Hematology & Blood Transfusion lab. rotation report summary

1
MUHIMBILI UNIVERSITY OF HEALTH
AND ALLIED SCIENCES
DEPARTMENT OF HEMATOLOGY AND BLOOD TRANSFUSION
BMLS IN HEMATOLOGY AND BLOOD TRANSFUSION
SCHOOL OF MEDICINE
YEAR II 2019/20
A HEMATOLOGY AND BLOOD TRANSFUSION LABORATORY
ROTATION REPORT SUMMARY
NAME: ISMAIL KAPITA MWARUKA
REG. NO: 2018 – 04 – 11593
ACADEMIC YEAR: YEAR II 2019/20
SUBMISSION DATE: 07TH
SEPTEMBER 2020

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TABLE OF CONTENTS
PHLEBOTOMY SECTION ......................................................................................................................4
LABORATORY INFORMATION SYSTEM..........................................................................................4
THE PHLEBOTOMY PROCEDURE......................................................................................................5
PRE-LABORATORY (SAMPLE RECEPTION) ....................................................................................6
HEMATOLOGY LABORATORY...........................................................................................................7
COMPLETE BLOOD COUNT (FULL BLOOD PICTURE)..................................................................7
Principle................................................................................................................................................7
Procedure ..............................................................................................................................................7
Red cell indices, leucocyte differentials and platelets estimation.........................................................8
ERYTHROCYTE SEDIMENTATION RATE (ESR) ...........................................................................10
Principle..............................................................................................................................................10
Materials needed.................................................................................................................................10
Procedure ............................................................................................................................................10
Reference ranges.................................................................................................................................10
Conditions...........................................................................................................................................10
COAGULATION TESTS.......................................................................................................................11
Prothrombin time (PT & PT-INR)......................................................................................................11
Principle..............................................................................................................................................11
Reference range ..................................................................................................................................11
Activated Partial Thromboplastin time (aPTT)...................................................................................11
Principle..............................................................................................................................................11
Reference range ..................................................................................................................................11
PERIPHERAL BLOOD SMEAR PREPARATION (PBS)....................................................................12
Wedge blood smear.............................................................................................................................12
Slide fixation.......................................................................................................................................13
Staining the blood film with Romanowsky (Leishman) stain.............................................................13
BONE MARROW BIOPSY & ASPIRATION ......................................................................................15
Procedure for bone marrow aspiration and trephine biopsy ...............................................................15
Procedure for staining Bone marrow films with Leishman stain........................................................16
Procedure for staining Bone marrow films with Perl’s Prussian blue (Iron) stain..............................16
INVESTIGATION OF HEMOLYTIC ANEMIA ..................................................................................16

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HEMATOLOGY CLINICAL RESEARCH LAB (HCRL) ..................................................................17
Sickle cell disease ...............................................................................................................................17
Diagnosis of Sickle cell disease..........................................................................................................17
SICKLE SLIDE TEST........................................................................................................................17
Hb GEL ELECTROPHORESIS .........................................................................................................18
SICKLE SCAN TEST ........................................................................................................................19
ISOELECTRIC FOCUSING (IEF) ....................................................................................................20
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)...............................................21
BLOOD TRANSFUSION UNIT..............................................................................................................22
DONOR REGISTRATION ....................................................................................................................22
HEMOGLOBIN LEVEL (Hb estimation)..............................................................................................22
DONOR COUNSELING........................................................................................................................22
BLOOD DONATION.............................................................................................................................23
Procedure for blood collection............................................................................................................23
TESTING FOR TTIs ..............................................................................................................................24
PREPARATION OF BLOOD COMPONENTS ....................................................................................25
Different blood components prepared at MNH-Blood bank...............................................................25
ABO & Rh BLOOD GROUPING..........................................................................................................25
Procedure for performing forward (cell) grouping by tube method...................................................25
CROSS-MATCHING (COMPATIBILITY TEST)................................................................................26
Procedure for performing cross-matching by immediate spin method...............................................26
ISSUING OF BLOOD PRODUCTS......................................................................................................26
REFERENCES ...........................................................................................................................................27

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PHLEBOTOMY SECTION
LABORATORY INFORMATION SYSTEM
Phlebotomy refers to all procedures done in a medical laboratory in order to collect whole blood
from specific veins or capillaries into correct containers that allow hematological examination. But
before these procedures are carried out the patient must correctly be registered into the MNH
Laboratory information system (Jeeva systems).1
These are the steps showing how to use laboratory information system to receive samples;
 Login into the system by a password and username
 On the main window click to select Sample collection & receiving
 Then record the MR number or patient’s name
 Mark the Pending button
 Click SHOW
 Tick the specific test you want to register, then click ACCEPT
 Click RECORD (if prompted click OK)
 Then choose the number of barcodes you need, then click OK to print out barcodes
 Head to the sample collection unit with the correct container(s) already labelled with
barcodes.
Knowing the correct container/tube(s) for blood sample collection is an essential thing for every
medical laboratory scientist. Furthermore, different tubes are used in different departments in the
Central Pathology laboratory (CPL). The following list summarizes the departments and the
common tubes used there;
 Haematology – Purple (lavender)2
& light blue3
top tubes
 Clinical chemistry – Red (plain), green4
& purple top tubes
 Microbiology – Red, purple & yellow top tubes and blood culture bottles
1
Visited on 25th
JUNE 2020
2
Contains EDTA anticoagulant
3
Contains Trisodium citrate anticoagulant
4
Contains Heparin anticoagulant

