Industrial Training Report

CHAPTER 1
HOSPITAL ORGANIZATION
1.1) History
Loh Guan Lye Specialist Centre is established in 1975, is a 250-bedded private
hospital.It is located in the heart of Georgetown,Penang, about 30 minutes from the
Penang International Airport and 20 minutes from the pristine
beaches of Batu Feriggi. The Centre offers up-to-date services and facilities in a
warm and caring environment. It is Loh Guan Lye Specialist Centre aspiration to be
a leader in the healthcare industry. Our mission is "Commitment to Customer
Service Excellence" and quality healthcare through continuous improvement and
the upgrading of technology and Human Resource development.
With its dedicated and caring team of Specialists, nurses and other healthcare
professionals, Loh Guan Lye Specialist Centre is committed to be
"Your Hospital of Choice".
1.2) Medical facilities that offered
A) Outpatient treatment facilities
The outpatient department has two sections that are the outpatient unit and the
Emergency unit. However, the facilities that offered by this department are
outpatient clinics, outpatient specialist clinic, Ambulance services and mortuary
vehicle, Emergency treatment services, and receiving referred cases or patients
from the Health Centre.
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B) In patient department
The in patient unit has 114 beds. It is divided into 3 other unit such as first ward for
the male, second ward for female and children as well as labour room as the third
ward.
C) Laboratory
The laboratory department offers 2 kind of supporting services which are laboratory
services and blood bank service. The services provided are clinical immunology
test, biochemistry test, serological test and blood transfusion test.
D) Radiology department
The services are normal x-ray and several contrast x-ray examination such as
intravenous urography and barium studies, CT scan and MRI.
E) Pharmacy department
The pharmacy department is a supporting department in this hospital. This unit
provides dispensary medicine and counseling to patients and clinical services.
1.3) Objective
To improve the quality of medical record services through effective and efficient
filing of patient’s records, preparation of medical reports and statistics as well as the
issuing of birth and death certificates.
1.4) Vision
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Towards a healthy community through intelligent, effective, friendly, efficient and
can be enjoyed by the community.
1.5) Mission
Encourage and increase health status of Hulu Selangor District community through
a fair and efficient treatment service using the latest technology and able to go along
the customers.
1.6) Customer service of Loh Guan Lye Specialist Centre Laboratory
Department
Every test results have to be reported as soon as possible maybe within a hour after
the quality control process without any technical problems. Specific tests that are
not done in the laboratory will be referred to other laboratory. Customer can refer to
the officer in charge regarding any problems.
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CHAPTER 2
PREANALYTICAL SECTION
2.1 Introduction
Preanalytical section functions as the common counter for the department. This
section is involved in receiving, checking and sorting of specimens before the
specimens are sent to the specific laboratories for analysis.
2.2 Request Form
2.2.1 A standard laboratory form is used for all categories of tests except otherwise
stated.
2.2.2 One set of carbonized forms (triplicate) should be used for one patient
requiring all categories of tests. (Haematology and Biochemistry) except
otherwise stated.
2.2.3 All request forms must be filled in completely and should be
accompanied by properly collected specimens.
2.2.4 Requests must specify name of tests required. Write all the tests required.
Marking ( √ ) in the box is discouraged.
2.2.5 All request forms must be signed by H.O, M.O or specialist.Name of
requesting doctor must be clearly written.
2.2.6 Clinical history and provisional diagnosis must be provided and clearly
written.
2.3 Rejection
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2.3.1 Request which do not fulfill the laboratory requirement will be rejected. Criteria
for rejection are:
a. Leaking specimen
b. Wrong container
c. Name of test not provided / not written
d. No specimen received for the intended test
e. Incomplete request form
2.3.2 Rejection shall notify through the Rejection Form
2.4 Specimen
2.4.1 Samples / specimen should be collected from the patients in the ward or
clinics and properly labeled.
2.4.2 All specimen containers for each patient should be put in one biohazard
plastic bag and stapled to the request form.
2.4.3 All specimens should be dispatched to the laboratory in appropriate containers
as specified.
2.4.4 All requests from the clinics must be accompanied by the despatch book.
2.4.5 Ward / clinic staffs or porters are required to wait at the counter waiting area
until all the specimens are checked by the laboratory staff.
2.5 Urgent request
2.5.1 Urgent requests must be justified by clinical history, diagnosis and reasons for
urgency.
2.5.2 The word URGENT must be stamped or written clearly (preferably in red) on
the request form at the upper right hand corner.
2.5.3 Urgent request must be separated from the non-urgent (other) requests.
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2.5.4 All urgent requests from the ward must be accompanied by dispatch book.
2.5.5 The ward / clinic staffs who send the urgent specimen are required to stamp
the time at the urgent counter and give / inform the laboratory staff in charge.
2.6 Reporting of result
2.6.1 All results shall be validated before they are dispatched.
2.6.2 All the reports shall be placed in the ‘pigeon holes’ located at receiving
counter of the laboratory for collection by the various wards and clinics. All reports of
urgent requests shall be informed by telephone and or dispatched immediately to
the person waiting.
2.6.3 Should there be no person waiting, the results shall be placed in the
respective ‘pigeon holes’ after being conveyed by telephone.
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CHAPTER 3
STAT SERVICE
3.1 Introduction
One of the services provided by the LSC Laboratory Department is a STAT service.
A test should be ordered STAT when the result of the test is required for immediate
patient management and if there is a delay in treatment, this might result in patient’s
morbidity and mortality. The service is provided on a 24 hours basis. The STAT
service be carried out immediately and the result will be informed to the requesting
doctor via phone.
3.2 Request Form
Request form for all STAT tests is the blood test request form. The form must be
stamp or written with the word STAT or URGENT. The request must be filled
completely and legibly with patient’s identification data, indication for requesting the
test, the time of sampling and the contact number of the requesting doctor or ward.
The requesting doctor must sign the form and the name of the requesting doctor
must be written legibly.
3.3 Receipt of specimens
3.3.1 During the office hours
All STAT test specimens will be received at the main counter (pre-analytical).
3.3.2 After the office hours
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The ward staff or doctor is required to send the specimen to the laboratory. The
ward staff or doctor is advised to press the bell at the counter to inform the
laboratory technician of the stat specimen. The ward staff or doctor is encouraged to
wait for the result.
3.4 Reporting of result
The result will be informed via phone to the requesting doctor as soon as the test is
completed and validated or the result will be given to the ward staff or doctor if they
wait for the result
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CHAPTER 4
ON CALL SERVICE
4.1 Other service offered by this hospital is on call service. On call service can be
defined by offering services after office hours and on off days to handle all
kind of test that listed in the ON CALL service list so the medical officer can
carry out the diagnosis and treatment even though after office hours and off
days.
4.2 All the Medical Laboratory Technologists (MLT) have to be ready to work
according the On Call service timetable.
4.3 The MLT will receive a call on service from the Medical officer in the inpatient
unit or from the emergency unit through the telephone operator.
4.4 The MLT will receive the specimen, request form and the on call service slip.
4.5 MLT has to record the arriving time and sign the On Call service book together
on the on call service form.
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CHAPTER 5
DISPATCHING SPECIMEN TO REFERENCE LABORATORY
Other service offered by this laboratory is dispatching specimen to the
reference laboratory so the test not done in the laboratory can be carried out there.
5.1 Specimen Preparation
5.1.1 All the specimen have to be checked by referring to the specimen receiving
procedure
5.1.2 The specimen that does not fulfill the requirement of the procedure must be
rejected according to the specimen rejecting procedure.
5.1.3 Specimen that needs serum has to be separated first before sending the
specimen to the laboratory.
5.1.4 The quantity for 24 hours urine specimen has to be measured and recorded
on the request form. The volume of urine needed has to be equal to the 50ml of
universal container.
5.2 Dispatching procedure
5.2.1 The specimen together with the form are placed into a BIOHAZARD beg.
5.2.2 All the samples are separated according to the each unit.
5.2.3 The sample that should be placed in the ice box without ice are blood
specimen, swab specimen, stool specimen, sputum specimen, Acid Fast bacteria
specimen, urine specimen.
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5.2.4 The dispatching should be done by recording the date and time in the
dispatching book as a evidence and should be signed by the officer on duty.
5.3 Result receiving procedure
5.3.1 All the results that received should be recorded in the reference laboratory
record book and send to related person or ward.
5.4 Reference laboratory
5.4.1 Pathology laboratory of GRIBBLES
5.4.2 Pathology laboratory of Hospital Kuala Lumpur
5.4.3 BP Lab Sdn Bhd.
5.4.4 IMR, Kuala Lumpur
5.4.5 Digestive Centre Kuala Lumpur.
5.4.6 AMAN allergy Laboratory, Kuala Lumpur
5.4.7 University Hospital of University Malaya
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CHAPTER 6
URGENT REQUEST PROCESS (SPECIMEN TURN AROUND TIME)
The objective of this chapter is to elaborate how the STAT request done
immediately so the report of the test can be released in a short time. The tests that
done for the STAT request are:
• Liver function test
• Renal profile test
• Blood glucose
• Amylase
• Total and direct Bilirubin
• Calcium
• Urine salicylate
• Blood crossmatch
• Prothrombin time/ a partial prothrombin time
• Full blood count
• Reticulocyte count
• Blood for Malaria parasite/ microfilaria parasite
• Urine FEME/ urine acetone
• Stool FEME/ occult blood
• Urine pregnancy test
• Coombs test
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6.1 The procedure
6.1.1 Receive the specimen and separate the STAT request from the routine test
6.1.2 Analysis of STAT specimen should done first for Biochemistry test and Clinical
test
6.1.3 Repeat the test if you have any doubts
6.1.4 Record the result on the request form and certify the result by signing.
6.1.5 Inform the result through the telephone to the wards. Record the time and the
name of the recipient on the request form.
6.1.6 Record the result in record book.
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CHAPTER 7
LABORATORY (Urine Section, Bacteriology)
7.1 Introduction
Clinical Pathology involves the laboratory evaluation of urine and stool in order to
identify diseases. It encompasses urinalysis and stool FEME.
7.2 Urine FEME
7.2.1 Objective
To detect abnormalities in urine using Clinitex 50-Urine Chemistry Analyzer
7.2.2 Materials
Clinitex 50-Urine Chemistry Analyzer and urine strips
7.2.3 Sample
Fresh urine
7.2.4 Procedure
1. Testing can be started only from READY FOR TEST screen. Be sure Reagent
Strip name on screen agrees with the strip being used.
2. Remove a Reagent Strip from the bottle and replace cap.
3. Dip a Bayer Reagent Strip into urine sample, wetting all pads.Immediately
remove strip.
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4. Press green key as you remove strip from urine. Drag the strip edge against
side of container as you remove it.
5. Blot by touching the edge of strip to paper towel to remove excess urine.
6. Place the reagent strip on test strip table within 10 seconds. Slide strip to end
as tough. Table is automatically pulled into the Analyzer.
7. Discard strip when test is finished. Gently wipe out table with a damp tissue, if
needed.
8. Report results in the request form. Request form will send to scientific officer
to validate.
9. After validated, record in a Record Book and dispatch.
7.2.5 Result
a) Colour
Normal, fresh urine is pale to dark yellow or amber in color. A red or red-brown
(abnormal) color could be from a food dye, eating fresh beets, a drug, or the
presence of either hemoglobin or myoglobin . If the sample contained many red
blood cells, it would be cloudy as well as red.
b) Glucose
The presence of glucose in urine (glycosuria) indicates that the filtered load of
glucose exceeds the ability of the renal tubules to reabsorb all of it. This usually
reflects hyperglycemia and should, therefore, prompt consideration of whether more
formal testing for diabetes mellitus is appropriate, e.g. by measuring fasting blood
glucose]. However, glycosuria is not always due to diabetes. The renal threshold for
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glucose may be lowered, for example in pregnancy, and glucose may enter the
filtrate even at normal plasma concentrations (renal glycosuria).
Normal value: Negative
c) Bilirubin
Bilirubin exists in the blood in two forms, conjugated and unconjugated. Only the
conjugated form is water-soluble, so bilirubinuria signifies the presence in urine of
conjugated bilirubin . This is always pathological.
d) Ketones
Ketones are the products of fatty acid breakdown. Their presence usually indicates
that the body is using fat to provide energy rather than storing it for later use. This
can occur in uncontrolled diabetes., where glucose is unable to enter cells (diabetic
ketoacidosis), in alcoholism (alcoholic ketoacidosis), or in association with
prolonged fasting or vomiting
e) Specific gravity
This is a semi-quantitative measure of urinary density, which in turn reflects
concentration. A higher specific gravity indicates more concentrated urine.
Assessment of urinary specific gravity usually just confirms the impression gained
by visually inspecting the colour of the urine. When urine concentration needs to be
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quantitated, most people will request urine osmolality, which has much wider
working range
Normal value: 1.015
f) Leucocytes
The presence of leucocytes in the urine suggests acute inflammation and the
presence of a urinary tract infection
g) pH (hydrogen ion concentration)
Measurement of urine pH is useful either in cases of suspected adulteration, e.g.
drug abuse screens, or where there is an unexplained metabolic acidosis (low
serum bicarbonate)
Normal value: <7.4
h) Protein
Proteinuria may signify abnormal excretion of protein by the kidneys (due either to
abnormally ‘leaky’ glomeruli or to the inability of the tubules to reabsorb protein
normally), or it may simply reflect the presence in the urine of cells or blood.
i) Urobilinogen
In the gut, conjugated bilirubin is broken down by bacteria to products known
collectively as faecal urobilinogen, or stercobilinogen. This too undergoes an
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enterohepatic circulation. However, unlike bilirubin, urobilinogenis found in the
systemic circulation and is often detectable in the urine of normal subjects.
Normal value: 3.2 µmol/L
j) Nitrite
This dipstick test depends on the conversion of nitrate (form the diet) to nitrite by the
action in the urine of bacteria that contain the necessary reductase . A positive
result points towards a urinary tract infection.
Figure 1: Clinitex-50 Urine Chemistry Analyzer
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7.3 Detection of urobilinogen in urine by manual
7.3.1 Principle
Urobilinogen is detected by the red color it gives with Ehrlich’s reagent.
7.3.2 Reagent
a) Ehrlich’s reagent
7.3.3 Materials
a) Test tube 75 mm by 12 mm
b) Plastic pipette
7.3.4 Sample
a) Fresh urine
7.3.5 Procedure
1. Pour 1 ml of urine in a test tube.
2. Add 1 drop of Ehrlich’s reagent in the test tube.
3. Observe for colour changes.
7.3.6 Results
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POSITIVE : Red colour appears (Urobilinogen is present)
NEGATIVE : Colour remains (Urobilinogen is absent)
7.3.7 Reporting format
Urobilinogen : Normal/Abnormal
7.3.8 Discussion
Urobilinogen is formed from bilirubin by the action of intestinal bacteria. The majority
is excreted in the faeces but a small amount is reabsorbed into the circulating blood.
Most of this urobilinogen is re-excreted by a normal liver. Thus a small amount of
urobilinogen in urine is normal. Urobilinogen is absent in biliary obstruction, reduced
in Antibiotic therapy which kills the intestinal flora and increased in liver tissue
damage which is an early warning of cirrhosis, acute viral hepatitis and hepatitis
associated with infectious mononucleosis (iceteric period) . Also in blood disorders
such as heamolytic anemias, malaria, congestive heart failure, extravascular
haemolysis, pernicious anemia and thalassemia.
7.4 Detection of bile salts in urine by manual
7.4.1. Principle
The presence of bile salts reduces the surface tension of the urine thus enabling the
sulphur powder to sink to the bottom of the container.
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7.4.2 Reagents
Flowers of sulphur
7.4.3 Sample
Fresh urine
7.4.4 Procedure
1. Pour 4.0 ml of urine in a test tube
2. Place the test tube on the rack.
3. Sprinkle a little finely powdered sulphur onto the surface.
4. Allow it to stand for 2 minutes and then see the bottom of the container.
7.4.5 Result
POSITIVE : The surface tension of the urine is lowered,
allowing the sulphur powder to sink to the
bottom of the container.
NEGATIVE : Sulphur powder floats on top of the urine.
Urine Bile Salt : POSITIVE / NEGATIVE
7.4.7 Discussion
Bile salts are synthesized by liver cells and excreted into the bile. They have the
property of lowering the surface tension of aqueous liquids which allows the
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emulsification of fats during digestion. Their presence in urine can, with the possible
