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Introduction
a. Hospital Profile
The University of Malaya was initially set up in Singapore in 1949 as an amalgamation

of the two premier institutions of higher education in colonial Malaya and Singapore,
Raffles College and King Edward VII College of Medicine to serve the needs of the
colony. Following independence of Malaya in 1957, two autonomous divisions of the
university, one in Singapore and one in Kuala Lumpur, were established in 1959. The
government of Malaya expressed a desire to change the status of the Kuala Lumpur
division to a national University and following appropriate legislation, the University of
Malaya was established in 1962. The Singapore Division was renamed the University
of Singapore.
In 1961, the Board of Education, University Malaya had proposed that a medical
institution be established to cater to the shortages of medical doctors in Malaya at that
time.The first Faculty of Medicine in Malaysia was established in the University of
Malaya in 1963. Instruction started in 1964 with the first batch of students.
On 5th August 1968, the spanking new University Malaya Medical Centre
(UMMC) was officially opened by His Majesty the Yang Dipertuan Agung, Duli Yang
Maha Mulia Seri Paduka Baginda Yang Dipertuan Agung Tuanku Ismail Nasarudin
Shah Ibni Alharmahum Sultan Zainal Abidin.
The University motto, "Ilmu Punca Kemajuan" (Knowledge is the Key to

Success) and the UMMC and Faculty of Medicine continues to strive to the best of
ability to live up to this ideal. University Malaya Medical Centre (UMMC) is one of the
organisations in the Ministry of Higher Education. Main objectives of UMMC are health
services, learning and research.
UMMC strive to provide the best services and treatment. Efforts that are
ongoing are to provide medical services, learning and research with the best efficiency
to our customer.
b. Organization Chart
c. Vision and mission of hospital
Vision
UMMC aims to be a world renowned medical centre providing highest quality
healthcare, medical training and research according to International Standards.
Mission
UMMC is committed to providing the highest quality healthcare, training and research
in tertiary medical services, community and patient welfare services.
d. Objective and function of every department
Pathology Department is divided into two main division which are Anatomic Pathology
Department and Medical Laboratory Division.
Besides providing clinical services to University Malaya Medical Centre

(UMMC), the department is also responsible in training tutors and lecturers for MBBS
degree, BDS, BBiomedSc and postgraduates courses, Masters in Pathology, Medical
Science Masters in Clinical Pathology, Medical Science Masters, medical doctors and
philosophical doctors under the Faculty of Medicine, University Malaya.
The department also conduct researches and studies in relevant medical fields.
The Division of Laboratory Medicine (LMD) is a clinical diagnostic laboratory of

national and international repute, providing quality service of the highest order to its
customers through relevant, effective and up to date laboratory diagnosis and
consultancy.
The facility is located on the 4th and 6th floors, Menara Timur, UMMC while a
satellite laboratory operates in the clinic area on the ground floor of the Primary
Medical Care (RUKA) Building.
1) THE CORE LABORATORY SERVICES

This is a composite of several disciplines providing routine diagnostic
service. These disciplines are grouped together on the basis of similar workflow in their
operations include:
- Routine Clinical Chemistry
- Fluids and Excretion
- Routine Haematology
2) Special Laboratory Services

Several areas function as specialized laboratories in view of the nature
of work, which are performed, requiring specific staffing and methodologies. These
include:
Endocrine Laboratory
This laboratory handles chemical and immunological tests that require
special techniques and include hormones and tumour markers.
Inborn Errors of Metabolism (IEM) Laboratory

Investigation of Inherited Metabolic Disorders or Inborn Error of
Metabolism including urine screening, amino acid and organic acid analyses, GAG
Quantitation and GAG electrophoresis and sweat test.
Immunology
This laboratory provides Immunology service including
immunochemistry, protein electrophoresis, auto antibodies screen, monoclonal
gamma globulin detection and quantitation of lymphocyte subsets.
Special Chemistry
Chemistry tests which are more complex and not provided on a 24-hour
basis are conducted here. This includes therapeutic drug monitoring (in conjunction
with Pharmacy Department), HbA1c and catecholamines.
Special Haematology
This laboratory handles Haematology tests that are more esoteric and
in-depth and include specific coagulation and haemoglobinopathy studies.
Bone Marrow Studies

These involve analysis of bone marrow samples including morphological
and immunophenotyping studies.
Molecular and Genetic Analysis Laboratory

This laboratory provides cytogenetics and molecular diagnostics for
various haematological and non-haematological disorders including histocompatibility
and immunogenetics testing.
Toxicology
This laboratory provides the services in the area of clinical Toxicology.
3) POLYCLINIC LABORATORY SERVICE

The division also operates a satellite laboratory, named The Polyclinic
Laboratory, located at the Rawatan Utama Building and serves all the outpatient areas.
It carries out basic Routine Haematology and Fluids and Excretion examination
including urinalysis and seminal assays.
PARASITOLOGY
LABORATORY
Department profile
Parasitology department University Malaya
The Department is responsible for teaching Medical Parasitology to the 2nd year pre-
clinical MBBS students.
With the rapid development in infrastructure and the introduction of new

courses, the Department of Parasitology also contributes in teaching other courses at
Diploma, Bachelor of Science and Master levels. Among the courses taught at the
Diploma level are Nursing and Medical Laboratory Technologist. At the first degree
level are the MBBS Phase II, Pharmacy and Biomedical Science courses. Courses at
the postgraduate level include the Master in Pathology (MPath) and Master in Public
Health (MPH). The Department has postgraduate students pursuing their Master by
research and PhD courses.
Since this Department was established, it has produced many Master and PhD
holders through research both locally and internationally. This Department has also
been involved in offering consultation and teaching to Royal College of Medicine Perak
(RCMP), Kolej Universiti Islam Malaysia (KUIM), UiTM and Institute of Medical
Research, Malaysia.
This Department is also a reference centre for parasitic diseases for

government and private hospitals from around the country. We hope, one day this
Department will be recognized as aNational Diagnostic Centre for Parasitic Infections.
The Department also provides services in detection of Cryptosporidium spp.

oocysts andGiardia spp. cysts in recreational and sewage treatment plants.
Changing and upgrading teaching and research methodologies have to be

continuously implemented in the department in order to meet the various demands. In
this context, the product and diagnostic analysis achieved would be the bench mark
(gold standard) that is acceptable by the clients.
Organization chart
HEAD OF DEPARTMENT
PROF DR. SURESH KUMAR GOVIND
PhD [NUS], MSc [Mal], BSc [Campbell]
PROFESSOR ASSOCIATE PROFESSOR
PROFESOR DATIN
Assc. Prof Dr.
PROF DR. INDRA A/p Assc. Prof Dr.
PROF DR. FONG Assc. Prof Dr. Init Zurainee Associate Prof.
DR. ROHELA VYTHILINGAM Prof Dr. Yvonne Veeranoot
MUN YIK Ithoi Mohamed Nor Dr. Lau Yee Ling
MAHMUD MSc [Liverpool], Lim Ai Lian Nissapatorn
PhD, MSc, BSc PhD, MSc and BSc Phd [Strathclyde, PhD, MMedSc,
MBBS,UM [MSia], PG MMed PhD, BSc [UKM] MCTM [Mahidol] ,
(Hons) [Mal] (Hons) [Mal] UK], BSc [Mal] BSc [Mal]
MPH & TM [USA] [Dundee], MBBS MBBS [Delhi]
taicc@um.edu.my
[Mal]
Name of test: Formol ether concentration for intestinal parasites
Principle:
The stool is emulsified in 10% formal saline (for fixation and preservation of parasites),
filtered to remove large particles and ether is added to remove fats and oils. After
centrifugation, a fatty plug, which may adhere to the inner walls of the tube, can be
seen at the interface of the two liquids. The ether layer, the fatty plug and the formalin
below it are discarded and the whole pallet retained for examination with bright field
microscopy. A stool in which organisms have been found previously in a concentrate
(positive control) is included for concentration with each day’s samples. If organisms
are found in the concentrate of the positive control, the method is deemed effective.
Parasites will settle more rapidly if the stool suspension is subjected to

centrifugation, however, partly digested food particles will also sediment more rapidly
and can mask the presence of parasites in the film examined. To overcome this
potential problem, larger food particles can be removed prior to centrifugation by
filtering the emulsified stool through a sieve with an aperture size large enough for
parasites to pass through, but which retains the larger food particles. As this process
is more efficient than sedimentation by gravity a smaller faecal sample (500 mg – 1 g:
the size of a pea) is sufficient for examination. Although centrifugation concentrates
the material more quickly, faecal debris which can obscure parasites is present. The
efficiency of detection is increased by adding formalin for fixation and preservation of
parasites, and ether to remove fats and oils. After centrifugation, a fatty plug, which
may adhere to the inner walls of the tube, can be seen at the interface of the two
liquids. The ether layer, the fatty plug, and the formalin below it are discarded and the
whole pellet retained for examination.
Many modifications to this procedure have been advocated, and the following
protocol, based on the method of Allen and Ridley, is typical of the method used in
diagnostic laboratories. Less distortion of protozoan cysts occurs with this method than
with zinc sulphate flotation. This method achieves a concentration of 15 to 50 fold;
dependent upon the parasite sought, and provides a good concentrate of protozoan
cysts and helminth eggs, which are diagnostically satisfactory. Disposable gloves are
worn for this procedure.
Internal quality control:

In order to ensure that this procedure is effective at concentrating parasitic organisms
on a daily basis, a positive control is added to each day’s work. The positive control is
the first sample to be examined microscopically, and if organisms are present in that
concentrate, the method used that day is deemed to be effective. In general, stools
containing Giardia cysts are used as the positive control, but stolls containing other
parasitic organisms which are concentrated by the formal-ether method can be
substituted when available.
Materials:
Disposable gloves, 15 ml conical glass centrifuge tubes, disposable wooden applicator
sticks, sieve (425 µm aperture size 38 mm diametera), 50 ml Pyrex beakerb, centrifuge
with 15 ml swing-out buckets, glass microscope slides (76 x 26 mm), coverslips (22x32
mm), diamond marker, bright field microscope with x10 and x40 objective lenses, 10%
formalin (10% of 40% formaldehyde in water), diethyl ether (or ethyl acetate), Lugol’s
iodine (iodine crystals 5.0 g, KI 10g, distilled water 100 ml. Dissolve KI in distilled water,
then add iodine crystals). Store in a dark glass bottle out of direct sunlight where it will
remain stable for several weeks.
Procedure:
Perform all steps (excluding centrifugation) in fume cupboard.
1. Wear gloves. Sample approximately 500 mg – 1 g faeces with an applicator
stickc and place in a clean centrifuge tube containing 4 ml of 10% formal
saline. If the stool is liquid, dispense about 500 ml into the centrifuge tube.
2. Break up the sample thoroughly and emulsify with an applicator stick.
3. Filter the resulting suspension through a sieve/gauze (two layers) into a
beaker and pour the filtrate back into the same tube a,d. Add 3 – 4 ml formal
saline.
4. Add 2.5 ml of diethyl ether (or ethyl acetate e) to the formalinized solution, seal
the neck of the tube with a rubber bung (or gloved thumb over the top of the
tube) and shake the solution vigorously for 30 sec. Invert the tube a few times
during this procedure and release the pressure developed gently by removing
the rubber bung (or your thumb) slowly.
5. Centrifuge the tube at 1500 rpm for 30 sec and continue with 2000 rpm for
another 30 secf.
6. Loosen the fatty plug with a wooden stick by passing the stick between the
inner walls of the tube and the plug.
7. Discard the plug and the fluid both above and below it by inverting the tube
allowing only the last one drops to fall back into the tube. Resuspend the
pellet by agitation.
8. Put one or two drops of the resuspended pellet onto a microscope slide, with
a Pasteur pipette, apply coverslip, and examine for the presence of parasites
using the x10 objective lens.g
9. Identify and definitive morphological features under the x40 objective. h
10. Assess the numbers of parasites present.i (if requested)
a 425 µm aperture size, 38 mm diameter.

b The skirt of the sieve should fit neatly into the rim of the beaker.
c The sample should include portions from the surface and from within a formed stool.
d Debris trapped on the sieve is discarded. Both the sieve and the beaker should be
washed thoroughly in running tap water between each sample.

e Ethyl acetate, although less flammable than diethyl ether is nevertheless flammable,
therefore the procedure should be performed in well ventilated areas, ensuring that
they contain no naked flames. Avoid prolonged breathing or skin contact.
f Centrifugation at speeds higher than 750 x g for long periods of time is not advised
since some helminth ova may rupture and collapse at higher centrifugal speeds.
g Too large a pellet is indicative of one or more of the following: centrifuging above the
recommended speed and/or time, insufficient shaking, taking too large a faecal sample.
h If protozoan cysts of the correct size and shape can be seen, but no diagnostic
inclusions can be diagnosed, add a drop or two of Lugol’s iodine either to the fluid at
the edge of the coverslip, and re-examine the preparation when the iodine has diffused
into the fluid under the coverslip (about 5 mins), or to the re-suspended pellet from
another concentrate prior to applying the coverslip. Lugol’s iodine stains nuclei and
glycogen masses in cysts yellow to brown. Lugol’s iodine preparations should be
viewed within 15 mins of preparation, otherwise over staining of cyst inclusions will
occur.
i Numbers can be recorded as scanty, moderate, or numerous.
Result:
Interpretation of result:
Name of test: Identification of nematode larvae in stool
Principle:
To differentiate between Strongyloides stercoralis larvae and hookworm.
Normally Strongyloides stercoralis larvae are the only parasitic larvae found in freshly
voided stools. These are usually the rhabditiform larvae (although the filariform larvae
can be found occasionally). If there is a delay in examining stools (e.g. postal delay,
etc.) embryonated eggs and larvae of hookworms can also present.
Therefore, for every stool sample that is positive for nematode larvae, a series
of stool culture technique is carried out. The culture methods that have to be done is
test tube cultures (Harada-Mori cultures)
Material:
Procedure:
Test tube cultures (Harada-Mori cultures)
1. Spread a thin (1 – 2 mm) film of faeces on one side of the middle third of a 13
x 120 mm strip of filter paper.
2. Place it in a 15 ml conical tip centrifuge tube containing about 3 ml distilled
water, forcing the filter paper to about the 1 cm level, and press the clean
slide against the side of the tube. Store the culture at room temperature (24 –
28°C) in the dark.
3. Examine culture for species confirmation and add water daily as needed to
keep the water level well above the bottom end of the filter paper up to 10
days.
The upward capillary flow of water through the paper and faecal film keeps the faeces
moist and carries soluble elements of the faeces to the top of the paper where they
either evaporate or accumulate in a dark deposit. On reaching the infective stage, the
larvae migrate downward against the upward flow of water. When they reach the water
pool, they sink to the bottom where they can be seen with a hand lens and taken up
with a pipette for microscopic examination.
Result:
The following table contains the distinguishing features of these larvae.
1st stage larva Infective (3rd) stage larva

Size Buccal cavity Oesophagus Size Morphology
Strongyloides 200 – 250 x 16 Short Large, bulbed 550 x 20 µm Filariform
stercoralis µm larvae
(rhabditiform) unsheathed
Hookworms 280 – 300 x 17 Long 660 – 720 µm Filariform
µm including larvae
(rhabditiform) sheath sheathed
Enterobius 145 x 10 µm N/A N/A
vermicularis
Name of test: Giemsa Stain
Principle:
Thin air dried blood films are stained in fresh Giemsa stain diluted in buffered water at
pH 7.2. Stained slides are differentiated using buffered distilled water, air dried and
examined under 10x, 40x, and 100x objective of bright field microscope.
Material:
Microscope slides, coverslip, pipette, coplin jars or similar, bright field microscope,
immersion oil, disposable gloves, methanol (94%), Giemsa stain, buffered (pH 7.2)
distilled water (buffered 150mM PBS, pH 7.2 can be used instead of buffered distilled
water), prepare fresh stain for every batch of smears
Procedure:
Undiluted Giemsa Stain Preparation

1. Giemsa powder 3.8g, methanol 250ml, glycerine 250ml.
2. Powder is placed in a mortar, grind thoroughly.
3. Add glycerine to mortar a little at a time, grinding and mixing with each
addition.
4. Add about half of methyl alcohol, continue to grind and mix.
5. Pour contents of mortar into a glass stoppered Pyrex bottle.
6. Pour remainder of methyl alcohol into mortar, mix and grind up residue of the
stain and add whole contents of the mortar into the bottle.
7. Incubate for 24 hours at 37°C giving bottle an occasional shake.
8. Filter through Whatman’s No. 1 filter paper before dispensing into bottles.
9. Giemsa stain is used diluted with buffered water (pH7.2).
- Preliminary fixation of peripheral blood film is necessary.
Staining method (for thin smear)

1. Fix air dried film in methanol for ½ - 1 minute.
2. Dilute Giemsa stain with buffered distilled water (pH7.2) in ratio of 1:3.
- equivalent to 3 drops of Giemsa stain to 1 ml of buffer.
3. Place slide with film side upwards on a horizontal staining rack (for large
number of slides, coplin jar can be used) and pipette 1.5 ml of stain carefully
to cover the film.
4. Stain for 35 minutes.
5. Rinse slide gently in running tap water for 3 – 4 seconds.
6. Place slides at an angle to drain and dry, do not blot.
7. Examine slide.
- Tail of PBF should be examine, RBC separated, one cell thick.