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THE PHLEBOTOMY PROCEDURE
In the phlebotomy section before performing sample collection it is recommended to have prepared
all the necessary instruments/items used in collection, this minimizes miscellaneous errors during
collection and ensures safety before performing any collection procedure. The following items
should be included in the phlebotomy tray before collection;5
 Request form and barcode for the specific test(s)
 Tourniquet
 Sterile disposable syringe (2/5/10 cc) or an Evacuated tube collection system
 Correct specimen containers (vacutainers)
 70% isopropyl swabs or 0.5% chlorhexidine or cotton wool socked in Methylated spirit
 Rack to hold specimen containers
 Puncture resistant disposal container
 Sterile gauze or cotton wool
 Sterile gloves
In the hematology clinical laboratory venous blood6
is usually recommended over capillary blood
due to multiple tests requiring a relatively large amount of blood sample. Also, if capillary blood
were to be used some test parameters would be affected while performing complete blood count.
The following are the steps for performing phlebotomy procedure;
1. The patient’s identity is checked and compared to what is written on the request form, and
labelling the evacuated containers with name and hospital ID barcode.
2. All necessary materials for performing blood collection are prepared prior.
3. A sterile, dry, disposable, plastic syringe of required volume is selected.
4. Using the index finger a suitable puncture area is located (where a vein can be felt).
5. A tourniquet is applied around the arm at approximately 4-5 finger widths above the
puncture site.
6. The patient is asked to form fist so that the veins become prominent.
5
Visited on 26th
JUNE 2020.
6
Usually drawn from the Median-cubital (1° choice), cephalic (2° choice), or basilic (3° choice) veins on the hand.

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7. The puncture site is disinfected using 70% isopropanol swab and left to dry for 5 seconds.
8. With the thumb of the left hand holding down the skin below the puncture site, a
venipuncture is made with the bevel (head) of needle facing upwards. The blood is
withdrawn steadily and gently to avoid induced hemolysis.
9. When sufficient blood is collected, the tourniquet is released from patient’s arm to avoid
hemoconcentration. The puncture site is pressed immediately with a piece of dry cotton
wool while removing the needle.
10. The syringe is plugged on the appropriate evacuated tube(s) and the blood is let to be pulled
by vacuum. The blood is then mixed gently with anticoagulant inside the tube to avoid
clotting.
11. The syringe is disposed in the sharps container without recapping.
PRE-LABORATORY (SAMPLE RECEPTION)
After the sample has been collected, it must be sent to pre-laboratory section where the
qualification of the sample will be done to check that it contains all required criteria before being
processed further. These criteria include sample quantity, correct tubes/container used, sample
condition (haemolysed or clotted blood). Very small samples or incorrect container may
necessitate sample rejection and re-collection. If the samples qualify to proceed they are
accepted in the Laboratory Information Systems (LIS) and sent to the hematology laboratory for
analysis.

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HEMATOLOGY LABORATORY
COMPLETE BLOOD COUNT (FULL BLOOD PICTURE)
Refers to analysis of the whole blood that provides information about the properties of red blood
cells, white blood cells and platelets. It is a routine test that an overview of a patient’s general
hematological status.
Cell dyn 3700A/3700B/Ruby or the SYSMEX machines are used in performing complete blood
counts by using the flow cytometry (Coulter’s) principle and impedance principle. (1)
At CPL, Cell dyn 3700A/3700B/Ruby machines are used in routine hematological analysis.
Principle
The cell dyn hematological analyzer has microchannels that separates two chambers containing
electrolyte solutions (diluent). As the diluted blood is drawn into each microchannel, each particle
causes a brief change to the electrical impedance of the liquid. The counter detects these
displacements as electrical resistance which is proportional to the volume of the particle passing the
microchannel. Hemoglobin concentration and related parameters are estimated only after hemolysis
of red cells due to dilution. Size and complexity (granulation) of the leucocytes can be detected by
forward and side scatter of the laser beam as the cells cross it.
Procedure
 Scanning the barcode containing hospital ID.
 Roll the EDTA tube with a steady motion to ensure the whole blood contents mix evenly
before processed.
 Sample aspiration7
 Dilution of the whole blood sample
 Analysis by Coulter’s principle
 Rinsing fluid components for the next sample
 Results reporting/printing.8
N.B: Complete blood count can’t be relied completely as a diagnostic or confirmatory test for most
hematological conditions. It can serve as merely a preliminary investigation among others done
to assess the whole blood status of a patient. (2)
7
Open system involves manual aspiration in 3700A/3700B, while closed system involves automatic aspiration of
multiple samples in Ruby version of Cell dyn.
8
Flags like RRBC, NRBC, BLAST, BAND, and many others may appear when incorrect aspiration is made or clotted
blood or too concentrated blood is used. Repeating the test may resolve the problem.

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Red cell indices, leucocyte differentials and platelets estimation
Red blood cells are the type of blood cell made in bone marrow and found in the blood. It contains
a protein called hemoglobin which carries oxygen from the lungs to all parts of the body.
Hemoglobin is a red protein responsible for transporting oxygen in the blood of vertebrate.
MCH is an average mass of Hemoglobin per red blood cells.
MCV is a measure of average volume of red blood cells.
Hematocrit (PCV) is the volume percentage of red blood cells in blood.
MCHC Is a measure of average Hemoglobin concentration in a given volume of a packed red cells.
RDW is a range of variation of red blood cells volume.
White blood cells are cells involved in body’s immune system defense.
Neutrophils are types of white blood cells with multiple lobes and contain granules.
Basophil is a granulocyte which is responsible for allergic reactions.
Eosinophils are bilobed granulocytes that fight parasites.
Monocyte is a large phagocytic cell with a simple oval nucleus without granules.
Lymphocyte is a form of small leukocyte with a single round nucleus responsible for viral
infections.
Platelet is a cytoplasmic extension of megakaryocytes responsible for blood clotting.
The following table 1.1 summarizes the above properties with more details on their reference
ranges and standard units. It further explains the possible hematological conditions where these
properties may rise or fall when analyzed by the Cell dyn 3700A/3700B/Ruby counter.9
N.B: Different hospitals and health centers may have different reference ranges.
9
Visited on 05th
JULY 2020.