exception of nephritis, always be regarded as indicative of liver damage or duct
obstruction]. In early stages of obstruction to bile passages, amount in blood
increases, and bile salts appear in urine. But with continuation of obstruction is
relieved. In health, peripheral blood contains small quantities of bile salts, since they
are absent from urine, they must be “thresh-hold substances”. In obstructive jaundice
there may be scratch marks that result from the itching which the bile salts evoke . It
has been suggested that this test is of little diagnostic value.
7.5 Detection of bile pigments in urine by manual
7.5.1 Principle
Bile pigments is detected by the green colour it gives with Trichure iodine.
7.5.2 Reagent
Trichure iodine
7.5.3 Sample
Fresh urine
7.5.4 Procedure
1. Pour 2.5 ml of urine in a test tube.
2. Add 2 – 3 drops of trichure iodine to the urine.
3. Observe colour changes.
7.5.5 Result
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POSITIVE : Green colour appears
NEGATIVE : No changes, colour remain.
Urine Bile Pigments : POSITIVE / NEGATIVE
7.6 Occult blood
7.6.1 Objective
To detect the present of blood in the feces for cancer cases
7.6.2 Reagent
Hema screen kit is used which have two control places and developing solution
7.6.3 Specimen
Feces
7.6.4 Procedure
1. Take some of the specimen using the applicator stick.
2. To develop Specimen test area, lift the flap and place 2 drops of Hema Screen
3. developer over each smear. Read results in 30-60 seconds
4. Apply one drop of developer onto Control Area. Blue color should appear within
5. 30 seconds on the positive and no changes should appear on the negative site.
7.6.5 Result
Normal values: Negative
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7.6.6 Interpretation of results
No color change : Negative
Light green color : Trace
Green color :+
Blue color : ++
7.7 Blood Film Malaria Parasites
7.7.1 Introduction
The definitive diagnosis of malaria infection is still based on finding malaria
parasites in blood films. In thin films the red blood cells are fixed so the morphology
of the parasitised cells can be seen. Species identification can be made, based
upon the size and shape of the various stages of the parasite and the presence of
stippling (ie bright red dots) and fimbriation (ie. ragged ends). However, malaria
parasites may be missed on a thin blood film when there is a low parasitaemia.
Therefore, examination of a thick blood film is recommended. With a thick blood
film, the red cells are approximately 6 - 20 layers thick which results in a larger
volume of blood being examined.
7.7.2 Reagents
• Field’s A
• Field’s B
7.7.3 Sample
Whole blood
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7.7.4 Procedure
1. Place a drop of blood on a microscope slide and spread to make an area of
approximately 1 cm2. It should just be possible to read small print through a thick film.
2. The film is air dried and NOT fixed in methanol.
3. The slide is dipped into Field's stain A for 3 seconds.
4. The slide is then dipped into tap water for 3 seconds and gently agitated.
5. The slide is dipped into Field's stain B for 3 seconds and washed gently in tap
water for a few seconds until the excess stain is removed.
6. The slide is drained vertically and left to dry.
7. Microscopic examination of the Field's stained thick blood film.
8. The end of the film at the top of the slide when it was draining should be looked
at. The edges of the film will also be better than the centre, where the film may be
too thick or cracked.
9. In a well stained film the malaria parasites show deep red chromatin and pale
blue cytoplasm.
10. White cells, platelets and malaria pigment can also be seen on a thick film. The
leucocyte nuclei stain purple and the background is pale blue. The red cells are lysed
and only background stroma remains. The occasional red cell may fail to lyse.
11. Schizonts and gametocytes, if present, are also easily recognizable.
12. A thick film should be examined for at least 10 minutes, which corresponds to
approximately 200 oil immersion fields, before declaring the slide negative.
7.7.5 Conclusion
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A thick blood film is recommended for routine diagnosis of malaria in addition to the
thin film and is particularly valuable in instances of low parasitaemia . However, in
order to correctly specify the parasite, examination of a thin film is required. If the
thick film is negative, it is unlikely that parasites will be found in the thin film.
7.8 Stains for acid-fast bacilli
7.8.1 Principle
Tuberculosis is a disease of global concern. Eight million new cases were detected
each year. The cornerstone of the diagnosis of tuberculosis is direct microscopic of
stained sputum specimens for tubercle bacilii. Between 5,000 and 10,000 tubercle
bacilii per millimeter of sputum are required for direct microscopy to be positive [8].
Usually sputum from patient with pulmonary tuberculosis (PTB) often contains
sufficiently large number of acid-fast bacilii and can be readily detected by direct
microscopy.
7.8.2 Reagents
• Carbolfuchsin
• 3 % hydrochloric acid-ethyl alcohol
• Methylene blue
7.8.3 Materials
a) Slide
b) Rack
c) Microscope
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d) Spirit lamp
e) Immersion oil
7.8.4 Sample
Sputum
7.8.5 Procedure
I. Smear preparation
1. Label a new, clean, unscratched microscopic slide at one end with relevant
patient identification number. Label with pencil for frosted part of the slide or label
with diamond stylus for normal slides.
2. Using a thin, disposable applicator stick (wooden) or bacteriological loop,
transfer an appropriate portion of the specimen to the slide.
3. Smear the specimen on a slide uniformly on oval-shape by smearing repeatedly
in coil-like patterns over an area approximately 3.0 cm by 2.0 cm in size.
4. Discard the applicator stick in disinfectant, and use a new one for each
specimen.
II. Ziehl’s Neelsen carbolfuchsin staining procedure
1. Place the fixed smear slide, on a staining bridge over the sink with smeared side
uppermost. Leave sufficient space between the slides to prevent the flow of solutions
from one slide to the next.
2. Cover the whole surface of the slides with carbolfuchsin which has been filtered
prior to use.
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3. Heat gently the slide till it is steaming using a low intermittent heat. Leave the
warm stain for 5 to minutes.
4. Rinse each slide individually in a gentle stream of running water until all free
stain is washed away.
5. Tilt the slide to drain off excess rinse water.
6. Cover whole slide with 3 % hydrochloric acid-ethyl alcohol and leave until
solution runs clear for 2-3 minutes.
7. Rinse each slide with gentle stream of running water.
8. Tilt the slide to drain off excess rinse water
9. Pour methylene blue to cover the whole surface of the slide and leave for 10 to
20 seconds.
10. Wash the slide with a gentle stream of running water.
11. Tilt and place the slide on the slide rack or piece of blotting paper to dry in the
air. Allow smear to air dry.
12. Observe under microscope
7.8.6 Procedures of recording
I. Recording after examination of one length or less
1. Examination of smeared stained slide should always commence at the center
of the smear beginning either on the rights or left hand slide of the smear.
2. If examination of the slide from the very beginning shows large number of
bacilli, it will not be necessary to examine the whole length when 50 bacilli have been
counted. Stop the examination and record as follows: 50/L (more than 50 bacilii
in one length)
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3. If 11 to 49 (more than 10) bacilli are counted at the end of one length, record
the actual number for example: 36/L (36 bacilii in one length)
4. II. Recording after examination of three lengths
5. If 0 to 10 acid-fast bacilii are observed at the end of one length, continue the
examination for up to three lengths. Three possibilities can occur:
6. 50 bacilii are observed, stop the examination and record as follows: 50 / > 1
L(50 bacilli in more than one length).
7. Less than 50 are observed, record the actual number counted: 29 / 3 L (29 in
three lengths)
8. No bacilii are observed in three lengths, records as follows: 0 / 3 L (0 bacilii in
lengths).
NEGATIVE (0 / 3 L)
POSITIVE (………../ 3 L)
7.9 Gram Stain
7.9.1 Principle
The Gram stain divides most bacteria into two major groups: gram positive and
gram negative. This differentiation is, made by the bacteria’s ability to bind and hold
dye crystal violet. Bacteria that bind the dye will stain a dark purple and are
designated as gram-positive bacteria]. Bacteria that cannot maintain the crystal
violet dye will be counterstained with safranin. These bacteria appear pink in the
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final preparation and are known as gram negative bacteria. Not all bacteria will stain
with the Gram stain and require special staining techniques for observation.
7.9.2 Reagents
a) Crystal violet
b) Gram’s iodine
c) Safranin
7.9.3 Materials
a) Slide
b) Microscope
c) Rack
d) Forceps
e) Bunsen burner
7.9.4 Sample
Sputum
7.9.5 Procedure
1. Heat a prepared smear to fix by holding it with forceps and passing through a
Bunsen burner flame 2 or 3 times.
2. When cool, place the smear on a staining rack. Flood the smear with the
crystal violet stain. Allow the stain to stand for 1 minute.
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3. Rinse the slide with a steady stream of water from a faucet/squeeze bottle. Tilt
the slide to remove excess water.
4. Flood the slide with Gram’s iodine and allowed to stain for 1 minute.
5. Rinse as in step (III).
6. Holding the slide with forceps, squeeze decolorizer onto the slide until no
more purple color runs off.
8. Flood the slide with safranin for 1 minute.
10. Remove excess water by standing on end. Wipe the back of the slide to
remove excess stain. Allow to dry completely before reading.
7.9.6 Result
POSITIVE : Gram positive organisms stain purple or
violet color and can be either bacilli or
cocci.
NEGATIVE : Gram negative organisms stain pink or red
color and can be either bacilli or cocci.
No Organism seen
A few/moderate gram positive/negative Cocci seen.
A few/moderate gram positive/negative bacilli seen.
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Figure 2: Gram stain
7.10 Field’s Stain Method for Thin Film
7.10.1 Introduction
This is a modification of the original Field’s stain to enable rapid staining of fixed thin
films. This method is suitable for malaria parasites, Babesia sp., Borrelia sp. and
Leishmania sp.
7.10.2 Reagents
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a) Field’s Stain “A”
b) Field’s Stain “B”
c) Methanol or methylated spirit
7.10.3 Materials
a) Forceps
b) Slide
c) Rack
d) Hair dryer
7.10.4 Sample
C.S.F., blood, sputum for eosinophil.
7.10.5 Procedure
1. Make a smear as for blood differential count.
2. Dry the film by waving to and from in the air or use a cool-air hair dryer till the
film is dry.
3. Fix the film by flooding the slide with pure methanol or pure methylated spirit for
two minutes. Then dry the film.
4. Dip in Field’s Stain “A” for 10 to 15 seconds and then wash in gentle running tap
smear.
5. Dip in Field’s stain “B” for 10 to 15 seconds and then wash in gentle running tap
water.
6. Return to Field’s Stain “A” for 10 to 15 seconds and then wash in gentle tap
water.
7. Finally dry the film and observe under the microscope.
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7.10.6 Percentage Calculation
Total cells counted = 200
Neutrophils noted = 150
Lymphocytes noted = 50
150 x 100 = 75 % Neutrophils
200
50 x 100 = 25 % Lymphocytes
200
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CHAPTER 8
BIOCHEMISTRY LABORATORY
8.1 Introduction
Biochemistry unit provides diagnostic and consultative services to Loh Guan Lye
Specialist Centre for patient management. The services cover analysis and
interpretation of biochemical changes in body fluids for diagnostic, monitoring and
screening of diseases.
8.2 List of services
The diagnostic services provided are:
• Emergency (STAT) service and 24 hour service
• Routine service
8.3 Specimen collection and preparation
8.3.1 Blood
Collect blood into a plain container or one with the appropriate anticoagulant /
preservative in it. Separate the plasma or serum (after the blood has been allowed
to clot as appropriate and transfer the specimen into a clean container.
8.3.2 Urine
24-hour Urine Collection
35
Most Quantitative assays are performed on urine specimen collected over 24 hours.
The 24-hour timing allows for circadian rhythmic changes in excretion at certain time
of day.
Procedure of collection:
1. The 24 hour urine bottle which contains preservative for the required test is
available at the pre-analytical counter and provided on request, with the
accompanying request form.
2. On the day of collection, the first urine voided must be thrown away. Time of
first urine voided is the start of timing for the 24 hour collection.
3. Collect the second and subsequent voided urine for 24 hours from the time
start into the 24 hour urine bottle.
4. At the end of 24 hour, the last urine voided is collected. For best result,
refrigerate if possible.
5. Label the bottle as directed and send immediately to the laboratory.
Figure 3: Abbott Architect ci8200
36
8.4 Test done using Architect ci8200
8.4.1 Liver Function Test
a) Alkaline Phosphatase (ALP)
Increases in ALP activity in liver disease are the result of increased synthesis of the
enzyme by cells lining the bile canaliculi, usually in response to cholestasis, which
may be either intra or extra hepatic .
Normal value: 80-280 g/L
b) Alanine aminotransferase (ALT)
ALT is widely used in clinical practice as a sensitive, albeit non-specific, index of
acute damage to hepatocytes irrespective of its aetiology . Causes of liver damage
include hepatitis, no matter the causative agent, and toxic injury which may
accompany any one of a large number of insults of the liver, including over dose.
Normal value: Men up to 40.0 U/L :Women up to 31.0 U/L
c) Bilirubin
Bilirubin is derived from haem, an iron-containing protoporphyrin mainly found in
haemoglobin .
Normal value: up to 17.0 µmol/L
d) Total protein
Changes in total protein concentration are common. An elevated total protein
concentration may mean the presence of a paraprotein. A decreased total protein
usually means that the albumin concentration is low.
37
Normal value: 55 – 82 g/L
e) Albumin
Albumin is the major plasma protein and is synthesized and secreted by the liver. It
accounts for about 50% or the total hepatic protein production. Albumin makes the
biggest contribution to the plasma oncotic pressure. If the albumin concentration
falls very low, oedema is the result .
Normal value: 35 – 46 g/L
8.4.2 Cardiac Enzymes
When myocardial cells die, they break up and release their contents. This is the
basis for the role of cardiac markers in myocardial infarction diagnosis. Historically,
the ‘cardiac enzymes’ commonly used to diagnose myocardial infarction included
crearine kinase (CK), aspartate aminotransferase (AST) and lactate dehydrogenase
(LDH) [1].
Normal value:
AST: Men up to 37.0 U/L, Women up to 31 U/L
LDH: 150 – 450 U/L
CK: Men (24 – 190 U/L), Women (24 -170 U/L)
8.4.3 Renal Profile
Serum creatinine and urea
38
The concentrations of creatinine and urea in serum samples are used as
convenient, but insensitive, measures of glomerular function [1].
Normal value:
Creatinine: 60 – 170 µmol/L
Blood Urea: 2.3 – 7.5 µmol/L
8.4.4 Serum Bilirubin
Bilirubin exists in the blood in two forms, conjugated and unconjugated [3].
Normal value:
Total bilirubin : 3 – 22 µmol/L
8.4.5 Serum calcium
The amount of calcium present in the extracellular fluid is very small in comparison
to that stored in bone. Even in the adult, calcium in bone is not static; some bone is
reabsorbed each day and the calcium returned to the extracellular fluid [1].
Normal value: 2.12 – 2.53 mmol/L
8.4.6 Serum Inorganic phosphate
Phosphate is abundant in the body and is an important intracellular and extracellular
anion. Much of the phosphate inside cells is covalently attached to lipids and
proteins [3].
Normal value: Adult (0.81 – 1.55 mmol/L),
Children (1.25 – 2.26 mmol/L)
39
8.4.7 Fasting Lipid serum
a) Cholesterol
b) Trigliseride
Normal value: 0.4 1.8 mmol/L
8.4.8 Uric acid
Nucleic acid contains bases of two different types, pyrimidines and purines. The
catabolism of the purines,adenine and guanine, produces uric acid. At physiological
hydrogen ion concentration, uric is mostly ionized and present in plasma as sodium
urate. An elevated serum urate concentration is know as hyperuricaemia . The
consequences of this are the medical condition, gout.
8.4.9 Blood glucose
a) Fasting blood glucose
Fasting blood glucose is regarded as diagnostic of diabetes, whether or not
hyperglycemic symptoms are present. The patient should be fasted overnight (at
least 10 hours).
b) Random blood glucose
40
If a patient complains of hyperglycemic (osmotic) symptomssuch as thirst,
frequency, polyuria, and nocturia, a random blood glucose may be used to diagnose
diabetes.
Normal value: <11.0 mmol/L
8.5 Modified Glucose Tolerence Test (MGTT)
8.5.1 Purpose
The Modified glucose tolerance test (MGTT) measures the body's ability to use a
type of sugar, called glucose, which is the body's main source of energy. An MGTT
is most commonly done to check for diabetes that occurs with pregnancy (gestational
diabetes) .
8.5.2 Reagent
75 gram of glucose
8.5.3 Sample
Whole Blood and urine
8.5.4 Procedure
1. Patient need to fast at least 8 hours before sample be taken.
2. Take fasting urine and blood sample from the patient.
3. Record on the request form.
4. Give 75 gram of glucose to the patient.
5. Order patient to rest at resting room.
41
6. After 2 hours, get sample of urine and blood again.
7. Perform test.
8. Record the result on the request form.
9. Validate the result and record in the Record Book.
8.5.5 Results
Fasting blood glucose: Non-diabetic: <6.0 , Diabetes: >7.0
Blood glucose after 2 hours: Non-diabetic: <11.0 , Diabetes: >11.0
Modified Glucose Tolerence Test
GLUCOSE (mmol/L) URINE SUGAR
FASTING ………………..
2 HOURS ………………..
8.6 TEST DONE USING ELECTROLYTE ANALYZER
8.6.1 Objective
To detect electrolytes in the blood such as sodium, potassium, and chloride
8.6.2 Reagent and preparation
• Prepared reagent
• Ilyte NA/K/CL Machine
• Biolyte Machine
42
8.6.3 Specimen
Fresh serum
8.6.4 Procedure
1. Read and follow the instruction as written on the screen
2. Written on the screen – “Analyze blood”
3. Written on the screen – “Open Probe”
4. Written on the screen – “Probe in sample”
5. Push the “YES” button
6. Written on the screen – “Sampling…Wait…Return the probe”
