Practical Attachment Phase 1 Report
Name: See Chen Yeh
I/D: BM080725756 (18)
Date: 05- 17 April 2010
Laboratory: Pantai Premier Pathology Pandan Indah, Hospital Pantai Indah
On-site supervisor: Miss Shanaliza
There are basically several disciplines in the lab, which are haematology, chemistry, blood bank, immunology &
microbiology and specimen reception.
Haematology
Test & Principle
Complete blood count (CBC)
-to determine haemoglobin (Hb),
total RBC, packed cell volume
(PCV), mean cell volume (MCV),
mean cell heamoglobin (MCH),
mean cell haemoglobin
concentration (MCHC), red cell
distribution width (RDW), white cell
count (Total WBC), platelet count
(PC), mean platelet volume (MPV),
differential count in peripheral blood
Differential count
- measures the number for each type
of white blood cells in the sample
and to detect the presence of any
abnormal or immature cells
- assess the body condition respond
to and eliminate infection
- normally expressed in percentage
Method
Automated machine with flow
cytometry function:
-run start up and daily QC
-check time and date of sample
-mix sample several times
before run cell count
-analyses results
-comments given based on
results and clinical history
-reference range varies among
different gender and age group
-automated method run by
machine with flow cytometry
function
-manual method will be
counting a total hundred of
white blood cells which are
neutrophils, lymphocytes,
monocytes, eosinophils and
basophils with microscope using
differential cell counter
Results Interpretation
-Total RBC: low= anaemia, hemolysis;
high=dehydration, polycythemia
-PCV measures amount of volume/space
of RBC : abnormal = anaemia or
polycythemia
-Hb measures amount of Hb in blood,
ability to carry oxygen: low= anaemia,
hemorrhage
-MCV, MCH, MCHC are red cell
indices, aid in diagnosing anaemia
-MCV measures size of RBC: Normal=
normocytic; Low= microcytic; High=
macrocytic
-MCH measures concentration of Hb in
average RBC
-MCH & MCHC: Normal=
normochromic; Low= hypochromic;
High= hyperchromic
-RDW shows the sizes or shape of RBC:
abnormal = aniso/ poikilocytosis
-PC/ thrombocytes count assesses
clotting function;
-Total WBC: high = infection
-Neutrophils: Increase= bacterial
infection, inflammatory diseases, bone
marrow disorders; Decrease= severe
infection, medications, chemotherapy
-Lymphocytes: Increase= Viral
infection, leukemia, bone marrow
cancer, radiation therapy; Decrease=
immune diseases, lupus
-Monocytes: Increase= infection,
inflammatory disorder, leukemia;
Decrease= Bone marrow injury/ failure
-Eosinophils: Increase= Allergic
disorders, skin inflammation, parasitic
infection;

Peripheral blood film (PBF)
-examination of RBC morphology,
WBC differential and platelets
distribution under microscope
-to detect and investigate
haematological disorder
-to detect parasites or abnormal cells
in blood
Reticulocyte count
-reticulocytes are immature RNA
containing red cells
-will produce a basophilic ribosomal
precipitate with New Methylene
Blue stain
-to determine the effectiveness of
erythropoiesis
- like PBF
Erythrocyte sedimentation rate
( ESR)
-non-specific screening test to
indicate presence of abnormal
plasma protein, inflammatory
disorder and tissue damage
-anticoagulated red cells will
produce sedimentation after allowed
to stand
-rate of fall of the red cells depend
on difference in specific gravity
between red cells and plasma
-put a 2-3 mm drop of blood
near the end of clean glass slide
-spread the blood with separator
using wedge smear technique
-allow the smear to air dry
-label slide with pencil
-stain with Leishman stain for 2
minutes
-mix with buffer pH 6.8 for 7
minutes
-wash under running tap water
-wipe clean on the opposite site
of smear and air-dry
-examine slide under
microscope
-pipette 200µl of blood sample
in a test tube
-add 3 drops of retic stain (New
Methylene Blue)
-mix by gentle shaking
-incubate at 37oC for 1 hour or
room temperature for 24 hours
-resuspend mixture by gentle
shaking and make a thin film
- label and air dry
--under 1000x magnification, 10
fields of approximately 100 red
cells are examined and number
of reticulocytes are counted
Automated:
-pour the blood from EDTA
tube into the ESR tube reaching
the indicated level
-label and put in the machine for
processing
Manual method for newborn:
-mix 240µl of blood and 60µl of
NaCl in the ESR container
-poke in the capillary tube into
the ESR container, ensuring no
bubbles trapped inside
-leave for 1 hour before read the
-Basophils: Increase= Leukemia, chronic
inflammation, hypersensitivity reaction
to food, radiation therapy
-good staining will reveal the white
blood cells with good morphology
-to note the size, shape of RBC (to
detect anaemia, sickle cell anaemia,
spherocytosis, etc.)
-to count the number of WBC with
differential count to detect infection
-to examine for platlet clumping
-to detect any ovals or eggs of parasites
-percentage of reticulocytes =
No. of reticulocytes X 100 %
No. of red cells
-normal range for adults/children = 0.2-
2.0% ; infants = 2.0-6.0%
-increase= haemolysis, blood loss,
anaemia under treatment
-decrease= red cell aplasia, renal
disease, marrow displacement, iron
deficiency anaemia, sideroblastic
anaemia, megaloblastic anaemia,
maturation disorder
-markedly elevated in multiple myeloma
macroglobulinaemia
-moderate elevation: rheumatoid
arthritis, chronic infection
-mild-moderate elevation: pregnancy
-ESR may be influenced by age, sex,
menstrual cycle, drugs ( corticosteroid,
contraceptive pills)
-presence of Rouleaux (stacking of red
cells) will induce higher red cell
sedimentation rate