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Table 1.1
Parameters S.I unit Reference range
Condition
Raise Fall
Total Red Cell (erythrocytes) (x1012
/L) Male 4.5-6.5, Female 3.9-5.6 Polycythemia Anaemias, pancytopenia
Haemoglobin (g/dL) Male 13.5-17.5, Female 11.5-15.5 Polychromatia Hemolytic anaemia,
pancytopenia
PCV (hematocrit) % Male 40-52, Female 36-48 Polycythemia Sickle cell anaemia
Mean cell haemoglobin (MCH) (pg) 27-34 Polychromatia Sideroblastic anaemia, iron
deficiency anaemia
Mean cell volume (MCV) (fL) 80-95 Megaloblastic or Non-megaloblastic
Anaemia
Microcytic anaemia, Thalassemia
Mean cell haemoglobin
concentration
(g/dL) 32-36 Polychromatia Iron deficiency, anaemia of
chronic disease
Reticulocyte count (x109
/L) 50-150 Acute blood loss (hemorrhage or
hemolytic anemia)
Aplastic anaemia, iron deficiency
anaemia
Total White cells (x109
/L) 4.0-11.0 Infections, Leukemia Chemotherapy, pancytopenia
Neutrophils (x109
/L) 1.8-7.5 Infections Septicemia, pancytopenia
Lymphocytes (x109
/L) 1.5-3.5 Viral infection Autoimmune disorders, cancer,
chemotherapy
Monocytes (x109
/L) 0.2-0.8 Chronic infection, autoimmune disease Septicemia
Eosinophils (x109
/L) 0.04-0.4 Parasitic infections Cushing’s syndrome,
intoxication
Basophils (x109
/L) 0.01-0.1 Allergic infection, Leukemia Severe allergic reaction,
pancytopenia
Platelets (x109
/L) 150-400 Disseminated intravascular coagulopathy,
Thrombocytosis
TTP (thrombotic
thrombocytopenic purpura

10
ERYTHROCYTE SEDIMENTATION RATE (ESR)
Refers to a non-specific test that measures a rate at which red blood cells in Trisodium citrate
anticoagulated whole blood descends in a standardized tube over a period of one hour.10
Principle
When an anticoagulated whole blood is placed in a vertical (Westergren) tube, the red blood cells
due to high molecular weight tend to settle slowly towards the bottom leaving a column of clear
plasma on top. The rate is then measured in mm per hour. (1)
Materials needed
 Westergren tube
 Trisodium Citrate anticoagulated blood
 Westergren stand
 Rubber bulb
 Timer
Procedure
 Mix 0.18ml anticoagulated blood with 0.25 ml of 3.8% Sodium citrate thoroughly
 Draw blood into the Westergren tube up to the mark “0” with the help of rubber bulb
 Wipe out blood from bottom of the tube
 Carefully transfer the tube to the Westergren stand and stand it vertically
 Record the distance in mm that the blood has descended per in 1 hour.11
Reference ranges
 Male: 0-9 mm/hour
 Female: 0-15 mm/hour
 Elderly: 0-20 mm/hour
Conditions
Increased ESR Decreased ESR
Anaemia Polycythemia
Pregnancy Sickle cell anaemia
Macrocytosis Hyper-viscosity
Table 1.2
10
Visited on 07th
JULY 2020.
11
Like Complete blood count, Erythrocyte sedimentation rate cannot be used as a diagnostic or confirmatory test
because it may be raised in different hematological conditions.

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COAGULATION TESTS
These are tests which measure the ability of blood to clot and how long it takes to clot. It can also
be used to assess the risk of excessive bleeding or thrombosis in the blood stream. At CPL a fully
automated machine ACL TOP-50012
is used to conduct coagulation tests like PT, aPTT and
coagulation factors analysis.
A prerequisite for this test is collection of whole blood sample in Trisodium citrate (blue top)
anticoagulant tube. EDTA anticoagulated blood may lack some coagulation factors or may have
platelet aggregation while sodium citrate is more efficient anticoagulant in preserving the plasma.
Prothrombin time (PT & PT-INR)
Refers to a test done to assess the extrinsic coagulation pathway activity.
Principle – When pre-formed thromboplastin and calcium chloride are added to citrated plasma at
37 ̊C, the plasma initiates coagulation process. The time taken for the clot to appear is called
prothrombin time. The clotting time in seconds is converted to the International Normalized Ratio
(INR), usually by reference to a table provided by the manufacturer of the reagent or from the
formula. (1)
Reference range = 12-14 seconds.
Activated Partial Thromboplastin time (aPTT)
Refers to a test done to assess the intrinsic coagulation pathway activity. This test can also be used
in monitoring patients under heparin therapy. (3)
Principle – In citrated plasma, the addition of a platelet substitute, factor XVII activator (Kaolin),
and calcium chloride at 37 ̊C allows for formation of a stable clot. The time required for the
formation of a stable clot is recorded in seconds and represents the actual aPTT result.
Reference range = 25-40 seconds.
Table 1.3
12
Visited on 09th
JULY 2020.
Condition for prolonged
PT & PT-INR
Liver disease, treatment with warfarin, DIC, HDFN.
Condition for
prolonged aPTT
Disseminated Intravascular Coagulopathy (DIC), Lack
of factors V, VIII, IX, X or XI, Vitamin K deficiency.