7. Close the probe and written on the screen – Measuring…Please wait…”
8. The calibration of the machine is done by the machine itself.
8.6.5 Results
1. The result will appear on the screen and printed on the paper.
2. Record the result in the Record Book and validate the results.
8.7 EXAMINATION OF CEREBROSPINAL FLUID(CSF)/BODY FLUID FOR
BIOCHEMISTRY TEST AND MICROSCOPICALLY.
8.7.1 Objective
To explain the procedure on how to examine CSF and body fluid to detect the
bacteria infection
43
8.7.2 Equipment
Microscope, centrifuge, Hair dryer, “Neubaur counting chamber”, Test tube, Plastic
pipette, Slide and cover slip.
8.7.3 Reagent and the preparation
• Toluidine Blue staining 0.05%
• Normal saline
• Gram staining
• Ziehl Nelsen staining
• Indian ink staining
8.7.4 Specimen
CSF specimen
8.7.5 First examination
Specimen that received is examined macroscopically for any turbidity, blood, color,
and coagulation. Record the results on the request form.
8.7.6 Diagnosis done in biochemistry lab
1. Interpretation on the specimen is done. For example, the interpretation of
glucose, protein and chloride.
44
2. Globulin confirmation
3. Insert 0.5 ml of Pandy reagent into the test tube
4. Insert 1 drop of specimen into the tube and look for the turbidity. If there is any
turbidity, it shows the present of globulin.
5. Record the results on the request form.
8.7.7 Cell count in the specimen
1. Drop 5 drop of specimen into the test tube by using disposable pipette.
2. Wipe the pipette on the gauze or tissue and using the same pipette, insert 5
drops of Toluidine Blue into the same tube.
3. Mix the specimen and place it on the neubaur counting chamber
4. Look under the microscope.
a) Count 4 mm square and record the number of white blood cells and
red blood cells
b) Calculation
I. The number of cell multiply by 5sel/ul
i. Normal value
1. RBC/mm3 = Nil
2. WBC/mm3 = 0-10
3. Big cells = Nil
5. Record the results in the request form
8.7.8 Detection of any organism
1. Centrifuge the specimen at 1500 rpm for 5 mins and remove the supernatant
2. Mix the sedimentation and do 2 smears on the slide and fix it carefully using the
hair dryer
45
3. Do the Gram staining for the first smear to detect bacteria.
4. Carry out the Ziehl Neelsen staining to detect acid fast bacilli.
5. Do the Indian Ink staining to detect the presence of Cryptococcus.
6. Place one drop of the specimen and Indian ink on the slide
7. Cover the slide using cover slip
8. Examine the slide under x10 follow by x40
9. Record as Cryptococcus positive or negative
1. Record the results on request form
2. Validate the results and record in the record book.
46
CHAPTER 9
HEMATOLOGY LABORATORY
9.1 Introduction
Hematology is the study of blood, and in a routine laboratory is concerned largely
with abnormalities of the blood. One of the primary functions is to detect anaemia
and assist in the diagnosis of the exact type of anaemia to enable the right
treatment to be given. Other important aspects of haematology are the identification
of disorders associated with abnormal proliferation of blood cell precursors (e.g.
leukaemia), the identification of inherited blood disorders (e.g. haemophilia) and the
monitoring and control of patient treatments.
9.2 Reticulocyte Count
9.2.1 Principle
Reticulocytes are juvenile red cells and they contain remnants of basophilic
ribonucleoprotein. This basophilic material has the property of reacting with certain
dyes such as Brilliant Cresyl Blue to form precipitate of granules or filaments. The
most immature reticulocytes are those with the largest amount of precipitable
material and the more mature reticulocytes are those with only a few dots or short
strands.
9.2.2 Reagent
47
Supravital stain
9.2.3 Materials
• 12 x 75 mm test tube
• Pipette
• Water bath
• Microscope
• Glass slide
9.2.4 Sample
Whole blood
9.2.5 Procedure
1. Add 2 drops of Supravital stain into a small glass tube.
2. Two drops of blood is added in.
3. Sample is mixed and incubated in water bath at 37oC for 15 – 20 minutes.
4. Smears are made and examined under microscope
9.2.6 Counting procedure
An area of film is chosen for the count where the cells are undistorted and where the
staining is good.1000 red cells are counted and % of reticulocytes present is
counted.
9.2.7 Calculation
Reticulocytes
= Number of reticulocytes counted x 100
48
1000
9.2.8 Result
Normal Value: 0.2 – 1.0 %
Reticulocytes…………………..%
9.3 Erythrocytes Sedimentation Rate (ESR)
9.3.1 Purpose
It is a measure of the presence and intensity of morbid process within the body. This
test is a useful supplement to clinical method, because it may be accelerated in
chronic disease and in localizes inflammatory disease .
9.3.2 Principle
Erythrocytes sediments or a specific rate in blood which has been rendered
incoagulable, this rate is determined primarily by the plasma proteins and the
number shape and surface properties of the erythrocytes.
9.3.3 Materials
• Stand
• Timer
• Sediplast pipette
49
9.3.4 Sample
Whole blood
9.3.5 Procedure
1. Venous blood is diluted is 31.3 gle tri sodium citrate in the proportion one part of
citrate to four part of blood.
2. Mix the sample well.
3. Impale the sediplast pipette in blood tube.
4. Place the tube exactly vertical and leave undisturbed for 1 hour, free from
vibration and draughts and not exposal to direct sunlight.
5. The height of the clear plasma above and upper limit of the column of
sedimenting red cells is then read to nearest mm.
9.3.6 Result
The normal sedimentation rate for woman is between 4 – 7 mm/hr and for men
it is 3 – 5 mm/hr.
ESR……………mm/hr
9.3.8 Discussion
Although it is frequently ordered, the erythrocyte sedimentation rate (ESR) is not a
useful screening test. It is only useful for diagnosing two diseases: temporal arthritis
50
and polymyalgia rheumatica (in which it may exceed 100 mm/hour).The clinical
usefulness of erythrocyte sedimentation rate (ESR) is limited to monitoring the
response to therapy in certain inflammatory diseases such as temporal arthritis,
polymyalgia rheumatica, rheumatoid arthritis . It can also be used as a crude
measure of response in Hodgkin's disease.
9.4 Glucose-6-Phosphate Dehydrogenase (G6PD)
9.4.1 Principle
The NADH produced in the reaction fluorescence under long way ultraviolet light. If
there is a marked deficiency of G6PD – DH or is this enzyme lacking entirely, no
fluorescence will be observed.
9.4.2 Purpose
Primary screening for the detection of G6PD in the newborns
9.4.3 Reagent
G6PD reagent
9.4.4 Sample
Whole blood dried on filter paper
9.4.5 Materials
• 75 mm by 12 mm test tube
• Hole puncture
51
• Water bath
• Whatman No.1 filter paper
• Hair dryer
9.4.6 Procedure
1. 0.1ml of working reagent is placed into a labeled test tube.
2. 6 mm of sample strip is punched into the test tube.
3. Test tube is incubated at 37oC for 1 minute.
4. Test mixture is spotted on Whatman No.1 filter paper and be allowed
to dry.
5. It is then examined under ultraviolet lamp for fluorescence present.
9.4.7 Interpretation
When the filter paper dried, view under a long wave UV lamp in a darkened room.
Specimen obtains from patients with normal or just slightly depressed G6P-DH
activity will show strong fluorescence. Failure to fluorescence after 10 minute of
incubation suggests a total lack or marked defiance of G6P-DH.
9.4.8 Result
Normal : Presence of fluorescence
Deficient : No presence of fluorescence
G6PD: NORMAL / DEFICIENT
52
9.4.10 Discussion
In some forms of G6P-DH deficiency, young erythrocytes manifest normal enzymes
activity. Blood from patients who have experienced a hemolytic crisis must first be
treated by the procedure of Herz et all. To separate the older erythrocytes from the
prevailing population of young ones, use 0.005ml of the suspension to obtain for the
assay. If the patients has received a blood transfusion, this test is clinically
significant only after 30 days have elapsed; as the donor’s erythrocytes generally
manifest a normal G6P-DH activity and can thus bias the result before the expiration
of this line. Commercially available UV lamps emitting long UV light are adequate for
the evaluation.
9.4.11 Conclusion
Neonatal jaundice with pathological hyperbilirubinemia develops more frequently in
cases of G6PD deficiency [9]. The early characterization of G6PD activity provides
an etiological diagnosis for neonatal jaundice, as well as the opportunity to give the
newborn's family information concerning hemolytic crisis prevention
9.5 Full Blood Count
9.5.1 Purpose
The Cell Dynn 3700 is a fully automated quantitative analyzer. It provides quick,
easy and accurate screening of the sample.
53
Figure 4: Cell Dyn 3700
9.5.2 Sample
Whole blood in EDTA tube.
9.5.3 Procedure
1. Blood received is checked for presence of clotting. If not clotted, sample is
labeled using the bar code. If sample is clotted, it is rejected and recorded in the
sample rejection book.
2. The labeled sample is checked using the Cell Dynn 300 automated machine.
3. Result obtained is recorded in the computer as well as in the book for
reference.
54
9.5.4 Discussion
In addition to counting, measuring, and analyzing red blood cells, white blood cells
and platelets, automated hematology analyzers also measure the amount of
hemoglobin in our blood and within each red blood cell. This information can be
very helpful to a physician who, for example, is trying to identify the cause of a
patient's anemia . If the red cells are smaller or larger than normal, or if there's a lot
of variation in the size of the red cells, this data can help guide the direction of
further testing and expedite the diagnostic process so patients can get the treatment
they need quickly.
9.6 Prothrombin Time Test (International Normalized Ratio)
9.6.1 Objective
To measure the prothrombin time of a patient whether the results are normal or not
and to control the heparin treatment
9.6.2 Reagent
• Neoplastine CI plus (freeze dried thromboplastin)
• Aqueuos solution (Calcium + sodium azide) act as activator
9.6.3 Reagent preparation
1. Mix the Bottle 1 together with Bottle 2
2. Place it at the room temperature for 30 minutes
55
3. Keep the mixed solution at 37 c for 8 hours, 20 c for 2 days and 2-8 c for 8
days.
9.6.4 Specimen
Whole blood
9.6.5 Procedure
1. Centrifuge the specimen to obtain the plasma at the speed of 2500 rpm for 2
minutes (the plasma can only be kept for 8 hours at 20 c)
2. Carry out the test using the Clot-1 machine
3. Keep the Reagent 1 at 37 c.
4. Insert a magnetic bar into the cuvette that placed in the cuvette
5. Pipette 0.2 ml Reagent 1 into the cuvette and place the cuvette into the reading
slot of the machine.
6. Press the reset button
7. Pipette 100ul of plasma into the cuvette and instant reaction will take place.
9.6.6 Manual method
1. Incubate the specimen in the waterbath
2. Place a empty test tube in the waterbath and pipette 100 ul plasma into the test
tube and together with 100 ul Reagent 1.
3. Mix the solution and look for any coagulation. Stop the stopwatch once
coagulation can be seen. Record the time.
Prothrombin time test:
56
Control:
P.R:
INR:
P.R(plasma ratio) = Prothrombin Time divide by Control
9.7 Activated Partial Thrombin Time
9.7.1 Princple
To detect the deficiency of coagulation factor in the congenital and acquired and to
control the heparin treatment
9.7.2 Reagent
• Reagent 1 contains cephalin (freeze dried Thrombin)
• Reagent 2 contains kaolin solution which acts as activator
• Calcium chloride solution 0.025M
• Waterbath
• Stopwatch
9.7.3 Specimen
Blood which collected in anticoagulant tube
9.7.4 Procedure
1. Place a test tube in waterbath. (If the Clot-1 is used, place the cuvette in the
machine that heated at 37 c)
57
2. Pipette 0.1 ml plasma into the tube or the cuvette together with 0.1 ml of
reagent 1
3. Mix and incubate at 37 c for 3 minutes.
4. Insert 0.1 ml 0.025 M calcium chloride and start the stopwatch and record the
time when the plasma freezes
5. (When using the Clot-1 machine, place the cuvette into the reading slot)
6. (Calibrate the machine by pressing the RESET button)
7. (Insert 0.1ml 0.025M Calcium chloride into the cuvette and the Clot-1 machine
will do the reading)
PTT:…………….sec
(NR: 30.0-44.0 sec)
9.8. Urine hCG (Pregnancy Test)
9.8.1 Principle
Pregnancy Test Strip is a rapid-chromatographic immunoassay for the qualitative
detection of human chorionic gonadotropin (hCG) in urine to aid in the early
detection of pregnancy. The test utilizes a combination of antibodies including a
monoclonal hCG antibody to selectively detect elevated levels of hCG. The assay is
conducted by immersing the test strip in a urine specimen and observing the
formation of colored lines. The specimen migrates via capillary action along the
membrane to react with the colored conjugate. Positive specimens react with the
specific antibody-hCG-colored conjugate to form a colored line at the test line region
of the membrane. Absence of this colored line suggests a negative result. To serve
58
as procedural control, a colored line will always appear at the control line region if
the test has been performed properly.
9.8.2 Reagents
The test strip contains anti-hCG particles and anti-hCG coated on the membrane.
9.8.3 Materials
• Test strips
• Package insert
9.8.4 Sample
Urine
9.8.5 Procedure
1. Bring the pouch to room temperature before opening it. Remove the test strip
from the sealed pouch and use it as soon as possible.
2. With arrows pointing toward the urine specimen, immerse the test strip
vertically in the urine specimen for at least 5 seconds.
3. Place the test strip on a non-absorbent flat surface, start the timer and wait for
red line(s) to appear. Read the results within 3 minutes.
POSITIVE : Two distinct red lines appear.
NEGATIVE : One red line appears in the control region.
INVALID : Control line fails to appear.
59
Urine for hCG hormone : POSITIVE
NEGATIVE
60
CHAPTER 10
BLOOD BANK
10.1 Introduction
Blood bank and transfusion services depend on voluntary donors to provide the
blood necessary to meet the needs of the patients they serve. To attract volunteer
donors initially and to encourage their continued participation, it is essential that
conditions surrounding blood donation be as pleasant, safe and convenient as
possible.
10.2 Blood Donor Selection
10.2.1 Objective
1. To ensure the potential donor is in good health.
2. To protect the recipient from any ill effects through transmission of disease or
drugs by blood
3. To protect the volunteer from any harm to his/her health
10.2.2 Procedure
1. The ultimate responsibility for the selection of donor nets with the head of
department. The immediate responsibility is that of the senior nurse in charge and the
medical officer in charge of the session.
2. Only people of good health may be accepted as donor.
61
3. Evaluate the volunteer’s medical history on the day of donation. Person with
features indicative of ill health or previous disease may only be accepted at the
discretion of a medical officer of the blood transfusion service.
4. If there is any doubt of the volunteer risk behavior he/she should be excluded as
a donor.
5. Qualitative selection criteria for whole blood donation
i. Age between 17 – 60 years of age
ii. Weight- minimum 45 kg
iii. Volume of donation – donor who weight more than
50 kg can safely donate 450 ml of blood. Donors who
weight between 45 and 50 kg can donate 250-300 ml
of blood.
iv. Hemoglobin: 12.5 g/dl
v. Blood pressure – minimum 110/70, maximum 130/90
vi. Frequency of donation (Female – every 4 month and
Male- every 3 month)
10.3 Hemoglobin Estimation
10.3.1 Objective
To estimate hemoglobin (Hb) level for blood donors semi quantitatively i.e >12.5 g
% or <12.5 g %
10.3.2 Materials
• Double pan balance
62
• 1 liter volumetric flask
• Uninometer
• Filter paper
• Needle (lancet)
• Pasteur pipette
• Screw cap container
10.3.3 Sample
Blood
10.3.4 Reagent
• Distilled water
• Cupric suplhate pentahydrate
10.3.5 Procedure
1. Prepare copper sulphate solution of specific gravity of 1.053 by dissolving 82
gram of CuSO4, 5H2O in 1 liter distilled water.
2. Disperse 40ml of copper sulphate solution in to a universal container. The
solution must be changed daily or each 25 tests.
3. Clean the site of skin puncture thoroughly with 70 % alcohol and wipe dry.
Puncture the finger with sufficient force using a sterile lancet, to allow free flow of
blood.
4. Collect the blood in a Pasteur pipette, avoiding air bubbles.
5. Allow a drop of blood gently in to the solution from a height about 1 cm above
the surface of the solution. (Cupric Suplhate Pentahydrate)
63
6. Observe for 15 sec. Watch for the sinking or floating of the drop of blood.
Record result >12.5g % or <12.5g %
7. If should be noted that is not a quantitative test and will only show that the
hemoglobin is equal to, above or below acceptable limits.
Hb > 12.5 g % / <12.5 g %
10.4 ABO and Rhesus (D) Grouping by Tile Method
10.4.1 Objective
To determine the ABO and Rh (D) grouping of the blood sample.
10.4.2 Reagents
• Antiserum A
• Antiserum B
• Antiserum AB
• Antiserum D
• Control sera
10.4.3 Materials
• Tile
• Applicator stick
• Pasteur pipette
64
I.
10.4.4 Procedure
1. Use pre labeled tile identifying sections for antiserum and cell to be used as
shown below.
Anti- Anti- Anti- O A B Anti- Control
A B AB Cell Cell Cell D
Table 1: Pre labeled tile
2. Drop 1 drop of antiserum to ABO cell in appropriate sections. e.g. Anti-A, Anti-B,
Anti-AB, Anti-D.
3. Drop 1 drop of blood sample for Anti-A, Anti-B and Anti-AB for ABO forward
grouping and Anti-D and Rh control for Rhesus (D) typing.
4. Mix cell and antiserum using clean applicator stick thoroughly over an area
about 2 cm diameter.
5. Keep cell and antiserum mixture in continuous gentle motion and observe for
agglutination. Grade the agglutination and record.
6. Care must be taken not to allow drying up to at the edges of cell suspension to
prevent false positive result
POSITIVE : Agglutination of RBCs
NEGATIVE : A smooth suspension of RBCs
10.5 ABO Grouping by Tube Method
65
10.5.1 Objective
To determine the ABO type of a blood donor or patient
10.5.2 Reagents
• 0.9 % normal saline
• Antiserum A, B, AB
10.5.3 Materials
• Test tube 75 mm by 12 mm
• Automatic cell washer
• Centrifuge
• Pasteur pipette
10.5.4 Procedure
1. Prepare 2-5 % suspension of test sample RBCs in saline solution (1 drop of
packed cells to 1ml of saline solution).
2. To each labeled tube (Anti-A, B, AB) add 1 drop of appropriate antiserum.
3. Add 1 drop of test cell suspension in each tube.
4. For reverse grouping, label 3 tubes for A, B and O cell respectively and 1 drop
of 5 % cell suspension of A cell, B cell and O cell in to corresponding tubes.
5. Add 2 drops of test serum to all ABO cell tubes.
6. Mix well and centrifuge the test tubes at 3000 rpm for 15 seconds.
7. Completely re-suspend the cells by agitation and examine for agglutination.
Grade the reaction.
66
10.5.5 Result
Anti- Anti- Anti- A B O Result
A B AB Cell Cell Cell

