Changes a. Establish a new mean and 2 SD reference range for each level of control using a minimum of 20 data points. Run the new control in parallel with the current control whenever QC is required. Note: Running all QC points consecutively is not recommended as this will not reflect changes in the age of reagents or controls, and may result in a reference range that is too narrow. Run QC on multiple days and multiple shifts. b. Compare the SD and mean of your current QC lot to that of the new lot. Generally there should not be any significant fluctuations. Ensure the CV is acceptable (5% or less). NOTES: Fibrinogen If the reagent being changed is for the fibrinogen assay, a new standard calibration curve must be performed before any preliminary testing proceeds. Compare old and new curves there should not be a significant change between the lot numbers. APTT If the laboratory performs APTTs on heparinized patients, a Heparin Therapeutic Range for APTT must be done using the same unfractionated heparin used for patients. Prothrombin Time 1. If the analyzer requires a PT Calibration when changing reagent lot number, this must be performed before any other testing. 2. Before implementing the new lot number of reagent, ensure that the new ISI and Mean Normal PT (MNPT) are entered in the appropriate areas of the analyzer and/or LIS (depending on how the INRs are calculated). 3. When doing the patient correlations (old vs. new lot of reagent) enter the new ISI and MNPT into the analyzer/LIS to enable comparison of INRs, instead of PTs (Prothrombin Times may not compare well if the ISI value has changed significantly).
Reference: Adapted from the Regina QuAppelle Health Region

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

TRANSFUSION MEDICINE

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

RETENTION OF TRANSFUSION MEDICINE RECORDS

The most recently published standards of the Canadian Society for Transfusion Medicine are referenced. Each transfusion service shall have in place an established system for record-keeping that is written and abides by the following: DOCUMENTS Issue Vouchers from CBS Correspondence Related to Blood Components Documents Related to Traceback or Lookback Daily Records for issue of Blood Components/Products Transfused Patient Data includes antibody investigation & resolution transfusion reaction investigation Non-transfused patient data Method Revision Blood Component & Product Final Disposition Records Tissue & Bone Banking Donor & Inventory Records Quality Control Records Include reagents, serological test controls, external proficiency testing Utilization Reports Blood component product inventory Direct & Stores Inventory Purchase & acquisition data Meeting & Related Documents Computer Program Validation & Exception List Workload Records/Reports In-service Education Documentation Test & Blood Product Request Forms

Indefinitely Indefinitely Indefinitely Indefinitely Indefinitely

5 years Indefinitely Indefinitely Indefinitely 5 years

5 years 5 years 5 years 5 years 3 years 3 years 13 months

SPECIMENS Donor Segments from transfused red cells 3 weeks Clotted and/or EDTA specimens, serum/plasma from transfused 1 to 3 weeks patients (pre-transfusion) Cord Blood 2 weeks All Other Patient Specimens 1 week Kleihauer-Betke Slides 1 year

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

PROCEDURE/METHOD STATISTICAL VALIDATION/WORK-UP GUIDELINES: TRANSFUSION MEDICINE

When initiating new methodology or implementing new testing, a validation process must be developed. Validation Process Object of the validation Purpose Manufacturers instructions/data References Description Standard Operating Procedure (SOP) Sample quantities and types known and unknown samples Patient population Test methods and conditions Isolated testing Parallel testing System stress points Testing personnel Training plan Training documents Data collection/documentation required Acceptance criteria Reviewing personnel Summary Results Performance characteristics/limitations Functionality Method SOP Training plan and documents Safety Sources of error Conclusion

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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

Test ABO typing

New Method Recommendations Known samples: -10 group O -10 group A -5 A1 positive -5 A1 negative -10 group B -10 group AB Unknown patient samples: -20 Known samples: - 10 Rh(D) positive - 10 Rh(D) negative Unknown patient samples: -20 Known samples (may be patient samples, cord blood samples or simulated samples): -5 DAT negative -5 DAT positive Unknown patient samples: -5 Known samples: -10 clinically significant antibodies -10 antibody negative samples Unknown patient samples: -20 Tested under routine laboratory conditions Testing shall include challenges to: -All shifts -Variety of personnel -Acceptable method variations (e.g. variation in incubation time)

Change in Methodology Recommendations Known samples: -10 group O -10 group A -5 A1 positive -5 A1 negative -10 group B -10 group AB Unknown patient samples: -20 Known samples: -10 Rh(D) positive -10 Rh(D) negative Unknown patient samples: -5 Known samples(may be patient samples, cord blood samples or simulated samples): -5 DAT negative -5 DAT positive Unknown patient samples: -5 Known samples: -10 clinically significant antibodies -10 antibody negative samples Unknown patient samples: -10 Tested under routine laboratory conditions Testing shall include challenges to: -All shifts -Variety of personnel -Acceptable method variations (e.g. variation in incubation time)

Acceptable level of result accuracy 100%

Rh(D) typing

100%

Direct antiglobulin test (DAT)

90%

Antibody screen

90%

All tests

Unknown patient samples shall be verified by a comparable validated method. Depending on laboratory test volumes, unknown patient samples may be obtained from another laboratory.

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