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PERIPHERAL BLOOD SMEAR PREPARATION (PBS)
One can never rely fully on results from an automated hematological analyzer due to many errors
and miscounts it may make during counting the whole blood components. Nucleated red cells
(NRBC) may be counted as lymphocytes and leucocytic Blasts may be mistaken for monocytes,
so a mere presence of flags in a patient’s CBC might be a good reason to perform a peripheral
blood smear that may give a more accurate hematological status of the patient.
At CPL the wedge technique is commonly used for peripheral blood smearing and Leishman
stain is used for routine staining of the made PBS.
Wedge blood smear13
Refers to the use of a glass slide (spreader) to smear a drop of blood on another slide into a thin
layer for microscopic examination when stained.
Materials needed
 EDTA anticoagulated venous or capillary blood14
 Spreader slide
 Clean glass slides
 Blood capillary tube or micropipette (10 µL) or Pasteur pipette
Procedure
 Label the frosted edge with patient name, MR number and date using a greasy pencil.
 Place a drop of blood (10 µL) just 1 cm above the frosted end.
 Place the slide on a flat surface, and hold the other end between your left thumb and
forefinger.
 With your right hand, place the smooth clean edge of a second (spreader) slide on the
specimen slide, just in front of the blood drop.
 Hold the spreader slide at a 30° angle, and draw it back against the drop of blood
 Allow the blood to spread almost to the edges of the slide.
 Push the spread forward with one light, smooth moderate speed. A thin film of blood in the
shape of tongue.
 Let the made blood film air dry.
13
Visited on 11th
JULY 2020.
14
Smears should be made within 1 hour of blood collection from EDTA specimens stored at room temperature to
avoid distortion of cell morphology.

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Characteristics of a good-made peripheral blood smear
 Tongue shaped with a smooth tail.
 Does not cover the entire area of the slide (almost 2/3rd
the slide area).
 Has both thick and thin areas with gradual transition.
 Does not contain any lines or holes.
Slide fixation
After air drying, the thin blood film is immersed in a water-free methanol jar that fixes it to
preserve the cell morphology. The blood film is kept in methanol for 3 minutes.
Staining the blood film with Romanowsky (Leishman) stain
This procedure is essential as it increases the contrast on the blood components for accurate
microscopic examination. The preferred and routinely used stain at CPL is Leishman stain. It is
a differential stain containing Eosin Y (acidic) & Azure B (basic) dyes. The acidic Eosin dye
stains the basic cellular components i.e. cytoplasm & haemoglobin to pink, while the basic Azure
dye stains the acidic cellular parts i.e. DNA & RNA to purple/dark blue.
Materials needed for Preparation of Leishman stain stock
 Leishman stain powder – 0.15 gm
 Absolute methanol – 100 ml
 Spatula or Measuring spoon
 Weighing paper
 Graduated cylinder
 Glass or plastic funnel
 Screw-capped, dark or amber glass bottle – 250 ml capacity
 Analytical balance or Weighing scale
 Mortar and pestle
Procedure for preparing Leishman stain stock15
 Weigh the 0.15 g of Leishman stain powder on an analytical balance or weighing scale.
Grind it in a Mortar and put the powder into the amber-bottle through a funnel.
 Gently pour in about 20 ml of absolute Methanol through the same funnel, ensuring that
all the dry stain is washed into the bottle.
15
Visited on 12th
AUGUST 2020.

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 Re-cap the bottle and shake it in a circular motion for 3 minutes to start dissolving the stain
crystals in Methanol solution. Warm the content at 50°C for 15 minutes in a water bath
with occasional shaking.
 Add the remaining amount of Methanol to the mixture through the funnel and make the
volume 100 ml, ensuring that even the last drop of the Methanol washes in the funnel into
the stain mixture.
 Tighten the screw-cap on the bottle and continue shaking for few minutes.
 Do not open the bottle and do not filter the content for about 1 week. After a week it is
ready to use. Filter the content before each use. (4)
 Label the bottle clearly as Leishman Stock Solution with the batch number, the name of the
personnel who prepared it, date of preparation and date of expiry, and document it in the
quality control log-book of MNH-CPL.
N.B: The Leishman stock solution should be stored in a cool and dry place away from direct
sunlight. The screw-cap of the bottle should be tightened to avoid absorption of water vapour
from the air.16
Procedure for staining Peripheral blood films with Leishman stain17
 The blood film is flooded with undiluted Leishman stain (drop-wise) for 3 minutes.
 Double volumes of buffered water are added and gently mixed with Pasteur pipette, then
left for 10 minutes.
 Running tap water is allowed to flow on the glass slide rinsing off the excess stain.
 Rub the back of the slide using a dry gauze.
 The slide is kept on the drying rack to air-dry, then examined microscopically.
16
If stored correctly, the Leishman stock stain solution is stable approximately for 90 days.
17
Visited on 24th
JULY 2020.