O O O + + O Group
O
+ O + O + O Group
A
O + + + O O Group
B
+ + + O O O Group
AB
Table 2: Results of agglutination
Positive : +
Negative: O
10.6 Rhesus (D) Grouping and Du Antigen Typing by Tube Method
10.6.1 Objective
To detect the pre sense of Rh (D) antigen in blood sample.
10.6.2 Reagents
• 22 % bovine albumin
• LISS
• AHG
67
• Coomb’s control cell
• Antiserum D
10.6.3 Materials
• Centrifuge
• 37oC water bath
• Pasteur pipette
• Microscope
• Glass slide
10.6.4 Procedure
1. Prepare 5 % cell suspension of test sample RBCs in saline solution (1 drop of
packed cell in 1 ml saline solution).
2. Label 2 tubes, 1 for Anti-D and the other for Rh control adds 1 drop of Anti-D
and Rh control to each of the pre labeled tubes.
3. Add 1 drop of 5 % cell suspension to both the tubes.
4. Mix and centrifuge at 3000 rpm for 15 seconds and examine for agglutination.
5. Incubate negative test and control at 37oC for 20 minutes and repeat step d.
6. For Du test, after incubation for 20 minutes at 37oC, proceed to negative tests
and the control with AHG. If agglutination is seen Du is positive.
7. Positive Du test indicates the presence of Du antigen.
68
10.6.5 Result
• Direct agglutination at room temperature and at 37oC with Anti-D means
positive for Rh(D) antigen.
• No agglutination at room temperature and at 37oC and negative AHG test is
negative for Rh(D) agglutination.
• Agglutination only at AHG test is positive for Ru antigen.
10.7 Anti-Human Globulin test (Coomb’s Test)
10.7.1 Objective
To detect human immunoglobulin and or complement bound to Human erythrocytes.
10.7.2 Reagents
• AHG
• LISS
• Coomb’s contro cell
10.7.3 Materials
• Test tubes 75 mm by 12 mm
• Centrifuge
• 37oC water bath
69
• Microscope
• Pasteur pipette
• Glass slides
10.7.4 Procedure
A. Direct AHG Test
1. Prepare 3 – 5 % red cells suspension from patient blood in saline (1 drop of red
blood cell saline).
2. Add 1 of above RBC suspension to an approximately labeled test tube and wash
red cells in saline a minimum of 3 times with saline.
3. Following the final wash, completely decant the supernatant saline and blot the
tube edges on the clean abs or bent tissue to ensure the removal of all residual saline
and obtain the resultant dry red cells button.
4. Centrifuge immediately for 15 second at 3000 rpm.
5. Gently dislodge recipient red cells button and examine for agglutination.
6. Grade and record result.
B. Indirect AHG Test
1. Label 2 tests tube 1 and 2.
2. Add 2 drops of serum to be tested in each tube.
3. Add 1 drop screening cells 1 and 2 to each tube respectively.
4. Add 2 drops of antibody enhancement medium (LISS).
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5. Mix the tube well and incubate at 37oC for 10 minutes, but incubation should not
exceed an hour when LISS is used. When 22 % bovine albumin is used incubation is
1 to 2 hours.
6. Centrifuge both tubes for 15 second at 3000 rpm and examine for hemolysis.
7. Record result
8. Wash the cell / serum mixture carefully with saline 3 times. Decant and drain
completely after last wash.
9. Add 2 drops AHG reagent to the cell sediment.
10. Mix well and centrifuge for 15 second at 3000 rpm.
11. Re-suspend the cells by gentle agitation and examine tubes macroscopically for
agglutination.
12. It is recommended all positive results be graded from 4+ to +/- each stage of
reading.
13. To all negative and weak positive test, add Coomb’s control cells to validate test
result.
10.7.5 Recommended grading of reactions strengths
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Score Grade Macroscopic Microscopic
Appearance Appearance
12 C Complete No free cells
agglutination a detected
single
agglutinate of
strongly reactive
red cells
10 +++ Strong reaction A few detected
1 or 2 large mussel of
agglutination agglutination cells
8 ++ Strong reaction, Large
a number of agglutination of
large smaller clumps,
agglutination and a scattering of
free cells
5 + Many small Many
agglutination agglutination of up
to 20 cells in
background of
small clumps and
free cells
3 +/- Weak Scattered
granularity in agglutination at 6-
cell suspension 8 cells, with
mostly free cells
1 W An even cell A few small

