Laboratory Medicine 2020 - Lab Manual

LABORATORY MEDICINE MANUAL
CONTENT PAGES
A. Anatomic Pathology and Cytology
Practical 1: Sputum for Cytology 1–3
Practical 2: Tissue Pathology – MI, PTB, Lung Cancer (Digital) 4–8
Practical 3: Tissue Pathology - Liver Diseases and GIT Diseases 9 – 14
Practical 4: Tissue Pathology Cervical Cancer and Prostate Cancer 15 – 19
B. Clinical Chemistry
Practical 1: Creatinine Clearance/eGFR & Order of Draw 20 – 29
Practical 2: Urinalysis 30 – 35
Practical 3: Glucose Tolerance Test (OGTT) 36 – 39
Practical 4: Point of Care Testing: Glucometer and Pregnancy Test 40 – 47
Practical 5: Point of Care Testing: Bilirubinometer 48 – 50
Practical 6: Immunological Assay: Flow Cytometry Analysis 51 – 58
& Serum Protein Electrophoresis
C. Hematology and Transfusion Medicine

Practical 1: Hemoglobin Measurement, Blood Grouping 59 – 62
Practical 2: Blood Smear Examination and White Cell Differential Count 63 – 68
(Manual and Digital)
Practical 3: Coagulation (PT) and Erythrocyte Sedimentation Rate (ESR) 69 – 73
D. Medical Microbiology
Practical 1: Blood Sample: Collection & Culture 74 – 76
Practical 2: Sputum from Pneumonia Patients 77 – 79
Practical 3: Urine from UTI Patient 80 – 81
Practical 4: Stool + Diarrhoea and Skin Swab 82 – 87
Practical 5: Parasites 88 – 95
Anatomic Pathology and Cytology
PRACTICAL 1
Sputum for Cytology
Learning Objectives
 To perform the observation of whole digitized sputum cytology slides

 To record results of sputum cytology slides.
 To discuss the results of sputum cytology slides.
 To describe the differential diagnosis of sputum cytology slides.
Introduction
Digital images are increasingly being used in the field of cytopathology for tele-education,
clinical consultation, telecytology, remote conferences, web-based learning, quality assurance,
and secondary applications such as image analysis. Today, digital images are used in a variety of
settings including education (E-education) such as cytopathology web pages, cytology
proficiency testing of Pap test slides. Digital imaging is beginning to replace the traditional
classroom with microscopes in medical education including cytopathology.
Whole slide imaging (WSI) is a digital imaging modality that uses computerized technology to
scan and convert pathology and cytology glass slides into digital images (digital slides) that can
be viewed remotely on a workstation using viewing software. The main advantage of WSI is that
the images are ready anywhere and are easily accessible. A potential disadvantage of using WSI
is that it initially may take a longer period of time to view cases as compared to glass slides. The
speed of loading digital images is dependent upon the speed of the user’s network and computer.
Sputum cytology refers to the examination of sputum (mucus) under a microscope to look for
abnormal or cancerous cells. Sputum, or phlegm, is the fluid that is secreted by cells in the lower
respiratory tract such as the bronchi and the trachea. It differs from saliva, in that it contains cells
that line the respiratory passages. Sputum cytology is increasingly being used in the evaluation
of lung lesions.
Topic: (1) Squamous cell carcinoma of lungs

(2) Adenocarcinoma of lungs
(3) Normal sputum cytology
1
Materials
 A whole slide images (digitized slides) of sputum cytology from a Thin Prep Pap Test
illustrating the viewer software provided by the vendor to allow for remote viewing and
manipulation of images by the cytopathologist.
An example of digital cytology image
Methodology
Instruction for use of Digital Pathology Image:

1. The software has been installed in the MRC1 computers.
2. The scanned slide images of the Pep stained slides are loaded in desktop.
3. Click on the slide image file, low power view appears. Use the cursor to pan (view) the
image.
4. Click on an area of interest.
5. Select image magnification by scrolling mouse wheel or select magnification size from
screen.
2
Findings
Sputum Cytology Slide 1
Review Questions
1. List the indications of sputum cytology test.

2. Describe the advantages of whole slide digital imaging (WSI) using.
3. Describe the pitfalls of sputum cytology test in lung cancer case.
3
PRACTICAL 2
Tissue Pathology - Myocardial Infarction, Atherosclerosis, Pulmonary
Tuberculosis, Lung Cancer

1. The DSA software has been installed in the MRC1 computers.
2. The scanned slide images of the following H&E stained slides are loaded in desktop/WBLE
including (1) Myocardial Infarction (2) Atherosclerosis (3) Tuberculosis of lung (4) Lung
carcinoma
image.
screen.
Case 1: Myocardial Infarction

A 63-year-old man had severe substernal chest pain radiating to his left jaw. He was hypotensive
and tachycardic. He had elevated creatine phosphokinase and troponin I. He was admitted to the
cardiac intensive care unit. He died 24 hours later of intractable ventricular tachycardia. The
Photograph shows the cross section of the left ventricle seen at autopsy. His myocardium at
autopsy is shown in the scanned slide image labeled 'myocardial infarct'.
Photograph: Cross section of the left ventricle
1. Describe the gross pathologic change seen in the Photograph.
2. Describe how early infarcts (3 to 6 hours old) can be detected by histochemical staining.
3. Identify the following histopathologic features in the digital tissue section of myocardial
infarction:
i. coagulation necrosis
ii. pyknosis
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iii. karyorrhexis
iv. karyolysis
v. hypereosinophilic cytoplasm
vi. inflammatory cellular infiltrate
4. Describe some of the ways by which infarcts have been classified by physicians and
pathologists.
5. What are the sites (location) of myocardial infarction?
6. What are the contrasting features of subendocardial and transmural infarcts?
7. What are the sequential myocardial changes in myocardial infarction?
8. What are the non-atherosclerotic causes of myocardial infarction?
Case 2: Atherosclerosis
A 61-year-old woman presented with lower extremity claudication, abdominal angina, and
hypertension. She died as a result of bowel infarction and resulting sepsis. The gross appearance
of her aorta at autopsy is shown.
Photograph: Cut open aorta
1. Describe the gross pathologic changes seen in the Photograph.
2. Identify the following histologic and histopathologic features in the digital tissue section
of atherosclerosis:
i. intima
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ii. media
iii. adventitia
iv. fibrous cap
v. central core
vi. foamy macrophage
vii. cholesterol cleft
viii. calcification
2. List some complications of atherosclerosis of abdominal aorta.
Case 3: Pulmonary Tuberculosis

A 35-year-old male construction site worker presented with a two month history of fever, cough
productive of blood stained sputum. His chest x-ray showed bilateral apical opacities. Sputum
specimen examination was done (Photomicrograph). Severe haemoptysis warranted resection of
the affected lung lobe. The specimen was sent for histopathologic examination. The digital tissue
section of the lung lesion is made available in the desktop/WBLE.
Photomicrograph: Ziehl-Neelsen stain of the Sputum
100x oil objective
1. Identify acid-fast bacilli in the Photomicrograph. What does the term 'acid fast' mean?
2. What is granuloma? What is tubercle?
3. What is the immunologic basis of the tissue pathologic changes seen in the lung lesion?
4. Identify the following histopathologic features of tuberculosis in the digital tissue section
of lung.
a. Epithelioid cells. These are so called because of their epithelial cell-like appearance.
They are modified macrophages/ histiocytes which are somewhat elongated cells
having slipper-shaped nucleus. The nuclear chromatin of these cells is vesicular and
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lightly-staining, while the cytoplasm is abundant, pale-staining with hazy outlines so
that the cell membrane of adjacent epithelioid cells is closely apposed. Epithelioid
cells are weakly phagocytic.
b. Multinucleate giant cells. Multinucleate giant cells are formed by fusion of adjacent
epithelioid cells and may have 20 or more nuclei. These nuclei may be arranged at the
periphery like the horseshoe (Langhans' giant cells). Giant cells are weakly
phagocytic.
c. Lymphoid cells. As a cell-mediated immune reaction to antigen, the host response by
lymphocytes (predominantly T cells) is integral to composition of a granuloma.
d. Plasma cells. Elliptical shape. Eccentric nucleus has cartwheel or clock-face
appearance. Cytoplasm is tinted blue.
e. Necrosis Necrosis may be a feature of some granulomatous conditions e.g. central
caseation necrosis in tuberculosis, so called because of its dry cheese-like appearance.
f. Fibrosis Fibrosis is a feature of healing by proliferating fibroblasts at the periphery of
granuloma.
The typical arrangement of the above is: there is central caseation necrosis,
epithelioid cells, Langhans giant cells (multinucleate giant cells with nuclei arranged
at the periphery in a horse-shoe distribution), cuff of lymphocytes and plasma cells,
proliferating fibroblasts at the periphery of granuloma.
5. List FIVE (5) lesions in the lungs and pleura in various types of pulmonary tuberculosis.
6. List the FIVE (5) causes of death in pulmonary tuberculosis.
Case 4: Lung Carcinoma

A 50-year-old businesswoman presented with a one month history of dry cough and chest pain.
She was a non-smoker. Her chest x-ray showed a right peripheral (subpleural) mass measuring
6cm diameter. The mass was resected. The Photograph shows the resected right lung.
Photograph: Right lung specimen
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1. Describe the gross pathologic features seen in the Photograph.
2. Describe the histopathologic features seen in the Digital Pathology Image labelled
'Carcinoma of lung 'accessible on the desktop/ WBLE
3. List the major histologic types of lung cancer.
4. (i) What is the cell of origin of small cell carcinoma?