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BONE MARROW BIOPSY & ASPIRATION
An invasive but indispensable procedure for collecting bone marrow specimen for hematological
investigations of its hematopoietic function. It is done as a confirmatory test to some conditions
like iron overload, megaloblastic anemia or iron deficiency anemia. It is also used in monitoring
treatment to certain hematological conditions and in staging of malignancies. (5)
The whole architecture (topographic detail) of the bone is preserved in a Trephine biopsy18, while
bone marrow aspiration19 gives quantitative and qualitative cytological details. The preferred
sites for specimen collection are Posterior superior iliac spine (PSIS) and the sternum20
. (3) (2)
Materials needed
 Surgical gloves
 70% isopropyl alcohol or 0.5% chlorohexidine or Iodine
 Lignocaine anaesthesia
 A stout Jamshid needle with a well-fitting stilette.
 A sterile syringe (5 or 10 cc)
 Clean glass slides
 Dry cotton wool
 A bone marrow aspiration/trephine request form with clinical history relevant for the
procedure.
Procedure for bone marrow aspiration and trephine biopsy
 After valid written consent has been taken from the patient, the patient is positioned in
lateral decubitus position and the site of puncture is identify (PSIS).
 Operator should wear surgical gloves and clean the skin at the site with 70% alcohol or
0.5% chlorohexidine or iodine.
 Infiltrate the skin, subcutaneous tissue and periosteum over the site with 5ml of lignocaine.
 With boring movement pass the Jamshid needle perpendicularly at center of PSIS.
 When bone has been penetrated, remove the stilette, attach 2 ml syringe and suck up
marrow contents (0.3 ml) for making smears immediately (at least 6 smears are made).
The slides are fixed in absolute methanol for 20 minutes after drying.
18
Visited on 27th
JULY 2020.
19
Visited from 13th
JULY 2020 to 27th
AUGUST 2020.
20
Contraindicated in children due to immature bone structures.

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 If large sample is needed for cytogenetics and immunophenotyping attach another 5 or
10 ml syringe and aspirate then keep in preservative free heparin container.21
 For Trephine biopsy, a hollow needle is inserted through the same incision about 1 cm
away from the aspirate site.
 And using boring movement, a core of bone and marrow is removed about 1.5 to 2cm long.
 Biopsy is fixed in 10% neutral buffered formalin for 6 hrs.
 Cover the puncture site with gauze and plaster strip.
Procedure for staining Bone marrow films with Leishman stain22
 After fixing the bone marrow slides for 20 minutes in absolute methanol, transfer the slides
on a staining rack and flood them with Leishman stain solution.
 Leave the stain for 3 minutes then add double volumes of buffered water and mix gently
by rocking the rack.
 Leave the mixture for 20 minutes then gently rinse the stain with running tap water.
 Rub the back-side of the slide to remove excess stains using a dry gauze.
 The slide is kept on the drying rack to air-dry, then examined microscopically.
Procedure for staining Bone marrow films with Perl’s Prussian blue (Iron) stain
 Place the bone marrow smear slides on a rack, flood with a ready-prepared mixture of equal
parts of Ferro-cyanide and hydrochloric acid solution for 30 minutes.
 Wash well in distilled water for 3 minutes, then counterstain with filtered eosin red stain
for 1 min.
 Rinse in distilled water and place the slides on the drying rack to air-dry. (3)
INVESTIGATION OF HEMOLYTIC ANEMIA
At MNH-CPL, common tests performed in investigating hemolytic anemia include; Complete
blood count (CBC), Peripheral blood smear (PBS), Reticulocyte count (Retics), Paroxysmal
Nocturnal Hemoglobinuria test (PNH), and Glucose-6-phosphate dehydrogenase deficiency test
(G6PD). These are of great importance in determining the cause of hemolysis of red blood cells.
21
This process marks the end of bone marrow aspiration.
22
Visited from 27th
JULY 2020 to 25th
AUGUST 2020.

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HEMATOLOGY CLINICAL RESEARCH LAB (HCRL)
Refers to an integration of the clinical components and researchers from various medical fields
which deals with studying hematological disorders. These disorders can be neoplastic (lymphoma
and leukemia) or genetic (e.g. Sickle cell disease and Thalassemia). Sickle cell disease (SCD) is
the epitome of research at HCRL constituting almost 75% of studies done.23
Sickle cell disease is a genetic disorder caused by a base substitution mutation of Thymine (T)
by Adenine (A) in the Hb gene on chromosome 6 hence translating valine instead of the normal
Glutamic acid. The resulting Hemoglobin polypeptide loses its flexibility causing the red cells to
sickle when deoxygenated.
Diagnosis of Sickle cell disease
Screening tests
Manual tests
Solubility test
Sickle slide test √
Rapid tests
Sickle scan √
Hemo-TypeSC
Confirmatory tests
Qualitative tests
Isoelectric focusing √
Hb (Gel) electrophoresis √
Quantitative tests
HPLC √
DNA analysis
Table 1.4
SICKLE SLIDE TEST
Is a qualitative screening test that utilizes the principle of creating hypoxic environment for red
cells with sickle hemoglobin (HbS) to exhibit sickling tendency which is detected microscopically.
Principle
EDTA anticoagulated blood when mixed with a chemical reducing agent (Sodium meta-bisulfite)
then covered by a coverslip and incubated at room temperature, the reducing agent deoxygenates
the Hemoglobin in red cells creating a hypoxic environment necessary for cells possessing HbS to
sickle.
23
Visited on 21st
JULY 2020.