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suspension clumps of 3-4 cells
Table 3: Grading of reactions strengths
10.8 Compatibility Test
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10.8.1 Objective
To get donor blood in which red blood cells are compatible to recipient’s blood and it
safe for transfusion.
10.8.2 Reagents
• 22 % bovine albumin
• AHG
• LISS
• Coomb’s contro cell
10.8.3 Materials
• Centrifuge
• 37oC water bath
• Microscope
• Pasteur pipette
• Glass tube
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10.8.4 Procedure
A. Technique 1: Saline Tube
1. Place 2 drops of patient serum in a 75 mm by 10 mm test tube and add 1 drop
of washed suspension of donor red cells.
2. Keep the tube at room temperature for a minimum of 5 minutes.
3. Centrifuge at 2000 rpm for 20 second.
4. Read macroscopically and observe for agglutination. If there is no agglutination
(e.g. negative reactions), examine microscopically and record result. During
emergency, this technique will detect major ABO grouping errors and the presence of
IgM antibodies.
B. Technique 2: Albumin Tube (immediate spin 37oC / AHG)
1. Place 2 drops of patient serum in a 75 mm by 10 mm test tube and add 1 drop
of 5 % suspension of donor red cells and 2 drops of 22 % bovine albumin and mix
well
2. Centrifuge at 3000 rpm for 15 second and examine for agglutination
macroscopically and record result.
3. Incubate the tube at 37oC in a water bath for ½ an hour (min-period) and up to 2
hours
4. Centrifuge the tube at 3000 rpm for 15 second and examine for hemolysis and
agglutination macroscopically.
5. Wash the donor cells 3 times in saline solution to remove serum manually or use
automated cell washed, to wash 3 times.
6. Agitate the button of donor cells and add 2 drops anti globulin (AHG) reagent.
7. Mix well and centrifuge at 3000 rpm for 15 second.
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8. Examine for agglutination macroscopically and microscopically.
9. No agglutination indicates the donor blood is compatible for the particular
patient. If agglutination is observed then the blood is incompatible and not suitable
for transfusion.
10. Add 1 drop of Coomb’s control cell (CCC) to all negative test and centrifuge at
3000 rpm for 15 second. A examine for agglutination is observed the test result is
invalid and must be repeated.
C. LISS Method
1. To 10 mm by 75 mm glass test tube, add 2 drops of patient serum, 1 drop of 5
% donor red cells and 2 drops of LISS reagent and mix well.
2. Incubate the tube at 37oC in a water bath for 10 minutes.
3. Centrifuge the tube for 15 second at 3000 rpm and examine for agglutination.
4. Wash cells in saline solution 3 times.
5. Decant saline completely after last wash.
6. Examine for agglutination macroscopically and record result. If no agglutination,
add Coomb’s control cell to validate test.
10.9 Veneral Diseases Research Laboratory
10.9.1 Principle
Syphilis is a veneral disease caused by spirochaete microorganism
Treponema pallidum. As the organism cannot be cultured on the artificial media, the
diagnosis of T.pallidum depends on the correlation of the clinical data with the
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detection of specific antibody by serological tests. Serological screening tests for
syphilis using cardiolipin and lecthinin as antigens are simple to perform but gives
rise to a small proportion of false positive results because the tests uses non
treponemal antigens. The test antigen is a modified form of VDRL antigen
containing microparticulate carbon which aids the macroscopic reading of results. A
reactive result if indicated by agglutination which is readily visible without the aid of
microscope. Weak reactive results can be easily and clearly distinguished from non
reactive patterns which display a macroscopically smooth and even appearance.
Test results are obtained in 8 minutes.
10.9.2 Specimen
Blood in a tube without anticoagulant and spin the blood at a high speed to obtain
the serum.
10.9.3 Reagent
• Antigen
• Positive control
• Negative control
• Dispensing bottle
• Dispensing Needle
• Test card
• Stirrer
10.9.4 Equipment
Mechanical Rotator
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10.9.5 Procedure
1. Pipette 50 ul of serum onto the ringed slide on the test card
2. Shake the dispensing bottle slowly and snap the dispensing needle to remove
the air bubbles
3. Hold the needle downwards and pipette a drop of antigen beside the serum
4. Mix the serum and the antigen carefully
5. Repeat the step 1-4 for the positive and the negative control
6. Rotate the test card using the mechanical rotator at the speed of 100rpm for 8
minutes.
7. Look for agglutination and if there is any agglutination, it is positive and if there
is no agglutination, it is negative.
10.9.6 Quantitative Method
1. Carry out the quantitative test on the positive serum
2. Pipette 50 ul of saline using the micropipette and place the saline on 5 different
rings on the test card
3. Mix the saline and serum using the same pipette and transfer 50 ul of the
solution to the second ring
4. Carry on the dilution by repeating the step 1-3
5. The dilution will be 1:2, 1:4, 1:8, 1:16 and 1:32
6. Using the applicator stick, mix and spread the dilution from the first ring to the
last ring.
10.9.7 Results
1. Consider the last dilution which contains agglutination as the positive result. If
the last dilution gives a positive answer, the dilution should be continued.
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2. Carry out the confirmation test of VDRL, TPHA (Trepenoma pallidum) test for
the positive result.
CHAPTER 11
CONCLUSION
The industrial training of 10 weeks had successfully completed on 21st June
2008. During the training period I had the opportunity to exposed to the real working
environment and improve the practical skill and good working characters such as
discipline, punctuality, humble attitude and willing to learn aggressively. In addition, I
also gained academic knowledge during the training. I also was taught to handle the
specimen safely as well as the methods to process the specimens until the result is
validated. This industrial training is essential for the students as it provides practical
training and academic knowledge to students but also develops and improves the
abilities of students so that the students are ready to face more challenges in the
future.
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REFERENCES
1. Allan Gaw, Michael J.Murphy, Robert A. Cowan, Dennis St. J. O’Reilly,
Michael J.Stewart, James Shepherd, “Clinical Biochemistry”, Churchill Livingstone,
Third Edition, 2004.
2. F.J. Baker & R.E. Silverton, “Introduction to Medical Laboratory Technology”,
Butterwoth & Co Ltd, Sixth Edition, 1985.
3. IMR Handbook, Institute for Medical Research, Fifth Edition, 2003.
4. Judith S.Heelan & Frances W.Ingersoll, “Essentials of Human Parasitology”,
William Brottmiller, Cathy L.Esperti, Darcy M.Scelsi, Delmar, 2002.
5. J.V.Dacie & S.M.Lewis, “Practical Haematology”, Churchil Livingstone, Fourth
Edition, 1974.
6. K.S.Leng, “Medical Office Laboratory Manual”, May 1980.
7. Lisa Anne Shimeld & Anne T.Rodgers, “Essentials of Diagnostic
Microbiology”, Delmar Publihers, 1998.
8. Monica Cheesbrough, “Medical Laboratory Manual for Tropical Countries,
Tropical Health Technology and Butterworth-Heineman, VolumeII Microbiology, 1985.
9. Sir John V. Dacie & S.M. Lewis, “Practical Haematology”, Churchill
Livingstone, Sixth Edition, 1984.
10. Virginia Vengelen, “Technical Manual”, American Association Of Blood
Banks, 12th Edition, 1996.
11. Lohguanlye.com
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12. Gribbles Pathology, Common test information
APPENDIX A
General Info
Full Blood Count
The interpretation of the indices measured is complex and the notes below are given
as a brief and general description only. If there are significant abnormalities detected
when measuring these indices then examination of a peripheral blood film is required
to complete the interpretation.
a. Haemoglobin (Hb)
Anaemia is defined as haemoglobin below normal for age and gender. Further
investigation of the cause of anaemia is guided by clinical features, blood film
and red cell indices (MCV, MCH, MCHC). The Hb is elevated in erythrocytosis.
b. Red Cell Count (RCC)
Interpretation is required to be made in conjunction with the other red cell
parameters below.
c. Packed Cell Volume (PCV)
PCV is reduced in anaemia and increased in erythrocytosis. In patients with
erythrocytosis the PCV correlates with blood viscosity.
d. Mean Cell Volume (MCV)
Macrocytosis (high MCV) is found in:
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o Megaloblastic, aplastic, dyserythropoietic and sideroblastic anaemias
o Myelodysplastic syndromes, myeloma; liver disease, alcohol excess
o Chronic hypoxic lung disease
o Myxoedema; following renal transplant
o Cytotoxic drug therapy particularly hydroxyurea, therapy with Zidovudine
(AZT).
Microcytosis (low MCV) is found in:
o Iron deficiency
o Anaemia of chronic disease
o Haemoglobinopathies (especially the thalassaemias).
e. Mean Cell Haemoglobin (MCH)
Arithmetically, MCH is Hb divided by RCC. The MCH is increased in macrocytic
anaemias and decreased in microcytic anaemias. If the MCH is significantly
abnormal then a blood film should also be requested.
f. Mean Cell Haemoglobin Concentration (MCHC)
Arithmetically, MCHC is Hb divided by PCV. There is a rough correlation
between low MCHC and hypochromia and between high MCHC and the
presence of spherocytes.
g. Red Cell Distribution Width (RDW)
The RDW may assist in the classification of anaemia, in association with the
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blood film, and the other red cell indices (MCV, MCH and MCHC). The RDW
gives a quantitative assessment of the degree of variation in red cell size /
volume (anisocytosis).
h. White Cell Count (WCC/TWDC)
The interpretation is required to be made in conjunction with the differential
count of white cells below.
i. Neutrophils
Neutrophilia (increased) may be caused by:
 Physiological stress e.g. physical, emotional, pregnancy.
 Infection especially bacterial or fungal.
 Inflammation e.g. connective tissue disease, arthritis.
 Tissue necrosis e.g. myocardial infarction, carcinoma.
 Blood loss - both acute and chronic.
 Haemolytic anaemia.
 Myeloproliferative disorders.
 Splenic antrophy/absence.
 Drug therapy e.g. corticosteroids, cytokines, lithium.
Neutropenia (decreased) may be caused by:
Decreased production
 Drug reactions such as cytotoxic drugs (usually pancytopenia) or
NSAID, Sulphonamides, Carbimazole, Clopidogrel, Clozapine.
83
 Bone marrow failure (usually pancytopenia), acute leukaemia,
myelodysplasia.
 Megaloblastic anaemia (usually pancytopenia)
 Chronic idiopathic neutropenia.
 Hereditary/Constitutional such as cyclic neutropenia,
Schwachman syndrome, Chediak-Higashi syndrome, Diamond-
Blackfan syndrome
Increased destruction and/or margination
 Autoimmune diseases such as SLE, rheumatoid arthritis
 Drug reactions e.g. Captopril, Penicillins
 Hypersplenism
 Haemodialysis
 Idiopathic
Decreased production and increased destruction
 Viral infection e.g. infectious mononucleosis, HIV
 Bacterial infections e.g. Septicaemia, typhoid fever (The
neutrophil count is variable in severe bacterial infection, but
neutropenia is common, especially in neonates and in patients
with Gram-negative septicaemia).
 Protozoal infection e.g. malaria
 Hairy Cell Leukaemia
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ii. Lymphocytes
Lymphocytosis (increased) may be caused by:
 Mononucleosis syndromes such as infectious mononucleosis,
CMV infection, Toxoplasmosis
 Other viral infections
 Chronic lymphocytic leukaemia, prolymphocytic leukaemia,
lymphoma (non-Hodgkin’s), Hairy Cell Leukaemia
 Splenic atrophy/absence following physical trauma
Lymphocytopenia (decreased) may be caused by:
 Bacterial infection
 Early stage of viral infection
 Carcinoma
 Irradiation
 Drugs especially corticosteroids and cytotoxics
 HIV infection
 Hodgkin’s disease
 Malnutrition
 Renal Failure
 SLE
 Cushing’s syndrome
 Sarcoidosis
 Protein losing enteropathy
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iii. Monocytes
Usually low numbers – monocytosis (increased) may be associated with acute
or chronic bacterial infection, carcinoma, Hodgkin’s disease, cytokine
administration, splenic atrophy/absence or monocytic leukaemias.
iv. Basophils
Usually low numbers – basophilia (increased) may be associated with
myeloproliferative disorders or in reactive disorders such as hypothyroidism,
ulcerative colitis, hypersensitivity states and together with idiopathic
hypereosinophilic syndrome.
v. Eosinophils
Usually absent or low numbers – eosinophillia (increased) is usually associated
with allergic reactions to drugs or conditions such as eczema, asthma, food
allergy and psoriasis. Eosinophillia may also be seen with some parasitic
infections and with Aspergillosis.
vi. Platelets
Thrombocytosis (increased numbers) may be caused by:
 Polycythemia Vera
 Post-Splenectomy syndrome
 Primary thrombocytosis
 Certain malignancies
 Early CML
 Anaemia
Thrombocytopenia (decreased numbers) may be caused by:
86
 Increased usage as in disseminated intravascular coagulation
 In association with haemorrhagic disease such as dengue or
haemolytic anaemia
 Idiopathic Thrombocytopenic Purpura (ITP)
 Leukemia
 Prosthetic heart valve
 Massive blood transfusion
 Chemotherapy
Erythrocyte Sedimentation Rate (ESR)
ESR is a non-specific indicator of inflammatory and neoplastic disease (C-reactive
protein is a more sensitive early indicator of an acute phase response). A normal ESR
does not exclude active disease.
The ESR increases with age, is raised in pregnancy and anaemia; mild to moderate
elevations should be interpreted with caution in these situations. It is increased in acute
and chronic inflammatory disease and in neoplastic disease.
The ESR may be very high (>100 mm in 1 hour) in multiple myeloma, tuberculosis and
temporal arthritis.
A low ESR (<1 mm in 1 hour) may be seen in polycythaemia rubra vera and sickle cell
disease.
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Renal Function Tests
a. Sodium
Sodium concentration is dependent on the state of hydration, body sodium
content and water shifts between plasma and other body fluid compartments.
Intravenous therapy with isotonic saline may cause hypernatraemia and volume
replacement with dextrose may cause hyponatraemia. Hyponatraemia occurs in
a small percentage of patients on diuretic therapy, particularly the elderly.
Severe hyperlipidaemia or hyperproteinaemia may cause
'pseudohyponatraemia'. Sodium is retained with mineralocorticoid excess and
lost with mineralocorticoid deficiency, gastrointestinal and renal loss, or
excessive sweating. Hyponatraemia as a result of fluid retention (dilutional
hyponatraemia) is seen in renal and cardiac disease and with SIADH. Urine
sodium estimation may assist in interpretation.
b. Potassium
Increased levels are usually found in acidosis, tissue damage, renal failure and
mineralocorticoid deficiency. Decreased levels are found in association with
loop or thiazide diuretic therapy, vomiting or diarrhoea, alkalosis, during
treatment of acidosis, and with mineralocorticoid excess. Haemolysis during
collection, delay in separation, refrigeration of unseparated blood, marked
leucocytosis and thrombocytosis, and muscle activity of limb immediately prior
to venepuncture may cause a misleading increase in potassium. As potassium
is commonly raised because of red cell leakage after venepuncture, it is