G6PD screening
-to screen for glucose-6-phosphate
dehydrogenase deficiency
-Glucose-6-P + NADP (G-6-
PDH)
gluconate-6-p + NADPH + H+
-glucose-6-Phosphate and NADP
will produce NADPH with the
presence of G6PD enzyme
-NADPH produced will fluoresce
with exposure of UV light
results
-
-a disk of dried blood-stained -the sample is put under long wave UV
paper of 3-5 mm diameter is light for examination
punched out -fluorescence indicates negative for
-the disk is put in small vial G6PD deficiency
containing 100µl of working -no fluorescence observed indicates
reagent positive for G6PD deficiency
-mix well and incubate for 10
minutes at 25oC
-a 10µl of the resulting solution
is taken an place on the paper
provided and air-dried

Prothrombin Time Semi-automated: -end point shown as solid gel clot

-determine activity of coagulation
factors II, V, VII and X for extrinsic
pathway
-monitor anticoagulant therapy
-addition of tissue thromboplastin to
normal anticoagulated plasma will
initiate coagulation
-solid gel clot forms within a
specified period of time
Activated partial thromboplastin
time (APTT)
-determine partial thromboplastin
time for factor VIII, IX, XI, XII etc
-used as pre-surgical screening for
intrinsic pathway factor deficiencies
-monitor heparin therapy
-coagulation of plasma triggered by
thromboplastin and calcium added
-time of formation of fibrin clot is
measured
-add 50µl of plasma with 100µl
of reagent in the chamber
Semi-automated:
-add 50µl of plasma with 50µl
of reagent in the chamber
-add in CaCl2 after indicated by
machine
-prolonged PT time may indicate
deficiency of factors II, V, VII and X or
occur in patient with oral anticoagulant
therapy
-report APTT in seconds
-prolonged APTT may indicate
deficiency/defect of factor VIII, IX, XI,
XII
-increase APTT may be caused by drugs
e.g. heparin, warfarin
-decrease APTT may be caused by
estrogen therapy in males or oral
contraceptive in females

The results have to be checked and commented by the personnel according to the gender and age of the patient. It is
important to interpret the results accordingly and logically. If the patient has previous set of measurement data,
comparison can be made to predict the possible causes for certain abnormalities of the readings. Before the results are
authorized and being sent to the doctor, troubleshooting of the abnormal results whether it is due to certain disease
condition or technical problem is necessary. For example, if the platelet of the patient is showing a decrease with an
alert of platelet clumping, microscopic examination of the patient’s blood smear has to be done to clarify the situation. If
it is not elevated due to platelet clumping, then the patient may be suspected with dengue haemorrhagic fever.

Chemistry
The general chemistry profiles of the specimens mostly are using fully automated machine. The common chemistry tests
conducted include liver function test, renal function test, lipid profile and glucose test. Glucose test is general diabetic
screening and monitoring test for hyperglycemia, hypoglycemia, diabetes and pre-diabetes. Elevation of blood glucose
may also due to chronic renal failure, Cushing syndrome, pancreatitis, hyperthyroidism or drugs. On the other hand,
decease of blood glucose may suggest adrenal insufficiency, hypothyroidism, insulin overdose, extensive heart disease,
alcohol or drugs.