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Procedure of Sickle slide test24
 Place one drop of patient blood a well labelled clean & dry slide.
 Add an equal volume of fresh Sodium meta-bisulfite solution, mix and cover with a cover
glass while excluding any air bubbles.
 Using a Pasteur pipette cover the edges of the coverslip with molten petroleum jelly.
 Place the slide(s) on a dump piece of blotting paper/gauze to prevent drying of the
preparation and let it incubate at 18-24 ̊C for 15 minutes.
 Examine microscopically at 40X objective. Report results as Sickle cell test Positive (if
crescent shaped RBCs are seen)/Negative.
Hb GEL ELECTROPHORESIS
Is used as a qualitative confirmatory test to identify variants and abnormal hemoglobin especially
in diagnosis of sickle cell disease. At HCRL it is currently phased out due to more advanced
technologies in the diagnosis of sickle cell disease.
Principle
Hemoglobin is a globin polypeptide. When hemoglobin are suspended in agarose gel, the gel acts
as a molecular sieve and when electric field is established across the gel, migration of the charged
hemoglobin polypeptides occurs under the influence of the electric field. Different hemoglobin
have different amount of charges and according to those charges and size, different chains move
at different speeds in the gel and separates. (6)
N.B: Proteins of the same kind always move the same distance for all individuals and that is why
we have to include controls in the test.
Results
 Available after 1 day at MNH-CPL.
 Normal results have no problem, but abnormally high amounts of some hemoglobin
variants indicates hemoglobinopathies i.e. HbS in high amounts means Sickle cell disease.
(see Fig. 01)
Fig. 01
24
Visited from 25th
JUNE 2020 to 28th
AUGUST 2020.

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SICKLE SCAN TEST25
Refers to a qualitative rapid screening test for sickle cell disease with very high specificity and
sensitivity (99%). It is a lateral flow immunoassay technique where specific antibodies against
various abnormal Hemoglobin are set to react with blood in a moving membrane. It comes as a kit
for identification of sickle cell disorder in hemoglobin A, S, and C variants.
Principle
A small amount of blood when haemolysed by pre-treatment buffer and allowed to flow through
the test cartridge, the sample will interact with antibody-conjugated colorimetric (dyed) detector
nanoparticles and travel to the capture zones where they may be detected as HbA, HbS, or HbC
depending on the hemoglobin variant present. (6)
Materials in the kit
 Sickle SCAN®
cartridges
 Pre-treatment buffer
 Capillary sampler (5 µL)
 Lancet
 Gloves
 Alcohol swabs
Procedure of Sickle SCAN®
test
 Make sure all the required materials for the test are prepared. Label the cartridge and Pre-
treatment buffer with patient ID.
 Perform a finger prick (by standard lab. Protocol) to obtain blood specimen by the
capillary sampler (5 µL).
 Open the pre-treatment buffer and dispense the blood specimen collected into the buffer.
Tightly re-cap and mix the contents by inverting 3 times.
 Remove the cap from pre-treatment buffer and immediately dispense 5 drops into the
Sickle SCAN®
cartridge. Allow the test to run for 5 minutes at room temperature. (6)
 Read results by viewing the detection window26
. (see Fig. 02)
Fig. 02
25
Visited on 13th
AUGUST 2020.
26
Test results that run over 10 minutes are invalid.

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ISOELECTRIC FOCUSING (IEF)27
This technique separates proteins according to their isoelectric points. Hemoglobin variants
polypeptides can also be separated electrophoretically on the basis of their relative contents of
acidic (-COOH) and basic (-NH3) residues. The Isoelectric point (pI) of a protein is the pH at
which its net charge is zero. At this pH, its ability to move under influence of electric field
(electrophoretic mobility) is zero. (7) For example the pI of HbA = 7, HbS = 7.2, HbF = 7, HbA2
= 7.4, and HbD = 7.2.28
Principle
A pH gradient is established in a gel before loading the sample. The sample is loaded and voltage
is applied. The proteins will migrate to their isoelectric pH, the location at which they have no net
charge. The proteins form bands that can be excised and used for further experimentation. (6)
Procedure for performing isoelectric focusing
 Carefully processed sample (from dried blood spot (DBS) or EDTA anticoagulated whole
blood) is loaded into small wells in the agarose gel on the isoelectric focusing machine.
 A stable pH gradient is established in the agarose gel by the addition of appropriate
ampholytes.
 With an applied electric field, proteins enter the gel and migrate until each reaches a pH
equivalent to its pI.29
 After 1 hour the gel is then fixed in 10% Trichloroacetic acid (TCA) solution for 10
minutes. The gel is then removed from fixative and rinsed by distilled water then dried in
a hot air oven.
 The gel is then stained by brilliant blue stain for 30 minutes then dried again in the oven.
 Results are read on an illuminated plate to identify different hemoglobin variants bands.
N.B: Proteins with different isoelectric points are thus distributed differently throughout the gel.
So HbS band will not be the same level as HbA band.
27
Visited on 27th
JULY 2020.
28
At HCRL, this test procedure can take up to 6 hours of active concentration.
29
Remember that when pH = pI, the net charge of a protein is zero.