88
important that unexpected hyperkalaemia is verified by a repeat (fresh)
sample if clinically indicated.
c. Chloride
Hyponatraemia and metabolic alkalosis are associated with hypochloraemia.
Hypernatraemia and metabolic acidosis, due to renal tubular acidosis or
bicarbonate loss, are associated with hyperchloraemia. An increased anion gap
indicates accumulation of an anion other than chloride (e.g. lactate,
hydroxybutyrate); this usually occurs with metabolic acidosis.
d. Urea
Increased levels are seen with reduced glomerular filtration due to renal or pre-
renal disease, bleeding into the gastrointestinal tract and hypercatabolic states.
Reduced values are seen in pregnancy, with water retention, with reduced
synthesis as a result of decreased protein intake, severe liver disease, or urea-
cycle defects.
e. Uric Acid
The likelihood of gout is low if the serum urate concentration is repeatedly
below 0.42mmol/L. The risk of developing gout is three times greater if the
serum urate concentration is consistently above 0.42 mmol/L. However, a
raised serum urate level alone is insufficient to diagnose gout. Impaired renal
function, pregnancy-induced hypertension, diuretics, fasting, hyperlactataemia,
hyperketonaemia and low dose salicylates can all produce increased urate
levels. Hypouricaemia is seen in patients with a low purine intake, in SIADH,
with hypouricaemic drugs (e.g. allopurinol) and in the rare condition of
xanthinuria.
89
f. Creatinine
Increased creatinine levels occur in conditions which decrease the glomerular
filtration rate. These may be pre-renal (e.g. hypovolaemia, hypotension), renal
or post-renal (e.g. obstruction). Levels are lower in patients with a reduced
muscle mass (e.g. the elderly) and this may conceal impairment of renal
function.
Please refer to MediTalk Client Circular No 14 - Routine Reporting of eGFR to
read about eGFR reporting.
g. Calcium
Total calcium should not be used for evaluation of patients. In most situations,
corrected calcium is used which accounts for the main binding protein's
(albumin) concentration. Ionised calcium is only required if complexed calcium
is likely to be very high (e.g. during massive transfusion), if pH is abnormal or if
an abnormality in calcium is marginal. Artefactual decrease in calcium occurs if
EDTA, unbalanced heparin or oxalate is used as an anticoagulant. As a
hypercalcaemic result may be artifactual (especially if potassium is
unexpectedly raised) it is important that unexpected reduced corrected
calcium results are verified by a repeat (fresh) sample if clinically
indicated.
h. Phosphate
Increased phosphate levels are found in response to low parathyroid hormone
levels (e.g. hypoparathyroidism, hypercalcaemia due to malignancy and other
non-parathyroid causes) and in renal failure. Decreased levels of phosphate are
usually found in patients with primary hyperparathyroidism, in some cases of
hypercalcaemia associated with malignancy, in renal tubular disorders and in
90
patients using magnesium and aluminium containing antacids. Levels may be
decreased during prolonged intravenous therapy if phosphate supplementation
is inadequate. Phosphate levels may also be decreased following a
carbohydrate-rich meal, due to cellular uptake of phosphate. Numerous other
conditions can affect serum phosphate levels. As a raised phosphate result
may be artifactual (especially if potassium is unexpectedly raised) it is important
that unexpected raised phosphate results are verified by a repeat (fresh)
sample if clinically indicated.
Liver Function Tests
a. Total Serum Protein
Total protein is measured together with albumin in order to calculate the
concentration of the globulin fraction. The interpretation of total protein
concentration depends on the levels of albumin and immunoglobulins, which
are the only proteins present in serum in sufficient concentration to significantly
alter total protein levels. Increased levels also occur after excessive venous
stasis during blood collection and in dehydration.
b. Albumin
Decreased levels may be associated with overhydration, chronic liver disease,
protein losing disorders (e.g. nephrotic syndrome, protein-losing enteropathy),
malnutrition, and shifts into the extravascular space (e.g. burns). Decreased
levels may also be seen as part of an acute phase response. Increased levels
may be seen with dehydration. Increases above the true level may occur with
91
excessive use of tourniquet for sample collection, and with some methods that
also measure acute phase reactants. Levels may be up to 15% higher if the
specimen is collected with the patient erect rather than supine. In severe
hypoalbuminaemia, non-immunological methods significantly overestimate the
level of albumin.
c. Globulin
Levels are increased with chronic inflammation, infection, autoimmune disease,
liver disease, and paraproteinaemia. Levels are decreased in protein-losing
enteropathy, humoral immunodeficiency and sometimes in the nephrotic
syndrome.
d. Total Bilirubin
Total bilirubin comprises unconjugated, conjugated and delta bilirubin, whereas
direct bilirubin comprises conjugated and delta bilirubin. In most cases total
bilirubin measurement only is adequate. High levels of total and direct bilirubin
are seen with hepatocellular disease or biliary disease (intra- or extra-hepatic).
Delta bilirubin, which is covalently bound to albumin, has a longer half-life in the
circulation than the other bilirubins and may cause bilirubin elevation for some
time after the others have returned to normal. Isolated elevation of
unconjugated bilirubin (that is elevated total bilirubin with normal direct bilirubin)
occurs when the rate of production exceeds the rate of conjugation.
It is seen in haemolysis and in megaloblastic anaemia, but the commonest
cause is the Gilbert syndrome, in which there is a non-pathogenic impairment of
bilirubin conjugation. Physiological jaundice in neonates is due to elevated
unconjugated bilirubin. Normal levels of bilirubin may be present in
92
uncomplicated cirrhosis, early in the course of fulminant liver failure, or with
hepatic metastases until the disease is advanced.
Yellow skin colour with normal bilirubin may be due to carotenaemia.
e. ALP
Increased levels in liver disease (particularly in association with cholestasis),
bone disease (with increased osteoblastic activity e.g. Paget’s disease), some
bony metastases (especially prostate and breast), and at times in malignancy
without liver or bone metastases (Regan isoenzyme). ALP may also be
elevated in some gastrointestinal diseases or due to a macroenzyme. Alkaline
phosphatase isoenzymes are rarely necessary to identify the source of an
elevated ALP. Marked but transient elevation of ALP may be seen in children,
probably attributable to viral infection. Abnormal dentition and fragile bones with
decreased ALP characterise the autosomal recessive disease
hypophosphatasia.
f. AST (SGOT)
Increased levels are found with hepatocellular disease.
The AST/ALT ratio is typically > 1 in alcoholic liver disease and < 1 in non-
alcoholic liver disease.
Although AST levels are increased with cardiac and skeletal muscle disease,
more specific tests are available in these situations.
Haemolysis during collection or refrigeration of unseparated blood may cause
an artefactual increase.
g. ALT (SGPT)
Increased ALT levels are associated with hepatocellular damage. ALT is more
93
specific for hepatocellular damage than AST or LD are and remains elevated
for longer, due to its longer half-life. The AST/ALT ratio is typically > 1 in
alcoholic liver disease and < 1 in non-alcoholic liver disease. ALT may be
slightly elevated in skeletal muscle disease but the degree of elevation is much
less than for AST and CK.
h. GGT
Increased levels are found in cholestatic liver disease and in hepatocellular
disease when there is an element of cholestasis. Levels are increased in
diabetes with chronic intake of excess alcohol and with certain drugs (especially
phenytoin) as a result of enzyme induction. Pancreatitis and prostatitis may also
be associated with increased levels. Levels may be normal early in the course
of acute hepatocellular damage e.g. acute viral hepatitis, paracetamol
hepatotoxicity.
Glucose
Please refer to Diabetes Mellitus & Glycaemic Control.
Top of the page
Lipid Studies
a. Total Cholesterol, Triglycerides, HDL and LDL
This profile is for the investigation of lipid status in suspected hyperlipidaemia
94
and in monitoring the efficacy of lipid lowering treatment. It also provides an
assessment of risk for atherosclerosis, especially coronary artery disease.
Low levels of HDL and high levels of LDL cholesterol are associated with an
increased risk of atherosclerotic vascular disease.
National guidelines generally specify specific targets. Desired lipid levels for
reducing the risk of cardiac disease vary greatly from country to country even
though most studies conducted by committees and working parties include
many racial groups. Gribbles is currently using the widely recognized ideal
targets for lipid levels as specified by the National Cholesterol Education
Program (NCEP) based on their Adult Treatment Program version III (ATPIII).
Full details, including the variations in desired levels according to the presence
of risk factors can be obtained here.
LDL levels are reduced for up to 8 weeks with acute illness (e.g. myocardial
infarction, acute infection) and assays should not be performed during this time.
b. Apolipoproteins
Apo A1 and apo B may be measured as an alternative to HDL and LDL
cholesterol respectively in the assessment of atherosclerosis risk factors, and
may offer better prediction of risk.
Apo B together with LDL cholesterol can be used to define
hyperapobetalipoproteinaemia, a condition associated with small dense LDL
and increased risk of atherosclerosis.
Apo A1 and apo B are very low in Tangier disease and abetalipoproteinaemia
(plus hypobetalipoproteinaemia) respectively.
Decreased apo A1 and increased apo B or apo (a) are associated with an
increased risk of atherosclerosis.
95
c. Lipoprotein (a)
Lp (a) is an independent risk factor for atherosclerosis and may be indicated in
the assessment of a patient with premature coronary or cerebral arterial
disease, especially if there is a suggestive family history.
Raised Lp (a) is associated with increased vascular risk. Homozygous apoE2
may result in type III hyperlipidaemia.
Urine FEME
Urine is subjected to the chemical screening tests below.
a. pH
Inability to acidify urine may indicate distal renal tubular acidosis.
b. Specific Gravity
This measures urine concentration.
c. Protein
> 300 mg/L suggests a glomerular protein leak or inflammatory exudate along
the urinary tract and should be followed up with a microalbumin test. Please
refer to Microalbumin for further information on this test.
d. Glucose
A positive result indicates hyperglycaemia at the time of urine formation, or
renal glucosuria.
Hypoglycaemic coma may be present when urine shows glucosuria from earlier
hyperglycaemia. Glucosuria is not a reliable indicator of gestational diabetes
and the test should not be used for this purpose.
96
e. Ketones
In a diabetic, positive ketones indicate ketoacidosis. If lactic acidosis is also
present, the ketone reaction may be inappropriately weak.
f. Bilirubin
A negative result in an apparently jaundiced person suggests unconjugated
hyperbilirubinaemia (haemolysis, Gilbert syndrome) or carotenaemia.
A positive result is found in hepatocellular or obstructive jaundice.
g. Blood
A positive test for blood may be due to red cells from inflammation, trauma, or
tumour of the renal tract.
Contamination of urine from vaginal bleeding may also be responsible.
If no red cells are seen on microscopy, it indicates haemoglobinuria or
myoglobinuria.
A negative test with red urine indicates presence of a coloured compound e.g.
beetroot, porphyrins.
h. Leucocytes
(Leucocyte esterase) - A positive result indicates the presence of neutrophils.
i. Microscopy
This is performed to identify and count the types of cells, casts, crystals and
other components, such as bacteria and mucous, that can be present in urine.
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RPR (VDRL)
RPR (rapid plasma regain) test is a simple, non specific test for syphilis. A positive test
result suggests either past or present exposure to syphilis. Biological false positives
may be found in pregnancy; transiently in measles, chicken pox; chronically in
cirrhosis, SLE, the phospholipid antibody syndrome, leprosy. All positive RPR tests are
routinely followed by a confirmatory and specific test for syphilis called TPHA.
Hepatitis
a. Hepatitis B surface Antigen (HBsAg)
Detected in the serum of patients with Hepatitis B virus infection 6 - 16 weeks
after exposure. If HBs Ag is positive then Hepatitis Be Antigen (HBeAg) should
be determined. HBeAg is an indicator of viral replication & infectivity of the
patient. The presence of HBe Antibodies may indicate reduced infectivity and
possible resolution of infection.
Hepatitis B core Antibody (Anti-HBc) develops soon after the appearance of
HBs Ag especially in patients with acute Hepatitis B infection and is present in
virtually all chronic Hepatitis B carriers. It is the only serological marker during
the “window period" of an acute infection and after previous Hepatitis B virus
infection.
The Hepatitis B virus results in a chronic carrier state in 90% - 95% of infections
acquired in childhood especially as a result of vertical transmission, and in 5% -
10% of adult infections.
98
b. Table 1: Interpretation of Hepatitis B results
Results Interpretation
Test
HBsAg Negative
Anti-HBcore Negative Susceptible
Anti-HBs Negative
HBsAg Negative
Anti-HBcore Negative Immune due to vaccination
Anti-HBs Positive
HBsAg Negative
Anti-HBcore Positive Immune due to natural infection
Anti-HBs Positive
HBsAg
Positive
Anti-HBcore
Positive
IgM anti Acutely infected
Positive
HBcore
Negative
Anti-HBs
99
HBsAg
Positive
Anti-HBcore
Positive
IgM anti Chronically infected
Negative
HBcore
Negative
Anti-HBs
HBsAg Negative
Four possible interpretations
Anti-HBcore Positive
possible*
Anti-HBs Negative
c.
* Four Interpretations:
1. May be recovering from acute HBV infection.
2. May be distantly immune, but the test may not be sensitive enough to detect
a very low level anti-HBs in serum.
3. May be susceptible with false positive anti HBcore
4. May be chronically infected and have an undetectable level of HBsAg
present in the serum.
100
For more information on Hepatitis B, please read our MediTalk Client Circular
No 10 - Hepatitis B.
d. Hepatitis C
The Hepatitis C Virus is a single-stranded RNA virus and is the cause of > 90%
of transfusion-associated hepatitis. The virus may be transmitted via the
parental route through blood or blood products. Sexual transmission, vertical
transmission and transmissions through the saliva are other possible routes of
infection.
Chronic infections occur in 50% - 70% of cases and 20% - 25% of these
develop antibodies. Hepatocellular carcinoma may develop from 7 to 23 years
after acute infections.
Anti-HCV antibody is detectable in < 50% of patients during an acute infection,
101
and in some patients do not develop at all. It may persist for many years
following resolution and in chronic diseases. Sero - conversion has been shown
to occur in 10 - 39 weeks following blood transfusion or 4 - 32 weeks after
clinical symptoms appear. Anti-HCV may be undetectable in some chronic
carriers.
Thyroid Function Tests
Table 2: Interpretation Matrix for Thyroid Function
High T4 Normal FT4 Low FT4
Mild thyroid failure