- Two slides should be examined before declare negative.
Staining method (for thick smear)
1. Dip the slide vertically into tap water for 1 minute. Then air dried.
- To hemolyze blood cells, so only parasites, ghost RBC, WBC remain on
slide.
3. Dilute Giemsa stain 1:3 with buffered distilled water (pH 7.2).
- equivalent to 60 drops of Giemsa stain into 20ml of buffered distilled water of
pH 7.2.
4. Place slide with film side upwards on a horizontal staining rack, and pipette
1.5 ml of stain carefully to cover the film.
- If large amount number of slides, coplin jar may be used.
5. Stain with Giemsa for 35 minutes.
6. Rinse slide gently under running tap water for 3 – 4 seconds.
7. Place slide at an angle to drain and dry, do not blot.
8. After slide is dried, examine the slide.
Result:
Microscopic examination: Malaria parasite ring form
Components Color
Chromatin Pink/red
Cytoplasm Purple/blue
Name of test: Slide preparation (Thin)
Principle:
1. To stain thin and thick blood films with Giemsa
2. To identify morphologies for all stages of Plasmodium spp.
3. To differentiate white blood cells from all stages of Plasmodium spp.
A drop of patient’s blood sample is spread across a glass slide with a cover slip. This
allows the blood film to be stained with dye and allows visualization of parasite
infestation under microscope. Due to the spreading action during preparation of blood
films (thin), it allows blood cells to spread out, causing blood cells to be distributed
evenly across glass slide, and at 1 blood cells thick. Blood cells can then be observed
easily for manifestation of parasite. Species of parasite can even be identified in a
good blood film preparation.
Material:
Glass microscope slides, mortar, wooden applicator sticks
Procedure:
1. Mix vacutainer containing blood with anticoagulant gently. Do not introduce any air
bubbles.
2. A wooden applicator stick was used, apply 1 drop of blood on a microscope slide.
3. Place a clean slide at an angle of 30° to the horizontal and bring it to the edge of
the drop of blood, when this slide touches the edge of the drop of blood, the blood
will spread along the width of it. Before the blood reaches the edges of the slide,
pull the drop along the length of slide. With the correct amount of blood being pulled
to a film, the film would form a good tail before the end of slide. Here, the film
should be 1 erythrocyte thick.
4. Prepare 2 slides.
5. PBF was left to air dry.
- Glass slide must be clean and free from dirt, grease, and fingerprints. Prior to use,
clean slides with methanol. Always handle slides by their edges.
Result:
Thin blood film preparation stained with Giemsa stain
Name of test: Slide preparation (Thick)
Principle:
A drop of patient’s blood sample is spread across a glass slide with an applicator stick.
This allows the blood film to be stained with dye and allows visualization of parasite
infestation under microscope. Thick blood film is prepared mainly for use of
parasitology screening. This is due to larger amount of blood used, giving an easier
identification of parasite infection
Material:
Glass microscope slide, marker, wooden applicator stick, disposable gloves
Procedure:
1. Mix the bottle containing blood with anticoagulant gently. Do not introduce any air
bubbles.
2. Using a wooden applicator stick, apply 1 drop of blood on a microscope slide.
3. The blood drop is spread with a circular motion using the edge of another glass
slide over an area approximately 2 cm in diameter or as big as a 5 cent coin.
4. Air dry slide at room temperature.
- Microscope slide must be clean and free from dirt, grease, and fingerprints. Prior to
use, clean slides in methanol. Always handle slides by their edges.
Result:
Microscopic examination: Thick blood film stained with Giemsa stain under
microscope (100x)
Name of test: Estimation of percentage of paraistemia in thin blood films
Principle:
Thin blood films stained with Giemsa stain is observed under microscope at 100x
magnification. Then, RBC infected with parasite is count. Estimation of parasitemia
can have several application, such as severity of infection, or in other way round,
recovery rate, and/or effectiveness of particular treatment or management.
Material:
Light microscope, think blood film stained with Giemsa stain
Procedure:
Areas of the thin blood film, where the erythrocytes are 1 cell thick, not overlapping
but touching, should be examined. The number of erythrocyte in a field (under x100)
should be counted and estimation made on 10 fields. In the enumeration of percentage
(%) parasitemia, an erythrocyte is regarded as being parasitized immaterial of whether
it is infected with one or more parasites.
The number of parasitized erythrocytes seen in peripheral blood (parasitemia) is very

important in cases of infection with Plasmodium falciparum. If the paraistemia exceeds
10%, exchange blood transfusion may be indicated. Estimation of parasitemia can
also provide indication of the effectiveness of treatment.
The number of parasitized cells should be counted in 10 fields and an average taken.
This figure should be divided by the average number of erythrocytes per field and
multiplied by 100, the figure derived being quoted as the percentage (%) of parasitized
erythrocyte.
𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑖𝑧𝑒𝑑 𝑒𝑟𝑦𝑡ℎ𝑟𝑜𝑐𝑦𝑡𝑒 𝑝𝑒𝑟 𝑓𝑖𝑒𝑙𝑑

𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑚𝑖𝑎 (%) = × 100
𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑒𝑟𝑦𝑡ℎ𝑟𝑜𝑐𝑦𝑡𝑒 𝑝𝑒𝑟 𝑓𝑖𝑒𝑙𝑑
- Alternatively, number of parasites in 1000 erythrocytes can be counted

- Parasitemia of under 1% are normally recorded as <1%
Result:
Name of test: Identification of microfilaria in blood
Principle:
1. Technique of staining blood films with Giemsa for microfilariae identification
2. Knott’s techniques for concentration of microfilariae in blood samples.
Material:
Glass slide, diamond marker, wooden applicator sticks, gloves
Procedure:
1. Mix vacutainer containing blood with anticoagulant gently. Do not introduce
any air bubbles.
2. Using a wooden applicator sticks, apply 1 drops of blood on microscope slide.
3. The drop of blood was spread with a circular motion using edge of another
glass slide over an area approximately 2 cm in diameter or as big as 5 cents
coin.
4. Dry slide at room temperature
- Microcscope slide must be clean and free from dirt, grease, and fingerprints. Prior to
use, clean slides in methanol. Always handle slides by their edges.
Staining
1. Dip the slide vertically into tap water for 1 minute. Then air dried.
- To hemolyze blood cells, so only parasites, ghost RBC, WBC remain on
slide.
3. Dilute Giemsa stain 1:3 with buffered distilled water (pH 7.2).
- equivalent to 60 drops of Giemsa stain into 20ml of buffered distilled water of
pH 7.2.
4. Place slide with film side upwards on a horizontal staining rack, and pipette
1.5 ml of stain carefully to cover the film.
- For large amount number of slides, coplin jar may be used.
5. Stain with Giemsa for 35 minutes.
6. Rinse slide gently under running tap water for 3 – 4 seconds.
7. Place slide at an angle to drain and dry, do not blot.
8. After slide is dried, examine the stained film using objective lens x10, x40, and
x100.
Result:
Name of test: Macroscopic Examination of Faeces
Principle:
Stools with evidence of blood and mucus may contain development or reproductive
stages of protozoa and are examined with the minimum of delay by direct microscopy
to ensure that these stages are still intact and motile. Where possible, blood or mucus
should be examined separately as they are more likely to contain trophozoites.
Examine the stool macroscopically and record whether it is formed, soft, unformed, or
liquid; whether it contains blood or mucus. Note the color of the stool.
Analyses of Faeces for the presence of protozoan parasites

Examine the stool macroscopically whether:
- It is formed, soft, unformed, or liquid.
- It contains blood or mucus.
- Note the color of the stool.
Stools with evidence or blood or mucus may contain the developmental or

reproductive stages of protozoa and are examined with minimum of delay by direct
microscopy to ensure that these stages are still intact and motile. Where possible,
blood or mucus should be examined separately as they are more likely to contain
trophozoite.
Formed stools without any evidence of blood or mucus are normally examined
following concentration within 24 hours of passage, and are stored at 4°C until then.
When long storage or transmit times, which can result in the deterioration of protozoan
morphology are anticipated, the use of a preservative should be considered.
Material:
Stool sample in appropriate urine container
Procedure:
Result:
Name of test: Identification of parasites (ova, cysts (including vegetative stages),
oocysts, larvae) in stool by direct microscopy
Principle:
A small sample of liquid (or emulsified formed) faeces is smeared onto a clean labelled
microscope slides and a coverslip is added. The entire sample is scanned under 10x
objective of a bright field microscope. Any object resembling an intestinal parasite is
examined under x40 objective. Lugol’s iodine can be used to enhance the
morphological appearance of some cysts, especially where cysts of the correct size
and shape which have no diagnosis inclusion are seen. Lugol’s iodine is not useful for
parasite other than cysts.
Material:
Gloves, microscope slides and coverslips, Pasteur pipettes and teats, disposable
wooden applicator sticks for emulsifying stool, bright field microscope with x10 and
x40 objective lenses, 150 mM NaCl, Lugol’s iodine (iodine crystals 10.0 g, KI 2.0 g,
distilled water 100 ml. Dissolve KI in distilled water, then add iodine crystals). Store in
a dark glass bottle out of direct sunlight.
Procedure:
1. Score reference number of specimen on a microscope slide, and use separate
microscope slide for each stool specimen. Place 1 drop of saline (about 50 µl) in
the center of the slide.
2. Remove a small sample of faeces (about 2 mg) with the tip of a clean applicator
stick, (or pipette if liquid) and emulsify the sample in saline by thorough mixing. For
liquid stools, mucus strands and exudates or pus can be mixed with saline on the
microscope slide. Ensure that the smear is of the correct transparency.
3. If smear is too thin or thick, parasites will be missed. An acceptable thickness can
be achieved when either the hands of your watch or print on newspaper page can
just be read when viewed through the preparation.).
4. Place a coverslip on top of the faeces suspension and examine the slide using x10
objective. If a cyst/oocyst like object is seen, examine it under x40 objective.
5. If objects of the correct size and shape, but no diagnostic inclusion can be seen,
place a drop of Lugol’s iodine at one edge of the coverslip and allow it to diffuse
under the coverslip and allow it to diffuse under the coverslip in order to stain the
putative parasites.
6. Lugol’s iodine stains the cytoplasm of the cyst yellow and the nuclei yellow to brown.
Lugol’ls iodinepreparation should be viewed within 15 minutes of preparation,
otherwise overstaining of cyst inclusion will occur.
Result:
Microscopic examination: Egg under microscope

Name of test: Isolation and in vitro culture of Blastocystis spp.
Principle:
Faeces sample suspected of containing Blastocystis spp. is introduced into Jone’s
medium and incubated for one day. The cultured fluid will be examined under bright
field microscopy for identification.
Material:
Stool sample in appropriate urine container
Jone’s medium
Procedure:
Prepration of Jone’s medium
1. Mix together the following substances:
a. Na2HPO4 1.244 g
b. KH2PO4 0.397 g
c. NaCl 7.087 g
2. Add and top up these mixture with 962.50 ml of distilled water.
3. Mix well.
4. Take out 12.5 ml and throw it away.
5. Autoclave the mixture.
6. Add 100 ml of 1% (1 g in 100 ml water) yeast extract.
~900 ml + 100 ml yeast = 1000 ml
7. Change the pH to 7.00.
8. Dispense the solution into sterile screw caped bottles and autoclaves them at
121°C/30 minutes. (Dispense the medium 90 ml to add 10% horse serum later
when to use them)
In vitro culture method

1. Place a small amount of faecal sample into 5 ml culture tubes containing 3 ml
of Jone’s medium (1.244 g of Na2HPO4; 7.087 g NaCl; 0.397 g of KH2PO4; 10%
horse serum, and yeast extract).
2. Place the culture tubes containing the faecal material at 37°C for 24 hours.
3. Examine the drop of the culture fluid under x40 magnification using light
microscope.
Result:
Name of test: Detection of Cryptosporidium spp. oocysts using modified Ziehl-Neelson
Principle:
Stool samples containing Cryptosporidium parvum oocysts can be air-dried onto
microscope slide and stained with cold carbol fuchsin. The oocysts are visualized
using a bright field microscope.
C. parvum positive faecal samples should be available when personnel are

familiarizing themselves with this staining technique. Smears from positive faecal
samples should be included each time this procedure is performed. Stool samples
containing Cryptosporidium parvum oocyst, obtained from routine submissions, can
be stored at 4°C in either 10% formalin or 2.5% K 2Cr5O7 (Potassium dichromate) for
reference purposes.
Material:
Microscope slides, coplin jar, light microscope, immersion oil
Methanol (94%), cold strong carbol fuchsin, 0.4% malachite green (w/v in distilled
water), 1% HCl (v/v) in methanol (94%)
Procedure:
Include a positive control slide each time performing this procedure
1. Fix the air dried direct or concentrated smeara,b in methanol for 5 mins.
2. Immerse the slide in cold strong carbol-fuchsin and stain for 5 – 10 mins.
3. Rinse the slide thoroughly in tap water.
4. Rinse with 33% acid alcohol (v/v) until the colour turns pale pink
(approximately 10 secs)
5. Rinse the slide in tap water.
6. Counter stain with 0.25% malachite green for 30 secs.
7. Rinse the slide in tap water.
8. Air dry the slide and scan the slide using x40 objective lens. Confirm the
presence of oocystsc,d under the oil immersion objective lens.
9, Measure the size and shape of the red-stained bodies.
a Moderately thick smears are recommended for this procedure.

b Cryptosporidium spp. oocysts can be concentrated using the formol-ether technique
of Allen and Ridley (see SOP PARAF 03) and stained with modified Ziehl Neelsen.
The use of modified formol-ether method has been advocated for stool samples which
contain only a few oocysts (e.g. follow up specimens from individuals who have
recovered) which is reported to be more sensitive than the method of Allen and Ridley.
c Cryptosporidium spp. oocysts stain red and appear as spherules (4 – 6 µm in
diameter) on a pale green background. The degree and proportion of staining varies
with individual oocysts. In addition, the internal structures take up the stain to varying
degrees. Some may appear amorphous whilst others may contain the characteristic
crescentic forms of the sporozoites. Yeasts and faecal debris stain a dull red. Some
bacterial spores may be acid fast, but these are too small to cause confusion.
d Isospora belli oocysts stain red and appear as large elongated ovoid bodies (20 – 30
x 10 – 19 µm), tapered at the end and containing either a granular zygote or two
sporoblasts, Cyclospora spp. oocysts stain pinkish red and appear as circular discs (8
– 10 µm in diameter) containing a central morula. The degree and proportion of
staining varies with individual oocysts. The unsporulated oocyst is seen generally in
stool samples.
Diagnostic features of Cryptosporidium parvum oocysts:

Oocysts are smooth, thick walled, colourless, spherical or slightly ovoid bodies,
containing, when fully developed (sporulated), four elongated, naked (i.e. not within a
sporocyst(s)) sporozoites and a cytoplasmic residual body. The modal size
measurement of C. parvum oocysts is 4.5 x 5.0 µm (range 4 – 6 µm).
Result:
Name of test: Detection of microsporidia spores in faecal smears using modified
trichrome stain
Principle:
Faeces is emulsified in 10% formalin, smeared onto a microscope slide and fixed in
methanol. The fixed smear is stained in modified trichrome stain for 10 minutes at
50°C, differentiated in acid – alcohol (4.5 ml glacial acetic acid in 95.5 ml of 90%
ethanol) for 10 seconds, rinsed in 95% ethanol, air dried and viewed under x100 oil
immersion lens of a bright field microscope.
Microsporidia can be sought in formed, unformed, and liquid faeces. Unformed
and liquid faeces are fixed by adding 3 volumes of 10% formalin to 1 volume of faeces.
Formed faeces are diluted in deionized water until liquid, and fixed in 10% formalin as
described before.
Material:
Microscope slide (76 x 26 mm), coplin jars or similar, absolute methanol, bright field
microscope with x100 oil immersion objective lens, diameter marker, trichrome stain
(chromotrope 2R [Gurr] 6 g, Fast green 0.15 g, phosphotungstic acid 0.7 g. Add the
reagents to 3 ml glacial acetic acid, mix and allow to stand for 30 minutes. Add 100 ml
distilled water), acid alcohol(add 4.5 ml acetic acid to 995.5 ml of 90% ethanol and stir
, 95% ethanol, 100% ethanol.
Procedure:
1. Score the reference number of specimen on a microscope slide, and use separate
microscope slides for each stool specimen. Place two drops (about 20 µl) of the
faeces-formalin slurry onto the microscope slide and make a smear with a wooden
applicator sticka.
2. Fix slide by immersion in absolute methanol for 4 minutes and air dry.
3. Immerse slide in Trichrome stain for 10 mins at 50°C/room temperature for 60 mins.
4. Rinse in acid alcohol (10 seconds).
5. Rinse in 95% alcohol.
6. Place in 95% alcohol for 5 minutes.
7. Place in 100% alcohol for 10 minutes.
8. Place in inhibisol (xylene) for 10 minutes.
9. Confirm the presence of spores under x100 immersion objective lens.
10. Measure size and shape of pinkish red stained bodies under x100 objective lens b.
a As for conventional demonstration of other protozoan parasites, the best results are
obtained when smears are of an optimal thickness. They should be neither too thick
nor too thin.
b. Spores stain pinkish red. Spores of microsporidia reported to infect human beings
range from 1.0 – 1.6 x 0.9 µm to 4.0 – 4.5 x 2.0 – 2.5 µm.
Result:
Microscopic examination: Microsporidia spp. spores stained with modified trichome
stain
Interpretation of Result:
Species Spore size
Encephalitozoon cuniculi 2.5 – 3.2 x 1.2 – 1.6 µm
Encephalitozoon hellem 2.0 – 2.5 x 1.0 – 1.5 µm
Encephalitozoon spp. 1.7 – 1.8 x 0.8 µm
Nosema connori 4.0 – 4.5 x 2.0 – 2.5 µm
Nosema corneum 3.7 x 1.0 µm
Nosema ocularum 3.0 x 5.0 µm
Microsporidium ceylonensis 3.5 x 1.5 µm
Microsporidium africanum 4.5 – 5.0 x 2.5 – 3.0 µm
Enterocytozoon bieneusi 1.0 – 1.6 x 0.9 µm
Pleistophora spp. 2.8 x 3.2 – 3.4 µm
Name of test: Trichrome staining for fresh specimen
Principle:
Faecal smears are fixed wet in Schudinn’s fixative for 1 hour (for PVA, faeces are
emulsified in PVA and the smear is made from faeces/PVA emulsion), fixed in 70%
alcohol, placed in 70% alcohol containing 4-5 drops of iodine for 1 min, returned to
70% alcohol and stained with trichrome stain for 2 – 6 mins. The slides are exposed
to an acidified alcohol solution, dehydrated, cleared in xylene and made into
permanent preparations. Slides are viewd under x10, x40, and x100 objectives of a
bright field microscope.
Material:
Reagents
Trichrome stain: chromotrope 2R 60 mg; light green SF 300 mg, phosphotungstic acid
700 mg; glacial acetic acid 1.0 ml, ditilled water 100 ml.
Schaudinn’s fixative, 70% alcohol containing 4 – 5 drops of iodine, acidified alcohol
solution (99ml of 90% ethyl alcohol and 1 ml glacial acetic acid), absolute alcohol,
xylene, DPX
Preparation of Schaudinn’s fixative