(ii) Describe some of their clinical characteristics.
(iii) How does immunohistochemistry help in the diagnosis of small cell carcinoma?
5. Name some sampling techniques for lung cancer.
6. What is liquid biopsy?
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PRACTICAL 3
Tissue Pathology - Diseases of Gastrointestinal Tract and Liver

2. The scanned slide image of
Liver diseases: (1) Fatty Liver (2) Liver cirrhosis (3) Liver carcinoma (4) Liver of
CVC (5) Liver of viral hepatitis (6) Metastasis of liver
GIT diseases: (1) Acute appendicitis (2) Adenocarcinoma of ? (3) Colon amoebic ulcer
(4) Stomach cancer (5) Signet ring cell carcinoma of stomach (6) is loaded in
desktop/WBLE.
3. Click on the slide image file, low power view appears. Use the cursor and pan (view)
around the image.
4. Click on any area of interest.
5. Select image magnification by scrolling mouse wheel or select magnification size.
Liver disease Case 1: Liver cirrhosis
A 42-year-old man presented with increased abdominal girth. He had received blood transfusions
7 years ago. Examination of the abdomen revealed a palpable liver and positive fluid wave test.
His total protein is 5 g/dL (nl 6 to 8.5 g/dL), albumin was 2.5 g/dL (nl 3.5 to 5 g/dL), AST is 150
U/L (nl <42 U/L), and ALT is 170 U/L (nl <48 U/L).
He developed uncontrollable haematemesis and died. The Photograph shows his liver at
autopsy.
Photograph: The liver at autopsy
Scale
5cm
9
1. i. Describe the gross pathologic change seen in the Photograph.
ii. State the diagnosis.
iii. Suggest the possible etiologies for the diagnosis.
2. Identify the following histopathologic features in the digital tissue section of liver
cirrhosis:
i. regenerative macronodules
ii. fibrous septa
iii. hepatic parenchyma
iv. bile ducts
v. mononuclear inflammatory cells
3. What caused his fluid wave?
4. What do the laboratory findings indicate?
5. What caused his death?
Liver disease Case 2: Liver carcinoma

A 61 -year-old woman presented with a 10-year history of malaise and intermittent jaundice.
Abdominal examination revealed right upper quadrant tenderness and hepatomegaly.
Liver biopsy was done. The result was reported as hepatocellular carcinoma. The Photograph
shows the resected liver specimen.
Photograph: Resected liver specimen
Scale:
5cm
1. Describe the gross pathologic change seen in the Photograph.
2. List THREE (3) macroscopic patterns of growth of hepatocellular carcinoma.
3. List FOUR (4) histologic patterns of hepatocellular carcinoma

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4. What is fibrolamellar carcinoma?
5. Identify the following histopathologic features in the digital tissue section of

hepatocellular carcinoma of the pseudoglandular (acinar) pattern:
a. Tumour cells resembling hepatocytes, having vesicular nuclei with prominent

nuclei
b. The cytoplasm is granular and eosinophilic
c. Tumour cells arranged in a ring-like pattern (pseudoglandular)
d. Central cystic space formed by degeneration and breakdown in solid trabeculae of
cells
e. Mitotic figures
6. What laboratory test (tumour marker) result might you expect to be elevated?
7. What are the risk factors for this neoplasm?
GIT disease Case 1: Pseudomembranous colitis

A 38-year-old woman presented with cramping abdominal pain, green foul-smelling diarrhea,
one week after being prescribed a course of antibiotics. Colonoscopy was performed
Colonoscopy findings are shown in Photograph 1. Photograph 2 shows the cut open
colectomy specimen.
Photograph 1: Colonoscopy finding
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Photograph 2: Cut-open colectomy specimen
1. Describe the gross pathologic features seen in the Photograph 1 and Photograph 2.
2. Identify the following histopathologic features seen in the Digital Pathology Image
labelled 'Pseudomembranous colitis' which is accessible on the desktop/ WBLE
i. Thin layer of fibrinopurulent debris (i.e. pseudomembrane) on the mucosa surface

ii. Pseudomembrane that is composed of a network of fibrin and mucus, in which
are entangled inflammatory cells and mucosal epithelial cells.
iii. Lamina propria that contains the inflammatory cell infiltrate, mainly neutrophils
iv. Submucosa that has congested capillaries and may show microthrombi
v. Inflammation that spreads laterally rather than deeply
3. What antibiotics can cause this?
4. What bacterial organism is likely present?
5. What toxins does this organism produce?
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GIT disease Case 2: Signet ring cell carcinoma, stomach
A 66-year-old woman presented with a 5-month history of nausea, vomiting, and a 5-kg weight
loss. She underwent endoscopy. There was loss of antral rugal folds and a 4-cm, irregular,
shallow ulceration. A biopsy specimen is obtained of the ulceration at its edge, and the
microscopic appearance is shown (Photomicrograph). Gastrectomy was performed
(Photograph).
Photomicrograph 1: Gastric biopsy
H&E stain, medium magnification
Phototograph: Gastrectomy specimen
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Photomicrograph 2: Signet ring cells
Histochemical stain: Mucicarmine
2. Identify the following histologic and histopathologic features seen in the Digital
Pathology Image labelled 'Signet ring cell carcinoma'' which is accessible on the
desktop/ WBLE
i. Gastric mucosa
ii. Submucosa
iii.Muscularis propria
iv. Serosa
v. Signet ring cell infiltration is seen all gastric layers (the signet ring cell is shown
in Photomicrograph 1)
vi. Desmoplasia
3. i. Describe the histologic appearance of a signet ring cell carcinoma.

ii. Name the histochemical stain that is used to highlight the cytoplasmic content of a
signet ring cell.
4. Give associated familial genetic factors.
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PRACTICAL 4
Tissue Pathology - Cervical Cancer and Prostate Cancer

2. The scanned slide images of
(1) CA Cervix
(2) CA Prostate
is accessible in desktop/WBLE.
3. Click on the slide image file, low power view appears. Pan (view) around.
4. Click on any area of interest.
5. Select image magnification by scrolling mouse wheel or select magnification size.
Case 1: Cervical Cancer

A 36-year old nulliparous female presented to the hospital with post-coital vaginal bleeding of
one month duration. There was no abdominal pain or urinary symptoms. Her menarche occurred
at age 12 and menstrual cycles have been normal ever since. She was sexually active with at least
six partners. She denied any history of smoking or alcohol. Colposcopic examination showed a
mass in the cervix. Biopsy of the mass was reported as well differentiated squamous cell
carcinoma. She underwent hysterectomy. The Photograph shows the cut section of the uterus.
Photograph: Cut section of the uterus.
Diagram illustrating fungating cauliflower-like growth
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2. Identify the following histopathologic features in the digital tissue section of the cervix
specimen:
I. Squamous carcinoma cells at the surface showing irregular downward
proliferation of malignant cells into the underlying stroma
II. Depending upon the grade of malignancy, the masses of squamous cells show
atypical features such as
 variation in cell size and shape,
 nuclear hyperchromatism,
 absence of intercellular bridges,
 individual cell keratinisation and
 atypical mitotic figures.
III. This case of well-differentiated squamous carcinoma has whorled arrangement of
malignant squamous cells forming squamous cell nests (whorls of squamous
cells), keratin pearls (pinkish masses of keratin without nuclei or cells). The
centres of these pearls consist of laminated, keratin material.
IV. Higher grades of squamous carcinomas (moderately differentiated and poorly
differentiated squamous carcinoma) however, have fewer or no keratin pearls and
may instead have highly atypical cells.
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Microscopic features of well-differentiated squamous cell carcinoma.
The dermis is invaded by downward proliferating epidermal masses of cells which show atypical
features. A few horn pearls with central laminated keratin are present. There is inflammatory
reaction in the dermis between the masses of tumour cells.
3. List the various histologic patterns (types) of cervical carcinoma.

4. Describe the clinical staging of carcinoma of the cervix uteri.
5. Describe cervical screening.
6. Describe the broad principles of The Bethesda system (TBS) of cytologic evaluation.
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Case 2: Prostate Cancer
A 72-year-old man had an 1-year history of increasing difficulty with urination, including
frequency, hesitancy and dribbling. On digital examination revealed a 5cm irregular firm nodule
of the prostate. The serum prostate specific antigen (PSA) was 14ng/mL. A biopsy of the
prostate nodule was reported as adenocarcinoma of prostate. Transurethral resection of the
prostate was done. The digitized slide shows the H&E stained slide of the prostectomy specimen.
1. Identify the following normal histologic features in the digital tissue section of the
prostate:
a. Pattern of glandular tissue separated by thick fibromuscular septa
b. Glandular acini forming papillary structures
c. Glandular epithelium consisting of 2 layers
d. A basal layer of low cuboidal cells
e. An inner layer of mucus secreting tall columnar cells
f. Fibromuscular tissue mass containing abundant smooth muscle fibres and nerve
bundles
g. Capsule
h. Corpora amylacea ("starch-like bodies") is a general term for small hyaline
masses found in the prostate gland.
2. Identify the following histopathologic features in the digital tissue section of the
prostate:
i. Architectural disturbance In contrast to convoluted appearance of the glands

seen in normal and hyperplastic prostate, there is loss of intra-acinar papillary
convolutions. The groups of acini are either closely packed in back-to-back
arrangement without intervening stroma or are haphazardly distributed.
ii. Stroma Normally, fibromuscular sling surrounds the acini, whereas malignant
acini have little or no stroma between them. The tumour cells may penetrate
and replace the fibromuscular stroma.
iii. Gland pattern Most frequently, the glands in well-differentiated prostatic
adenocarcinoma are small or medium-sized, lined by a single layer of
cuboidal or low columnar cells. Moderately-differentiated tumour have
cribriform or fenestrated glandular appearance. Poorly-differentiated tumours
have little or no glandular arrangement but instead show solid or trabecular
pattern.
iv. Tumour cells The outer basal layer seen in the normal or benign acini is lost. The
tumour cells may be clear, dark and eosinophilic cells. Clear cells have foamy
cytoplasm, dark cells have homogeneous basophilic cytoplasm, and
eosinophilic cells have granular cytoplasm. The cells may show varying
degree of anaplasia and nuclear atypia but is generally slight.
v. Invasion One of the important diagnostic features of malignancy in prostate is the
early and frequent occurrence of invasion of intra-prostatic perineural spaces.
Lymphatic and vascular invasion may be present.
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3. List the histologic types of prostate cancer.
4. Describe Gleason's histologic grading.
5. Describe TWO (2) biochemical serum tumour markers employed for diagnosis and
monitoring the prognosis of prostate cancer.
6. Describe the spread of prostate cancer.
7. Describe the TNM staging of prostate cancer.
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Clinical Chemistry
PRACTICAL 1
PART A: Creatinine Clearance
Learning Outcomes
1. To describe the procedure for creatinine clearance