In vivo/in vitro
(primary)
artefact
(also termed
Pituitary Primary
High TSH subclinical
hyperthyroidism hypothyroidism
hypothyroidism and
[TSHoma]Thyroid
diminished thyroid
Hormone resistance
reserve)
Normal As above. Sampling Normal (in patients Pituitary or
TSH within 6 hours of taking thyroxine, hypothalamic
thyroxine dose. TSH>3mU/L may hypothyroidism.
indicate subtle under- Severe non-thyroidal
102
replacement) illness
Subclinical
hyperthyroidism
Subtle thyroxine over-

Pituitary or
Hyperthyroidism (for replacement
hypothalamic
this diagnosis, TSH Thyroid
Low TSH hypothyroidism
must be suppressed autonomy(multinodular
Severe non-thyroidal
rather than just low) goitre or autonomous
illness
functioning thyroid
nodule)
Non-thyroidal illness
a. Hyperthyroidism may be caused by:
o Graves disease
o Multinodular goiter
o Autonomously functioning single thyroid nodule (adenoma)
o Thyroiditis e.g. subacute, postpartum or lymphocytic
o Factitious hyperthyroidism i.e. thyroid hormone ingestion
o Functioning thyroid carcinoma (follicular carcinoma)
o HCG-mediated e.g. hyperemesis gravidarum or trophoblastic disease
o Foetal and neonatal hyperthyroidism (TSH-receptor-antibody-mediated)
o Struma ovarii
o TSH secreting pituitary tumour
103
o Partial (pituitary-selective) thyroid hormone resistance
b. Hypothyroidism may be caused by:
o Autoimmune lymphocytic thyroiditis e.g. atrophic thyroiditis, classic
Hashimoto’s disease
o Post ablative therapy (i.e. radioiodine (RAI) therapy, thyroidectomy)
o Transient e.g. subacute thyroiditis, postpartum thyoriditis, early post-
ablative therapy (RAI, subtotal thyroidectomy)
o Drug induced e.g. thionamide, lithium, amiodarone, interferon, drugs
that interfere with thyroxine absorption in treated hypothyroidism (iron
salts, cholestyramine, sucralfate)
o Iodine associated i.e. iodine-deficiency disease or iodine-induced
o Infiltrative e.g. Reidel’s thyroiditis, scleroderma, amyloid disease,
haemochromatosis
o Neonatal or congenital e.g. thyroid agenesis/ectopia, genetic disorders
of TSH, TSH receptor, thyroid peroxidase, thyroglobulin, pendrin and
transplacental passage of blocking TSH-receptor antibody
o Secondary e.g. pituitary or hypothalamic disease
o Thyroid hormone resistance
For more information on Thyroid Function tests, please read our MediTalk Client
Circular No 3 - Interferences in the Measurement of TSH and Free Thyroid Hormones.
Top of the page
104
Rheumatoid Factor
Rheumatoid factor is elevated in patients with rheumatoid arthritis as well as in a
number of other inflammatory conditions such as TB, syphilis and malignancy. There is
now a more specific test for rheumatoid arthritis called anti-CCP (anti-cyclic citrullinated
peptide antibody). In patients who are known to have rheumatoid arthritis or those who
demonstrate unexpectedly raised levels of RF, a measurement of anti-CCP is
recommended.
HIV
a. HIV Ag/Ab Screening Assay
The HIV screening assay performed by LSC is known as a “combo” assay. It
detects not only antibodies to HIV which are generally produced 3 to 4 weeks
after exposure, but also p24 antigen which can be present in detectable
amounts at 2 weeks post exposure. This combination enables detection of an
infected individual at the earliest possible moment by minimising the “window
period” between infection and detection.
b. Western Blot Assay(IMR)
This is a confirmatory assay for HIV I/II antibody. A positive Western Blot is
generally regarded as conclusive for a HIV infection. Negative tests do not
necessarily rule out HIV infection, because there is an interval between HIV
infection and the appearance of measurable anti-HIV antibodies (called the
105
"window period"). If the screening test is positive and Western Blot is negative,
then the Western Blot should be repeated in 2 – 3 weeks.
c. HIV Viral Load Assay(GRIBBLES)
Viral load means the quantity of HIV RNA present in the blood. This assay
measures the amount of HIV RNA in a small amount of blood.
This test is commonly ordered due to indeterminate HIV antibody/antigen test
(where both Western Blot and viral load would be suggested), for initial
evaluation of newly diagnosed HIV infection, before initiation or change of
antiretroviral drug therapy and to monitor patient response to drug therapy.
HIV RNA is often detected using this assay before the screening assay or the
Western Blot assay shows positive results.
Cancer Markers
Tumor markers are substances either produced by neoplastic tissue (e.g. CA 125) or
are normally occurring organ specific substances which as a result of neoplastic
activity are released into the circulation (e.g. PSA).
a. AFP (Alpha Fetoprotein)
Raised AFP may be associated with viral hepatitis, cirrhosis and neoplasia
(especially hepatoma). A repeat of this marker in a few weeks is suggested to
see if there is a rising titre. Most elevations found in non-neoplastic disease are
often transient, whereas with neoplastic disease they remain elevated or rise
continuously.
106
b. ß2M (ß2 Microglobulin)
ß2M is the light chain of the HLA-A,-B and-C major histocompatibility complex
antigens and occurs on the surface of nucleated cells. It is abundant on
lymphocytes, monocytes and on many tumour cell lines. Elevated serum levels,
in the presence of normal glomerular filtration rate suggest increased
production as may be seen in lymphoproliferative disorders such as multiple
myeloma, chronic lymphocytic leukaemia, Hodgkin’s disease, non Hodgkin’s
lymphoma, SLE, rheumatoid arthritis, Sjogren’s syndrome, Crohn’s disease and
certain viral infections, including CMV, non A and non B hepatitis and infectious
mononucleosis. As ß2M is of low molecular weight it is considered a sensitive
means for diagnosing proximal tubule dysfunction with increased urinary
excretion observed in a wide variety of conditions including upper urinary tract
infections, kidney transplantation and nephrotoxicity resulting from exposure to
heavy metals such as cadmium and mercury as well as cyclosporine,
aminoglycoside or cis-platinum therapy.
c. CA 125 (Cancer Antigen 125)
Approximately 75% of patients with ovarian carcinoma have elevated levels of
CA 125. It is however, also found in non malignant conditions such as
pericarditis, cirrhosis, severe hepatic necrosis, endometriosis, first trimester
pregnancy, and ovarian cysts. Mild elevations may be seen during
menstruation. Non ovarian malignancies which are reported to show elevations
include uterine carcinoma, hepatoma, pancreatic adenocarcinoma, lung and
endometrial cancer.
d. CA19.9 (Cancer Antigen 19.9)
CA 19.9 is elevated in pancreatic and gastrointestinal malignancies. It is also
107
elevated in a number of benign conditions including cholecystitis, cirrhosis,
renal failure and may be mildly elevated in normal individuals.
e. CA 15.3 (Cancer Antigen 15.3)
Patients with confirmed breast cancer frequently have raised levels of CA 15.3.
Elevated levels are also associated with non mammary malignancies such as
lung, colon, pancreas, hepatoma, ovary, cervix and endometrium. It is also
raised in some benign conditions of the ovary and breast. A repeat of this
marker in a few weeks is suggested to see if there is a rising titre. Most
elevations found in non-neoplastic disease are often transient, whereas with
neoplastic disease they remain elevated or rise continuously. A negative test
does not necessarily exclude the presence of disease
f. CEA (Carcinoembryonic Antigen)
Borderline values of CEA may occur due to heavy smoking and chronic
inflammation. Elevated CEA may be associated with primary colorectal cancer
or other malignancies including breast, gastrointestinal tract, liver, lung, ovarian,
pancreatic and prostate cancers. A repeat of this marker in a few weeks is
suggested to see if there is a rising titre. Most elevations are found in non-
neoplastic disease and are often transient, whereas with neoplastic disease
they remain elevated or rise continuously.
g. Catacholamines
Urinary catacholamine excretion is used in the diagnosis of
phaeochromocytoma (increased adrenaline and/or noradrenaline),
neuroblastoma and ganglioneuroma (increased dopamine). Excretion of
adrenaline and noradrenaline is usually increased in phaeochromocytoma,
108
particularly after a hypertensive paroxysm. Excretion may also be increased in
malignant hypertension and with severe stress. Some drugs may interfere with
the assay – please note on the request form the drugs patient may be taking.
h. EBV EA & EBV VCA IgA
There is a strong and constant association of undifferentiated carcinoma of the
nasopharynx with the Epstein - Barr virus (EBV). Reactivation of the virus in the
nasopharynx is accompanied by synthesis of EBV related IgA antibodies, and is
believed to lead to carcinogenesis in some patients. The most useful cancers
markers for the NPC are Anti-VCA (Viral Capsid Antigen) IgA and Anti-EA
(Early Antigen) IgA. Combined use of the VCA and EA specific antibody is
recommended for the diagnosis of NPC, due to the sensitivity of VCA, and
increased specificity of EA. High titres and rising titre are particularly significant,
and NPC should be actively excluded in these patients.
i. PSA (Prostatic Specific Antigen)
PSA is produced in the prostate, and is useful in the diagnosis of prostatic
cancer as well as for monitoring patients for tumour recurrence and
metastases. The diagnostic sensitivity increases when used together with
measurement of free PSA (fPSA).
Studies have shown that the ratio of free/total PSA is useful to further
distinguish those patients with abnormal prostate glands. Cancer patients tend
to produce more bound PSA and therefore have a low ratio while those with
benign disease produce more of the free form and have an elevated ratio. To
calculate the ratio the laboratory performs a measurement of total PSA and a
separate measurement for “free” PSA.
Free/total PSA ratio is of most benefit for those patients with values of total PSA
109
within the range of 2-10 ug/L. At levels below 2 ug/L the ratio does not give
meaningful information and at levels above 10 it is already highly likely that the
patient has prostatic carcinoma.
Note that between the ranges of 7 - 25% of free/total PSA there is still an
overlap of clinical findings. There is no advantage in measuring free PSA by
itself.
Although free/total PSA enhances the ability to select patients for further
investigations there is still no single biochemical test which will rule in or rule
out carcinoma of the prostate. In patients in whom there is a high index of
110
suspicion, free/total PSA appears to offer advantages over PSA measurement
alone, especially in those patients with mild elevations in total PSA.
Top of the page
Diabetes Mellitus & Glycaemic Control
Glucose reference ranges are in line with guidelines of the American Diabetes
Association. These guidelines have been adopted by the Malaysian Endocrine Society.
(Reference Diabetes Care 27:S5-S10, 2004)
A brief summary of the guidelines is:
a. Fasting Glucose(Ideally after 10 hour fast)
o Fasting glucose < 5.6 mmol/L → Normal fasting glucose
o Fasting glucose 5.6 – 6.9 mmol/L → IFG (Impaired fasting glucose) and
recommend 75g OGTT
o Fasting glucose > 7.0 mmol/L on more than 1 occasion or on 1 occasion
together with symptoms of diabetes → Diabetes
b. Random Glucose
o Random glucose = 11.1 mmol/L on more than 1 occasion or on 1
occasion together with symptoms of diabetes → Diabetes
o Random glucose is 5.6 – 11.0 mmol/L, then recommend 75g OGTT.
111
c. Glucose Tolerance Test (75g)
o 2 hour post load glucose < 7.8 mmol/L → Normal glucose tolerance
o 2 hour post load glucose 7.8–11.0 mmol/L → IGT (Impaired glucose
tolerance)
o 2 hour post load glucose = 11.1 mmol/L → Diabetes
Criteria for the diagnosis of diabetes mellitus
Symptoms of diabetes plus casual plasma glucose concentration = 11.1 mmol/L.
Casual is defined as any time of day without regard to time since patient’s last meal.
The classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight
loss.
OR
FPG = 7.0 mmol/L. Fasting is defined as no caloric intake for at least 8 hrs.
OR
2 hour post load glucose = 11.1 mmol/L during an OGTT. The test should use a
glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water.
Gestational Diabetes
a. One-step approach
Perform a diagnostic OGTT without prior plasma or serum glucose screening.
The one-step approach may be cost-effective in high-risk patients or
populations and diagnosis is as noted under diagnosis of Gestational Diabetes
Mellitus (GDM) with a 75 g glucose load is as noted above.
112
b. Two-step approach
Perform an initial screening by measuring the plasma or serum glucose
concentration 1 hour after a 50g oral glucose load (glucose challenge test -
GCT) and perform a diagnostic OGTT on that subset of women exceeding the
glucose threshold value on the GCT.
Note: For the glucose challenge test (GCT), the patient is not required to fast
and is given a 50g glucose load. Only one blood is drawn at 1 hour post
loading.
When the two-step approach is used, a glucose threshold = 7.8 mmol/L
identifies ~ 80% of women with GDM, and the yield is further increased to 90%
by using a cutoff of = 7.2 mmol/L.
HbA1c Classifications
The HbA1c guidelines used are those adopted by the Malaysian Endocrine and
Metabolic Society and are the same guidelines as that adopted by the International
Diabetes Federation in 2002. The Singapore Endocrine Society has also adopted the
same guidelines.
Haemoglobin A1c (%) Glucose Control Index
< 6.5** Good control
6.5 - 7.5 Diabetic with satisfactory control
> 7.5 Diabetic with poor control
113
Note:** HbA1c less than 6.5% does not necessarily exclude diabetes.
For more information on Diabetes Mellitus, please read our MediTalk Client Circular No
12 - Diabetes Mellitus (Targets for Control).
Top of the page
Microalbumin
A serious prognostic sign of this complication of diabetes is the development of a
persistent proteinuria or macroproteinuria. Before this occurs, there is detectable
microproteinuria. This stage of diabetic renal disease is referred to as incipient diabetic
nephropathy. Macroalbuminuria is irreversible whereas it has been suggested that, in
well-controlled diabetes, microalbuminuria is reversible. It is thus apparent that
microalbuminuria is an important predictor of diabetic nephropathy.
The term “microalbuminuria”, which has been used to describe albuminuria, is
somewhat misleading, as the albumin is of normal molecular mass. Urine albumin is
elevated in the nephrotic syndrome, in other conditions with increased glomerular
permeability (e.g. glomerulonephritis) and in urinary tract inflammation.
In a spot urine (first void morning sample) the normal albumin creatinine ratio (ACR)
range is <3.5 (female) and <2.5 (male) mg albumin/mmol creatinine, while ratios in the
range 3.5–25.0 mg/mmol are consistent with microalbuminuria. ACR values of >25.0
mg/mmol are likely to reflect overt proteinuria. In a timed urine collection (24 hour or
overnight 8 hour collection), microalbuminuria is defined as being an albumin excretion
114
rate (AER) of 20–200 ug/min measured. Excretion rates of >200 ug/min are considered
to be evidence of macroalbuminuria, and this is most likely irreversible.
There are no special dietary requirements prior to sample collection. A first void
morning specimen is the sample of choice for spot albumin:creatinine ratio (ACR). It is
important to remember that exercise increases excretion rate and so at least 1 hour of
rest is advised before collection of spot urine. The albumin excretion rate utilises either
a 24 hour or overnight 8 hour timed urine collection. If an 8 hour collection is used it is
important that the collection includes the first morning void. It is essential that the
period of collection be accurately noted on the request form.
The recommended procedure for an overnight urine collection is as follows:
a. Prior to going to bed empty the bladder and note the time (do not save this
urine).
b. Save all the urine passed during the night into the container provided.
c. On rising in the morning empty the bladder again (saving this urine) and note
the time.
Top of the page
eGFR
Please refer to MediTalk Client Circular No 14 - Routine Reporting of eGFR for eGFR
information.
Top of the page
115
Helicobacter pylori
Testing Regime
116
Top of the page
Dengue
There are 4 serotypes of the dengue virus and infection by any of the serotypes leads
to a similar spectrum of illness and lasting immunity to the infecting serotype but not to
the other three. The individual non-protective antibodies cross-react with the other
serotypes.
Laboratory diagnosis of dengue infection includes virus isolation, antigen detection,
serology, and molecular methods. In addition, a clinical diagnosis of DHF can be made
in the presence of fever, haemorrhagic tendencies, thrombocytopenia (platelets =
100,000/cmm) and haemoconcentration. Virus isolation and antigen detection are most
successful during viraemia which usually last 3 - 5 days and coincides with fever.
IgM antibodies appear around the 5th day of the fever and last for 2 - 3 months. IgG
antibodies are detected from the 14th day in primary infections and 2nd day in
secondary infections and are usually detectable for life. Three serological tests are
widely used:
a. HI (Hemagglutin Inhibition Test) → Detects a mixture of IgM and IgG
antibody and cross reacts with other serotypes. Paired titres are tested 7 days
apart.
b. IgM antibody enzyme immunoassay → False positive due to cross-reaction
with other viruses may occur. Sensitivity is in the region of 80% and specificity
99%.
117
c. Dengue Blot Test → Rapid test for IgG antibody, but is usually negative in
primary infections.
118