Liver function test

This test is used to detect any abnormalities of the liver. However, due to its limited sensitivity and specificity, any
detected abnormalities have to be interpreted together with clinical presentation or medical history. Increase in total
serum protein may indicate liver dysfunction, viral hepatitis. Serum albumin will decrease in chronic liver disease.
However, hypoalbuminemia is not specific as it can occur in protein malnutrition, nephritic syndrome. Increase level of
globulin may indicate biliary cirrhosis, obstructive jaundice. An increase albumin to globulin (A/G) ratio may indicate
underproduction of immunoglobulin whereas a decrease may suggest multiple myeloma or autoimmune diseases,
cirrhosis. For liver cell injury, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are used.
ALT is specific for liver and will raise parallel with AST in liver cell damage. AST will also present in order organ such
as heart and muscle. Elevation of AST will indicate hepatocellular disease, myocardial infarction or muscle disease.
Increase of both enzymes will indicate hepatocellular damage. For detecting cholestasis, serum alkaline phosphatase
(AP) and gamma-glutamyltransferase (GGT) are used. AP will increase in cholestasis, hepatic metastasis and primary
biliary cirrhosis. GGT can help to determine whether increased AP is of hepatic origin. GGT will increase in
hepatocellular damage and cholestasis where the ALT is increased greater than GGT. Nonetheless, elevation of GGT
could also due to alcoholism and some drugs. Hyperbilirubinemia can lead to jaundice which can be categorized into
pre-hepatic, hepatic and post-hepatic jaundice. Pre-hepatic jaundice occurs when the bilirubin formation is excessive
that the liver is unable to conjugate, causing serum unconjugated bilirubin to increase. Hepatic jaundice will take place
when there is hepatocellular damage due to viral infection, drugs, alcoholism. This will lead to increase of conjugated
and unconjugated serum bilirubin. For post-hepatic jaundice, conjugated serum bilirubin will increase due to the
obstruction in canaliculi or bile duct.

Renal function test

Creatinine is a breakdown product of creatine and its serum level will increase in acute tubular necrosis, dehydration,
diabetic nephropathy and glomerulonephritis and kidney failure. Blood urea nitrogen (BUN) is by-product of protein
metabolism and will increase in kidney dysfunction non-specifically. Serum creatinine is a more specific indicator of
kidney dysfunction compared to BUN. Serum electrolytes such as sodium, potassium, chloride, calcium, phosphate, uric
acid which involve kidney regulation are measured to determine the renal function. Hyperkalemia could indicate
acidosis, acute/ chronic renal failure whereas hypokalemia may suggest alkalosis. Hyponatremia may be caused by
kidney diseases which cause protein loss e.g. nephritic syndrome or elevated level of anti-diuretic hormone.
Hypernatremia could occur due to Cushing syndrome or diabetes insipidus or hyperaldosteronism. Hyperchloremia
normally will suggest dehydration, Cushing’s syndrome or kidney disease, metabolic acidosis. Hypochloremia is
probably due to prolonged vomiting, emphysema or metabolic alkalosis. Hypophosphatemia may associate with
diuretics, rickets and osteomalacia, hypothyroidism. Hyperphosphatemia may be caused by kidney failure,
hypoparathyroidism, diabetic ketoacidosis. High serum uric acid may suggest gout, higher risk of kidney stones. Low
serum uric acid may due to liver or kidney diseases, toxic compounds.

Lipid profile
It measures and determines the risk of having coronary heart diseases such as atherosclerosis, stroke or myocardial
infarction. Common parameters include total cholesterol, high density lipoprotein cholesterol (HDL), low density
lipoprotein cholesterol (LDL) and triglycerides. Higher total cholesterol, LDL, triglycerides may indicate higher risk of
having heart diseases. However, higher HDL could indicate lower risk of developing heart diseases. Many factors
contribute to the abnormal results such as cigarette smoking, excessive alcohol consumption, diabetes, genetic diseases.
In general, the patient’s background such as age, sex, medical history, clinical presentation, life-style behavior may
affect the risk of developing heart disease.

Enzymes
Creatine kinase (CK) is used to detect heart attack or other muscle injury. CK-MB is one of its 3 isoenzymes which is
specifically increase in myocardial infarction or heart diseases.
Lactate dehydrogenase (LDH) is used to determine the cause and location of tissue damage and also to monitor its
progression. It increases in many conditions due to its widespread tissue distribution. Elevation of LDH could indicate
heart attack, hemolytic anemias, kidney or liver diseases, pancreatitis, muscular dystrophy or lymphoma.