2. To calculate the creatinine clearance
3. To discuss the major errors that can limit the accuracy of the creatinine clearance as a
measure of glomerular filtration rate (GFR)
Introduction
Creatine is synthesized in the liver and stored in muscle which contains about 98% of total body
store of creatine. Therefore, the creatine pool is related largely to muscle mass, which is affected
mainly by gender and age. A relatively constant amount of creatine is degraded to creatinine
each day. Creatinine is released into the circulation and excreted from the body by glomerular
filtration. In health, the serum creatinine concentration is relatively constant and maintained
within narrow limits. Its level is determined by the balance between rate of production and rate
of excretion. Therefore, increased levels are seen in acute muscle disease and in impaired renal
function. The latter explains the routine measurement of serum creatinine as an indicator of renal
functional status. The clearance of creatinine by the kidneys is another measure of renal function.
The renal clearance of a substance can be defined as the volume of plasma that is completely
cleared of that substance per unit time (mL/min) by the kidney. The clearance of a substance that
is filtered and not reabsorbed or secreted by the renal tubules provides a measure of the GFR.
Materials
 Items for 24-Hour urine collection including a urine collection container (a kidney dish),
a urine storage container, and a name tag)
 Items for collection of blood sample
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Methods
Instruction for 24-Hour Urine Collection
The 24-hour urine collection is required for evaluation of the function of the kidneys and for the
measurement of the 24-hour excretion of specific products in the urine. For accurate results, it is
important to collect all the urine for exactly 24 hours.
1. Start the 24-hour urine collection by voiding directly into the toilet (i.e. at around 0700-
0730, Sunday). DO NOT save this urine. Note and record the date and time on the
storage container. For the next 24 hours, urinate first into the collection container, and
then pour the urine into the storage container. The last collection should be the first void
urine at 0700-0730 hour the next day.
2. If there is a need to use more than one container during the 24-hour collection period, use
one container at a time. When the first container is full, continue collection using the next
container.
3. Send the urine to the laboratory as soon as possible after the last collection. To prevent
leaks, make sure the lid is on tightly, and that the container is transported upright inside a
plastic bag. If the weather is warm and the duration of transportation is prolonged,
transport the urine container on ice or in a cooler.
4. A blood sample for measurement of serum creatinine will be collected at the time of
receipt of the 24 hour urine specimen.
*** Important Note:
 You are given a urine collection container (a kidney dish), a urine storage container,
and a name tag. You may need more than one storage container to contain all of your
urine for the 24-hour period.
 Make sure each storage container has a name tag with your full name and hospital
number written on it. If your container is not labeled properly, you may be asked
to repeat the 24-hour collection.
 Keep your storage container refrigerated throughout the 24-hour collection period
until you bring it back to the laboratory. If you do not have access to a refrigerator,
your collection should be kept on ice or in a cooler.
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Calculation of Creatinine Clearance
1. Measure and record the volume of the of 24-hour urine collection.

2. Measure the serum and urine creatinine concentrations.
3. Record the laboratory results in the worksheet.
4. Calculate the creatinine clearance (ml/min) using the following formula
Ccr = UV
P
where U = Urine creatinine concentration

V = Urine flow rate in 24 hours
P = Serum creatinine concentration
CREATININE CLEARANCE WORKSHEET
Patient I.D./Name: ____________________________ Date: __________________
REFERENCE
Unit TEST RESULT
RANGE
Volume of 24-Hour
mL
urine sample
Volume of 24-hour urine sample in ml
Urine flow rate (V) mL/min
/ 1440
Concentration of
µmol/L 62 – 125 µmol/L
serum creatinine (P)
Concentration of urine
mmol/L
creatinine (U)
Calculation:
22
Review Questions
1. It is noted that the calculated creatinine clearance overestimates the actual GFR by 10 –
20%. Explain why?
2. List three (3) major errors that can limit the accuracy of creatinine clearance as an
estimate of GFR.
3. Shorter duration of urine samples (i.e. 6-Hour and 12-Hour) can be used to calculate the
creatinine clearance but tend to give less accurate results. Explain why?
4. State the causes of increased serum creatinine and the limitation of serum creatinine as an
indicator of impaired kidney function.
5. What is the clinical application for the measurement of creatinine clearance?
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PART B
Estimated Glomerular Filtration Rate (eGFR)
Learning Outcome
1. To calculate the estimated GFR (eGFR) using the equations given

2. To explain the impact of various factors on eGFR
Introduction
Estimating kidney function is part of the routine medical practice (i) for assessing overall health
and (ii) as an aid in evaluation and monitoring renal function. Knowledge of the kidney
functional status is important for estimating drug dosage, and for preparation of patients for
invasive diagnostic or therapeutic procedures. Until recently, the estimation of the glomerular
filtration rate (GFR) by creatinine clearance is considered the best overall index of kidney
function in health and disease. However, in the past decade, several equations have been derived
to provide estimates of GFR based on the serum creatinine level. These equations take into
consideration various factors that can influence the GFR including gender, ethnicity, body size
and age.
Procedure:
1. Obtain the information required for determining eGFR:

a. Serum Creatinine result
b. Age of Subject
c. Sex of Subject
d. Ethnic origin of subject
2. Record the laboratory result in the worksheet.

3. Calculate the eGFR using the formulae provided.
4. Compare the results obtained
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EQUATIONS FOR ESTIMATION OF GFR
1. Modification of Diet in Renal Disease (MDRD) Equation
186 x [Scr (µmol/L)/88.4]-0.203 x 0.742 (if female) x 1.21 (if African-Caribbean)

Result in ml/min/1.73 m2
Reference range > 90 ml/min / 1.73 m2 (for M and F)
2. Cockcroft-Gault Equation
eGFR = [(140 - age in years) x (Wt in kg) x 1.23] / Serum creatinine (μmol/L)
(Multiply result by 0.85 if female)
Result in ml/min
Reference range > 60 ml/min
3. CKD Epidemiology Collaboration (CKD-EPI) equation
GFR = 141 X min(Scr/κ,1)α X max(Scr/κ,1)-1.209 X 0.993Age X 1.018 [if female] X 1.159

[if black]
Where Scr is serum creatinine (mg/dL), κ is 0.7 for females and 0.9 for males, α is –0.329
for females and –0.411 for males, min indicates the minimum of Scr/κ or 1, and max
indicates the maximum of Scr/κ or 1.
Result in ml/min/1.73 m2
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CREATININE CLEARANCE WORKSHEET
REFERENCE
Unit TEST RESULT
RANGE
Volume of 24-Hour
mL
urine sample
Volume of 24-hour urine sample /
Urine flow rate (V) mL/min
1440
Concentration of serum
µmol/L 62 – 125 µmol/L
creatinine (Pcr)
Concentration of urine
mmol/L
creatinine (Ucr)
Age years
Body weight kg
Body height cm
Results:
1. Creatinine clearance (obtained from PART B)
2. eGFR [Modification of Diet in Renal Disease (MDRD)]
3. eGFR [CKD-EPI equation]
Review Questions
1. Compare the results of creatinine clearance, eGFR by the MDRD and eGFR by the CKD-
EPI equation
2. Describe the impact of age on eGFR?
3. Describe the impact of ethnicity on eGFR?
4. Describe the impact of body weight on eGFR?
Reference
1. Levey AS, Coresh J, Greene T, Stevens LA, Zhang YL, Hendriksen S, Kusek JW, Van
Lente F. Chronic Kidney Disease Epidemiology Collaboration. Using standardized serum
creatinine values in the modification of diet in renal disease study equation for estimating
glomerular filtration rate. Ann Intern Med. 2006 Aug 15;145(4):247–54.
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2. Levey AS, Greene T, Kusek JW, et al. A simplified equation to predict glomerular
filtration rate from serum creatinine (Abstr). J Am Soc Nephrol 2000; (11):155A
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PART C: Blood Collection - Order of Draw
Learning Objectives
1. To name the anticoagulants of different blood collection tubes

2. To explain the purpose of different types of additives and anticoagulants in the blood
collection tubes
3. To name the collection tubes required for common laboratory tests
4. To state the order of draw for multiple blood tube sampling
5. To explain the rationale of the order of draw
Introduction
When collecting blood specimens by venepuncture into evacuated tubes, the order of draw
should ALWAYS be followed to prevent erroneous results due to additive crossover. It is very
important that blood samples are not tainted, contaminated or handled in a manner that would
affect the quality or accuracy of the blood analysis. Incorrect order of draw can lead to
misdiagnosis of health condition or diseases.
Materials
 Blood collecting tubes with different anticoagulants and additives

 Tube rack
Method
1. Examine the blood collecting tubes.

2. Complete the following table.
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Tube Type
Order of
(Anticoagulant Applications
Draw
/ Additive)
Review Questions – Refer to the colour chart and answer the following questions
1. Why is it important to follow order of draw when collecting multiple blood samples?
2. What will happen if the ‘light blue top’ tube is filled after the ‘lavender’ and ‘dark green
top’ tube?
3. What will happen if the ‘light gray top’ tube is filled before the ‘lavender top’ tube?
4. Which is the most commonly used blood collecting tube for routine haematology tests?
Reference:
1. Estridge BH, Reynolds AP. Basic Clinical laboratory Techniques, 5th ed. Delmar: Cengage
Learning; 2008.
2. WHO Guideline on Drawing Blood: Best Practice in Phlebotomy. Geneva: WHO Document
Production Services; 2010.
29
Clinical Chemistry
PRACTICAL 2
PART A
I) Urinalysis: Physical Examination
Learning Objective
To describe the physical characteristics of urine
Introduction
The physical characteristics of urine are colour, transparency or clarity, specific gravity and
odour.
Materials:
Urine collecting bottle
Method
1. Obtain a fresh (recently voided) urine sample.