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CHAPTER 1

Loh Guan Lye Specialist Centre is established in 1975, is a 250-bedded private

hospital.It is located in the heart of Georgetown,Penang, about 30 minutes from the

Penang International Airport and 20 minutes from the pristine

a leader in the healthcare industry. Our mission is "Commitment to Customer

Service Excellence" and quality healthcare through continuous improvement and

the upgrading of technology and Human Resource development.

professionals, Loh Guan Lye Specialist Centre is committed to be

"Your Hospital of Choice".

1.2) Medical facilities that offered

A) Outpatient treatment facilities

outpatient clinics, outpatient specialist clinic, Ambulance services and mortuary

vehicle, Emergency treatment services, and receiving referred cases or patients

from the Health Centre.

test, biochemistry test, serological test and blood transfusion test.

intravenous urography and barium studies, CT scan and MRI.

The pharmacy department is a supporting department in this hospital. This unit

provides dispensary medicine and counseling to patients and clinical services.

issuing of birth and death certificates.

can be enjoyed by the community.

1.6) Customer service of Loh Guan Lye Specialist Centre Laboratory

the officer in charge regarding any problems.

section is involved in receiving, checking and sorting of specimens before the

specimens are sent to the specific laboratories for analysis.

2.2 Request Form

requiring all categories of tests. (Haematology and Biochemistry) except

2.2.3 All request forms must be filled in completely and should be

accompanied by properly collected specimens.

Marking ( √ ) in the box is discouraged.

2.2.5 All request forms must be signed by H.O, M.O or specialist.Name of

requesting doctor must be clearly written.

for rejection are:

c. Name of test not provided / not written

d. No specimen received for the intended test

e. Incomplete request form

2.3.2 Rejection shall notify through the Rejection Form

clinics and properly labeled.

plastic bag and stapled to the request form.

2.4.3 All specimens should be dispatched to the laboratory in appropriate containers

until all the specimens are checked by the laboratory staff.

2.5 Urgent request

the request form at the upper right hand corner.

2.6 Reporting of result

2.6.1 All results shall be validated before they are dispatched.

urgent requests shall be informed by telephone and or dispatched immediately to

the person waiting.

respective ‘pigeon holes’ after being conveyed by telephone.

doctor via phone.

3.2 Request Form

must be written legibly.

3.3 Receipt of specimens

3.3.1 During the office hours

3.3.2 After the office hours

wait for the result.

3.4 Reporting of result

wait for the result

according the On Call service timetable.

unit or from the emergency unit through the telephone operator.

on the on call service form.

DISPATCHING SPECIMEN TO REFERENCE LABORATORY

Other service offered by this laboratory is dispatching specimen to the

5.1 Specimen Preparation

rejected according to the specimen rejecting procedure.