Urinalysis
Urinalysis is used to screen and detect any substances or cellular material in urine related to various metabolic and
kidney disorders. The test is done using test strips and measured the changes by automatic analyzer in order to be time
effective. If any substances are increased and indicated by machine, microscopic examination is performed to confirm.
Colour and clarity of the urine are measured to detect any abnormalities. Colour of urine is varied from very pale yellow
or colourless to very dark/amber due to various diseases, drugs or food consumption. However, other abnormal colour
could also indicate problem with kidney such as red colour urine may indicate blood in urine due to urinary system
problem. The urine clarify could be clear, slight cloudy, cloudy, or turbid. Cloudy urine may contain erythrocytes,
leukocytes, bacteria. Chemical tests for protein, blood, leukocytes, specific gravity may be abnormal in patients with
renal failure. Proteinuria may indicate kidney disease, inflammation of urinary tract infection (UTI). Glucosuria may
due to diabetes mellitus, liver disease, pregnancy or hormonal disorders. Hemoglobinuria and hematuria may indicate
UTI, kidney diseases, trauma, and drug. Positive testing of leucocyte esterase, nitrile in urine may indicate UTI e.g.
bladder or kidney infection. Presence of bilirubin in urine may indicate hepatitis or biliary obstruction and jaundice.
High concentration of urobilinogen may suggest hepatitis, cirrhosis or hemolytic anemia. Microscopic examination is
performed to confirm the abnormal results shown by the machine. It is normally screen for erythrocytes, leucocytes,
epithelial cells, microorganisms such as bacteria, trichomonas or yeast, and various casts or crystals. RBC and WBC are
seen in urine with inflammation whereas abundant epithelial cells may be seen with inflammation or malignancies.
Presence of bacteria, parasite or yeast may indicate infection. Casts such as granular, fatty or waxy casts may be found
with kidney disorder. Crystals such as crystalline uric acid, calcium oxalate or amorphous phosphate may be identified
with their shape, colour and the urine pH.

Blood bank
Blood group typing is one of the common tests to be performed over the lab. Basically, the lab practices two types of
blood grouping methods which are tile and tube method. The principle is to detect the presence or absence of the A/B
antigens on red blood cells with corresponding antibodies. Three types of antibodies which are anti-A, anti-B and anti-
AB are used. This is known as forward grouping and will simultaneously be confirmed with reverse grouping. In
reverse grouping, the patient’s serum will be tested against known A, B and O red cells. Also, the Rhesus grouping will
also be done by testing the presence or absence of Rh D antigen on the red cells using anti-D test serum. Tube method
involves forward and reverse grouping whereas tile method is only used for forward grouping which is done by another
lab personnel. This is to triple confirm the accuracy of the blood group typing and minimize the human error.
For forward grouping, the procedure will be adding a drop of whole blood to a drop of anti-A, anti-B, anti-AB and anti
D IgM respectively. For reverse grouping, a drop of patient’s serum is added with a drop of known red cells A, B and O
respectively. Forming agglutination indicates there is reaction with the respective reagent antibodies or blood antigens.

Interpretation of blood group typing results

ABO Forward Reverse
Anti A Anti B Anti AB A cell B cell O cell
A P N P N P N
B N P P P N N
O N N N P P N
AB P P P N N N
*P = positive for agglutination; N= negative for agglutination.
Rh D positive is found agglutination with anti D IgM.
Immunology & Microbiology
Dengue IgG and Ig M test kit
This test is to detect the presence of antibodies against dengue virus in serum, plasma or whole blood. It can detect all 4
dengue serotypes by using a mixture of recombinant dengue envelope proteins. It is an immunochromatographic test
where the recombinant dengue virus is bound to a nitrocellulose membrane to detect the dengue Ig G and Ig M
antibodies. The sample is added to the sample well and followed by buffer into round well. This allows the sample and
antibody-gold conjugate to migrate. During the migration, the relevant anti-human Ig G and/or anti-human Ig M will be
captured and generate a coloured line. Results are valid when the test control line is positive with a coloured line.

Helicobacter pylori Ig G test kit

This is a qualitative membrane based immunoassay for detection of H.pylori antibodies in serum/ plasma. Anti-human
IgG is immobilized in the test line region. The specimen is added and will react with H.pylori antigen coated particles,.
Upon migrating chromatographically, it reacts with immobilized anti-human IgG. Presence of H.pylori antibodies will
show a coloured line in the test line region. Result is valid when coloured line is appeared at the control line region.