2. Record the identity of the specimen on the worksheet (report form).
3. Mix by swirling the urine gently. Pour approximately 10 ml of urine into a clear
centrifuge tube.
4. Observe the colour of the urine and record on the worksheet.
5. Note the odour of the urine. If unusual, record in the comment section.
6. Observe and record the transparency of the urine.
7. Save the sample for chemical examination
30
Results
PHYSICAL EXAMINATION WORKSHEET
Name: ____________________________ Date: ____________________

Specimen I.D. ______________________
Observation Result Reference Value

1. Transparency (appearance) ( ) Clear Clear
( ) Hazy (slight cloudy)
( ) Cloudy (turbid)
( ) Milky (opalescent)
( ) other _________________
2. Colour: Pale yellow to amber
3. Specific gravity: 1.005-1.030
Comment:
_____________________________________________________________________________
_____________________________________________________________________________
Review Questions
1. What gives urine its normal colour?

2. Name three metabolic conditions in which abnormal urine odour can occur.
3. Name three causes of cloudy condition.
4. Name four abnormal urine colours and give a possible cause for each.
31
PART A
II).Urinalysis: Chemical Examination
Learning Objectives
 To state 10 urine chemical tests routinely included in the urine test strip
 To explain the principle of each test
 To perform the chemical examination of urine using a reagent strip
 To state the quality assessment procedures in the chemical examination of urine
Introduction
Chemical testing by urine strips is part of routine urinalysis. Reagent strips consists of thin
plastic strip with reagent pads attached. By dipping the reagent strips in urine, chemical reactions
on each reagent pad is visible within a specified time. Strips used in routine urinalysis usually
include tests for pH, protein, bilirubin, blood, nitrite, ketone, urobilirubin, glucose, leukocyte
esterase, and sometimes gravity (SG).
Materials
 Urine collecting bottle

 Reagent strips
 Disposable plastic Pasteur pipette
 Waste bin
 Timer
Methodology
1. Obtain a urine specimen.

2. Mix the urine by gentle swirling.
3. Use a plastic Pasteur pipette to aspirate some urine from well-mixed urine sample. Apply
the urine slowly onto the urine strip. Make sure all pads are moistened.
4. Tap urine strip on container edge to remove excess urine. Blot the edge of the strip
quickly on absorbent paper. Begin timing.
5. Observe reagent pads and compare color to color chart on reagent strip reader at the
appropriate time internal.
6. Record results on the urinalysis worksheet.
32
Results
CHEMICAL EXAMINATION OF URINE
Name: __________________________ Date: ___________________

Specimen I.D. ____________________
RESULT
REAGENT STRIP REFERENCE VALUE
OBSERVED
Glucose Negative
Bilirubin Negative
Ketones Negative
Blood Negative
Ph 4.5-8.0
Protein Negative, trace
Urobilirubin 0.1 to 10 mg/dl (or EU/dL)
Nitrite Negative
Leukocyte esterase Negative
Specific gravity 1.005-1.030
Comment:
_____________________________________________________________________________
Review Questions
4. List the 10 chemical tests routinely performed on urine using the reagent strip method.
5. Name a condition that can cause the following substances to be increased in urine:
a. Protein
b. Ketones
c. Glucose
d. Bilirubin
e. Nitrite
f. Microalbumin
33
PART A
III) Urinalysis : Microscopic Examination
Learning Objectives
 To prepare urine specimen for microscopic examination

 To perform microscopic examination of an urine sediment
 To list cells, casts and crystals that can be found in urine sediment and give their
significance
Introduction
Microscopic examination of urine sediment is the third part of routine urinalysis. The test may be
omitted in some laboratory if the physical and chemical examinations are normal. Microscopic
examination can provide helpful information about infection, disease or trauma in the urinary
tract.
Materials
 Urine collecting bottle

 Glass slide and cover slip
 Disposal plastic Pasteur pipette
 15 ml centrifuge tube
 Benchtop centrifuge
 Microscope equipped with Polaroid set
Methodology
1. Obtain the urine specimen.

2. Pour 15 ml of well-mixed urine into a clean 15 ml centrifuge tube.
3. Centrifuge urine at 2000 rpm for 5 minutes.
4. Pour off the supernatant carefully. Leave approximately 0.5 ml of urine in the tube.
5. Resuspend urine sediment by tapping the bottom of the tube.
6. Place 1 drop of resuspended urine onto a clean glass slide.
7. Place a cover slip over drop of urine.
8. Observe the slide under microscope.
9. Record the observation.
34
Results
MICROSCOPIC EXAMINATION OF URINE
Specimen I.D.____________________________ Date: _______________
REFERENCE VALUE
White blood cells 0-4
Red blood cells 0-4
Epithelial cells Occasional (higher in female)
Casts Occasional, hyaline
Type present
Yeast (circle result) negative 1+ 2+ 3+ 4+ Negative
Bacteria (circle result) negative 1+ 2+ 3+ 4+ Negative
Mucus (circle result) negative 1+ 2+ 3+ 4+ Negative to 2+
Amorphous deposition ___ none seen ___ present
Crystal ___ none seen ___ present
Type present
Comment:
_____________________________________________________________________________
_____________________________________________________________________________
Review Questions
1. Explain the importance of the microscopic urine test.

2. Name four types of cells that can be seen in urine sediment.
3. Explain how casts are formed.
4. Name eight crystals that can be seen in normal urine sediment.
35
Clinical Chemistry
PRACTICAL 3
75g 2-Hour Oral Glucose Tolerance Test (OGTT)
Learning Outcome
1. To describe the OGTT procedure

2. To perform the OGTT
3. To state the criteria for diagnosis of Type 2 diabetes and glucose intolerance in Malaysia
4. To describe the algorithm for the screening of diabetes mellitus in Malaysia
Introduction
The glucose tolerance testing (GTT) is used to evaluate the ability to regulate glucose
metabolism and is indicated when random/fasting blood glucose testing alone is insufficient in
establishing or ruling out the diagnosis of diabetes mellitus.
The reference range of serum or plasma glucose is less than 11.1 mmol/L (140 mg/dL) at 2 hours
after a 75-g glucose load.
Indications and Contraindications for OGTT
Indications
The OGTT is indicated in following conditions:
 Patients having symptoms suggestive of diabetes mellitus, but fasting blood sugar value is
inconclusive (between 100-126 mg/dl).
 During pregnancy, excessive weight gaining is noticed, with a past history of big baby (more
than 4 kg) or a past history of miscarriage.
 To rule out benign renal glucosuria.
Contraindications
There is no indication of doing OGTT in following conditions:
 Person with confirmed diabetes mellitus.
 OGTT has no role in follow up of diabetes. It is indicated only for the initial diagnosis.
 The test should not be done in acutely ill patients.
36
Diagnosing Diabetes
 Diabetes may be diagnosed on the basis of one abnormal plasma glucose (random ≥11.1
mmol/L or fasting ≥7 mmol/L) in the presence of diabetic symptoms such as thirst,
increased urination, recurrent infections, weight loss, drowsiness and coma.
 In asymptomatic people with an abnormal random plasma glucose, two fasting venous
plasma glucose samples in the abnormal range (≥7 mmol/L) are recommended for diagnosis.
 The World Health Organization (WHO) now recommends that glycated haemoglobin
(HbA1c) can be used as a diagnostic test for diabetes.
 OGTT is not recommended as a screening test for diabetes mellitus.
Materials
 75g anhydrous glucose dissolved in 250-300ml water

 Sterile, disposable lancets
 Glucometer
 Well-fitting, non-sterile gloves
 Alcohol hand rub
 70% alcohol swabs
 Sterile gauze or cotton-wool ball
 Laboratory specimen labels
 Biohazard sharps container
 Biohazard container
Methodology
Instruction for the subject:
The subject should eat normally and maintain an adequate carbohydrate/starch intake (at least
150g/day, including potatoes, rice, pasta, bread etc.) and continue normal physical activity for at
least three days prior to the test.
The subject should fast overnight for at least 10 to 16 hours before the test (do not smoke and
drink caffeinated drink, only water is permitted for drinking) e.g. time of test: 9am then fast
from 11pm the night before.
Note: The test should not be performed when the subject is unwell, as this will affect the results.
37
Procedure
1. Record the presence of factors that influence interpretation of the results of the test
including medication, inactivity, infection and others.
2. Ask the subject to sit and rest for at least 5 minutes before the phlebotomy or blood
taking.
3. Take blood for measurement of fasting blood glucose. Mark tube as fasting blood glucose
(0-Hour sample).
4. Give the subject 75 g of anhydrous glucose. The glucose should be dissolved in 250 - 300
ml water and should be consumed over the course of 5 minutes (not more that 5 minutes)
followed by a further 100 ml water. Immediately after ingestion of glucose, record the
time.
Other acceptable alternatives:
• Lucozade – 394 ml (73 kcal/100 ml formulation)
• Maltodextrins of appropriate volume to provide 75 g carbohydrate (e.g. Calsip 150 ml)
5. The subject should remain sedentary (i.e. seated in waiting room) and should not smoke,
eat or drink for the duration of the test.
6. Take 2nd blood sample at 2-hour. Mark tube as 2-Hour sample.
7. If venous blood is used - send the two blood samples together to the lab. Select ‘Oral
Glucose Tolerance Test’ on the Test Order Form and note the identity of the blood
samples (i.e. 0-Hour and 2-Hour samples).
[If glucometer is used – measure blood glucose at the point of blood taking and record
results accordingly].
38
Review questions
1. State the criteria for diagnosis of (i) Type 2 diabetes and (ii) glucose intolerance
(impaired glucose tolerance, impaired fasting glucose & gestational diabetes mellitus) in
Malaysia.
2. Describe the algorithm for screening diabetes mellitus in Malaysia. Illustrate your answer
with a flow chart.
(Please refer to Clinical Practice Guideline of Malaysia)
75g 2-hr OGTT Worksheet
Reference range
Blood Sampling Time Test Results (mmol/L)
(mmol/L)
0 Hour <6.1
2 Hour <7.8
Comment on Results
____________________________________________________________________________
____________________________________________________________________________
39
Clinical Chemistry
PRACTICAL 4
PART A
Point of Care Testing: Glucometer
Learning Outcome
1. To measure the capillary blood glucose level using a glucometer