1. Mercuric chloride(HgCl2)
2. 95% ethyl alcohol
3. Glycerin
4. Glacial acetic acid
Prepared reagents
1. Saturated mercuric chloride solution
a) Dissolve 80 to 90 g mercuric chloride in 1000 ml, distilled water by heating
b) Allow solution to cool (excess mercuric chloride crystallized out)
c) Filter solution into a glass stoppered bottle and store until needed.
2. Mix 600 ml saturated mercuric chloride solution with 300 ml 95% ethyl alcohol and
15 ml glycerin. Store until needed.
3. Immediately prior to use, add 5 ml glacial acetic acid to every 100 ml of stock
solution to be used.
Glass microscope slide (76 x 26 mm), coverslips, bright-field microscope with x10, x40,
and x100 (oil) objective lenses, coplin jars, disposable gloves.
Procedure:
1. Prepare fresh faecal smears. Without allowing them to dry, immerse slides in a
coplin jar containing in Schaudinn’s fixative for 1 hour. For PVA, faeces are
emulsified in PVA and the smear is made from the faeces/PVA emulsion.
2. Place slides in 70% alcohol containing 4 – 5 drops of iodine for 1 minute.
3. Place slides in 70% alcohol for 1 minute.
4. Place slides in 70% alcohol for 1 minute.
5. Stain with Trichrome stain for 2 – 8 minutes.
6. Place slides into acidified alcohol solution (add 1 drop of glacial acetic acid in 10
ml alcohol) for 10 – 20 seconds.
7. Rinse twice with 95% alcohol.
8. Dehydrate smear in absolute alcohol (100% alcohol) for 1 minute.
9. Clear in xylene for 1 minute.
10. View under the x10, x40, and x100 (oil) objective lenses.
Result:
HAEMATOLOGY
LABORATORY
Department profile
Besides providing clinical services to University Malaya Medical Centre (UMMC), the
department is also responsible in training tutors and lecturers for MBBS degree, BDS,
BBiomedSc and postgraduates courses, Masters in Pathology, Medical Science
Masters in Clinical Pathology, Medical Science Masters, medical doctors and
philosophical doctors under the Faculty of Medicine, University Malaya.
Medical Laboratory Division
Medical Laboratory Division is responsible in diagnostic services for patients

and doctors, conducting researches for techniques in diagnostic tests, disseminating
information on services offered to clients of the department. The division also provides
continuous advisory and technical training for UMMC student and staff.
The division also provides diagnostic services for Chemical Pathology (clinical
chemistry, endocrine, immunology, fluid and excretion, therapeutic drug monitoring,
specialised chemistry), Haemathology (routine haemathology, specialised
haemathology, coagulation, bone marrow test), Inborn Errors of Metabolism,
cytogenetic, molecular genetic, immunogenetic and transplantation.
Organization chart
DIRECTOR
YM PROF. DR. TUNKU KAMARUL ZAMAN BIN TUNKU ZAINOL ABIDIN
SPECIAL GRADE A
DEPUTY DIRECTOR (CLINICAL SERVICES)

ASSOC. PROF. DR. NAZIRAH BINTI HASNAN
PREMIER GRADE C
HEAD OF DEPARTMENT
ASSOC. PROF. DR. NAZARINA BINTI ABD RAHMAN
ASSOCIATE PROFESSOR GRADE DU54
MEDICAL LAB DIVISION

DR. HEMALATHA SHANMUGAM
Name of test: Full blood count
 Haemoglobin (Hb)
 WBC
 RBC
 Platelet
 Reticulocyte
 Haematocrit (Hct)
 Packed cell volume (PCV)
 Mean cell volume (MCV)
 Mean cell haemoglobin (MCH)
 Mean cell haemoglobin concentration (MCHC)
Principle:
The XN series utilises 3 primary analysis principles
 Fluorescence Flow Cytometry Method (FCM) – a semiconductor laser (639nm).
 Hydrodynamic Focussing DC detection – RBC and PLT analysis.
 SLS Haemoglobin Method – cyanide-free HGB analysis
Fluorescence Flow Cytometry

Sample dilutions are passed through the middle of the flow cell by sheath flow
technology or “hydrodynamic focussing”. As cells pass through the laser beam,
Forward scattered light (FSC) is generated. A fluorescent dye is included in the
reaction channel, causing the side scattered light to split by a dichroic mirror, into
fluorescent light (FL) and side scattered light (SSC)
 Forward Scattered Light provides information on cell volume.

 Side Scattered Light provides information on internal structure e.g. granularity
and lobularity.
 Side Scattered Fluorescent Light provides information on the RNA/ DNA
content of the cell.
Each cell that passes through the laser generates a unique cell signature. Cell
scatter properties are classified by the Sysmex XN Adaptive Flagging Algorithm based
on Shape recognition (AFLAS) in the WDF channel. This information is then
graphically displayed in the scattergrams on the IPU.
AFLAS uses a human-like recognition of the WDF scattergram, by not only

looking at the numbers of cells, but the shape of each cluster, their position, angle,
size, length and width.
Each cell produces a unique “cell signature” or position on the scattergram, as

it passes through the laser beam.
If the analyser cannot clearly separate the cell population clusters, the clusters
are greyed out and the associated flagging messages will be displayed on the IPU.
Hydrodynamic Focussing Direct Current (DC) Method (RBC/ PLT)

The RBC and PLT dilution is injected into the RBC PLT detector. The sample
dilution passes through the middle of the aperture, assisted by the hydrodynamic
focussing principle where laminar flow ensures that cells are not counted twice.
As cells pass through the aperture, they cause an electrical resistance, which
is recorded as an impedance pulse. The size of the cell is proportional to the pulse
height. The RBC and PLT histograms are generated from this detection principle.
Sodium Lauryl Sulphate (SLS) Haemoglobin Method

The SLS-HGB method has been adapted to provide a cyanide-free method of
haemoglobin analysis and demonstrates excellent correlation with the ICSH reference
method.
The RBC is lysed and Haemoglobin is liberated. In the reaction chamber the
Sodium-LaurylSulphate reagent (Sulfolyser) is added.
The Hydrophobic portion of SLS reacts with GLOBIN, and a conformational
change occurs exposing the HAEM unit. Fe2+ is oxidised to Fe3+. The hydrophilic
group of SLS binds with Fe3+ forming a stable reaction product.
The SLS-HGB concentration is measured at light absorbance, 555 nM, and is
calculated by comparison with the absorbance of a blank diluent sample prior to HGB
analysis.
Material:
Sysmex XN-9000
Patient’s blood in EDTA vacutainer (lavender top)
Procedure:
Sampler analysis
1. Check RN, patient name, test ordered on barcode and patient specimen.
2. Analyser and sampler ready.
3. Select the test if the sample without order
4. Load sample rack in the right sampler pool.
5. The sampler analysis automatically starts
6. Rack is remove after analysis.
Manual analysis
1. Make sure instrument is ready.
2. Press mode switch on analyzer to eject tube holder.
3. Change analysis mode, select ‘whole blood’.
4. Mix sample and place sample in sample tube holder.
5. Press start switch on analyzer.
6. Remove sample after analysis.
7. Press mode switch after manual analysis.
Result:
Result produced by haematology analyzer
ANALYTES UNIT MEN WOMEN

-12
Red blood cell (RBC) x10 /L 4.5 - 5.5 3.8 - 4.8
Haemoglobin (HGB) g/L 130 - 170 120 - 150
Packed cell volume (PCV) L/L 0.40 - 0.50 0.36 - 0.46
Mean cell volume (MCV) fL 77 - 97
Mean cell haemoglovin (MCH) Pg 27 - 32
Mean cell haemoglobin concentration (MCHC ) g/L 315 - 360
Reticulocyte (RETIC) % 0.5 2.5
-9
White blood cell (WBC) x10 /L 4 - 10
Red cell distribution width (RDW – CV) % 11.6 - 14.0
Red cell distribution width (RDW - SD) fL 39.0 - 46.0
-9
Neturophil (NEUTRO) x10 /L 2.0 - 7.0 (40 - 80%)
Lymphocyte (LYMPHO) x10-9/L 1.0 - 3.0 (20 - 40%)
-9
Monocyte (MONO) x10 /L 0.2 - 1.0 (2 - 10%)
-9
Eosinophil (EOSI) x10 /L 0.02 - 0.5 (1 - 6%)
-9
Basophil (BASO x10 /L 0.02 - 0.1 (<1 - 2%)
-9
Platelet (PLT) x10 /L 150 - 400
Erythrocyte sedimentation rate (ESR) mm/hr <21 <29
Reference:
Dacie J. V and Lewins . M Practical Haematology, 9th Edition
Nathan and Oski Elsevier 6th Edition, 2003
Instrument: Sysmex XN-9000

Name of test: Thin blood film preparation
Principle:
Thin blood film is prepared for microscopic examination, it aids diagnosis of any blood
cells related abnormalities in patient such as leukemia. Abnormalities of blood cells
can be visualized through staining of dried thin blood film with Wright’s stain.
Material:
Glass slide, cover slip (use as spreader), blood specimen in EDTA vacutainer
(lavender top), Bayer HealthCare Hematek 2000
Procedure:
1. Label patient’s RN on glass slide on the frosted area, and place slide on a flat
surface.
2. Using an applicator stick, place few drop of blood, about 2 mm in diameter
approximately 5 millimeters from the frosted area of glass slide.
3. Using a cover slip, place it just in front of the blood drop.
4. Hold the spreader cover slip at a 30° angle, draw it back against the drop of
blood.
5. Wiggle slightly to allow blood to spread across the edges of the slide.
6. Push the spread forward with one light, smooth, and fluid motion. A thin film of
blood in the shape of a bullet with a feathered edge will remain on the slide.
7. Allow blood film to air dry, then load slide into autostainer.
8. Autostainer stain thin blood film with Wright’s stain.
9. MLT responsible for blood film microscopy will examine stained thin blood film
and comment on results.
Result:
Thin blood film preparation with Wright stain
Autostainer: Bayer HealthCare Hematek 2000

Name of test: Supravital staining
Principle:
Supravital staining is a method of staining used in haematology to diagnose blood
abnormalities. In CDL of UMMC, supravital staining is used mainly for screening of
thalassemia, by using new methylene blue. RBC found in patient with HbH disease
demonstrate a signature golf ball appearance after stained with new methylene blue.
Specimen can then proceed with further test such as hemoglobin electrophoresis.
Material:
Patient’s blood in EDTA vacutainer (lavender top), glass slide, clean plastic tube,
disposable pipette
Procedure:
2. With a disposable pipette, add 1 drop of patient blood and 1 drop of new methylene
blue solution into a clean plastic tube.
3. Plastic tube is incubate for 1 hour at 37°C
4. Thin blood film was prepared from the mixture of blood cells and new methylene
blue.
5. Thin blood film was left to air dry then observe under microscope.
Result:
Supravital stain with new methylene blue
Supravital stain with new methylene blue demonstrates HbH inclusion found on rbc.
Name of test: Hb A2
Principle:
The BIO-RAD VARIANT II TURBO utilizes principles of ion-exchange high-
performance liquid chromatography (HPLC). The samples are automatically diluted on
the VARIANT II TURBO Sampling Station (VSS) and injected into the analytical
cartridge. The VARIANT II TURBO Chromatographic Station (VCS) dual pumps deliver
a programmed buffer gradient of increasing ionic strength to the cartridge, where the
hemoglobins are separated based on their ionic interactions with the cartridge material.
The separated hemoglobins then pass through the flow cell of the filter photometer,
where changes in the absorbance at 415 nm are measured. An additional filter at 690
nm corrects the background absorbance. The VARIANT II TURBO Clinical Data
Management (CDM™) software performs reduction of raw data collected from each
analysis. Two-level calibration is used. A sample report including retention times of
detected peaks and a chromatogram are generated by CDM for each sample.
Material:
BIO-RAD VARIANT II TURBO (with β-thalassemia Short Program), patient’s blood
sample in EDTA vacutainer (lavender top)
Reagents:
1. Elution bufers (1,2): sodium phosphate bufer.,
2. Wwhole blood primer: lyophilized human red blood cell hemolysate with gentamicin,
tobramycin, and EDTA as preservative.
3. HbA2/F calibrator/diluent set: lyophilized human red blood cell hemolysate with
gentamicin, tobramycin, and EDTA as preservative analytical cartridge. Diluent
contains deionized water.
4. Wash/diluent solution: deionized water.
5. Control: normal (HbF 1-2%, HbA2 1.8–3.2%) and abnormal (HbF 5–10%, HbA2 4–
6%) controls
Procedure:
2. Vacutainer is placed on specimen rack specific for BIO-RAD VARIANT II TURBO.
3. Load specimen rack into analyzer.
Result:
Peak of haemoglobin in interpret based on normal ranges given. Patient can be
identified either have major thalassemia or a carrier.
Intrument: BIO-RAD VARIANT II TURBO

Name of test: Hb electrophoresis
Principle:
The HYDRAGEL HEMOGLOBIN(E) assay is based on the principle of electrophoresis
separation on agarose gel at alkaline pH (pH 8.5).
Prior to performing the test, it is mandatory to prepare the samples (hemolyzed

washed red blood cells).
The HYDRASYS system is a semi-automated multi-parameter instrument. The

automated steps include processing of the agarose gel in the following sequence:
 Sample application
 Electrophoretic migration
 Drying
 Staining with amidoblack dye
 Destaining
 Final drying
Hemoglobin is a complex molecule composed of two pairs of polypeptide chains. Each

chain is linked to the heme, a tetrapyrrolic nucleus (porphyrin) which chelates an iron
atom. The heme part is common to all hemoglobins and their variants. The type of
hemoglobin is determined by the protein part called globin. Polypeptide chains α, β, δ
and γ constitute the normal human hemoglobins:
 hemoglobin A =α2β2
 hemoglobin A2 =α2δ2
 fetal hemoglobin F =α2γ2
The α-chain is common to these three hemoglobins.
The hemoglobin spatial structure and other molecular properties (as that of all
proteins) depend on the nature and the sequence of the amino acids forming the
chains. Substitution of amino acids by mutation is responsible for formation of
hemoglobin variants which have different surface charge and consequently different
electrophoretic mobilities, which also depend on the pH and ionic strength of the buffer.
The resulting qualitative (or structural) abnormalities are called hemoglobinopathies.
Decreased synthesis of one of the hemoglobin chains leads to quantitative (or
regulation) abnormalities, called thalassemias.
The assay is performed on the hemolyzate from washed red blood cells. The
hemoglobins are separated by electrophoresis on alkaline gels and the fractions are
visualized by staining with amidoblack. The dried gels are ready for interpretation.
Material:
Sebia HYDRASYS 2 SCAN
Procedure:
SAMPLES FOR ANALYSIS
Sample collection and storage
Fresh anticoagulated blood samples are recommended for analysis. Common
anticoagulants such as those containing EDTA, citrate or heparin are acceptable ;
avoid those with iodoacetate. Blood must be collected according to established
procedures used in clinical laboratory testing. If needed, store samples at 2 to 8 °C for
up to 5 days.
Sample preparation (standard procedure)

 Mix the collection tube before taking the blood to prepare.
 Centrifuge anticoagulated blood at 5 000 rpm for 5 minutes.
 Discard the plasma.
 Wash the red blood cells (RBC) 2 times with 10 volumes of saline ; great care
must be taken when processing volumes of red blood cells smaller than 10 µL.
 Discard the excess of saline over the red blood cells pellet and vortex them
before taking 10 µL to hemolyze.
 Hemolyze 10 µL packed red cells with 130 µL Hemolysing Solution.
 Vortex for 10 seconds and incubate 5 minutes at room temperature.
NOTES:
 To prepare hemolysate from subjects, mildly anemic (approximately 10
g/dL Hb) or severely anemic (< 7 g/dL Hb), the volume of packed RBC
may be increased to 15 µL and 20 µL, respectively. The staining intensity
will thus increase but relative concentrations of individual fractions will
not change.
 The hemolyzate need not be filtered or centrifuged.
 The SEBIA’s hemolysing solution does not affect the unstable
hemoglobin Bart’s.
Sample preparation for hemoglobin H detection