VDRL Test for Syphilis

This is a non-treponemal test for qualitative detection of syphilis using serum/plasma. It measures the IgG/ IgM
antibody produced responding to lipoidal material released from damaged host cells, also the lipoprotein-like material
released by the spirochaetes. The use of VDRL carbon particle in this test enhances the macroscopic reading of result.
It allows positive results with weak agglutination can be distinguished from negative results which display a smooth and
even appearance.

Latex Serology test for Rheumatoid Factor detection

It is a rapid latex agglutination test kit to detect rheumatoid factor (RF) in serum. RF can be found in patients with
rheumatoid arthritis. It is an autoimmune disease where the IgM antibodies are directed against patient own IgG. The
RF latex particles are coated with purified human gamma globulin. The latex suspension is mixed with serum on a slide.
Clear agglutination will be observed in serum with elevated RF levels within 2 minutes.

Specimen reception
In this department, the lab personnel have to process the specimens received from hospital or outside clinics. The
specimen is first checked together with the order form. The specimen must be labeled and correspond with the name
written on the order form. Correct specimen with suitable container which corresponds to the ordered test is processed,
by labeling a barcode with different test codes written on the specimen container. Then the order form is passed to
registration counter whereby the specimen is registered into the lab integrated information system. Meanwhile, the
specimen is passed to relevant department to be processed. Unlabeled, wrongly-labeled specimens will be rejected.
Also, insufficient specimen, specimen with incorrect container, faulty specimen such as clotted blood in EDTA tube will
be rejected.

Blood collection with venepuncture is performed by lab personnel frequently. Thus, various tubes for different blood
tests have to be familiarized.
Vacutainer Principle & Function
EDTA (Ethylenediamine tetraacetic acid) -EDTA as anticoagulant
(lavendar cap) -chelates calcium ion forming soluble complex, prevent blood clotting
-haematology test

Sodium citrate tube -sodium citrate as anticoagulant

(light blue cap) -bind to calcium ion forming insoluble salt- calcium citrate
-coagulation studies e.g. prothrombin time, partial thromboplastin time
-correct citrate to blood ratio is important as it is in liquid form and will
 affect the clotting time

Heparin tube (green cap) -contain sodium, lithium, or ammonium heparin

-prevent formation of thrombin, thus fibrin
-enzyme and chemistry studies
-chromosome and HLA studies

Serum separation tube (gold cap) -contain clot activator

-gel at bottom will separate blood from serum after centrifugation
-chemistry, immunology, serology studies

Sodium fluoride- Potassium oxalate tube -sodium fluoride will inhibit glycolytic enzymes which cause glucose
(grey cap) metabolism
-potassium oxalate is anticoagulant which precipitating calcium
-glucose test

Plain tube (red cap) -no anticoagulant

-glass surface will activate clotting naturally
-blood bank testing e.g. cross-matching
-routine chemistry test, immunology, serology test

Trace element free tube (dark blue cap) -with or without anticoagulant
-for trace elements studies, toxicology and certain nutrient determinations

The specimens will be kept for one week for back-up purposes in the refrigerator of about 2-8 oC. Later, it will be
disposed as biohazard waste. Results for blood bank will keep for 10 years whereas for blood test e.g. CBC will keep for
3 months.

As safety precaution, gloves and lab coat are worn during the specimen processing and all specimens shall be treated as
health-hazardous to reduce transmission of laboratory infectious diseases. Other universal precaution such as thorough
hand-washing is essential to minimize risk of being infected.

Conclusion
The quality control of the machine is done internally and externally. It is to ensure the accuracy of the results is up to the
standard. The internal QC is done on daily basis prior the start of the day whereas the external QC is done on monthly
basis. Different reagents will be used according to the type of QC to be carried out. If there are any abnormalities in the
QC report, the personnel have to report to the lab manager and calibration/ maintenance service is needed.

In short, medical laboratory technologist has to be alert, attentive, flexible, fast, timely, accurate, precise and efficient in
conducting his duty. Also, he/she must not be over-dependent on the machine as troubleshooting of abnormal results is
pivotal. Interpretation and comment of results are important as they will affect the diagnosis and treatment of the
patient. Teamwork and communication among the healthcare personnel within the hospital are essential to provide
accurate and quality services to the patients.