2. To interpret the results generated by the glucometer
3. To state the limitations of blood glucose measurement using glucometer
4. To discuss the factors affecting the results generated by the glucometer
Introduction
The blood glucose monitoring system comprises the meter (glucometer) and test strips. It is
intended to be used for quantitative blood glucose in fresh capillary blood. The system is suitable
for self-testing as well as for professional use. People with diabetes can use the system to self-
test and –monitor their blood glucose. Healthcare professionals can use the system to check
patients’ blood glucose and they can use it in suspected cases of diabetes and in emergency
diagnostics.
Materials
 Tray
 Blood glucose meter (glucometer)
 Test strips
 Lancet and lancing device
 Alcohol swab
 Dry swab
 Non-sterile gloves
 Receiver
 Sharp bin
40
Procedure:
A. Planning and Preparation:

 Check the patient’s medical record or nursing plan of care for monitoring schedule.
 Identify the purpose of blood glucose testing.
 Prior to performing the procedure, introduce self and verify the patient’s identity.
 Explain the procedure to the patient.
 Perform hand hygiene
B. Implementation and Rationale:

Implementation Rationale
1. Turn on the monitor. Enter the 1. Use of identification number allows for
patient’s identification number, if electronic storage and accurate
required, according to facility policy. identification of patient data.
2. Don non-sterile gloves. 2. Gloves protect the nurse from exposure
to blood or body fluids.
3. Prepare lancet using aseptic technique. 3. Aseptic technique maintains sterility.
4. Remove test strip from the vial. Recap 4. Immediate recapping protects strips from
container immediately. exposure to humidity, light, and
discoloration.
5. Insert the strip into the meter 5. Correctly inserted strip allows meter to
according to directions for that specific read blood glucose level accurately.
device. 6. Matching code numbers on the strip and
6. Check that the code number for the glucose monitor ensures that the machine
strip matches code number on the is calibrated correctly.
monitor screen
41
Inserting the strip into the glucometer Matching code number on the strip
with glucometer
7. Gently rub side of finger toward 7. Massage encourages blood to flow to
puncture site. the area.
8. Cleanse the skin with an alcohol swab. 8. Alcohol cleanses the puncture site.
Allow skin to dry completely. Alcohol can interfere with accuracy of
results if not completely dried.
9. Hold lancet perpendicular to skin and 9. Holding lancet in proper position
pierce site with lancet. facilitates proper skin penetration.
10. Encourage bleeding by lowering the 10. An appropriate-sized droplet
hand, making use of gravity. facilitates accurate test results.
11. Lightly stroke the finger, if necessary, 11. Squeezing can cause injury to the
until sufficient amount of blood has patient and alter the test result.
formed to cover the sample area on the
strip. Take care not to squeeze the
finger, not to squeeze at puncture site,
or not to touch puncture site or blood.
12. Gently touch a drop of blood to the test 12. Smearing blood on the strip may result
strip without smearing it. in inaccurate test results.
42
Holding lancet perpendicular to skin
and piercing site
Gently touching a drop of blood to

the test strip
13. Apply pressure to puncture site with a 13. Pressure causes hemostasis. Alcohol
cotton ball or dry gauze. Do not use stings and may prolong bleeding.
alcohol wipe. 14. Patient to be fully informed of actions
14. Read blood glucose results. Inform and any potential changes.
patient of test result. 15. Proper disposal prevents exposure to
15. Turn off meter, remove test strip and blood and accidental needle stick.
dispose of supplies appropriately.
Place lancet in sharps container. 16. Removing gloves properly reduces the
16. Remove gloves and dispose risk for infection transmission and
appropriately. contamination of other items.
17. Hand hygiene reduces the
17. Perform hand hygiene. transmission of microorganisms.
C. Evaluation
1. Patient’s blood glucose level is measured accurately without adverse effect.

2. Patient participates in monitoring and the patient verbalizes comfort with the procedure.
43
D. Documentation
1. Document blood glucose level.

2. Document patient assessments, any intervention related to glucose level, and any patient
teaching.
3. Report abnormal results and/or significant assessments.
Result
Sample ID Glucose level (mmol/L) Remark
Normal range:
7.8 – 11.1 mmol/L
Review Question
1. State THREE (3) clinical conditions that are associated with increased blood glucose
level.
2. State the advantages of using glucometer.
3. State factors that influence the blood glucose level.
4. State and discuss the factors affecting the results of a glucometer.
44
PART B
Urine Human Chorionic Gonadotropin (hCG) Test
Learning Objectives
 To explain the principle of modern pregnancy tests designed to detect human chorionic
gonadotropin (hCG)
 To perform a test for urinary hCG
 To discuss the possible causes of false-positive and false-negative results in urine hCG
test
 To discuss factors that must be considered when interpreting hCG test results
Introduction
Urine pregnancy tests are based on the detection of human chorionic gonadotropin (hCG) in
urine. Urine tests for hCG are designed to be sensitive, to be easy to perform and interpret and to
give rapid results.
Materials
 Urine samples of a pregnant woman

 Urine sample of a non-pregnant woman
 Disposal plastic Pasteur pipette
 Pregnancy test reagent strip
Methodology
Using One-Step Test Device
1. Placed the test device on a clean and level surface

2. Hold dropper vertically and transfer 3 full drops of urine (approximately 100 µl) to the
specimen well of the test device and then start the timer. (Refer to figure below)
3. Wait for the red line(s) to appear. The result should be read at 3 minutes. It is important
that the background is clear before result is read.
45
Figure shows the proper technique used to load the urine sample into the test (specimen) well
46
Results
Sample Y Sample Z
Illustration of the
result
Interpretation of
the result
Review Questions
1. Explain the principle of modern pregnancy tests designed to detect human chorionic
gonadotropin (hCG).
2. When does hCG first appear in pregnancy? When does it disappear?
3. What condition can cause a positive hCG test?
4. What can cause a false negative hCG test?
47
PRACTICAL 5
Measuring of Total Serum Bilirubin Using Bilirubinometer
Learning Objectives
 To describe the principle of the total serum bilirubin (TSB) test

 To perform the TSB test using Bilistick System Reader
 To interpret the TSB results
 To describe the limitations when performing the TSB test
Introduction
Total Bilirubin assay is used for the quantitative analysis of total bilirubin in whole blood or
serum of adults and neonates. Bilirubin is the orange-yellow pigment derived from senescent red
blood cells. It is extracted and biotransformed in the liver. Bilirubin is screened and monitored
for liver disorders such as jaundice or liver diseases such as cirrhosis. Total bilirubin
measurements are also used in the diagnosis and treatment of haematological disorders. Total
bilirubin measures the entire concentration of bilirubin in the blood.
The Bilistick System is a Point-of-Care diagnostic device that measures the Total Serum
Bilirubin (TSB) concentration in whole blood. It consists of the Bilistick Reader, a hand held
rechargeable battery reflectance reader; Bilistick Test Strips and Bilistick Sample Transfer
Pipettes. The test requires the collection of a small amount of blood sample (25µL) directly from
a finger-tip or a tube by using the Bilistick Sample Transfer Pipette.
Bilistick Reader
48
Bilistick Test Strips Bilistick Sample Transfer Pipettes
Materials
 Whole blood sample

 Bilistick reader
 Bilistick test strip
 Bilistick sample transfer pipette
Method
1. Place the Bilistick reader on the table and press the power On/Off button. (Never use the
reader in your hand)
2. Extract the Bilistick test strip from the bag. (Do not touch the nitrocellulose membrane)
3. Insert the Bilistick test strip in the reader with longitudinal movement until you hear a
‘click’ sound.
4. Prepare the Bilistick sample transfer pipette to collect the blood sample.
5. Perform the finger-tip prick and load the pipette. (Load the pipette by capillary, do not
squeeze the bulb)
6. Load the blood sample on Bilistick test strip. (Do not touch the strip with the pipette edge or
create bubbles to avoid haemolysis)
7. Press the ‘M’ Button to start measuring the bilirubin concentration.
8. Wait the measuring process ends to get the bilirubin result.
9. Read the result and the measuring time would be <2 minutes.
10. Remove the Bilistick test strip from the reader.
11. Dispose blood filled strip and pipette as hazardous waste.
49
Results
Sample ID TSB (mg/dL or umol/L) Remark
Limitations:
Haemolysis of the sample falsely decreases the direct bilirubin result.
Reference ranges:
0 – 2 weeks : 0.2-11.7 mg/dL
2 weeks – 10 years : 0.2-0.9 mg/dL
>10 years : 0.2-1.0 mg/dL
Review Questions
1. State THREE (3) clinical conditions that are associated with increased TSB level.
2. State the serious risk condition of the neonates with increased TSB level.
3. State the quality assessments for TSB lest and its limitation.
50
PRACTICAL 6
Immunological Assay
PART A
Flow Cytometry Analysis
Learning Objective
 To determine the percentage of mature T, B and NK lymphocyte populations as well as

CD4+ and CD8+ T-cell subset ratios in peripheral whole blood sample in a single-tube
format.
Introduction
Compositions (percentages and absolute count) of different lymphocyte subsets can provide
valuable information on immune status for the patients with various medical conditions including
cancer (i.e. leukemia), autoimmunity and immunodeficiency. For example, percentage and
absolute count of CD4+ lymphocytes are useful for monitoring disease progression and therapy
efficiency in patients infected with Human Immunodeficiency virus (HIV). In addition, abnormal
percentages of CD8+ have been associated to certain forms of autoimmunity.
Materials
 Peripheral whole blood

 BD Multitest 6-Color TBNK Reagent containing CD3 FITC, CD16/56 PE, CD45 PerCP-
CyTM5.5, CD4 Pe-CyTM7, CD19 APC and CD8APC-Cy7
 1X Lysing solution
 Washing buffer (1X PBS buffer supplemented with 0.2% bovine serum albumin)
 Centrifuge
 FACSCanto II Flow Cytometer
 FACSCanto Clinical Software
51
Method
1. Label the 5 ml centrifuge tube with corresponding sample ID.