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HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)30
Refers to an enhanced version of column chromatography with a high resolving power used at
HCRL as a quantitative and qualitative confirmatory test for various hemoglobinopathies
especially Sickle cell disease. At HCRL, the VARIANT nbs-1 machine is used to perform HPLC
in a fully automated mode.
Principle
HPLC works by the partitioning of solutes in a column principle. Different hemoglobin
polypeptide variants are pushed into fine forms that partition differently in the liquid column in
the cartridges. Voltage is then used to illuminate the different variants of hemoglobin, this voltage
is proportional to the amount/quantity of the specific hemoglobin variant. A graphical presentation
of absorbance of the hemoglobin variants is made and printed. (7)
Phases of HPLC
i. Mobile phase – here high pressure forces (pumps) the sample mixture (Hemoglobin pool)
through a Calcium silicates containing solvent and into the standard cartridge.
ii. Stationery phase – here very high pressure is applied to a standard cartridge containing
chromatographic packing material.
As the sample passes through the column it interacts between the two phases above at different
rates due to different polarities in the analytes (hemoglobin variants). Different hemoglobin
variants will have different retention times. (see Fig. 03)
N.B: 360 samples can be analyzed per 1 run
which takes almost 6 minutes. Specimens can
be EDTA anticoagulant whole blood or dried
blood spot (DBS).
Fig. 03
HPLC result graph from a sickle cell (+) patient
30
Visited on 28th
AUGUST 2020.

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BLOOD TRANSFUSION UNIT
DONOR REGISTRATION31
Here the donors’ particulars (like full name, address, date of birth, and contact details) and
demography (i.e. place of birth, marital status, and employment status) are filled in the donation
request form prior to preliminary hemoglobin level estimation and donor counseling. Details of
the last donation are also taken to identify first time donors.
The donors’ weight, blood pressure and pulse rate are also measured to ensure the donor has all
necessary donation criteria (>50kg, 110/70mmHg – 125/80mmHg, 70 – 75 beats/min
respectively). Failure to meet these criteria leads to deferring/postponement of donation.
HEMOGLOBIN LEVEL (Hb estimation)
The copper sulphate (CuSO4) solution is used as a rapid test for measuring hemoglobin quantity
in the donors’ blood prior to donor counseling. Adult hemoglobin being a globin polypeptide has
a specific density greater than the copper sulphate solution hence in an adequate amount
(>12.5g/L) in red blood cells, the hemoglobin makes the donor red cells to descend and sink down
the CuSO4 solution. (3)
Half-way sinking or floating of the drop of blood leads to postponement/deferring of donation
process due to an inadequate amount of hemoglobin in donor red cells. (1)
DONOR COUNSELING32
Here an informed written consent has to be obtained from the donor who provides the required
answers to a set of 38 questions posed by a counselor concerning his/her general ability to donate,
clinical history of sexually transmitted diseases or malignancy diseases, number of sexual partners,
the purpose for donating (replacement or volunteering), time when last meal taken and many
others.
The clinician also observes the physical state of the donor (is he/she afraid, does he/she have
indications of high blood pressure, is he/she confident on the procedure?).
When a donor succeeds in all requirements by providing accurate and sufficient information on
his/her health status, he/she will be directed towards blood donation room. Otherwise he/she is
deferred/postponed.
31
Donor registration and Hb level estimation both visited on 24th
AUGUST 2020.
32
Visited on 26th
AUGUST 2020.

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BLOOD DONATION33
The donor now undergoes a phlebotomy procedure to collect sufficient whole blood in standard
polyvinyl carbon blood bags with Citrate Phosphate Dextrose Adenine (CPDA-1) anticoagulant.
One blood unit (450 ml) is collected from each donor. A well-trained personnel should perform
the procedure while observing carefully the donor’s body gestures and fainting symptoms. (1)
Materials needed
 Gloves
 Tourniquet
 70% ethanol (alcohol)
 Dry cotton wool
 CPDA-1 standard blood bag (450 ml) with stopper
 Red top (plain) evacuated tube
 Pieces of adhesive tape
Procedure for blood collection
 Label the blood bag and plain tube with donor blood unit number/barcode then lie the
donor in a good position and select a large well situated vein for the venipuncture, usually
near the bend of the elbow.
 Clean very well the required part of the arm with cotton wool and 70% ethanol (alcohol).
Wipe dry with a clean swab of cotton wool.
 Take the blood collecting bag and place it well in a stand or beside the bed, close the stopper
before performing venipuncture.
 Make the venipuncture with needle bevel facing upwards in the line of the vein, open the
stopper to allow blood to flow into the blood bag. Apply a strip of adhesive tape on the
needle to stabilize the flow.
 When the blood enters the bag, gently mix it with the anticoagulant by lifting and tilting
the bag for 3 times.34
Wait for the blood to fill while observing the donor for any indications
of fainting or adverse reaction35
. (1)
33
Visited on 26th
AUGUST 2020.
34
Do not squeeze the bag tightly as it may damage red cells.
35
If any adverse reaction like dizziness or fainting occurs, STOP the blood collection process immediately.

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 When the bag fills up, weigh the blood bag. When the bag is 500 – 600gm i.e. 450 – 495ml,
this indicates completion of blood donation.
 Close the stopper again and remove tourniquet. While pressing on puncture site with a dry
cotton wool, steadily remove the adhesive tape and the needle. Immediately plug the needle
in the plain tube. Collect enough sample in the red tube and close the stopper.
 Mix the blood bag 3 more times while asking on the status of the donor (how is he/she
feeling?). Proceed emptying the strip blood into the bag and mix thoroughly with
anticoagulant.
 After making sure the bleeding has stopped on puncture site, cover with a pad of cotton
wool and adhesive tape.36
TESTING FOR TTIs37
Here the donated blood is screened for transfusion transmitted infections (TTIs) like syphilis,
hepatitis B virus, hepatitis C virus, and HIV. This is according to the standard testing protocol at
MNH-Blood bank unit.
The new Syphilis rapid immunoassay test kits are very efficient in detecting treponema specific
antigens in the donors’ blood by the antibody-conjugated colorimetric (dyed) detector
nanoparticles that travel to the capture zones where they may be detected as syphilis positive
when a test-band appears. (see Fig. 04)
Results can be read after 5 minutes.
Fig. 04 Results from a syphilis (+) donor
For HIV, HBV & HCV antibodies, they can be screened/detected by a chemiluminescent
microparticle immunoassay (CMIA) principle of immunoassays in the Abbott Architect®
analyzer. The serum of donor(s) are loaded into special tubes and given identity numbers then
processed by the Architect analyzer for about 1 hour in a fully automated manner. Results are
printed or can be uploaded to the Laboratory information system (MNH-LIS).
36
Not to be removed in less than 3 hours.
37
Visited on 26th
AUGUST 2020.

of 27
PREPARATION OF BLOOD COMPONENTS38
It is in rare instances when whole blood transfusions are made (e.g. auto-transfusion in elective
surgery), most of the time patients at MNH require only specific blood products/components. So
this necessitates the process of separation of blood components into different bags with the same
blood unit number in order to transfuse the right product to the right patient in need.
The process involves ultra-centrifugation and squeezing out the desired product into a different
bag. The bags are labelled by the donors’ blood bag unit number.
Different blood components prepared at MNH-Blood bank
 Packed red cells (PRBC)
 Fresh frozen plasma (FFP)
 Platelets39
 Leuco-reduced PRBC40
ABO & Rh BLOOD GROUPING41
Both patients’ and donors’ blood groups should be typed in order to establish correct blood
transfusion and minimize the likelihood of transfusion reactions occurring. At MNH-Blood bank
unit only forward (cell) grouping is performed. Here unknown donor and patients’ red cells are
reacted with known Anti-sera to determine antigens present on their red cell surfaces (A, B, AB,
or O and Rh+ or Rh-).
Procedure for performing forward (cell) grouping by tube method
 Label the test tubes according to the number of samples to be tested.
 Add a drop of Anti-sera to each test tube by starting with Anti A, B and D respectively.
 Add a drop of blood sample given in each test tube.
 Mix the blood sample given and Anti-sera for 10 minutes.
 Observe for agglutination or clumping macroscopically.
N.B: If there is doubt on results, call a fellow lab personnel or observe under the microscope for
agglutination of red cells. (see Table 1.5)
38
Visited on 26th
AUGUST 2020.
39
To prevent clumping/aggregation they are always kept on a shaker machine and stored there for 5 days only.
40
Especially for patients undergoing kidney transplantation at MNH.
41
Visited on 23rd
AUGUST 2020.

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Anti-A Anti-B Anti-D Group
CONTROL
Cell A + - + A+
Cell B - + + B+
Cell O - - + O+
Forward Grouping
D1 cell + - + A+
D2 cell - + + B+
D3 cell - - + O+
P cell - + + B+
KEY D = Donor
+ Agglutination P = Patient
- No agglutination
Table 1.5
CROSS-MATCHING (COMPATIBILITY TEST)42
In order to be sufficiently accurate that a blood product from a certain donor won’t initiate a
transfusion reaction in the recipient, a major cross-matching should be performed between the
donor cells or other blood products (platelets43
) to ensure no floating antibodies are in the patient’s
serum against the donor’s red cell antigens. At MNH-Blood bank, this procedure is a must before
issuing of the blood product is done, even if forward grouping gave compatible donor and
patient’s blood groups.
Procedure for performing cross-matching by immediate spin method
 Mix one drop of donor red cells in 2 drops of patient serum in a clean & dry tube.
 The patient’s serum with donor cell are centrifuged immediately at 1000rpm for 60
seconds.
 Absence of hemolysis or agglutination indicates compatibility.
ISSUING OF BLOOD PRODUCTS44
This is the final and essential step prior to blood transfusion. A compatible blood product with an
identity card bearing patient ID and other important details must be registered and documented
correctly in the issuing register of MNH-Blood bank. This includes the name and signature of
the laboratory personnel who performed the process and counter checking if details in the request
form match with those on the blood product identity card.
42
Visited on 23rd
AUGUST 2020.
43
Platelet cross-matching not performed at Muhimbili currently.
44
Visited on 23rd
AUGUST 2020.

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REFERENCES
1. Cheesbrough M. District Tropical Practice in Laboratory Countries Part 2 Second Edition.
2nd ed. CAMBRIDGE UNIVERSITY PRESS; 2006. 442 p.
2. Ciesla B. Hematology in Practice. 2012. 386 p.
3. Bain BJ, Bates I, Laffan MA, Lewis SM. Dacie and Lewis. 2011. 11–15 p.
4. BATRA S. Preparation of Leishman Stain in Laboratory | Hematology Practicals [Internet].
2018 [cited 2020 Sep 7]. Available from: https://paramedicsworld.com/hematology-
practicals/preparation-of-leishman-stain-in-laboratory/medical-paramedical-studynotes
5. Victor, Hoffbrand H M. Hoffbrand’s Essential Haematology 7th edition [Internet]. 7th ed.
WILEY Blackwell; 2011. 382 p. Available from: www.wiley.com/buy/9781118408674
6. Nelson DL, Cox MM. PRINCIPLES OF BIOCHEMISTRY 4th edition. 4th ed.
7. L. Jeremy M. John. Biochemistry 5th edition. 5th ed. © W. H. Freeman and Company and
Sumanas, Inc.; 1515 p.

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