 Mix the collection tube before taking the blood to prepare.
 Centrifuge anticoagulated blood at 5 000 rpm for 5 minutes.
 Discard the plasma.
 Wash the red blood cells 2 times with 10 volumes of saline ; great care must be
taken when processing volumes of red blood cells smaller than 10 µL.
 Discard the excess of saline over the red blood cells pellet and vortex them
before taking 40 µL to hemolyze.
 Hemolyze 40 µL packed red cells with 100 µL Hemolysing Solution.
 Vortex for 10 seconds and incubate 5 minutes at room temperature. - Centrifuge
hemolyzate at 10 000 rpm for 5 minutes.
 The analysis is performed on the supernatant of this hemolyzate ; then, follow
the procedure with "7 / 15 Hb" migration program.
PROCEDURE
The HYDRASYS system is a semi-automated multi-parameter instrument. The
automated steps include processing of HYDRAGEL agarose gels in the following
sequence: sample application, electrophoretic migration, drying, staining, destaining
and final drying. The manual steps include handling samples and gels, and setting up
the instrument for operation.
I. MIGRATION SET UP
1. Switch on HYDRASYS instrument.
2. Place one applicator on a flat surface with the well numbers in the right-side-up
position (Fig. 1).
 Apply 10 µL hemolyzed sample in each well. Load the applicator within 2
minutes.
 Place the applicator into the wet storage chamber with the teeth up (handle
it by the plastic tooth protection frame).
 Let the samples diffuse into the teeth for 5 minutes after the last sample
application.
3. Open the lid of the Migration Module and raise the electrode and applicator carriers.
4. Select "7/15 Hb" migration program.
5. Remove buffered strips from the package ; handle them by the plastic ends.
Engage the punched ends of the strip's plastic backing to the pins on the electrode
carrier ; the strip's plastic backing must face the carrier (Fig. 2).
6. Unpack the HYDRAGEL agarose gel plate.
 Place its plastic side down on a tissue or filter paper to remove water
droplets.
 Roll quickly and uniformly one thin filter paper onto the gel surface to absorb
the excess of liquid. Remove the paper immediately.
WARNING: Do not leave the filter paper for a too long contact with the gel
to avoid its dehydration.
 Streak 150 µL ethylene glycol (EG) for HYDRAGEL 7 HEMOGLOBIN(E), or
300 µL for HYDRAGEL 15 HEMOGLOBIN(E), across the lower third of the
frame printed on the Temperature Control Plate of the migration module.
IMPORTANT: The temperature control plate must be perfectly clean and dry
before aplying the ethylene glycol solution.
 Place the gel plate (the gel side up) with its edge against the stop at the
bottom of the printed frame (Fig. 3).
 Bend the gel and ease it slowly down onto the EG streak (Fig. 3). Ensure
that no air bubbles are trapped, EG is spread underneath the entire gel plate
and the gel is lined up with the printed frame.
7. Lower both carriers down. In this position the buffered strips do not touch the gel.
DO NOT FORCE THE CARRIERS ALL THE WAY DOWN
8. Remove the applicator from the wet chamber. Handle it by the protection frame.
 Snap off the applicator teeth's protection frame.
 "7 / 15 Hb" migration program: Place the applicator into position No. 4 on
the carrier.
 "7 / 15 Hb F-S" migration program: Place the applicator into position No. 3
on the carrier.
IMPORTANT: The numbers printed on the applicator must face the operator
(Fig. 4).
9. Close the lid of the migration module.
10. Start the procedure immediately by pressing the green arrow "START" key on the
left side of the keyboard.
IMPORTANT: Make sure that the ventilation air inlet on the right side of the
instrument is not blocked.
MIGRATION - DESCRIPTION OF THE AUTOMATED STEPS

 The two carriers are lowered so that buffered strips and applicator contact the
gel surface.
 Sample applicator carrier rises up.
 Migration is carried out under 340 V constant at 25 °C, controlled by Peltier
effect, until 65 Vh have accumulated (for about 12 minutes, "7 / 15 Hb" migration
program) or until 85 Vh have accumulated (for about 15 minutes, "7 / 15 Hb F-
S" migration program).
 The electrode carrier rises to disconnect the electrodes.
 The temperature of the control plate rises to 50 °C for 15 minutes to dry the gel.
 An audible beep signals that the migration module lid unlocks. The plate
temperature remains at 50 °C until the lid is opened. Then, the temperature
keeps decreasing until it reaches 25 °C (in less than 5 minutes) after which a
new migration run may start.
NOTE: The migration module lid remains closed during all migration steps.
II. GEL PROCESSING SET-UP

1. Open the lid.
2. Remove the applicator and discard.
3. Raise both carriers, remove the buffered strips by their plastic ends and discard.
4. Remove the dried gel film for further processing.
5. Wipe very carefully the electrodes and the temperature control plate with a soft
tissue well soaked with water. Make sure that the electrodes and the plate are well
dried before re-use.
IMPORTANT: The electrodes have to be cleaned systematically after each use.
6. Open the Gel Holder. Lay it flat and position the dried gel (with gel side facing up)
into the grooves of the two rods and close the holder. Make sure that the film is
correctly positionned inside the holder (Fig. 5).
7. Place the gel holder into the Gel Processing / Staining Module. IMPORTANT:
Before starting the gel processing / staining program check the following:
 the staining container is filled with 300 mL of staining solution ;
 the destaining container contains at least 1 liter of destaining solution ;
 the waste container is empty.
For reagent line connection: refer to the information displayed on the screen
of the instrument (select key: REAGENT LINES).
IMPORTANT: Do not forget to block up the unused lines.
8. Select "PROTEIN(E)/ß1-ß2/Hb" or "Hb" staining program from the instrument
menu and start the run by pressing the "START" key (green arrow on the right side
of the keyboard).
During staining, destaining and drying steps, the compartment remains locked.
After cooling step, an audible beep signals that the compartment unlocks (the
ventilation is maintained until the gel holder is removed).
III. GEL PROCESSING COMPLETION

1. Remove the gel holder from the compartment, open it and remove the dried gel.
NOTE : After gel staining / destaining and before densitometry / scanning, a gel
may be put through an additional wash step, if needed, to further clarify the gel
background and to remove any residual stain that may appear as blue spots. Wash
the gel using the "WASH ISOENZ/GEL" program.
2. If needed, clean the back side (the plastic support side) of the dry film with a damp
soft paper.
3. Scan using a densitometer / scanner by selecting the appropriate scanning
program. When using HYRYS or DVSE densitometers, position the A 2 fraction on
the 5 mm mark of the scanning plate: the background zero is made between the
A2 and carbonic anhydrase fractions at the lowest point.
NOTE: To assure the most accurate and consistent results, do the following:
 Adjust the scan length to include the entire electrophoretic pattern (≈ 30 mm).
 If necessary, position the minima on both sides of A2 fraction (if not, let the
minima where they are automatically positioned).
It is a good practice to read the stained gels without delay. For future reference,
they can be stored in a protective cover in a dry, dark place away from sources of
heat and visually interpreted within at least 3 months.
Graphical procedure of operating sebia HYDRASYS 2 SCAN

Result:
1. Qualitative abnormalities: Hemoglobinopathies
Most hemoglobinopathies are due to substitution by mutation of a single amino acid in
one of the four types of polypeptide chains. The clinical significance of such a change
depends on the type of amino acid and the site involved. In clinically significant disease,
either the α-chain or the ß-chain is affected.
More than 200 variants of adult hemoglobin have been described. The first abnormal
hemoglobins studied and the most frequently occuring have an altered net electric
charge, leading to an easy detection by electrophoresis.
There are four main abnormal hemoglobins which present a particular clinical interest:
S, C, E and D.
The HYDRAGEL 7 / 15 HEMOGLOBIN(E) kits are intended for the preliminary
identification of hemoglobinopathies and thalassemias. Once an abnormal pattern is
indicated, its identity should be confirmed by appropriate discriminatory tests (e.g.,
electrophoresis on acidic agarose gels).
Hemoglobin S
Hemoglobin S is the most frequent. It is due to the replacement of one glutamic acid
(an acidic amino acid) of the ß-chain by valine (a neutral amino acid). Its
electrophoretic mobility is therefore slowed down. On alkaline buffered HYDRAGEL 7
/ 15 HEMOGLOBIN(E), hemoglobin S migrates between A and A 2 fractions.
Hemoglobin C
One glutamic acid of the ß-chain is replaced by lysine (a basic amino acid): its mobility
is strongly reduced. On HYDRAGEL 7 / 15 HEMOGLOBIN(E), C, E and A2 are
superimposed. When this fraction is > 15 %, hemoglobins C and E must be suspected.
Hemoglobin E
One glutamic acid of the ß-chain is replaced by lysine: hemoglobin E migrates exactly
like hemoglobin C on HYDRAGEL 7 / 15 HEMOGLOBIN(E). Unlike hemoglobin C, it
does not separate from hemoglobin A in acidic buffer [HYDRAGEL 7 / 15 ACID(E)
HEMOGLOBIN(E)]. This property allows to differentiate E and C.
Hemoglobin D
One glutamic acid of the ß-chain is replaced by glutamine. On HYDRAGEL 7 / 15
HEMOGLOBIN(E), this hemoglobin migrates exactly like hemoglobin S. Unlike
hemoglobin S, hemoglobin D does not separate from hemoglobin A in acidic buffer
[HYDRAGEL 7 / 15 ACID(E) HEMOGLOBIN(E)] ; this property allows to differenciate
S and D.
2. Quantitative abnormalities: Thalassemias
Thalassemias constitute a quite heterogeneous group of genetic disorders
characterized by decreased synthesis of one type of the polypeptide chains.
The molecular mechanism of this decrease has not been fully described.
There are two types of thalassemia syndromes:
Alpha-thalassemias
They are characterized by the decrease of synthesis of the α-chains, consequently
affecting the synthesis of all normal hemoglobins.
The excess of synthesis of the ß- and γ-chains in relation to α-chains induces the
formation of tetrameres without any α-chain :
 hemoglobin Bart = γ 4,
 hemoglobin H = ß 4.
Beta-thalassemias They are characterized by the decrease of synthesis of the ß-
chains. Only hemoglobin A synthesis is affected.
Therefore hemoglobin F and hemoglobin A2 percentages are increased with respect
to hemoglobin A.
3. Migration patterns
Instrument: sebia HYDRASYS 2 SCAN

Name of test: Thrombin time, prothrombin time, partial thromboplastin time
Instrumentation Laboratory ACL TOP 500 CTS coagulation analyzer are capable of
analyse following parameters:
PT, APTT, Fibrinogen, Thrombin Time, Intrinsic Factors, Extrinsic Factors, D-Dimer,
Antithrombin, Protein C, Protein S, Factor V Leiden (APC-R), Lupus Anticoagulant,
von Willebrand Factor, Plasminogen, Plasmin Inhibitor, Heparin (UF and LMW), HS-
CRP
Principle:
The ACL TOP system provides results for both direct hemostasis measurements and
calculated parameters. The ACL TOP performs the following types of tests:
Coagulometric (Turbidimetric) Tests

Chromogenic (Absorbance) Tests
Immunological Tests
The following describes the operating principles for each of the three types of tests.
Coagulometric (Turbimetric) Measurements

The principle of coagulometric clot detection is used to measure and record the
amount of time required for a plasma specimen to clot. The technique assesses
coagulation endpoints by measuring change in optical density.
Chromogenic (Absorbance) Measurements Chromogenic tests use the colorimetric

principle of measuring absorbance of light by the solution in a cuvette. The amount of
light that reaches the photodector is converted into an electrical signal that is
proportional to enzyme activity.
Immunological Measurements
The principle of immunological measurement is used to directly measure and record
the concentration of an analyte in a sample (and not its activity) by measuring change
in optical density. Although similar to the turbimetric method, the immunological
method relies on the formation of antigen-antibody complexes to affect light
transmission. The ACL TOP testing process is fully automated, with on-board QC and
maintenance, enhancing performance and productivity. Please see below for more on
system features and benefits.
Material:
1. Patient blood specimen in vacutainer contained buffered sodium citrate for
coagulation determination (light blue top).
2. Instrumentation Laboratory ACL TOP 500 CTS Hemostasis Testing Systems
Procedure:
1. Patient’s name, RN, barcode, and test ordered was checked and make sure tally
on both patient sample and order form.
2. Vacutainer is then place on centrifuge bucket and balanced with empty vacutainer
if required.
3. Patient’s sample is centrifuge at 3500 rpm for 10 minutes.
4. After centrifuge, vacutainer is unload from centrifuge bucket, and placed into rack
specified for ACL TOP 500 CTS coagulation analyzer.
5. Pressing button below the desired track on analyzer grant access to the particular
track for sample rack loading.
6. Barcode reader located on the front of the system automatically scans barcode on
vacutainer when sample rack was loaded into analyzer.
7. A rubber curtain ensures the area is always maintains at optimal temperature
during analysis.
8. When rack is loaded correctly, it would latched in place. Once latched in place, LED
for the tract turns green.
9. During analysis, LED turns orange indicated the rack is locked in position and not
movable.
10. Upon complete analysis, LED turns to green. This indicates that rack can be
removed and a new rack of samples can be loaded.
Result:
Results generated by coagulation analyzer
Coagulation analyser: Instrumentation Laboratory ACL TOP 500 Hemostasis Testing

Systems
Name of test: May-Grunwald Giemsa Stain
Principle:
May-Grünwald Giemsa (MGG) staining is a mix of two neutral stains:
 A May-Grünwald stain composed of an acidic stain (eosin) and a basic stain
(methylene blue).
 A Giemsa stain composed of eosin and another metachromatic basic stain:
azure of methylene.
The first stain induces an orthochromatic staining on cell components (pink or orange
dye for acidophilic components and blue or purple for basophilic and neutrophil
components). The second induces a metachromatic staining: red dye for azurophil
components.
May-Grünwald Giemsa (MGG) staining is used in hematology to differentiate and

count different blood cell populations on cellular preparations (cytology).
Material:
Reagents
Absolute methanol
Phosphate buffer pH6.8±0.1
Diluted May-Grunwald stain (Working solution)

- Dilute 2:5.5 May-Grunwald stock buffer (20 ml stain top up to 55 ml with buffers)
Diluted Giemsa stain (Working solution)

Dilute 1:11 Giemsa stock:Buffer (5 ml stain top up to 55 ml with buffer)
Procedure:
1. Fix air dried smears in methanol for 20 minutes at room temperature. Discard
methanol.
2. Incubate with diluted May-Grunwald for about 14 minutes.
3. Discard May-Grunwald stain.
4. Incubate in diluted Giemsa stain for about 14 minutes.
5. Discard Geimsa stain and quickly rinse with phosphate buffer 3 times.
6. Differentiate in phosphate buffer for 30-40 seconds.
7. Dry slides with hair dryer using cold air followed by hot air for about 1 hour.
8. Meanwhile rinse staining jars, cylinders and flasks with water and left to dry.
9. Once slides are dried, mount them with coverslip using Ultramount.
10. Stick on the generated patient’s bar code labels accordingly.
11. Examine smear microscopically.
Result:
Microscopic examination: MGG stain (40x)
Component Color
Acidophilic Pink or orange
Basophilic/neutrophil Blue or purple
Azurophilic Red
Name of test: Peroxidase stain
Principle:
Myeloperoxidase splits H2O2 in the presence of chromogenic electron donor (DAB)
and forms an insoluble reaction product. The reaction product is stable, insoluble, and
non diffusible.
Material:
Included in test kit (DakoCytomation)
1. DAB + chromogen
2. DAB + substrate buffer
Peroxidase Fixative
Acetone 30 ml
Glutaraldehyde 25% 60 ml
Distilled water 200 ml
Reaction mixture:
1 drop (about 20 µl) of DAB chromogen
1 ml of substrate buffer
Procedure:
1. Fix air dried smears at room temperature with cold peroxidase fixative for 1
minute.
2. Wash with distilled water.
3. Dry smears
4. Incubate in reaction mixture for 6 minutes.
5. Rinse gently with distilled water.
6. Dry in cool air.
7. Counterstain with Giemsa for 30 minutes.
8. Wash in tap water two times.
9. Differentiate with buffer.
10. Dry the slides with hair dryer using cold air followed by hot air for about 1 hour.
11. Meanwhile, rinse staining jar and cylinders with water and leave to dry.
12. Once slides are dried, mount them with coverslips using Ultramount. Stick on
generated patient’s bar code labels
13. Examine smear microscopically.
Result:
Microscopic examination – PAS stain (40x)
 In IHC and ISH procedures, the DAB Substrate-Chromogen yields a brown
reaction end-product at the site of the target antigen or nucleic acid.
 Early myeloblasts are negative, with granular positivity appearing progressively
as they mature.
 Dark brown granules in cytoplasm of granulocytes and monocytes.
 Monocytes exhibit weaker and more scattered staining properties than
granulocytes.
 RBC will stain diffusely brown because haemoglobin has pseudo-peroxidase
activity. Hence, act as internal control.
 Eosinophil granules stain strongly and they are cyanide resistant MPO positive.
 Auer rods stain well with DAB.
 Plasma cells and lymphoblast are negative.
 Peroxidase activity is present in basophil but not demonstrable by DAB.
Name of test: Iron staining
Principle:
Material:
A) Acid cyanide solution
0.2N HCl 25 ml
2% Potassium Ferrocyanide 25 ml
Mix and heat at 50°C.
B) Safranin 0.1% (w/v) aqueous
Procedure:
1. Fix air dried smears in methanol for 20 minutes.
2. Dry smears with cold air.
3. Heat acid-cyanide mixture to approximately 50°C. Pour into staining jar.
4. Incubate slides for at least 10 minutes.
5. Wash well in running tap water for about 2-3 minutes.
6. Counterstain with 0.1% safranin for about 30 seconds.
7. Wash with running tap water.
8. Dry slides with hair dryer using cold air followed by hot air for about 1 hour.
9. Meanwhile rinse staining jars, cylinders, and flasks with water and leave to dry.
10. Once slides are dried, mount with coverslips using Ultramount. Stick on
generated patient’s bar code labels accordingly.
11. Examine the smear microscopically.
Result:
Microscopic examination - Iron staining (40x)

Cells Color
RBC Pink/red
Iron deposits Dark blue granules
Name of test: Periodic acid Schiff stain
Principle:
In hematology, the PAS reaction is an important method for the identification of
lymphatic cell elements. Apart from peroxidase and esterase reactions, it is one of the
three basic cytochemical staining methods important for differential diagnosis, which
are regularly carried out in acute cases of leukemia. Smears already stained by the
Pappenheim method can additionally be stained with PAS which can be removed by
1 % periodic acid again.
Periodic acid cleaves neighboring carbon carbon bonds in polysaccharides
(glycogen) when hydroxyl groups are attached to both carbon atoms. The alcoholic
groups are then oxidized to aldehydes, which can subsequently be clearly revealed
with Schiff’s reagent (fuchsin sulfurous acid), producing a red stain.
The kit can be used in combination with either Schiff’s reagent to yield a vibrant
red stain or Schiff’s reagent Intense, which produces an even more intense red stain.
The shorter reaction times are more apparent when Schiff’s reagent Intense is used.
Material:
Patient’s bone marrow smear
Included in Merck Diagnostics test kit

Reagent 1: Periodic acid
Reagent 2: Potassium disulfide
Reagent 3: Hydrochloric acid
Preparation of staining solution:

Solution A: Dissolve contents of 1 bottle of Reagent 1 in 60 ml of distilled water and
transfer into a staining cuvette.
Solution B: Dissolve the contents of 1 bottle of Reagent 2 in 60 ml of distilled water,
transfer to a staining cuvette, add 2 ml of Reagent 3 and mix. All the reagent solutions
are colourless and stable for 3 hours.
LEUCOGNOST fixing solution

Mayer’s Haemalum solution
Schiff’s reagent
Aquatex mounting agent
Procedure:
Sample preparation
The sampling must be performed by qualified personnel.
Use thin, air-dried blood or bone-marrow smears that have been stored not longer
than three days.
The smears must be dried in air for at least 30 minutes and be fixed according to the
relevant instructions prior to the actual cytochemical reaction
Fix air-dried blood or bone-marrow smears in LEUCOGNOST
1 – 3 minutes
fixing mixture
Rinse with running tap water 10 seconds
Air-dry
After fixing the smears can be stored in the refrigerator for up to 3 days.
1. Immerse air dried smears in LEUCOGNOST fixing solution 1-3 mins

2. Wash under running tap water. Dry in cool air. 10 secs
3. Place in solution A 30 mins
4. Wash with distilled water. Dry in cool air. 10 secs
5. Place in solution B. 1 mins
6. Wash with distilled water. Dry in cool air. 10 secs
7. Stain in Schiff’s reagent. 20-25°C, incubate in dark 30 mins
8. Wash with distilled water, dry in cool air. 10 secs
9. Place in solution B 2 mins
10. Place in distilled water. 3 mins
11. Stain with Mayer’s Haemalum solution 3 mins
12. Wash under running tap water. 3-5 mins
13. Air dry, mount with Aquatex and a cover glass. Label accordingly. 15 mins
Examine microscopically.
Result:
Microscopic examination – PAS stain (100x)
 Specimens treated with Schiff’s reagent Intense exhibit a more intense stain.
 All polysaccharide and in particular glycogen containing structures are stained
bright red.
 Blast populations that at least partly show a characteristic coarse grained PAS
positive granulation generally belong to the lymphatic series.
 Leukemia blasts of the myeloid series are diffuse to fine grained, sometimes
also coarse plaqued and PAS positive.
 In contrast, normal myeloblasts, eosinophilic ones and cells of the unaffected
red blood cell series are PAS negative.
 Promyelocytes, monocytes, basophilic ones and the entire neutrophilic
development series result in a diffuse red coloration that are stained bright red
with increasing maturity.
 Erythroblasts in erythroleukemia and some extremely hyper regenerative
anaemias can demonstrate a conspicuous PAS reaction.
Name of test: G6PD screening
Principle:
This fluorescence spot test is based on reduction of NADP (nicotinamide adenine
dinucleotide phosphate) to NADPH, which fluorescence s under long wavelength ultra
violet (UV) light. The appearance of fluorescence reveals NADPH formation indicating
presence of normal G6PD activity, while G6PD deficient samples shows little or no
fluorescence.
Material:
Specimen: 0.5 ml of EDTA blood
Equipment:
- UV chamber with long wavelength (320 – 420 nm) lamp
- 37°C water bath
- Paper/eyelet punch
- Hair dryer
Reagent:
1. Tris-HCl buffer 0.75 M pH7.8
2. GSSG (gluthathion oxidized) 0.008 M
3. NADP or TPN (triphosphoridine nucleotide) 0.0075 M
4. G6P (glucose-6-phosphate) 0.01 M
Reagent preparation
1. Tris-HCl buffer 0.75 M, pH 7.8
1.1. 0.75 M Tris
Tris(hydroxyl methyl) aminomethane 90.855g/L
[2-amino-2-(hydroxymethyl) propane-1, 3-diol] molecular weight: 121.14
1.2. 1 N HCl
Add 42.55 ml concentrated HCl very slowly into deionized water and top up to
500 ml.
1.3. Tris-HCl buffer 0.75 M, pH 7.8
0.75 M Tris 250 ml
1 N HCl 125.6 ml
Adjust pH to 7.8 and make up to 1 L with water.
2. GSSG
0.098 g (98 mg) in 20 ml Tris-HCl buffer.
3. TPN
0.1148 g (114.8 mg) in 20 ml Tris-HCl buffer
4. G6P
0.00608 g (60.8 mg) in 20 ml Tris-HCl buffer
Working reagent
Freshly mix equal volumes of GSSG, TPN, and G6P
Procedure:
1. The blood is collected on 3 mm filter papers, strip of 8x80 mm size.
2. Allow it to dry.
3. Punch a disc of 6 mm from the sample strip with an eyelet punch.
4. Place the disc into the tube containing 0.1 ml of working reagent.
5. Incubate at 37°C for 15 minutes.
6. Spot the solution on to a filter paper (Whatman no. 1)
7. Allow it to dry.
8. Examine under long UV lamp for fluorescence.
Quality control
A known intermediate and deficient sample is used as controls. It is included in each
batch of G6PD screening test.
Limitations
The fluorescent spot test is limited by nature of fluorescence pattern assessment,
particularly with intermediate levels of enzyme activity.
Result:
Blood spot visualised under UV light
Normal : Fluorescent
Intermediate : Fluorescent intensity reduced
Deficient : no fluoresence
G6PD deficiency clinically presents as a drug-induced hemolytic anemia, neonatal
jaundice, favism or chronic non-spherocytic hemolytic anemia. The disorder is sex-
linked with full expression in hemizygous males and homozygous females. The female
heterozygous is characterized by a mosaic population of red cells containing both
normal and deficient G6PD activity in individual erythrocytes. This phenotype results
from inactivation of an X chromosome
Name of test: D-Dimer
Principle: During blood coagulation, fibrinogen is converted to fibrin by activation of

thrombin. The resulting fibrin monomers polymerize to form a soluble gel of non-cross-
linked fibrin. This fibrin gel is then converted to cross-linked fibrin by thrombin activated
Factor XIII to form an insoluble fibrin clot. Production of plasmin, the major clot-lysing
enzyme, is triggered when a fibrin clot is formed. Fibrinogen and fibrin are both cleaved
by fibrinolytic enzyme plasmin to yield degradation products, including cross-linked
fibrin degradation products (XL-FDP). Degradation products from cross-linked fibrin
also contain D-dimer protein molecule. Therefore, XL-FDP is a specific marker of
fibrinolysis. ActiScreen XL-FDP is a rapid immunoagglutination assay utilizing latex
beads coupled with a highly specific monoclonal antibody. XL-FDP present in plasma
sample binds to the coated latex beads, which results in visible agglutination occurring
when the concentration is above the upper limit of detection for the assay.
Material:
Reagents
Immunoagglutination Reagent (white cap): 1 x 2.0 ml dropper bottle containing a
0.83% suspension of latex particles coated with mouse monoclonal anti-XLL-FDP
antibody, 10 mg/ml BSA and 0.1% sodium azide.
Positive control (yellow cap): 1.x 0.6 ml dropper bottle containing solution of purified
human XL-FDP fragments, 5 mg/ml BSA and 0.1% sodium azide.
Negative control (black cap): 1 x 0.6 ml dropper bottle containing a buffer solution
containing 5 mg/ml BSA and 0.1% sodium azide.
Buffer: 1 x 20.0 ml 10 mM phosphate buffer solution with 0.1% sodium azide.
Patient’s blood sample in EDTA vacutainer (lavender top)
Procedure:
A. Qualitative method
1. Take one disposable test card. Avoid touching the reading surface. Mark, or make
note of, positions on the test card for specimens and, as needed, for positive and
negative controls.
2. Hold the immunoagglutination reagent dropper bottle vertically and place one drop
of the reagent within a well on a test card. Avoid touching the surface of the test
card.
3. Accurately pipette 20 µl of undiluted plasma, or one drop of control solution, inside
the same well next to the drop of immunoagglutination reagent.
4. Mix the immunoagglutination reagent and test samples with a stirrer until the latex
is uniformly distributed.
5. Rock the test card gently by hand for exactly 3 minutes.
6. At exactly 3 minutes, check for agglutination under a strong light source. (NOTE:
If test reading is delayed beyond 3 minutes, the latex suspension may dry out giving
a false agglutination pattern. If this is suspected, the specimen must be retested).
7. Discard the test card and stirrer into a biohazard container and do not re-use.
B. Semi-quantitative method
1. Prepare doubling dilutions of the test plasma with buffer solution as follows
- 1:2 dilution: Add 100 µl of plasma to 100 µl of buffer.
- 1:4 dilution: Add 100 µl of 1:2 dilution to 100 µl buffer.
- 1:8 dilution: Add 100 µl of 1:4 dilution to 100 µl buffer.
2. Repeat this procedure to extend the dilution series as desired. Test each dilution
as described in the qualitative method (Procedure A).
Result:
A. Qualitative method
For the qualitative method protocol, the following pattern of results should be obtained.
Note: All values in ng/ml and mg/L are approximate
Undiluted plasma XL-FDP concentration
Negative Less than 200 ng/mL (0.20 mg/L)
Positive Greater than 200 ng/mL (0.20 mg/L)
B. Semi-quantitative method
Approximate levels of XL-FDP for specimen dilutions are shown in the table below. As
with all semiquantitative tests, some variability in dose-response can be expected.
XL-FDP Sample dilution
Levels ng/mL (mg/L) Undiluted 1:2 1:4 1:8
< 200 (< 0.20) - - - -
200 – 400 (0.20 – 0.40) + - - -
400 – 800 (0.40 – 0.80) + + - -
800 – 1600 (0.80 – 1.60) + + + -
1600 – 3200 (1.60 – 3.20)* + + + +
‘+’ = agglutination, ‘-’ = no agglutination

* Levels of XL-FDP greater than 3200 ng/mL (3.20 mg/L) can be estimated by further
dilutions beyond 1:8.
Positive control Negative control

BIOCHEMISTRY
LABORATORY
Name of test:
Principle:
Material:
Procedure:
Result:
Name of test: Routine biochemistry test
Principle:
 Photometric
Photometer: 14 fixed wavelengths (340, 410, 451, 478, 505, 545, 571, 596, 658,
694, 751, 805, 845, and 884 nm)
Assay methods: Endpoint, rate reaction, 2-point rate, multi-point homogeneous
immunoassay
 Ion-selective electrode (ISE)
1. The first reagent (R1) for a test is aspirated from reagent tray 1 and dispensed by
the reagent probe into the cuvette in the reaction tray.
2. Samples on the sample tray or rack handler are aspirated and diluted by the dilution
probe, then dispensed into cuvettes in the dilution tray.
3. The dilution mixer stirs the diluted sample.
4. The sample probe dispenses the required amount of diluted sample into the RRV
cuvettes (the reagent is already in the cuvettes).
5. The system can use the remaining diluted sample in the DTT cuvettes for additional
tests on a workorder, a rerun, dilution, or reflex testing.
6. The reaction mixer 1 mixes the first reagent and the sample.
7. The second reagent (R2) for a test is aspirated from reagent tray 2 and dispensed
by the reagent probe into the cuvette in the reaction tray.
8. The reaction mixer 2 mixes sample and reagent 1 and reagent 2.
9. The reaction takes place for the amount of time designated in the assay.
10. The spectrophotometer obtains the concentration data every six seconds.
11. For each measurement, the RRV moves the cuvettes in front of the
spectrophotometer.
12. Cell blank measurements are performed at each wavelength.
13. The RRV cuvettes are washed when measurement is complete.
14. When the analysis is complete, the lamp energy is checked at each wavelength
The reagent probes (RPP1 and RPP2) aspirate reagent from the reagent trays (RTT1
and RTT2) and dispense it into reaction tray (RRV) cuvettes for analysis, according to
specified conditions. Reagent pumps (RP1 and RP2) handle the aspiration and
dispensing functions.
The ISE measures the amount of sodium (Na), potassium (K), and chloride (Cl) in
serum or urine samples through voltage measurement by ion-selective electrodes.
The sample-dilution probe (DPP) aspirates the sample for electrolyte analysis. The
electrolyte analysis uses buffer as reagents.
In a two-stage process, the buffer voltage is measured, then the sample voltage is
measured. The difference between these voltages, the reference voltage, and the
temperatures of the liquids determine the concentration of Na, Cl, and K in the sample
.
Material:
Patient’s blood specimen in gold top vacutainer (with clot activator and gel for serum
seperation)
Siemens ADVIA 2400 chemistry analyzer
Siemens Aptio automation
Procedure:
1. Patient’s name, RN, test ordered was checked and make sure tally on both
specimen vacutainer and barcode.
2. Barcode was then placed on vacutainer.
3. Cap of vacutainer was removed and placed in sample rack specific for ADVIA 2400
chemistry analyzer.
4. The rack is then load into Siemens APTIO automation system.
5. Sample is automatically centrifuge, analyzed, and stored.
Result:
Routine chemistry analyzer: SIEMENS Healthineers ADVIA 2400 Chemistry System
Name of test: Urine screening
Principle:
Reflectance photometry: with 4 different wavelengths (465, 528, 560 and 615 nm)
Refractometry: SG testing
Turbimetry: clarity testing
Specific gravity (SG): The test detects the ion concentration of the urine. In the
presence of cations, protons are released by a complexing agent and produce a color
change in the indicator bormothymol blue from blue via blue-green to yellow.
pH: The test paper contains the indicators methyl red, phenolphthalein and
bromothymol blue and reacts specifically with H+-ions. The most frequent pH values
of fresh urine from healthy subjects lie between 5 and 6.
Leukocytes (LEU): The test reveals the presence of granulocyte esterases. These
esterases cleave an indoxyl ester, and the indoxyl so liberated reacts with a diazonium
salt to produce a violet dye. Bacteria trichomonads or erythrocytes present in the urine
do not affect the reaction.
Nitrite (NIT): The test is based on the principle of the Griess test and is specific for
nitrite. The reaction reveals the presence of nitrite and hence indirectly nitrite-forming
bacteria in the urine by a pink-to-red coloration of the test patch. Even a slight pink
coloration is indicative of significant bacteuria.
Protein (PRO): The test is based on the principle of the protein error of a pH indicator.
It is particularly sensitive to albumin, Quinine, quinidine, chloroquine, tolbutamide and
an elevated pH (up to 9) do not affect the test.
Glucose (GLU): The glucose determination is based on the specific glucose-

oxidase/peroxidase reaction (GOD/POD method). The test is independent of the pH
and specific gravity of the urine and is not affected by the presence of ketone bodies.
Ketone bodies (KET): This test is based on the principle of Legal’s test and is more
sensitive to acetoacetic acid than acetone.
Urobilinogen (UBG): A stable diazonium salt reacts almost immediately with

urobilinogen to give a red azo dye. The test is specific for urobilinogen and is not
susceptible to the interfering factors know n to affect the Ehrlich’s test.
Bilirubin (BIL): The test is based on the coupling of bilirubin with a diazonium salt. Even
the slightest pink coloration constitutes a positive, i.e. pathologic, result. Other urinary
constituents produce a more or less intense yellow coloration.
Blood (ERY/Hb): The peroxidase-like action of hemoglobin and myoglobin specifically
catalyzes the oxidation of the indicator by means of the organic hydroperoxide
contained in the test paper to give a blue-green coloration.
Compensation area (COMP): This white area, which is not impregnated with reagents,
allows instrumental compensation for the intrinsic color of the urine while testing
leukocytes, nitrite, protein, glucose, ketone bodies, urobilinogen and bilirubin
Material:
Patient’s urine sample in appropriate urine container
Cobas U 601 urine analyzer
Procedure:
1. Urine specimen received in urine container, together with barcodes with registered
test.
2. Check RN, patient name, and barcode number on both specimen container and
barcode, make sure they are tally.
3. Place barcode on a clean plastic centrifuge tube.
4. Urine from urine container was poured into a plastic centrifuge tube until the 10 mL
mark.
5. Centrifuge tube with urine specimen was then placed into rack specific for Cobas
U 601 analyzer.
6. The rack loaded with centrifuge tube was then placed into input buffer of Cobas U
601 analyzer.
7. Analysis would be ran as soon as the rack is loaded, as the analyzer equipped with
sensors.
Result:
Name of test: Urine Microscopy
Principle: To ‘sieve out’ urines requiring microscopic examination, the following

parameter must be positive, ie: leukocytes, erythrocytes, protein, nitrite, and pH of
>7.0. Blood, kidney, lower genitourinary tract and external contamination contribute
formed elements to the urine. These are RBC, WBC, epithelial cells, bacteria, yeast,
mucus, fat globules, spermatozoa, crystals, casts and artifacts.
Microscopic examination is done to identify these insoluble materials present in

urine specimens.
Material:
Microscope, counting chamber, centrifuge, capillary tubes
Procedure:
Collection of urine
Clean mid-stream urine or random urine sample should be sent to laboratory within 2
hours from time of collection.
Urine microscopy
1. Use a marked conical centrifuge tube, top mark is at 10 ml, bottom mark is at 1 ml.
2. Mix the urine by swirling the container or by gentle inversion several times if the
container is full.
3. Pour urine into centrifuge tube until it reaches the top mark.
4. Centrifuge at 1500 rpm for 5 minutes.
5. Aspirate off the supernatant urine until it reaches the bottom 1 ml mark.
6. Resuspend the 1 ml sediment by gentle agitation.
7. About 1 drop of resuspended sediment is transferred to a counting chamber.
8. Count the RBC and WBC in one large square using a 40x objective and report as
cells/µl.
9. Examine the deposit further under 10x and then 40x objective for any casts,
crystals, and other urine deposits. Report findings as occasional (occ), moderate
(+), or numerous (++) or (+++), or packed field.
Result:
occ 1 - 5 under high power field
+ 6 - 10 under high power field
++ 11 - 15 under high power field
+++ > 15 under high power field
Packed field When RBC and WBC count exceed 250 cells/µl or cannot be
counted in one microscopic field (40x)
Frank blood If fresh blood or blood was obviously visible in urine, do not
proceed with microscopy.
Blood stained If urine is reddish in color, perform microscopic examination.
Calculation of RBC and WBC in cells/µl

Depth of Fuchs-Rosenthal chamber : 0.2mm
Area of 16 small squares : 1.059 mm
Volume of 16 small squares (1 big squares) : 0.2 cubic mm (µl)
Concentration of urine (from 10 ml to 1 ml) : 10
𝑥
𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑖𝑜𝑛 = ÷ 10 (𝑥 = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑐𝑜𝑢𝑛𝑡𝑒𝑑)
0.2
Dysmorphic RBC
Dysmorphic RBC, if detected, shall be reported as a percentage of total RBC counts.
1. Casts need to be identified and reported as hyaline, granular, WBC, RBC,
epithelial, fatty, or waxy casts
2. Crystals identified will be reported, for example, uric acid, CaOX, amorphous
phosphate, etc.
3. If Trichomonas spp., sperm, or yeast are identified, will be reported.
Limitations
- Ideally, specimen for routine urinalysis should be examined while fresh. If this is not
possible, then it should be refrigerated until exam. Specimen left at room temperature
will soon begin to compose, mainly due to presence of bacteria in sample.
- Centrifugation speed must be maintained so that any cells and casts present will not
be denatured.
- The centrifuge tubes used must be thoroughly washed so as to prevent cross
contamination and also to prevent lysis of cellular component in urine deposits.
Reference ranges
Men/Child RBC 0 - 3/µl
WBC 0 - 3/µl
Women RBC 0 - 3/µl
WBC 0 - 10/µl
Clinical applications
Examination of urinary sediment is of great value in detecting asymptomatic subclinical
cases or UTI, particularly pyelonephritis. It is also invaluable in detecting very early
cases of glomerulonephritis. The urinary sediment may yield info of a prognostic
nature. Fatty casts may be found in urine before other clinical feature of nephrotic
syndrome develop casts in urine of patient at rest may give warning many years in
advance of the onset of serious CVD, or renal disease.
Name of test: Fluid FEME
Principle:
Material:
Microscope, counting chamber, centrifuge, capillary tubes
Procedure:
1. Mix sample thoroughly by gentle agitation.
2. About 1 drop of resuspended sediment is transferred to a counting chamber.
3. Count the RBC and WBC in one large square using a 40x objective and report as
cells/µl.
4. Examine the deposit further under 10x and then 40x objective for any casts,
crystals, and other urine deposits. Report findings as occasional (occ), moderate
(+), or numerous (++) or (+++), or packed field.
Result:
Cryptococcus spp.
occ 1 – 5/cover slip
+ 6 – 15/cover slip
++ > 15 under high power field
Mononuclear cells 500 cells/µl

- Viral/aseptic meningitis, tuberculosis, or fungal meningitis
- Acute bacteria meningitis, cell count usually over 1000 cells/µl, mostly
polymorphonucleus leukocyte.
- Normal CSF have concentration of glucose about ½ - ⅔ compared in blood
- Low glucose – bacteria meningitis, viral infection, TB, carcinomatous and fungal
meningitis.
- CSF protein high in many situation, thus non specific.
- contamination with blood, can high
- inflammatory conditions due to Guillain-Barre syndrome or tumours
- CSF EP can be useful for diagnosis for multiple sclerosis.
Reference ranges
Colour = Colourless
Clarity = Clear
Microscopic
Cell count RBC : negative
WBC (lymphocyte only) : Adults 0 – 5/µl
WBC (lymphocyte only) : Newborn up to 30/µl
Organism negative
Cryptococcus spp. negative
Biochemistry
Glucose : 2.5 – 5.0 mmol/l
Protein : 0.15 – 0.45 g/l
Synovial fluid
- Glucose and uric acid concentration are equivalent to blood plasma
- Total protein and immunoglobulin concentration can vary from ¼ - ½ of in plasma.
Fat globules
- Colourless – Can be stained with bile pigment
Neutral fat dissolve readily in cold ethanoic solution (ethanol). Sudan III in
concentrated ethanoic solution stains neutral fat orange to red.
Sudan III : 1 g Sudan III powder

100 ml absolute alcohol
Limitations
Laxative such as liquid paraffin may result in false positive.
Clinical applications
- Found in patient lacking lipase enzyme/bile duct obstruction.
Name of test: Pregnancy test
Principle: The hCG one step pregnancy test device (urine) is a rapid chromatographic
immunoassay for the qualitative detection of human chorionic gonadotropin in urine to
aid in the early detection of pregnancy. The test uses two lines to indicate results. The
test line utilizes a combination of antibodies including a monoclonal hCG antibody to
selectively detect elevated levels of hCG. The control line is composed of goat
polyclonal antibodies and colloidal gold particles. The assay is conducted by adding a
urine specimen to the specimen well of the test device and observing the formation of
colored lines. The specimen migrates via capillary action along the membrane to react
with the colored conjugate. Positive specimens react with specific antibody-hCG-
colored conjugate to form a colored line at the test line region of the membrane.
Absence of this colored line suggests a negative result. To serve as a procedural
control, a colored line will always suggest a negative result. To serve as a procedural
control, a colored line will always appear in the control line region. If the control line
does not appear, the test result is not valid.
Material:
Reagents
- anti-hCG particles and anti-hCG coated on the membrane.
Materials
 Test devices
 Droppers
 Patient’s urine sample in appropriate urine container
Procedure:
1. Allow the test, urine specimen and/or controls to reach room temperature (15 -
30°C) prior to testing.
2. Bring the pouch to room temperature before opening it. Remove the test device
from sealed pouch and use it as soon as possible.
3. Place the test device on a clean and level surface. Hold the dropper vertically
and transfer 3 full drops of urine (approx. 100 µL) to the specimen well (S) of
the test device, and then start the timer. Avoid trapping air bubbles in the
specimen well (S).
4. Wait for the colored line(s) to appear. The result should be read at 3 minutes.
Do not interpret the result after 10 minutes.
Note: A sample hCG concentration below the cut-off level of this test might
result in a weak line appearing in the test region after the 3 minute read time. A
line in the test region seen after the 3 minute read time could be indicative of a
low hCG level in the sample. If such results are seen, it is recommended that
the test be repeated with a new sample in 48 – 72 hours or that an alternate
confirmation method is use.
Result:
Positive: two distinct colored lines appear. One line should be in the control line region
(C) and another line should be in the test line region (T).
Note: The intensity of the color in the test line region (T) may vary depending on the
concentration of hCG present in the specimen. Therefore, any shade of color in the
test line region (T) should be considered positive.
Negative: One colored line appears in the control line region (C). No apparent colored
line appears in the test line region (T).
Invalid: No line appears in the control line region (C). If this occurs, read the directions
again and repeat the test with a new test. If the result is still invalid, stop using the test
kit immediately.
Name of test: Hormonal studies
Principle:
Siemens IMMULITE 2000 XPi uses assay-specific antibody or antigen-coated
polystyrene beads as the solid phase.
A bead is dispensed into a specially designed Reaction Tube, which serves as
the vessel for the incubation, wash, and signal development processes.
After the sample is incubated with an alkaline phosphatase-labeled reagent, the
reaction mixture is separated from the bead by spinning the Reaction Tube at high
speed along its vertical axis. The fluid is transferred to a Coaxial Sump Chamber,
which is integral to the Bead/Tube Wash Station. Four discrete washes occur within
seconds, allowing the Reaction Tubes to be processed sequentially with uniform timing.
The bead remains in the Reaction Tube with no residual unbound label.
The bound label is then quantified using the dioxetane substrate to produce
light. Light is emitted when the chemiluminescent substrate reacts with the alkaline
phosphatase label bound to the bead. The amount of light emitted is proportional to
the amount of analyte originally present in the sample. This light emission is detected
by the Photomultiplier Tube (PMT) and results are calculated for each sample.
Material:
Patient’s blood sample in SST vacutainer (gold top)
Siemens IMMULITE 2000 XPi
Procedure:
2. Place barcode sticker to a plain plastic tube according to test ordered.
3. Place vacutainer into centrifuge bucket and balance.
5. Transfer serum into plastic centrifuge tube.
6. Place plastic tube into sample rack.
7. Load sample rack into analyzer.
Result:
Instrument: Siemens IMMULITE 2000 XPi

Name of test: Thyroid function test
Principle:
 Sandwich ELISA – Direct chemiluminisence
 Competitive ELISA – Indirect chemiluminiscence
 Chemiluminescence using Advanced Acridinium Ester
v
v
Material:
Patient’s blood sample in SST vacutainer (gold top)
Siemens ADVIA Centaur XPT Immunoassay system
Procedure:
9. Place barcode sticker to a plain plastic tube according to test ordered.
10. Place vacutainer into centrifuge bucket and balance.
12. Transfer serum into plastic centrifuge tube.
13. Place plastic tube into sample rack.
14. Load sample rack into analyzer.
Result:
Instrument: Siemens ADVIA Centaur XPT Immunoassay System

Name of test: Everolimus
Everolimus is a macrolide immunosuppressant derived by chemical modification of the
natural product rapamycin. Rapamycin is produced by certain strains of Streptomyces
hygroscopicus.
Immunosuppressivve treatment stratergies are aimed at prevention of T cell

activation and/or proliferation. Everolimus acts as a proliferation inhibitor. On a cellular
level, everolimus inhibits in general, growth factor involved. Inhibition is reversible
since everolimus is not a cytotoxic compound. Everolimus inhibits the T cell by
inhibiting G0 to G1 phase. Calcineurin inhibitors, cyclosporine (CSA) and Tacrolimus,
prevents the activation of T cells by inhibiting G0 to G1 phase transition. The different
modes of action for everolimus and calcineurin inhibitors such as cyclosporine provide
adequate rationale for the pharmacodynamics synergy.
Careful blood concentration guide dosing is recommended as an aid in patient

management with clinical use of Everolimus. The preferred matrix is whole blood
because at therapeutic concentration the compound is predominately partitioned into
erythrocytes. Liquid chromatography couple to mass spectrometry has been used to
measure concentration of everolimus in blood.
Principle:
The QMS Everolimus assay is a homogenous particle enhanced turbidimetric
immunoassay. The assay is based on competition between drugs in the sample and
drug coated onto a microparticle for antibody binding sites of the Everolimus antibody
reagent. The everolimus coated microparticle reagent is rapidly agglutinated in the
presence of the anti-everolimus antibody reagent and in the absence of any competing
drugs in the sample. The rate of absorbance changes is measured photometrically.
When a sample containing everolimus is added, the agglutination reaction is partially
inhibited slowing down the rate of absorbance. A concentration dependent classic
agglutination inhibition curve can be obtained with maximum rate of agglutination at
the lowest everolimus concentration and the lowest agglutination rate at the highest
everolimus concentration.
Material:
Procedure:
Extraction procedure for samples, calibrators, and controls
Extracts must be run immediately after extractions
1. Prepare micro-centrifuge tubes for extraction of samples, calibrators, and
controls.
2. Mix samples, calibrators, and controls well by inversion.
3. Accurately pipette 350 µl of calibrators, controls, samples to be assayed into
the appropriate micro-centrifuge tube.
4. Accurately pipette 350 µl of methanol into each micro-centrifuge tube.
5. Accurately pipette 50 µl of QMS everolimus precipitation reagent into each
micro-centrifuge tube.
6. Cap each micro-centrifuge tube and immediately to prevent evaporation, then
vigorously vortex at the highest speed for at least 10 sec. It may be necessary
to invert the tube and remix to ensure complete mixing. After mixing, the sample
should change from red to brown color.
7. Place the tubes in a micro-centrifuge tube and centrifuge for at least 8 minutes
at 1300g.
8. After centrifuge, pipette at least 350 µl of each supernatant into new micro-
centrifuge tube and cap immediately to minimize sample evaporation.
9 Dispose of extracts after analysis. Retesting of samples requires fresh
extractions.
10. Begin the analyzer calibration or assay process immediately to minimize
sample evaporation.
11. Dispose of extracts after analysis. Retesting of samples requires fresh
extractions.
Result:
Name of test: Urine toxicology
Principle: A deuterated internal standard (D5-Diazepam) is added to urine and QC

samples to serve as an internal quality control. Urine sample is then hydrolysed by
beta glucuronidase solution (enzyme hydrolysis) and incubated at 60°C for 2 hours.
Drugs of interest are recovered by solid phase extraction, with the use of Oasis SPE
MCX cartridge. At the end of extraction, two fractions are collected, one contains acidic
and neutral compounds, and another one contains basic compounds. Both fractions
are evaporated to dryness by N2 (g) and reconstitute in 20% methanol prior analysis.
Screening is performed using an MRM-IDA (Information Dependant Acquisition)

workflow. This workflow consist of an MRM survey scan that contains MRM transition
for 200 different drugs and their metabolites.
If a signal is detected, an ion trap enhanced product ion (EPI) MS-MS spectrum
is acquired, and this spectrum can be searched against an MS-MS library for
identification and confirmation.
Cliquid Drug Screen and Quant software are used for data acquisition,
processing and reporting.
Material:
Sample requirement
Urine sample is required.
- Sample type: a ‘clean-catch’ (mid-stream urine). Hands and genital area should be
washed clean and wiped dry prior to urination.
- Sample container: sterile urine container
- Volume requirement: at least 5 ml of urine.
- Delivery: all samples collected must be sent to the lab immediately by ward/clinic
staff. Samples should not be sent by patient, personally. If immediate delivery of
samples is not available, store samples in refrigerator or -20°C freezer prior to delivery.
- Store samples in -20°C freezer upon receipt in the lab .
- Sample rejection criteria: follow laboratory standard acceptance and rejection criteria.
At average, drugs in urine are stable for about 3 days at room temperature, 14 days if
refrigerated and longer period if frozen. Reject if samples received exceed the stability
limits.
Equipments and consumables

- ABSciex 3200 QTRAP tandem mass spectrometer
- Shimadzu Prominence UFLC system
- Oasis MCX SPE cartridge
- Oven
- Heating block
- Centrifuge
- Vortex
- Glass test tubes with stopper/cap, 10 ml
-Autosampler vials (2 ml, screw cap) and vial inserts (250 µl glass with polymer feet)
- Thermo Scientific Titan Syringe Filter-PVDF (0.2 µm)
- Microcentrifuge tubes, 1.5 ml
Reagents:
1) Mobile phase
a) Mobile phase A: water + 10 mM ammonium formate
b) Mobile phase B: acetonitrile : methanol (50:50, v/v)
c) (Filter and sonicate mobile phases before uses ensure there is no precipitate in
mobile phase A)
2) β-glucuronidase, from Helix Pomatia (> 85,000 units/ml)
a) Sigma Aldrich
b) Store in 2 - 8°C. When stored at 2 - 8°C, enzyme retains activity for at least 1
year.
3) Sodium acetate buffer (0.1 M, pH 4.0).
a) Weigh 0.246 g of sodium acetate anhydrous (MW: 82.03 g/dl). Dissolve in ~20
ml of deionized water. Using 6N hydrochloric acid, adjust pH to 7.5. Top up to
30 ml with deionized water. Store at room temperature. Stable for 1 month.
4) Sodium phosphate buffer (0.1 M, pH7.5)
a) Weight 0.426 g of disodium hydrogen orthophosphate (MW: 141.96 g/dl).
Dissolve in ~20 ml of deionized water. Using 6N hydrochloric acid, adjust pH to
7.5. Top up to 30 ml with deionized water. Store at room temperature. Stable
for 1 month.
5) SPE (solid phase extraction) reagents
a) Methanol
b) Deionized water
c) 0.1N hydrochloric acid (stable for 1 year)
i) Prepare 1 N hydrochloric acid (in 50 ml)
Using a volumetric glass pipette, slowly add 4.17 ml of 12 N Hcl, into
deionsed water to final volume of 50 ml.
ii) To prepare 0,1 N hydrochloric acid
Using a volumetric glass pipette, slowly add 10 ml of 1 N HCl into deionized
water to final volume of 100 ml.
d) Methanol : ammonium hydroxide (95 : 5, % v/v)
- Prepare just before use. Slowly add 1 ml of ammonium hydroxide solution
(25%), to 19 ml of methanol.
- 1% hydrochloric acid (stable for 1 year): Slowly add 2.7 ml of 37% HCl into
deionized water to final volume of 100 ml
- 20% methanol (stable for 1 month): Add 10 ml of methanol to 40 ml of
deionized water.
6) Internal standard
D5-Diazepam, 1 mg/ml (Cerilliant, TX) solution. Prepare as working stock (5 µg/ml)
in methanol. Store in -20°C. Stable for 2 months.
7) External standards
a) Drugs of Abuse (Cenrriliant, TX), 1 mg/ml each
i) Amphetamine
ii) Methamphetamine
iii) 3,4-methylenedioxymethamphetamine
iv) 3,4-methylenedioxyamphetamine
v) Morphine
vi) Codeine
vii) 6-acetylmorphine
viii)Ketamine
ix) Diazepam
x) Lysergide
8) Blank (neg) sample
Urine donated from an identified person is screened to ensure its integrity (drug
free). Store at 2-4°C.
Quality control
Internal QC
- For each batch of sample processing, include one negative and one positive internal
QC material, process as the same condition as the samples.
Review of QC results
- IQC neg: Absence of any drugs of interest. Only internal standard peak should be
detected at its respective time.
- IQC pos: Presence of any drug from external stander mixtures. All external standard’s
peak and internal standard peak should be detected at their respective retention time.
- Acceptance of sample/QC run: Internal standard peak area is > 1.0x10 5.
External QC
Laboratory participates in RCPA QAP for urine toxicology. 12 samples per cycle,
process as the same condition as the samples. Results is submitted, through the
website, before the stated due date.
Procedure:
1. Preparation of instruments
1.1. Start-up procedure
i. HPLC
ii. Mass spectrometer
iii. Analyst software
2. Sample preparation and extraction
2.1. Enzyme hydrolysis
i. Label test tube (sample/QC) with sample number accordingly.
ii. Transfer 0.5 ml of urine/QC sample into glass tube, then add 50 µl of
internal standard solution (D%-Diazepam, 5 µg.ml).
iii. Add 0.5 ml of 0.1 M sodium acetate (pH 4.0) buffer. Vortex and mix well.
iv. Add 53 µl of β-glucuronidase enzyme (approximately 4500 units), and
vortex. (Preferably handle in dark room, away from source of light).
v. Wrap glass tubes with aluminium foil.
vi. Incubate in oven at 60°C for 2 hours.
vii. When incubation completed, leave samples at room temperature to cool
down for approximately 15 to 20 minutes.
viii. Proceed with solid phase extraction.
2.2. Solid phase extraction by mixed mode cation exchange cartridge
i. Set up SPE cartridge to vacumm manifold chamber. Place a tube for
waste fluid collection.
ii. Precondition the cartridge with 2 ml of methanol, immediately followed
by 2 ml of deionized water.
iii. Switch on vacumm pump, and allow it to pass through at rate of 5 ml/min.
iv. Adjust flow rate to 1 ml/min and load hydrolyzed urine/QC samples.
v. Wash sorbent by passing through 1 ml of 0.1 N hydrochloric acid at flow
rate of 5 ml/min. Let dry fro 3 minutes.
vi. Release vacumm and discard waste fluid. Place a collecting tube and
turn on vacumm. Adjust flow rate to 1 ml/min.
vii. Elute first fraction (acidic and neutral compounds) by passing through 2
ml of methanol.
viii. Release vacumm, and place another collecting tube.
ix. Turn on vacumm. Elute second fraction (basic compounds) by passing
through methanol ammonium hydroxide (95:5, v/v) solution, 1 ml by 1
ml (total volume of 2 ml).
x. BASIC FRACTION only: add 50 µl of 1% hydrochloric acid, vortex and
mix well.
xi. Evaporate both fractions to dryness under nitrogen stream on heat
block at temperature of less than 40°C.
2.3. Sample reconstitution
i. Reconstitute both fractions with 2000 µl of 20% methanol.
ii. Filter into labelled microcentrifuge tubes by using syringe filter (0.2 µm).
Spin at 3500 rpm for 3 minutes.
iii. Transfer filtrate into an insert and place insert into labeled vial. Ensure
no air bubble is trapped inside the insert. Ready for analysis.
2.4. Reporting
i. Pathologist checks chromatograms and mass spectrum with library to
confirm presence of drugs in acidic and basic extracts.
ii. Entry of the drugs present in the LIS is done by pathologist. If no drugs
of interest are detected, it is reported as ‘no drug detected’.
iii. Results are validated by pathologist.
2.5. Test limitation
False negative results can occur due to low drug concentrations in urine,
tampering, dilute urine, prolonged time since last use, metabolic factors and in
other situations.
For positive screening results, an extensive medication history including
prescription, non-prescription, and herbal medications should be obtained from
patient medication histories are important in order to anticipate false positive
as well as differentiate between drugs used for legitimate medical purposes
and drugs of abuse.
2.6. Storage and archiving
i. Keep samples frozen, in their original containers, at -20°C for at least 1
year
ii. Keep reports indefinitely.
Result:
HISTOPATHOLOGY
/CYTOLOGY
LABORATORY
Department profile
Besides providing clinical services to University Malaya Medical Centre (UMMC), the
department is also responsible in training tutors and lecturers for MBBS degree,
BDS, BBiomedSc and postgraduates courses, Masters in Pathology, Medical
Science Masters in Clinical Pathology, Medical Science Masters, medical doctors
and philosophical doctors under the Faculty of Medicine, University Malaya.
Anatomic Pathology Division
Anatomic Pathology Division provides diagnostic services in histopathology and

cytopathology to UMMC.
Types of services provided by Histopathology Lab are histopathology routine

services based on H+E colouring, special colouring, immunohistochemistry, and in-
situ hybridization. Histopathology Lab also provides prompt diagnostic
services via intraoperative frozen section diagnosis. Clinical and specialist consultant
in this division involve in consultation services for hospitals under the Ministry of
Heallth and private sectors in kidney biopsy, cardiac transplant, muscles, neurologic
and etc.
Gynaecology and non-gynaecology smear test together with Fine Needle

Aspiration are among the services provided by Cytopathology Lab. Pathology
Specialists conduct Fine Needle Aspiration for UMMC patients of Fine Needle
Aspiration Clinic held twice a week.
Organization chart
DIRECTOR
YM PROF. DR. TUNKU KAMARUL ZAMAN BIN TUNKU ZAINOL ABIDIN
SPECIAL GRADE A
DEPUTY DIRECTOR (CLINICAL SERVICES)

ASSOC. PROF. DR. NAZIRAH BINTI HASNAN
PREMIER GRADE C
HEAD OF DEPARTMENT
ASSOC. PROF. DR. NAZARINA BINTI ABD RAHMAN
ASSOCIATE PROFESSOR GRADE DU54
ANATOMIC PATHOLOGY
HISTOPATHOLOGY LAB CYTOPATHOLOGY LAB
DEVISION
DR. DIANA ONG BEE LAN DR. TOH YEN FA
PROF. MUN KEIN SEONG
Name of test: Tissue processing
Principle:
The Leica TP1020 is an automatic tissue processor designed for laboratory
applications. It is used for the fixation, dehydration and infiltration of histological tissue
samples with fixatives, alcohol, solvents and paraffin wax.
The reagent stations numbered 1 - 10 are used to contain reagents. Station 10
may be replaced with an optional third wax bath. Stations 11, 12, and if used 10, are
heated, temperature controlled wax baths that can be filled with either wax pellets or
molten paraffin wax.
Embedding cassettes used to hold the tissue samples, are placed into the
tissue basket. The basic instrument is designed for a single tissue basket. An optional
second basket can be added. The basket, or baskets, are moved clockwise from
station to station.
To ensure thorough infiltration the basket containing the tissue samples is
agitated, by moving up and down, at each station. This function can be switched off at
any time.
During processing as the tissue basket moves from station to station there is a
delay period of sixty (60) seconds during which time the basket is suspended above
the station. Excess liquid can drip down during this process. This ensures that there is
minimal reagent carryover from station to station.
All instrument functions are activated through the control panel. Real time is
displayed via LCD. The instrument can be operated in manual and automatic
processing mode. Automatic processing is controlled via 9 different programs which
can be individually set up, altered and edited.
If a power failure occurs, the specimens are protected from drying out - even
when overnight processing has been selected, since in case of a power failure the
tissue basket will always be immersed into a station. Once the power is restored,
processing will be resumed where it had been interrupted. After a long-term power
failure, critical excess immersion time in a station will be visually displayed
Material:
Instrument: Leica TP1020 Tissue Processor
Grossed and/or trimmed tissue enclosed in plastic cassette
Procedure:
1. Insert basket into first container, which contains 10% neutral buffered formalin.
2. Select profile and start the processing.
3. Tissue processing will be completed by the morning of following day.
Processing step in Leica Carousel TP 1020

1. Formalin 10%
2. Formalin 10%
3. Alcohol 95%
4. Alcohol 95%
5. Alcohol 100%
6. Alcohol 100%
7. Alcohol 100%
8. Toluene
9. Toluene
10. Paraffin wax
11. Paraffin wax
12. Paraffin wax
Result:
Instrument: Leica Carousel TP 1020

Name of test: Tissue embedding
Principle:
Tissue sample that have been retrieved from Leica tissue processor is ready to embed
in paraffin wax.
Material:
Tissue sample
Tissue cassette
Instrument: SAKURA Tissue-Tek TEC 5 Tissue embedding console system
Procedure:
1. Open cassette to view tissue sample and choose a mold that best corresponds to
the size of the tissue. A margin of at least 2 mm of paraffin surrounding all sides of
the tissue gives best cutting support. Discard cassette lid.
2. Put small amount of molten paraffin in mold, dispensing from paraffin reservoir.
3. Using warm forceps, transfer tissue into mold, placing cut side down, as it was
placed in the cassette.
4. Transfer mold to cold plate, and gently press tissue flat. Paraffin will solidify in a
thin layer which holds the tissue in position.
5. When the tissue is in the desired orientation add the labeled tissue cassette on top
of the mold as a backing. Press firmly.
6. Hot paraffin is added to the mold from the paraffin dispenser. Be sure there is
enough paraffin to cover the face of the plastic cassette.
7. If necessary, fill cassette with paraffin while cooling, keeping the mold full until solid.
8. Paraffin should solidify in 30 minutes. When the wax is completely cooled and
hardened (30 minutes) the paraffin block can be easily popped out of the mold; the
wax blocks should not stick. If the wax cracks or the tissues are not aligned well,
simply melt them again and start over.
Result:
Trimmed tissue sample embedded in paraffin wax
Instrument: SAKURA Tissue-Tek TEC 5 Tissue embedding console system

Name of test: Tissue sectioning
Principle:
Tissue sectioning is done through use of a microtome. After mold is remove from tissue
block, it can be trimmed down and section into thin ribbons. Ribbon of sections is then
adhere to a glass slide, then the section can be stained either routine or special stain.
Material:
Glass slide, water bath, microtome, tweezers, tissue block, heat plate.
Instrument: Leica RM2235 Rotary Microtome
Procedure:
1. Chill paraffin-embedded tissue blocks on ice before sectioning. Cold wax allows
thinner sections to be obtained by providing support for harder elements within the
tissue specimen. The small amount of moisture that penetrates the block from the
melting ice will also make the tissue easier to cut.
2. Fill a water bath with tap water and heat to 40 – 45 °C.
3. Place the blade in the holder, ensure it is secure and set the clearance angle. The
clearance angle prevents contact between the knife facet and the face of the block.
Follow the microtome manufacturer’s instructions for guidance on setting the
clearance angle.
4. Insert the paraffin block and orientate so the blade will cut straight across the block.
5. Carefully, approach the block with the blade and cut a few thin sections to ensure
the positioning is correct. Adjust if necessary.
6. Trim the block to expose the tissue surface to a level where a representative
section can be cut. Trimming is normally done at a thickness of 10µm.
7. Cut sections at a thickness of about 4-5 µm (you will probably need to discard the
first few sections as they are likely to contain holes caused by trimming).
8. Using tweezers, pick up the ribbons of sections and float them on the surface of
the water in the water bath so they flatten out. Use the tweezers to separate the
sections.
9. Use microscope slides to pick the sections out of the water bath and place on a
heat plate for about 20 minutes.
10. Tissue section on glass slice can then be stain.
Result:
Thin tissue section from tissue block
Instrument: Leica RM2235

Name of test: H&E staining
Principle:
Tissue section is stained with H&E stain, which stands for haematoxylin and eosin.
Both dye have different affinity towards different organelles within a cell, thus staining
oragnelles with different color, providing contrast and visualization. This helps
identification of normal and abnormal cells.
Acidic components within a cell would attract basic dye (haematoxylin), staining
component such as nucleus, giving dark blue/purple color to nucleus. While basic
components would attract acidic dye (eosin), staining component such as cytoplasm,
giving pink/red color for cytoplasm.
Material:
Leica Autostainer XL
Procedure:
1. Slides with tissue section was placed into rack specified for Leica Autostainer XL.
2. Rack is then load into loading bay of Leica Autostainer XL.
Steps Reagent Time

1 Xylene 2 minutes
2 Xylene 2 minutes
3 Xylene 2 minutes
4 100% alcohol 2 minutes
8 Water 1 minute
9 Haematoxylin 10 minutes
10 Water 2 minutes
11 Acid alcohol 0.25% 5 seconds
12 Water 1 minute
13 Blue buffer 10 seconds
14 Water 3 minutes
15 80% alcohol 10 seconds
16 Eosin 6 minutes
21 Xylene 2 minutes
22 Xylene 2 minutes
23 Xylene 2 minutes
24 Xylene -
Result:
Microscopic examination: H&E stain
Nuclei : Blue/black
Cytoplasm : Varying shades of pink
Muscle fibres : deep pink/red
RBC : Orange/red
Fibrin : Deep pink
Instrument: Leica Autostainer XL

Name of test: PAP stain
Principle:
PAP stain consist of several dye which is use to stain different organelles within a cell.
Dyes consists of basic or acidic properties, PAP stain consist both acidic and basic
dye.
- Haematoxylin: Nuclear stain which stains nuclear of cell blue.

- Orange Green: Acidic counterstain (cytoplasmic stain) which stains matured
and keratinized cells. The target structures are stained orange in different
intensities.
- Eosin Azure: This is the second counterstain which is a polychrome mixture of
eosin Y and light green SF.
Eosin Y: Gives pink color to cytoplasm of mature squamous cells, nucleoli, cilia
and RBC.
Light green SF: Stains blue to cytoplasm of metabolically active cells like
parabasal squamous cells, intermediate squamous cells and columnar cells.
Material:
Leica Autostainer XL
Procedure:
Reagent Time Function

1 80% alcohol 15 seconds Hydration
4 Water 3 minutes Hydration
5 Haematoxylin 4 minutes Nuclear stain
6 Running water 3 minutes Washing
7 0.5% HCl 1 second Differentiation
8 Running water 6 minutes Washing
9 50% alcohol 1 miunute Dehydration
10 70% alcohol 20 seconds Dehydration
13 Orange G (OG-6) 4 minutes Cytoplasmic stain
14 95% alcohol 5 seconds Differentiation
15 EA-50 6 minutes Cytoplasmic stain
16 95% alcohol 1 second Differentiation
17 Absolute alcohol 20 seconds Dehydration
18 Absolute alcohol 4 minutes Dehydration
19 Absolute alcohol 5 minutes Dehydration
20 Xylene 2 minutes Clearing
21 Xylene 5 miunutes Clearing
22 Xylene 1 dip Clearing
23 DPX Mounting
Result:
Microscopic examination: PAP stain (40x)
Nuclei : Blue
Acidophilic cells : Red
Basophilic cells : Blue green
Erythrocytes : Orange red
Keratin : Orange red
Superficial cells : Pink
Intermediate and parabasal cells : Blue green
Eosinophil : Orange red
Candida spp. : Red
Trichomonas : Grey green
Instrument: Leica Autostainer XL

Name of test: Liquid-based cytology (ThinPrep 2000) smear preapration
Principle:
The ThinPrep 2000 system is intended as a replacement for the conventional method
of Pap smear preparation for use in screening for the presence of atypical cells,
cervical cancer, or its precursor lesions (Low Grade Squamous Intraepithelial Lesions,
High Grade Squamous Intraepithelial Lesions), as well as all other cytologic categories
as defined by The Bethesda System for Reporting Cervical/Vaginal Cytologic
Diagnoses.
Material:
Specimen type: cervical smear, fine needle aspirations.
 ThinPrep Processor Instrument (Model TP 2000)
 2 filter Caps
 PreservCyt® Solution vial
 2 spare filter seal O-rings
 ThinPrep Pap Test Filter for Gynecologic Applications
 ThinPrep Microscope slides
 Fixative vials
Procedure:
1. ThinPrep process begins with the patient’s gynecologic sample being collected by
the clinician using a cervical sampling device which, rather than being smeared on
a microscope slide, is immersed and rinsed in a vial filled with PreservCyt®
Solution.
2. The ThinPrep sample vial is then capped, labeled, and sent to a laboratory
equipped with a ThinPrep 2000 processor.
3. At the laboratory, the PreservCyt sample vial is placed into a ThinPrep 2000
processor and a gentle dispersion step breaks up blood, mucus, non-diagnostic
debris, and thoroughly mixes the cell sample.
4. The cells are then collected on a ThinPrep Pap test filter specifically designed to
collect diagnostic cells.
5. ThinPrep 2000 processor constantly monitors the rate of flow through the ThinPrep
Pap test filter during the collection process in order to prevent the cellular
presentation from being too scant or too dense.
6. A thin layer of cells is then transferred to a glass slide in a 20-mmdiameter circle.
7. The slide is then automatically deposited into a fixative solution.
Graphical procedure of operation of ThinPrep2000

Result:
Instrument: Hologic ThinPrep 2000

Name of test: Frozen section
Principle:
Frozen section (cryosection) is used for rapid microscopic analysis/diagnosis of a
specimen/disease. Usually used with oncologic surgery. Rapid diagnosis can guide
intra-operative patient management
Frozen section can provide rapid, gross, microscopic diagnosis to identify an
unknown pathologic process, identify extent of disease/evaluate margins, identify
metastases or simply identify a tissue. Frozen section can also be done to confirm that
pathological tissue is present for diagnosis on permanent sections.
Frozen section is commonly paired up with rapid H&E stain (a modified H&E
stain) for rapid diagnosis.
Material:
Fresh patient tissue sample, glass slide, rapid H&E stain, Leica Frozen Section
Compound (FSC), Leica Frostbite (rapid coolant)
Instrument: Leica CM 1860
Procedure:
1. Obtain frozen tissue, preferably frozen in liquid nitrogen. It is imperative that the
tissue be frozen as quickly as possible in order to avoid ice crystal formation
resulting in artifact and poor morphological preservation.
2. Make sure the cryostat is at proper operating temperature -20° C to -30° C. Place
a small amount of FSC on a cryostat object disk (make sure the disk is at room
temp. before mounting the specimen).
3. Position the frozen specimen in the center of the object disk and place the disk on
the cryobar in the cryostat to begin the quick freeze process.
4. Using Leica Frostbite, spray around the periphery of the object disk, as the FSC
freezes it will begin to turn from a clear gel to white solid substance.
5. Before the disk is frozen solid add enough FSC to cover the top the specimen and
quickly place a heat extractor on top of the specimen. The heat extractor serves
two purposes, (1) rapidly freezes the OCT and tissue and (2) produces a flat
embedded surface for easy cutting.
6. Spray with Leica Frostbite if necessary to expedite the freezing process.
7. Place the object disk in the microtome object disk holder and tighten the set screw
or clamp.
8. Make sure that there is enough clearance between the block and the microtome
knife.
9. Move the block toward the knife edge. Make sure the ratchet is disengaged from
the micrometer gear. Turn the flywheel with the right hand and begin turning the
gross adjust wheel (on the down stoke) slowly with the left hand. Face off enough
FSC until a full section of the specimen is visible.
10. Engage the ratchet on the micrometer gear, cut and discard the first two or three
sections.
11. Have the proper fixative (95% ETOH for H&E) and slides ready. Turn the flywheel
with the right hand. As the block comes in contact with the knife edge the section
will move down the blade and begin to curl. Hold the section down with as little
force as possible and guide in along the blade using a paint brush in the left hand.
Continue the cut until a full specimen section has been obtained, but stop before
passing through the remaining FSC. One edge of the section is held flat with the
paint bush and the other with the knife edge.
12. Pick up the slide with the right hand and turn it so that the top side is facing toward
the knife blade.
13. Carefully lower the slide onto the blade, keeping the slide parallel to the section.
As the tissue comes into contact with the slide the FSC and tissue will melt causing
the tissue to adhere to the slide.
14. Position of tissue section on glass slide is marked with a diamond pen. Patient RN
and name, together with initial of staff performing frozen section is written with
pencil at the frosted section of glass slide.
15. Tissue section is then ready to be stain with rapid H&E.
Result:
Instrument: Leica CM 1860

Name of test: Periodic acid schiff (PAS/PAS-D)
Principle:
PAS method works by exposing the tissue to periodic acid. Periodic acid acts as
oxidizing agent which oxidizes compounds having free hydroxyl group (-OH group) or
amino/alkylamine group resulting in dialdehydes. These dialdehydes when exposed
to Schiff’s reagent, an insoluble magenta colored complex is formed. A suitable basic
stain is used as counter stain.
Material:
Tissue section on glass slide, DPX, coplin jar, tap water, forceps,
Schiff’s reagent, Harri’s haematoxylin, sodium acetate, periodic acid solution
Procedure:
1 Deparaffinise and hydrate to water

2 1% aqueous periodic acid solution 5 minutes
3 Distilled water Rinse
4 Schiff’s reagent 20 minutes
5 Running water 5 minutes
6 Harri’s haematoxylin 1 minute
7 Differentiate
8 Blue in sodium acetate Wash
9 Dehydrate, clear, and mount
Result:
Mucin - Red, diasterase resistant
Glycogen - Red, diasterase sensitive
Name of test: Ziehl-Neelsen stain
Principle:
Organisms such as Mycobacteria are extremely difficult to stain by ordinary methods
like Gram Stain because of the high lipid content of the cell wall. The phenolic
compound carbol fuchsin is used as the primary stain because it is lipid soluble and
penetrates the waxy cell wall. Staining by carbol fuchsin is further enhanced by steam
heating the preparation to melt the wax and allow the stain to move into the cell. Acid
is used to decolorize nonacid-fast cells; acid-fast cells resist this decolorization. The
ability of the bacteria to resist decolorization with acid confers acid -fastness to the
bacterium. Following decolorization, the smear is counterstained with malachite green
or methylene blue which stains the background material, providing a contrast colour
against which the red AFB can be seen.
Acid alcohol can also be used as decolorizing solution, resistant organisms are
referred to as Acid Fast Bacilli (AFB) or Acid Alcohol Fast Bacilli (AAFB).
Material:
Carbol fuchsin, methylene blue, alcohol,
Procedure:
1 Deparaffinize
2 Blot until dry
3 Carbol fuchsin (preheat at 60 °C) 30 minutes
4 Place slides in hot carbol fuchsin 30 minutes
6 Differentiate until section is pale pink
- 0.5% acid alcohol
7 Running water Rinse
8 Methylene blue Flood slide using a
disposable pipette, few
seconds
9 Running water Rinse
10 Dry at 60 °C 20 minutes
11 Dehydrate rapidly 1 dip
- 100% alcohol
12 Clear and mount
Result:
Acid fast bacilli : Red
RBC : Light red
Background : Pale blue
Name of test: Reticulin stain (Gordon & Sweet)
Principle:
The Gordon and Sweet's silver staining method is used to demonstrate reticular (retic)
fibers. This method relies on the impregnation of retic fibers with silver through
oxidation and reduction. The tissue is first oxidized using potassium permanganate to
enhance subsequent staining. It is then sensitized using an iron alum solution that
targets and binds to the tissue element (retic fibers). The retic fibers are then
impregnated by an ammoniacal silver solution that removes and replaces the
sensitizer. The silver solution is reduced by 10% formalin so that a visible metallic tone
highlights the retic fibers. The metallic silver is then toned and converted to metallic
gold using gold chloride solution, thereby providing better chemical stability, fiber
contrast, and clarity. Unreduced silver and excess gold are removed via a 5% hypo
(sodium thiosulfate) solution. The tissue section may then be counter-stained with
nuclear fast-red or light-green.
Material:
Acidified potassium permanganate, 2% oxalic acid, distilled water, 2.5% ferric
ammonium sulphate, ammonical silver, 10% neutral buffered formalin, 0.2% gold
chloride, 5% sodium thiosulphate, nuclear fast red
Procedure:
1 Deparaffinize, hydrate to water

2 Acidified potassium permanganate 5 minutes
3 2% oxalic acid 1 minute, till colourless
4 Distilled water 3x, 1 minute each time
5 2.5% ferric ammonium sulphate 15 – 20 minutes
6 Distilled water 3x. 1 minutes each time
7 Ammonical silver 1 minutes
8 Distilled water Rinse till silver clears
9 10% neutral buffered formalin 1 minute
11 0.2% gold chloride until colour turns grey
12 5% sodium thiosulphate 3 minutes
13 Distilled water 1 minute
14 Nuclear fast red 15 – 20 minutes
Result:
Reticulin : Black
Nucleus : Red
Name of test: Van Gieson
Principle:
Van Gieson is used to differentiate between collagen and smooth muscle in tumours
and to demonstrate the increase of collagen in diseases.
When using combined solutions of picric acid and acid fuchsin, the small
molecules of picric acid penetrate all of the tissues rapidly, but are only firmly retained
in the close textured, red blood cells and muscle. The larger molecules of ponceau S
displaces picric acid molecules from collagen fibres, which have larger pores, and
allow the larger molecules to enter.
Material:
Celestin blue, Harri’s haematoxylin, acid alcohol, Van Gieson
Procedure:
1 Deparaffinize and hydrate to water

2 Celestin blue 5 minutes
3 Harri’s haematoxylin 5 minutes
4 0.5% acid alcohol 1 dip
6 Van Gieson 5 minutes
7 Dehydrate, clear, mount
Result:
Collagen : Red
RBC/muscles : Yellow
Name of test: Nitro blue tetrazolium (NBT)
Principle:
Nitro blue tetrazolium chloride belongs to the tetrazol dyes.These dyes are used in
histology, especially for histochemical methods.
Material:
Tetrazolium working solution
Procedure:
1 Rinse fresh heart slices in running water
2 Buffered tetrasolium working solution (37 °C) 30 minutes
- water bath
3 Infarcted muscle Remains pale
4 Fix in 10% formalin or discard
Result:
Viable myocardium : Blue
Infarcted myocardium : White
Name of test: Oil red o
Principle:
Staining with oil-soluble dyes is based on the greater solubility of the dye in the lipoid
substances than in the usual hydroalcoholic dye solvents.
Oil red O is a Sudan staining commonly used in histology to highlight lipids. It
is a lychrosome (liposoluble stain) and an azo compound use to visualize neutral
triglycerides and lipids in frozen histological slides. This staining enable hydrophobic
lipids identification in an aqueous medium. The lychrosome move from the solvent to
the tissue lipids by physical interaction. Oil red O staining is not working on paraffin
tissue sections because, in that conditions, it can damage the tissue lipids.
Material:
605 aqueous triethyl phosphate, oil red o, haematoxylin
Procedure:
1 Frozen section
2 Fresh or fixed tissue only
3 60% aqueous triethyl phosphate 1 dip
4 Oil red o 20 minutes
5 Distilled water 1 minute
6 Haematoxylin 1 minute
7 Distilled water 5 minutes
8 Dry gently on hotplate
9 Mount in aquamount
Result:
Lipid : Red to pink globules
Name of test: Masson trichome (MT)
Principle:
Masson’s trichrome staining is used to discriminate collagen fibers from muscular
tissues on histological slides. This staining is a mix of 3 stains:
- A purple nuclear stain (hematoxylin)
- A red cytoplasmic stain (a mix of xylidin ponceau and acid fuchsin)
- A collagen fiber stain: light green or anilin blue
The discrimination between collagen fibers and the other components of tissue
matrices is linked to a difference in permeability. Indeed, the red acidic cytoplasmic
stain labels the tissue acidophilic components like collagen fibers. When treated with
the Phosphotungstic Acid, the less permeable components retain the red stain, while
the red diffuses from the loose texture fibers of collagen and at the same time is
replaced by the light green dye.
Material:
Bouin’s fixative, Celestine blue, Harri’s haematoxylin, acid alcohol, biebrich scarlet-
acid fuchsin, 5% aqueous phototungstic acid, light green
Procedure:
1 Deparaffinize and hydrate to water

2 Bouin’s fixative at 56 °C 60 minutes
3 Running water Wash till colourless
4 Celestine blue 6 minutes
6 Running water 1 minute
7 0.5% acid alcohol 2 dip
8 Running water 1 minute
9 Biebrich scarlet-acid fuchsin 15 minutes
10 5% aqueous phosphotungstic acid 15 minutes
12 Light green 5 minutes
Result:
Muscle : Red
RBC : Red
Fibrin : Red
Collagen : Green
Name of test: ATPase
Principle:
The calcium method for ATPase demonstration, employing solutions of different pH
values, have been used primarily to distinguish muscle fiber types. Muscle fibers may
be broadly categorized as type 1 ("slow, red muscle, oxidative") and type 2 ("fast, white
muscle, glycolytic"). Type 2 muscle fibers are further subdivided as 2a (glycolytic), 2b
(glycolytic/oxidative), and 2c which we believe to be fibers that are changing types due
to disease or injury. The way this stain is believed to work is as follows. The
preincubation pH inactivates the myosin-ATPase enzyme of specific fiber types. The
remaining active enzyme is attached to a calcium atom which is replaced by a cobalt
and finally precipitated as a black insoluble compound by the ammonium sulfide.
Material:
Tissue section on glass slide, coplin jar, tap water, forceps
0.01M sodium barbital, 0.18M calcium chloride, 0.1M HCl, 2% ammonium sulphide,
2% cobalt chloride, 1% calcium chloride,
Procedure:
1 8 unstained frozen sections required 2 section/slide

2 Place all alkaline pH slides (pH 9.4, 10.1) into their 30 minutes
respective coplin jar
3 After 15 minutes, place acidic pH slide 4.2 into its coplin jars 5 minutes
4 Tranfer acidic pH slides 4.2 into buffer solution pH 9.4 Rinse
5 Then, immediately transfer slides 4.2 into incubating solution 20 minutes
pH 9.4 (pre-warmed to 37 °C)
6 When step 2 has 5 minutes left, put acidic pH slides 4.6 and 5 minutes
4.8 into their respective coplin jar.
7 Transfer acidic pH slides 4.6 and 4.8 and alkaline pH slides Rinse
10.1 into buffer solution pH9.4
8 Then, immediately transfer acidic pH slides 4.6 and 4.8 and 10 minutes
all alkaline pH slides 9.4, 10.1 into incubating solution pH 9.4
9 Wash all slides in 1% calcium chloride (pour out incubating 3x, 3 minutes
solution and replace with 1% calcium chloride)
10 Transfer all slides into 2% cobalt chloride 2x, 2 minutes
11 Wash all slides in 0.01M sodium barbital 6 changes
12 Wash all slides in tap water
13 Place all slides in 2% ammonium sulphide 60 seconds
14 Rinse sin tap water
15 Dehydrate, clear and mount
Result:
Fibers Light Intermediate Dark
Alkaline
Type 1 - Type 2
(pH 9.4, 10.1)
Acidic
Type 2A Type 2B Type 1, Type 2C
(pH 4.2, 4.6, 4.8)
Name of test: NADH-Tetrazolium reductase
Principle:
"Diaphorase" is a term given to flavoprotein enzymes that have the property of
transferring hydrogen from reduced nicotinamide adenine dinucleotide (NADH) to
various dyes. The hydrogen transfer reduces the dye. Usually tetrazolium compounds
function as the hydrogen acceptor when diaphorases are being demonstrated
histochemically, and the product of the reduction is the water-insoluble formazan
pigment. Commonly used tetrazoliums include nitro blue tetrazolium (NBT). Enzymatic
activity releases hydrogen from the substrate, and the released hydrogen is
transferred to the tetrazolium. With the addition of hydrogen, the tetrazolium is
converted to purple-blue formazan pigment marking the site of enzyme activity.
Material:
0.2 M Tris buffer at pH 7.4, 0.1% aqueous nitro blue tetrazolium, 0.5 M cobalt chloride,
distilled water, 0.1 NaOH, NADH
Procedure:
1 Working solution 1 – 2 drops
2 Incubate at 37 °C 20 – 30 minutes
3 Check with microscope
4 Cold formol calcium at 4 °C 10 minutes
5 Rinse in distilled water
6 Mount with glycerol
Result:
Muscle fibre architecture: Shades of grey to black (Type 1 darker than Type 2)
Name of test: Acid phosphatase
Principle:
Napthol acid phosphate is hydrolyzed by acid phosphatases present in tissue, and
napthol derivatives are produced, The naphthol derivatives couple with an unstable
diazonium salt, hexazonium pararosaniline, to produce a red azo dye to mark the sites
of enzyme activity.
Material:
Naphthol AS-BI phosphate, dimethyl formamide, veronal acetate, 0.2M barbital
acetate, sodium acetate, sodium barbitone, distilled water, sodium nitrite,
pararosaniline hydrochloride, concentrated HCl
Procedure:
1 Formol calcium at 4 °C 10 minutes
2 Wash in running water 5 minutes
3 Incubating solution at 37 °C 45 – 60 minutes
4 Running water Few minutes
5 Haematoxylin Few seconds
6 Mount in glycerol
Result:
Histiocytes, macrophages, necrotic areas : Pink to red
Background : Colourless
Name of test: Gomori trichrome
Principle:
Gomori's one-step trichrome is a staining procedure that combines the plasma stain
(chromotrope 2R) and connective fiber stain (fast green FCF) in a phosphotungstic
acid solution to which glacial acetic acid has been added.
Material:
Chromotrope 2R, Fast green FCF, phosphotungstic acid, glacial acetic acid, distilled
water, 1M NaOH
Procedure:
1 Air dry frozen section 10 – 20 minutes
3 Rinse in tap water 3 minutes
4 Gomori trichrome mixture 20 – 60 minutes
5 Rinse in tap water
6 Dehydrate, clear, mount
Result:
Muscle fiber : Blue green
Nemaline rods : Red
Ragged red fibers : Red
APPENDIX
Parasitology request form (front)
Parasitology request form (back)
CDL request form (front)
CDL request form (back)
CDL rejection form
Histology / Cytology rejection form

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Introduction

The University of Malaya was initially set up in Singapore in 1949 as an amalgamation

The University motto, "Ilmu Punca Kemajuan" (Knowledge is the Key to

Besides providing clinical services to University Malaya Medical Centre

The Division of Laboratory Medicine (LMD) is a clinical diagnostic laboratory of

1) THE CORE LABORATORY SERVICES

2) Special Laboratory Services

Inborn Errors of Metabolism (IEM) Laboratory

Bone Marrow Studies

Molecular and Genetic Analysis Laboratory

3) POLYCLINIC LABORATORY SERVICE

Parasitology department University Malaya

With the rapid development in infrastructure and the introduction of new

This Department is also a reference centre for parasitic diseases for

The Department also provides services in detection of Cryptosporidium spp.

Changing and upgrading teaching and research methodologies have to be

PROFESSOR ASSOCIATE PROFESSOR

Parasites will settle more rapidly if the stool suspension is subjected to

Internal quality control:

a 425 µm aperture size, 38 mm diameter.

washed thoroughly in running tap water between each sample.

The following table contains the distinguishing features of these larvae.

1st stage larva Infective (3rd) stage larva

Undiluted Giemsa Stain Preparation

- Preliminary fixation of peripheral blood film is necessary.

Staining method (for thin smear)

- Tail of PBF should be examine, RBC separated, one cell thick.

Microscopic examination: Malaria parasite ring form

The number of parasitized erythrocytes seen in peripheral blood (parasitemia) is very

𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑖𝑧𝑒𝑑 𝑒𝑟𝑦𝑡ℎ𝑟𝑜𝑐𝑦𝑡𝑒 𝑝𝑒𝑟 𝑓𝑖𝑒𝑙𝑑

- Alternatively, number of parasites in 1000 erythrocytes can be counted

Analyses of Faeces for the presence of protozoan parasites

Stools with evidence or blood or mucus may contain the developmental or

Microscopic examination: Egg under microscope

In vitro culture method

C. parvum positive faecal samples should be available when personnel are

a Moderately thick smears are recommended for this procedure.

Diagnostic features of Cryptosporidium parvum oocysts:

Preparation of Schaudinn’s fixative

Medical Laboratory Division

Medical Laboratory Division is responsible in diagnostic services for patients

DEPUTY DIRECTOR (CLINICAL SERVICES)

MEDICAL LAB DIVISION

Fluorescence Flow Cytometry

 Forward Scattered Light provides information on cell volume.

AFLAS uses a human-like recognition of the WDF scattergram, by not only

Each cell produces a unique “cell signature” or position on the scattergram, as

Hydrodynamic Focussing Direct Current (DC) Method (RBC/ PLT)

Sodium Lauryl Sulphate (SLS) Haemoglobin Method

ANALYTES UNIT MEN WOMEN

Instrument: Sysmex XN-9000

Thin blood film preparation with Wright stain

Autostainer: Bayer HealthCare Hematek 2000

Supravital stain with new methylene blue

Intrument: BIO-RAD VARIANT II TURBO

Prior to performing the test, it is mandatory to prepare the samples (hemolyzed

The HYDRASYS system is a semi-automated multi-parameter instrument. The

Hemoglobin is a complex molecule composed of two pairs of polypeptide chains. Each

Sample preparation (standard procedure)

Sample preparation for hemoglobin H detection

MIGRATION - DESCRIPTION OF THE AUTOMATED STEPS

II. GEL PROCESSING SET-UP

III. GEL PROCESSING COMPLETION