2. Add 20 µl of peripheral whole blood to the respective 5 ml centrifuge tube.
3. Add 20 µl of BD Multitest 6-Color TBNK Reagent to the sample. Mix gently by vortex.
Incubate the sample at 4C for 15 minutes in dark.
4. Add 1 ml of 1X lysing solution and mix gently by vortex. Incubate the sample at room
temperature for 10 minutes in dark.
5. Add 1 ml of washing buffer. Invert the tube a few times to mix. Centrifuge the sample at
1500 rpm for 5 minutes. Carefully decant and discard the supernatant.
6. Repeat Step 5. Resuspend the pellet with 400 µl of washing buffer.
7. Acquire and analyze the sample with FACSCanto Clinical Software.
Results
Referring to the Laboratory Report, complete the table below:
Parameter Lymphocyte Subset Identity Percentage Value / Absolute Count

Lymph Event
CD3+
CD3+CD8+
CD3+CD4+
CD3+CD4+ CD8+
CD16+C56+
CD19+
CD45+
CD4+/ CD8+ Ratio
Review Questions:
1. What do you expect on the CD4 cell count, CD4:CD8 ratio and CD4 percentage of a
patient with progressive HIV infection?
2. What is the implication of increased CD16/46 cell count or percentage?
52
Example of BD FACSCanto Laboratory Report:
BD FACSCanto Laboratory Report obtain from BD FACSCanto Laboratory Report obtain from
a BD Multitest 6-color assay with BD TruCount a BD Multitest 6-color assay without BD
Tubes TruCount Tubes
Reference:
 BD Multitest 6-Color TBNK Reagent: Product Information Sheet

 BD Multitest 6-Color TBNK Reagent: Application Note
53
Part B
Serum Protein Electrophoresis
Learning Objective
 To identify and determine the electrophoretic pattern of serum proteins

 To discuss the implication of the outcome of the electrophoretic pattern of serum proteins
Introduction
There are more than hundred over types of individual proteins found in plasma. Each of these
proteins has a specific set of functions and subject to specific variations in concentration under
different physiologic and pathologic conditions. Plasma proteins interact with virtually all body
tissues and play important roles in many physiologic functions.
Serum protein electrophoresis is a method to separate proteins into multiple bands based on the
size and charge of the proteins. After that, protein bands are analyzed by densitometer to
calculate the concentration of protein in each band. Serum protein electrophoresis is commonly
used to distinguish hyperglobulinemia caused by either an innate and/or acquired immune
response or neoplastic transformation.
Materials
 Densitometer
 Applicator
 Sample well plate
 Aligning base
 Cellulose acetate support medium
 Chamber and wicks
 Staining set
 Evaporation hood
 Barbital buffer pH 8.6
 Porceau S stain
 5 % acetic acid
 Clearing solution: 125 mL of reagent grade methanol plus 50 Ml double distilled water
 Specimens (fresh unhaemolysed serum; urine and CSF)
Methods
1. Preparation of the gel tray

2. Preparation of the electrophoresis chamber
3. Sample application
4. Electrophoresis
54
5. Staining and visualization of the protein bands
6. Evaluation of the protein bands using densitometer
[Detailed illustration of the protocol is available at the following link:
https://www.youtube.com/watch?v=IcjREcjIpAY]
Results
1. Relative position of the protein fractions
AB CDE
Identify the protein fractions labelled in A – E above.

Protein Fraction Identity
A
B
C
D
E
55
2. Identify some commonly known proteins from a normal serum protein graph.
Give TWO (2) examples of commonly known proteins for protein fraction labeled B
– E.
Protein Fraction Examples

B i
ii
C i
ii
D i
ii
E i
ii
3. Quantify the various protein fractions in a sample

Total Protein = 8.2 g/dL
Protein Relative Quantity of protein fraction (g/dL) Reference values
Fraction percentage (g/dL)
(%)
A 59 3.63 – 4.91
B 5 0.11 – 0.35
C 8 0.65 – 1.17
D 10 0.74 – 1.26
E 19 0.58 – 1.74
*** Relative percent and g/dL of various protein fractions may be computed
automatically using a computer accessory with the densitometer.
56
Review Question
1. If plasma is used for electrophoresis, an extra peak will be detected. What would cause this
extra protein peak to occur in electrophoresed normal plasmas?
2. How does haemolysis affecting the serum protein electrophoresis result?
3. Suggest a clinical condition
Serum Protein Electrophoresis pattern Associated Interpretation

Medical
condition
1
57
4
58
Haematology and Transfusion Medicine
PRACTICAL 1
PART A
Haemoglobin Estimation
Learning Objectives
 To describe the principle of haemoglobin (Hb) estimation by using haemoglobinometer
 To perform haemoglobin estimation by using haemoglobinometer
Materials
 Haemoglobinometer (DiaSpect Hemoglobinometer T)
 Whole blood sample
 Measuring micro cuvette
 Disposable pipette
Principle
Type of measurement based on photometric method with innovative, microprocessor controlled
sensor system and wave length range 400nm-900nm. The analyser is factory calibrated against
ICSH reference method.
59
Method
1. Fill 10 ul of whole blood into measuring microcuvette by using disposable pipette.

2. Open the power and sensor lid.
3. Put this microcuvette onto sensor.
4. Close the sensor lid.
5. Read the result and the measuring time would be 1-2 seconds.
6. Reopen the sensor lid and dispose blood filled cuvette as hazardous waste.
Reference ranges:
 Adult Male : 13.5-17.5 g/dl

 Adult Female : 11.5-15.5 g/dl
60
PART B
Blood Grouping
Learning Objectives
 To describe the principle of ABO blood grouping

 To state the importance of ABO blood grouping
 To describe steps in ABO blood grouping
 To perform direct blood grouping
 To interpret the ABO blood grouping results
Introduction
The ABO blood grouping system was discovered around 1900 by Karl Landsteiner. All
individuals can be classified into one of the four major groups: A, B, AB or O. ABO grouping is
based on the reciprocal relationship between the antigens (designated A and B) on the red cells
and the naturally occurring antibodies in the serum.
Materials
 Whole blood specimen
 Commercial anti-A
 Commercial anti-B
 70% alcohol
 Microscope slides
 Timer
 Applicator sticks
Method
1. Obtain two slides, label A and B on each of the slide.

2. Place one drop of anti-A serum on slide A and one drop of anti-B serum on slide B. Do
not allow dropper to touch the slide.
3. Dispense one drop of whole blood on each slide using glass pipette. Do not allow the
pipette to touch the slide.
4. Mix the blood and the anti-serum using a disposable applicator stick on each slide A and
B.
5. Rock the slide gently for 2 minutes and observe for agglutination.
6. Record agglutination results on ABO worksheet below.
7. Determine the blood group.
8. Repeat steps 1 to 7 with other blood samples.
61
9. Discard all specimens appropriately. Disinfect the workplace with 70% alcohol.
ABO Worksheet
Name: Date:
AGGLUTINATION RESULTS* INTERPRETATION
Specimen I.D.
Anti-A Anti-B ABO GROUP
*Record results as: + = agglutination; 0 = no agglutination
Review exercise
State the blood type of the following blood grouping results:
*** Anti-D - Rh grouping reagent

***Control - normal saline
62
PRACTICAL 2
PART A
Blood Smear: Preparation and Staining
Learning Objectives:
 To describe steps to prepare a blood smear
 To describe steps to stain a blood smear
 To carry out blood smear preparation and staining
 To describe the purpose of staining the blood smear
 To describe the information obtained from a stained blood smear
 To state the safety precaution to be taken when preparing and staining a blood smear
Introduction:
The examination of a stained blood smear is a part of the routine examination of blood. Staining
is applied to the blood smear to facilitate viewing of the cellular components of the blood.
Materials:
Whole blood specimen
70% alcohol
Blood stain reagents: Wright’s Stain and buffer
Absolute methanol
Mounting reagent
Glass slides
Coverslip
Slide drying rack
Staining jar
Microscope
Pencil
Timer
Methodology:
Preparation of a blood smear
1. The frosted-end of a glass slide is labelled with the name of the subject, date and time of
collection.
2. Using a glass pipette and add a drop of blood on the slide.
3. Bring a clean spreader slide (clear glass slide), held at a 45° angle, toward the drop of blood
on the specimen slide.
4. Wait until the blood spreads along the entire width of the spreader slide.
5. While holding the spreader slide at the same angle, push it forward rapidly and smoothly.
63
6. Allow the blood smear to air dry quickly.
7. Place the air-dried blood smear in absolute methanol for 30-60 second and allow to air dry
completely before staining.
Staining of blood film
8. Place the dried smear on the staining rack.

9. Flood the smear with Wright’s stain and leave it on for 2 minutes.
10. Add buffer drop by drop to the stain until buffer volume is equal to that of the stain. Gently
mix the buffer and stain without touching the surface of the blood film on the slide. A
metallic sheen will appear on the surface of the staining solution mixture.
11. Leave to stand for 3 minutes
12. Rinse the slide with (distilled) water for 30 seconds
13. Dry the slide in a tilted position; do not blot-dry
14. Mount a cover glass.
15. Observe the slide under microscope.
16. Identify the cellular components of the blood film and complete the table below.
64
Cell Type Schematic Drawing Description
Red blood cell
Platelet
Monocyte
Leukocyte Neutrophil
Eosinophil
65
Basophil
Lymphocyte
66
PART B
White Cell Differential Count (Manual and Digital)
Learning Objectives:
 To describe the purpose of the white blood cell differential count
 To perform the white blood cell differential count
 To state the importance of the white blood cell differential count
Introduction:
The examination of a stained blood smear is part of the routine examination of blood. Staining is
applied to the blood smears to enable the technologist to view the cellular components of the
blood and identify any abnormalities present.
Methodology:

1. The software has been installed in the MRC1 computers.
2. The scanned slide images of the peripheral blood film slides are loaded in desktop.
image.
screen.
6. Scan the slide to observe the white blood cells using the 40 X objective.
7. Count 100 to 200 consecutive white blood cells (WBC). Moving the slide so that
consecutive microscopic fields are viewed. Refer to coning patter illustrated above.
8. Record on the total number of WBC and the differential WBC counts of each type.
9. Observe the red blood cells (RBC) in at least 10 fields. Note the haemoglobin content and
record as either normochromic or hypochromic.
10. Observe the size of the RBC. Record as normocytic, microcytic or macrocytic.
11. Observe the platelet in at least 10 fields.
67
BLOOD SMEAR EVALUATION WORKSHEET
Cell count, cells Average Cell

Cell Type Percentage, %
1 2 Count
White Blood Cell (WBC)

Lymphocyte
Neutrophil
Monocyte
Eosinophil
Basophil
Abnormality
Size ( ) Normocytic; ( ) Microcytic; ( ) Macrocytic

Red Blood Cell (RBC) Colour ( ) Normochromic; ( ) Hypochromic
Abnormality
Number ( ) Normal; ( ) Increased; ( ) Decreased

Platelet
Morphology
Reference ranges (Adult)
Type of Cells Differential Count Reference Value Absolute Count Reference Value
(%) (Cells/µl)
Neutrophil (seg) 50-65 2250-7150
Neutrophil (band) 0-7 0-770
Eosinophil 1-3 45-330
Basophil 0-1 0-110
Monocyte 3-9 135-990
Lymphocyte 25-40 1125-4400
Platelet An average of 7-20 platelet per oil immersion field is consider normal
68
PRACTICAL 3
PART A
Coagulation Test (Prothrombin Time, PT)
Learning Objectives
 To describe the extrinsic coagulation pathway
 To describe the principle of the prothrombin time (PT) test
 To perform the PT test with manual tilt-tube technique
 To calculate the INR value
 To list the safety precautions when performing the PT test
 To describe the quality assessment procedures for prothrombin time test
Introduction
Prothrombin time (PT) is one of the most common coagulation screening tests performed in
clinical laboratory. It measures, as a whole, the activity of the coagulation factors I, II, V, VII,
and X. The test measures the clotting time of the sample in the presence of calcium and
thromboplastin. Nowadays, coagulation tests including PT, APTT and TT are performed using
automated or semi-automated instruments. Nevertheless, manual tilt-tube technique is still
employed in laboratories to cross-check the reliability of the automated techniques. International
normalized ratio (INR) value is a better way to report PT results. The INR value is used to
standardize PT reporting among different laboratories to compensate for varying sensitivities of
different lots of thromboplastin reagent [International sensitivity index (ISI) is assigned to each
lot of reagent]. INR value is calculated using formula below:
Materials
 Whole blood sample, Plasma sample
 Glass tubes
 Plastic pipette
 Water bath at 37°C
 Stopwatch
69
Methodology
1. Collect 9 ml of whole blood into a citrate tube.

2. Centrifuge the blood at 2500 rpm for 10 minutes.
3. Transfer the plasma into a 15 ml centrifuge tube using a plastic pipette. Place the plasma
sample in the water bath. The plasma sample must be processed within 8 hours.
4. Label the glass test tube with the specimen identity (ID).
5. Add 200 µl of thromboplastin-CaCl2 reagent into each glass using plastic pipette.
6. Place the glass tubes in the water bath.
7. Add 100 µl of plasma. Mix the plasma and the reagents immediately by shaking; start the
stopwatch.
8. Allow the tube to stay in the water bath for 10 seconds.
9. Work quickly – pick up the tube, wipe the outside with tissue, start tilting the tube in
good light.
10. Stop the timer at the first sight of thickening of the moving liquid and record the time in
second (s) in the table below.
Results
Sample ID Clotting time (s) INR value
***Mean normal PT of the facility = 10.5 seconds

***ISI = 1.2
Review Questions
1. State THREE (3) clinical conditions that are associated with prolonged PT.
2. State the reference value for prothrombin time.
3. State THREE (3) quality assessments for PT test.
70
PART B
Erythrocyte Sedimentation Rate (ESR)
Learning Objectives
 To describe the Erythrocyte Sedimentation Rate (ESR)
 To perform the Erythrocyte Sedimentation Rate (ESR)
Introduction
The Erythrocyte Sedimentation Rate (ESR) measures the sedimentation of red cells in plasma.
Although the examination of Erythrocyte sedimentation Rate (ESR) is a non-specific test, its
measurement is clinically useful in disorders associated with increased production of acute phase
proteins.
Materials
 Trisodium citrate/ Heparin/ EDTA
 Westergren tube/ Wintrobe tube
 Teat or mechanical device
 Tube stand
 Test tube
 Micropipette
Methodology
I. WESTERGREN METHOD
1. The recommended tube is a straight glass tube and 30 cm in length & 25 mm in diameter.
The bore must be uniform to 0.05 mm throughout. A scale graduated in mm extends over
the lower 20 cm (0-200 mm). The tube must be clean and dry.
2. Trisodium Citrate is used as the anticoagulant diluent solution.
3. The test is performed on venous blood diluted accurately in the proportion of 1 volume of
citrate to 4 volumes of blood.
4. The sedimentation rate is reduced in stored blood. The test should thus be carried out
within 2 Hours of collecting the blood, although a delay of up to 6 hours is permissible
provided that the blood is kept at 4oC.
71
5. Mix the blood sample thoroughly and then draw it up to the Westergren tube to 200 mm
mark by means of a teat or a mechanical device (mouth suction should NEVER be used).
6. Place the tube exactly vertical and leave undisturbed for 60 minutes in a location that is
free from vibration and draught and away from direct sunlight.
7. Read to the nearest mm the height of the clear plasma above the column of sedimentated
cells. The measurement in mm is the ESR (Westergren/1hr).
Reference Ranges (Westergren Method):

Adults:
 Men under 50 years old: less than 15 mm/hr
 Men over 50 years old: less than 20 mm/hr
 Women under 50 years old: less than 20 mm/hr
 Women over 50 years old: less than 30 mm/hr
Children:
 Newborn: 0 to 2 mm/hr
 Newborn to puberty: 3 to 13 mm/hr
72
II. WINTROBE METHOD
1. Wintrobe tube is 110 mm in height.

2. Venous blood anticoagulated by Heparin or EDTA is used.
Reference Ranges:
 Male : 0-9 mm/1st hr

 Female : 0-20 mm/1st hr
73
Medical Microbiology
PRACTICAL 1 - 3
Learning Objectives: At the end of the sessions students should be able to
A) describe
 the type of specimen(s) to collect for the diagnosis of a specific infection
 the appropriate timing of specimen collection
 the optimum mount of specimen to collect
 the type of specimen container to use
 the correct transportation and storage of specimen before testing
 the information to enter into the specimen requisition form
 when to expect results
 what results to expect
B) interpret test results

C) discuss the utilisation of test results
Introduction
In the management of a patient with an infection, laboratory tests are often necessary to confirm
the clinical diagnosis and suggest appropriate antimicrobials for therapy. The procedures used in
a routine diagnostic laboratory include microscopy, culture, antimicrobial susceptibility testing,
serology, and molecular identification and prediction of antimicrobial susceptibility. A clear
understanding of test principles and limitations is necessary for the correct interpretation and
optimal use of test results.
Materials
 Case histories
 Laboratory diagnostic tools and reagents
 Bacterial and fungal cultures
 Stained and unstained smears for microscopy
 Test requisition forms
 Sample test reports
74
Methodology
For each case scenario,

 discuss the rationale for the choice of specimen(s) and laboratory test(s)
 describe test principles
 describe pre-analytical handling of specimens
 fill in the specimen requisition form
 carry out the procedure(s) specified for each case
 interpret test results
 discuss the utilization of test results in patient management
75
PRACTICAL 1
Blood sample : Collection & culture
Case 1. A young man known to be HIV-positive presents with high fever. A blood
culture is done.
Demonstrations:
 Aseptic skin preparation for blood culture
 Use of the BD BACTEC 9000 series Continuous Monitoring Blood Culture
system
Procedures to do on the mannikin:

 Aseptic skin preparation
 Venepuncture
 Collection of blood for culture
Discussion:
 Suitable sites for venepuncture

 Timing of blood collection
 Amount of blood to collect
 Type of blood container(s)
 Transportation and storage of blood specimen before testing
 Information to fill in the specimen requisition form
 When to expect results?
 What other investigations to request for?
 Advantages and limitations of the Continuous Monitoring Blood Culture system
76
PRACTICAL 2
Sputum from Pneumonia Patients
Case 2. Mr. TAR has been coughing out yellow and thick sputum. His doctor’s diagnosis
is community-acquired pneumonia.
Materials provided:
 Sputum sample
 Agar plate cultures
 Reagents for Gram and ZN staining
 Glass slides and coverslips
 Wooden applicator sticks
 Inoculation loops
 Normal saline
 Immersion oil
Demonstrations:
A) Gram staining procedure
Steps Staining reagents Holding time

1 - Flood fixed smear with Crystal violet One minute
- Drain off crystal violet and rinse off with
distilled water
2 - Flood with Lugol’s Iodine (Mordant) One minute
- Rinse off iodine with distilled water.
3 - Hold the slide at an angle and drop 95% Until the alcohol leaving the
ethyl alcohol onto it slide no longer has a purple
tint.
- Rinse with distilled water.
4 - Flood the slide with Safranin 2 – 3 minutes.
- Rinse with distilled water and blot dry
77
B) Ziehl Neelsen staining procedure
Step Reagent Holding time
1. - Flood fixed smear with Carbol fuchsin Heat intermittently, without

boiling the stain, for five minutes
- Rinse with water
2 - Add acid alcohol drop by drop Until red colour stops streaming
from the smear
- Rinse thoroughly with water.
3 - Apply Methylene blue or Malachite 1 minute

green counterstain
- Rinse with water and blot dry.
Procedures to carry out:

 Describe the sputum specimen collected.
 Describe the bacterial growth on the sputum culture plates.
 Make direct smears from the sputum for Gram and ZN staining and describe what
you see under the light microscope.
 Describe the macroscopic and microscopic appearance of the pathogen isolated.
Sputum bottle Fixed and stained smear
78
Gram smear microscopy ZN smear microscopy
Discussion:
 Methods of sputum collection

 Timing of sputum collection
 Type of specimen container
 Transportation and storage of specimen before testing
 What are the likely pathogens in sputum?
 How to distinguish sputum from saliva?
 What further investigations are necessary for the management of this patient?
79
PRACTICAL 3
Urine from UTI Patient
Case 4. Mrs Ummah complains of pain on micturition. Her urine is sent to the laboratory
for investigations.
Demonstrations:
 The urostrip urine viable count
 The standard loop urine viable count
 Cell counts with the Neubauer chamber
Materials provided:
 Urine collection container
 Midstream urine collection kit
 Urine test strips
 Urostrips
 Standard loops
 MacConkey and BloodAgar plates
 Lactose-fermenters and non-lactose-fermenters on MacConkey agar
 Neubauer chamber
 Cover slips

 Collect urine A (from student volunteer) for microscopy, urine strip testing and
urine viable count determination
 Collect urine B (from patient) for microscopy, urine strip testing and urine viable
count determination
Neubauer chamber for cell counts
80
Urine test strips Urine viable count with standard loop inoculation
Discussion:
 Methods of urine collection and their drawbacks

 Amount of urine to collect
 Types of urine containers
 Transportation and storage of urine before testing
 Information to fill in the specimen request form
 What factors affect the interpretation of urine viable counts?
 What factors are taken into consideration when deciding on the need for antibiotic
therapy for the patient?
81
PRACTICAL 4
Stool + Diarrhea and Skin Swab
Case 4(A). A toddler presents with a 3-day history of watery diarrhoea and abdominal
pain. A stool examination is done.
Materials provided:
 Container for stool collection

 Stool cultures on MacConkey, XLD (Xylose Lysine Deoxycholate) and TCBS
(Thioglycolate Citrate Bile Salt) agar
 Slides on parasite ova and cysts
Demonstration:
 Microscopy of stool sample for ova and parasites
 Hanging drop microscopy

 Microscopic examination of the slides provided
 Macroscopic and microscopic examination of the stool cultures
Discussion:
 Methods of stool collection
 Transportation and storage of specimen before testing
 Likely pathogens in stool
 Methods for the identification of pathogens in stool
 Differentiation of pathogens from the normal flora in stool
82
Parasitic forms in stool
Bacterial growth on different agar
83
Case 4(B). A patient presents with a gaping post-laparotomy wound.
Demonstrations:
 Collection of wound swab, pus aspirate and tissue for microbial culture
Wound Swab Collection Procedure:

 Perform hand hygiene and wear PPE
 Clean the wound using sterile water or saline. Irrigate the wound surface using
syringe and needle to flush out contaminating and colonizing bacteria
 Moisten the sterile swab (if the wound surface is dry) with normal saline
 Use a zigzag motion whilst simultaneously rotating the swab stick between the
fingers
 Sample the whole wound surface
 Place the specimen straight into the transport medium and label the container
 Complete the request form with relevant clinical information
 Place the specimen and request form into the Biohazard bag and send straight to
the laboratory for microbial culture
Collection of wound swab
84
Tissue Sampling Procedure:
 Obtain tissue sample from the deep part of the wound or base of the wound and
place it into a sterile screw capped container with few drops of saline to keep it
moist.
Pus Aspirate Procedure:

 Using a syringe, aspirate about 5ml of pus and syringe into a sterile screw-capped
bottle.
 If there is little pus, use a sterile swab to collect a sample from the cavity of the
wound and immerse the swab in the transport tube with transport medium.
 Send the specimen immediately to the lab.
Discussion:
 Compare the reliability of a wound swab vs pus aspirate vs wound tissue for the
diagnosis of wound infection
 What pathogens are likely to be in a hospital-acquired wound infection?
 What should be done to recover anaerobic pathogens in the wound specimen?
 What is the interpretation of a mixed growth from the wound specimen?
85
Case 4 (C). A farmer complains of a progressively expanding rash on his lower leg. A
swab is taken from the affected skin for microbial culture.
Demonstrations:
 Yeasts and molds on Sabouraud dextrose agar (SDA) plates
 KOH wet mount microscopy
 Lacto-phenol cotton blue (LPCB) wet mount microscopy
 Slide culture for fungal growth
 Subculturing a fungal colony
LPCB wet mount KOH wet mount

Scrapings taken from the edge of the lesion are
mixed with a drop of KOH on a slide and viewed
under low power beneath a cover slip.
Fungal culture on SDA Fungal slide culture
86
Discussion:
 Characteristic features of a fungal skin infection

 Factors affecting the macroscopic and microscopic features of fungi
 Advantages of slide culture for the identification of fungi
 Differentiation between fungal contamination and fungal infection
 Rapid diagnostic methods for fungal infections
Pneumocystis jirovecii cysts

stained with methenamine silver
ITS-based molecular identification

of fungal spp.
87
Medical Microbiology
PRACTICAL 5
Parasites
Learning objectives: At the end of the session student should be able to

1. Examine a blood film for identification of malaria parasites
2. Examine a blood film for identification of microfilaria
3. Examine a stool specimen for identification of intestinal parasites
Introduction
Human malaria parasites include:

1. Plasmodium falciparum
2. Plasmodium vivax
3. Plasmodium malariae
4. Plasmodium ovale
5. Plasmodium knowlesi
Microscopic examination of thin and thick blood film for malaria parasites
Material
 Demonstration slides of malaria parasites
Method
Collection of specimen
i) Blood drop from finger prick
ii) Venepuncture
Time for collection

 For P. falciparum = a few hours after the febrile paroxysm reaches its peak.
 For other malaria parasites = both during the febrile and afebrile periods.
88
Microscopic Examination of Malaria Parasites in Thin Blood Film
Ring Forms of P. falciparum Ring form of P. vivax
Ring Forms of P. ovale Ring form of P. malariae
89
Ring form of Plasmodium knowlesi
Plasmodium falciprum P. falciparum (gametocyte)

(Ring form + gametocyte form)
90
Different Species of Malaria Parasites with Different Stages
91
Microscopic Examination of Blood Film for Microfilaria
Introduction
Filariasis or elephantiasis
Adult worm = Wuchereria bancrofti
= found in lymphatic vessels and lymph nodes of man.
Embryo = Microfilaria of Wuchereria bancrofti
= found in peripheral blood
Materials
 Demonstration slides of microfilaria
Method
Collection of Specimen
i) Blood drop from finger prick
ii) Venepuncture
Time for collection

The blood should be collected between 10.00 pm and 2.00 am
Elephantiasis of Legs Microfilaria in Blood Film
92
Microscopic Examination of Stool Specimen for Intestinal Parasites
Materials
 Demonstration slides of intestinal parasites
Method
Specimen: Stool
Entamoeba histolytica (Cyst form) Entamoeba histolytica (Trophozoite form)
Entamoeba histolytica (Cyst + trophzoite forms)
93
Ascaris lumbricoides (egg) Ascaris lumbricoides (egg) Ascaris lumbricoides (egg)
Ascaris lumbricoides (adult) Ascaris lumbricoides (adult)
Trichuris trichiura Egg) Trichuris thichiura (adult male & female)
94
Hookworm (egg) Hookworm (egg)
Hymenolepis nana (egg) Hymenolepis nana (egg)
H. nana (scolex)
95

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LABORATORY MEDICINE MANUAL

C. Hematology and Transfusion Medicine

 To perform the observation of whole digitized sputum cytology slides

Topic: (1) Squamous cell carcinoma of lungs

An example of digital cytology image

Instruction for use of Digital Pathology Image:

Sputum Cytology Slide 1

Sputum Cytology Slide 2

Sputum Cytology Slide 3

1. List the indications of sputum cytology test.

Instruction for use of Digital Pathology Image:

Case 1: Myocardial Infarction

Photograph: Cross section of the left ventricle

1. Describe the gross pathologic change seen in the Photograph.

5. What are the sites (location) of myocardial infarction?

6. What are the contrasting features of subendocardial and transmural infarcts?

7. What are the sequential myocardial changes in myocardial infarction?

8. What are the non-atherosclerotic causes of myocardial infarction?

Photograph: Cut open aorta

1. Describe the gross pathologic changes seen in the Photograph.

2. List some complications of atherosclerosis of abdominal aorta.

Case 3: Pulmonary Tuberculosis

Photomicrograph: Ziehl-Neelsen stain of the Sputum

100x oil objective

2. What is granuloma? What is tubercle?

6. List the FIVE (5) causes of death in pulmonary tuberculosis.

Case 4: Lung Carcinoma

Photograph: Right lung specimen

3. List the major histologic types of lung cancer.

4. (i) What is the cell of origin of small cell carcinoma?

5. Name some sampling techniques for lung cancer.

6. What is liquid biopsy?

Instruction for use of Digital Pathology Image:

Liver disease Case 1: Liver cirrhosis

Photograph: The liver at autopsy

3. What caused his fluid wave?

4. What do the laboratory findings indicate?

5. What caused his death?

Liver disease Case 2: Liver carcinoma

Photograph: Resected liver specimen

1. Describe the gross pathologic change seen in the Photograph.

2. List THREE (3) macroscopic patterns of growth of hepatocellular carcinoma.

3. List FOUR (4) histologic patterns of hepatocellular carcinoma

5. Identify the following histopathologic features in the digital tissue section of

a. Tumour cells resembling hepatocytes, having vesicular nuclei with prominent

7. What are the risk factors for this neoplasm?

GIT disease Case 1: Pseudomembranous colitis

Photograph 1: Colonoscopy finding

i. Thin layer of fibrinopurulent debris (i.e. pseudomembrane) on the mucosa surface

3. What antibiotics can cause this?

4. What bacterial organism is likely present?

5. What toxins does this organism produce?

Photomicrograph 1: Gastric biopsy

H&E stain, medium magnification

Phototograph: Gastrectomy specimen

Histochemical stain: Mucicarmine

1. Describe the gross pathologic features seen in the Photograph.

3. i. Describe the histologic appearance of a signet ring cell carcinoma.

4. Give associated familial genetic factors.

Instruction for use of Digital Pathology Image:

Patient I.D./Name: __________ Date:

Patient I.D./Name: __________ Date:

Name: ________ Date:

Name: ________ Date: _

Specimen I.D.______________ Date: _

Patient I.D./Name: __________ Date: