MATERIALS & ROUTINE TESTING IN THE BLOOD
BANK LABORATORY
BLOOD COLLECTION BAGS
• All collection bags have a sterile interior, including the
needle and all attached tubing, and contain an
anticoagulant
-preservative solution.
• The ideal collection system consists of a closed
system. The closed system is constructed of a primary
bag and one or more satellite bags.
• The interconnected bags allow for transfer of
components from the original bag without exposing the
blood product to air. This maintains sterility of the
components and allows the red cells to retain the
expiration date of the whole blood.
• An open system, as it implies, opens the airtight
system of the original collection container during
component preparation. This process exposes the
components to an increased risk of contamination and
changes the expiration date of the components.
RED CELL ANTICOAGULANT AND PRESERVATIVE
USED IN BLOOD BAGS
• Citrate: It prevents coagulation by interfering with
calcium-dependent steps in the coagulation cascade.
• Dextrose: The dextrose provide nutrient for red cells
and support the generation of ATP by glycolysis thus
enhancing red cells viability and extending shelf life.
• Acid–citrate–dextrose: It contains citric acid, sodium
citrate, and dextrose. It has a shelf life of 21 days. It is
now no longer use for red cells preservation, as other
solutions are available with extended shelf life of red
cells.
• Citrate–Phosphate–Dextrose: Shelf life of red cells is
extended to 28 days. CPD is now most commonly used.
• Citrate–Phosphate–Dextrose–Adenine-1 (CPDA-1):
Citrate phosphate dextrose adenine solution was
developed in 1968 and shown to permit whole blood
storage for 5 weeks. The citrate prevents coagulation by
binding or chelating to calcium; phosphate acts as a
buffer and, hence, maintains the pH of the blood.
Dextrose serves as substrate for the blood cells, while
adenine maintains high ATP level in the RBC.
• Most blood collection bags (adult) contain 63 mL
CPDA anticoagulant which is sufficient to anticoagulate
and ensure the viability of blood cells in 450 mL ± 10%
blood for up to 28–35 days when the blood is stored at
2–8 °C.
• Addition of adenine is associated with improved
synthesis of ATP, allowing longer shelf life of 35 days.
• Saline–Adenine–Glucose– Mannitol (SAGM): After
taking blood donation in CPD and separating red cells
from plasma and platelets, SAGM is added to the
packed red cells. The resulting red cells have flow
characteristics equivalent to plasma-reduced blood and
a storage life of 35–42 days.
• SAGM additive solution provides optimum red cell
viability.
• Sodium chloride provides isotonicity.
• Adenine maintains ATP for red cell viability. BLOOD TYPING METHODS
• Glucose supports red cell metabolism (nutrition).
• Mannitol helps reduce red cell lysis.

WHY STORE BLOOD AT 2–6 °C?

• If blood is not stored between +2 and +6 °C,
its oxygen-carrying ability is greatly reduced.
• Another important reason for storing blood
between +2 and +6 °C is to keep the blood unit free
from the growth of any bacterial contamination. If
blood is stored above +6 °C, the chances of
contamination with bacteria increase.
 HEMATOLOGY LABORATORY
Peripheral Film Preparation
Types of Films
Manual wedge technique.
It is the most convenient and most commonly
used method for making peripheral blood films.
This technique requires at least two 3-inch x 1-
inch (75-mm x 25-mm) clean glass slides.
High-quality, beveled edge microscopic slides
with chamfered (beveled) corners for good
lateral borders are recommended.
One slide serves as the film slide, and the other
is the pusher or spreader slide.
A drop of blood (about 2 to 3 mm in diameter)
from a finger, heel, or microhematocrit tube
(nonheparinized for EDTA-anticoagulated blood
or heparinized for capillary blood)is placed at
one end of the slide.
The size of the drop of blood is important; too
large a drop creates a long or thick film, and too
small a drop often makes a short or thin blood
film.
The pusher slide, held securely in the dominant
hand at about a 30- to 45-degree angle, is
drawn back into the drop of blood, and the
blood is allowed to spread across the width of
the slide.
It is then quickly and smoothly pushed forward
to the end of the slide to create a wedge film.
Features of a Well-Made Wedge Peripheral Blood
Film
1. The film is two-thirds to three-fourths the length of
the slide
2. The film is finger shaped, very slightly rounded at the
feather edge, not
bullet shaped; this provides the widest area for
examination.
3. The lateral edges of the film are visible.
4. The film is smooth without irregularities, holes, or
streaks.
5. When the slide is held up to the light, the thin portion
(feather edge) of the
film has a “rainbow” appearance.
6. The whole drop of blood is picked up and spread.

Staining of Peripheral Blood Films
Slides must be completely dry before staining or
the thick part of the blood film may come off
the slide in the staining process.
Pure Wright stain or a Wright-Giemsa stain
(Romanowsky stain) is used for staining
peripheral blood films and bone marrow
smears.
Methanol in the stain fixes the cells to the slide.
Oxidized methylene blue and eosin form a
thiazine-eosinate complex, which stains neutral
components.
Because staining reactions are pH dependent,
the buffer that is added to the stain should be
0.05 M sodium phosphate (pH 6.4) or aged
distilled water (distilled water placed in a glass
bottle for at least 24 hours; pH 6.4 to 6.8).
Free methylene blue is basic and stains acidic
(and basophilic) cellular components, such as
ribonucleic acid (RNA).
Free eosin is acidic and stains basic (and
eosinophilic) components, such as hemoglobin
and eosinophilic granules.
Wright Staining Methods
Slides are placed on the rack, film side facing
upward.
The Wright stain is poured directly from the
bottle through a filter onto the slide. It is
important to flood the slide completely.
Stain should remain on the slide at least 1 to 3
minutes to fix the cells to the glass.
Then an approximately equal amount of buffer
is added to the slide. A metallic sheen (or green
“scum”) should appear on the slide if mixing is
correct
The mixture is allowed to remain on the slide
for 3 minutes or more (bone marrow smears
take longer to stain than peripheral blood
films).
When staining is complete, the slide is rinsed
with a steady but gentle stream of neutral -pH
water, the back of the slide is wiped to remove
any stain residue, and the slide is air -dried in a
vertical position.
Peripheral Film Examination
The laboratory professional evaluates the
platelet and WBC count and differential, along
with WBC, RBC, and platelet morphology.
Microscopic Examination
10X objective examination.
At the low power magnification, overall film
quality, color, and distribution of cells can be
assessed. The feather edge and lateral edges
should be checked quickly for WBC distribution.
40X high-dry or 50X oil immersion objective
examination.
At either of these magnifications, it is easy to
select the correct area of the film in which to
begin the differential count and to evaluate
cellular morphology.
100X oil immersion objective examination.
Provides the highest magnification on most
standard binocular microscopes.
The WBC differential count generally is
performed using the 100X oil immersion
objective.
Performing the differential normally includes
counting and classifying 100 WBCs to obtain
percentages of WBC types.
The RBC, WBC, and platelet morphology
evaluation and the platelet estimate also are
executed under the 100X oil immersion
objective lens.
RBC inclusions, such as Howell-Jolly bodies, and
WBC inclusions, such as Döhle bodies, can be
seen easily if present.
Reactive or abnormal cells are enumerated
under the 100X objective as well.
If present, nucleated RBCs (NRBCs) are counted
and reported as NRBCs per 100 WBCs.

BLEEDING TIME DUKE & IVY METHOD
Principle of the test: The test is performed by using a
sterile blood lancet to make a measured skin puncture
in the earlobe or forearm. The time is started
immediately after the puncture. Touching a piece of
filter paper to the cut every 30 seconds until bleeding
ceases and record the time and reported as bleeding
time
A. Duke Method:
1. Obtain a piece of filter paper and stopwatch.
2. Moisten a piece of cotton with 70% alcohol or
Povidone iodine and thoroughly cleanse the ball of the
patient’s middle or ring finger.
3. Allow the skin to air-dry.
4. Make a puncture wound 2-4mm deep in the earlobe
or finger with a disposable blood lancet.
5. Start the stopwatch immediately.
6. Being careful not touch the puncture site. Blot the
filter paper every 30 seconds, until the bleeding stops.
7. Record the bleeding time.
Note: Should be the blood flow more than 15 minutes.
Discontinue the test and report the test as “greater
than 15 minutes”
Reference Range: 1 to 3 minutes
B. Standardized Ivy Method:
1. Obtain a piece of filter paper, stopwatch, and
sphygmomanometer.
2. Position the patient’s arm with the volar surface
exposed.
3. Place the sphygmomanometer on the upper arm.
4. Moisten a piece of cotton with 70% alcohol or
Povidone iodine solution and thoroughly cleanse the
puncture site.
5. Apply the cuff and inflate to 40mmHg, and hold at
this exact pressure for the duration of the test.
NOTE: the time between inflation and incision should
be 30 to 60 seconds.
6. After applying the pressure cuff and preparing the
test site. Make three (3) same punctures with a
disposable lancet (1mm wide and 3mm deep)
Caution: Avoid subcutaneous veins.
7. Start the stopwatch immediately.
8. After 30 seconds, wipe the flow of blood with the
filter paper. (Bring the paper close to the incision, but
do not touch the paper directly to the incision, so as not
to disturb the formation of the platelet plug.)
9. Blot each site with filter paper every 30 seconds
thereafter, until no blood stains the paper.
10. Stop the timer when only clear fluid is absorbed
onto the filter paper. The bleeding time is determined
to the nearest 30 seconds.
11. Release the pressure of the sphygmomanometer
12. Record the bleeding time for each of the puncture.
13. Get the average of the bleeding time in minutes and
seconds.
Reference Range: 3 to 6 minutes Borderline: 6 to 11
minutes
CLOTTING TIME SLIDE, WRIGHT’S, AND LEE &
WHITE METHOD
A. Slide Method
1. Disinfect site of puncture with 70% ethyl alcohol. Air
dry.
2. Puncture to a depth of 3mm using a blood lancet.
3. Start timer as soon as the first drop of blood appears.
4. Transfer the three drops of blood onto a clean glass
slide. Careful not to touch the skin.
5. Pass the tip of the lancet through the first drop of
blood every 30 seconds and not for theformation of
fibrin strands. Repeat with second drop and then the
third drop.
6. Stop the timer immediately as soon as fibrin strands spilling the content (that is, until the blood is
are seen clinging at the tip of the lance onthe third completely clotted).
drop. 6. Record the time it took the blood in test tube #1 to
Reference Range: 2-4 minutes clot.
7. Thirty seconds after the blood in test tube #1 had
B. Wright’s Method or Glass Capillary Method clotted, proceed with tube #2, and repeat the preceding
1. Apply 70% alcohol to the clean finger with cotton procedure tilting the tube every 30 seconds, until it is
swab. Allow it to dry naturally. completely clotted. Record the result. Repeat this
2. Prick the finger with usual aseptic precautions. procedure for test tube #3.
Immediately start the stopwatch. 8. Since agitation and handling, speed up coagulation,
3. Dip one end of the capillary into blood drop gently the coagulation time of test tube #3 is the result to be
without pressure. reported.
4. Allow to fill the capillary with blood by lowering the Reference Range: 7-15 minutes
end of fitted capillary. (Do not stuck the blood around ¾
of its length undipped.
5. After about two minutes start snapping off small
lengths of the tube, at intervals of 15 seconds, each
time noting whether the fibrin thread is formed
between the snapped ends.
6. Repeat breaking at regular time intervals, till fibrin
thread appears at the broken end of capillary tube. Do
not pull away the catted pieces.
7. Record the time interval between pricking finger and
first appearance of fibrin thread at the broken ends of
capillary tube. That is clotting time of blood.
Reference Range: 4-9 minutes

C. Modified Lee and White Clotting time

Principle of the Test: The coagulation of blood is the
length of time required for a measured amount of blood
to clot under certain conditions. The test is based on the
fact that when venous blood is put into a glass tube
(foreign surface), it will form a solid clot. The time
required for this response is a measure of the overall
intrinsic and common pathways of coagulation.
1. Label three, 13 x 10mm test tubes with Px’s name,
and number them.
2. Perform aseptically a venipuncture using a 20-gauge
needle syringe and withdraw 4 mL of blood.
3. After obtaining the blood, remove the needle from
the syringe, and carefully place 1 mL of the blood in test
tube #3, then 1 mL in tube #2, and 1 mL to tube #1. The
last 1 mL must be discarded (follow universal safety
procedures on the disposal of needles, etc.). Start the
stopwatch as soon as the blood is placed in tube #3.
4. Place the three tubes in a 37° C water bath.
5. At exactly 5 minutes, tilt test tube #1 gently to a 45-
degree angle. Repeat this procedure every 30 seconds
until the test tube can be completely inverted without
 COMMON SEROLOGICAL TEST
Abbott HIV-1/2 Ag/Ab COMBO
• This is an immunochromatographic test for the
simultaneous and separate qualitative detection of free
HIV-1 p24 antigen and antibodies to HIV-1 and HIV-2.
• The test device is a laminated strip that consists of a
sample pad containing monoclonal biotinylated anti-
HIV-1 p24 antibody, a conjugate pad containing
monoclonal anti-HIV-1 p24 antibody colloidal selenium
and HIV-1 and HIV-2 recombinant antigen-colloidal
selenium, and a nitrocellulose membrane with an
immobilized mixture of recombinant and synthetic
peptide HIV-1 and HIV-2 antigens in the Lower Test
Area, immobilized streptavidin in the Upper Test Area,
and an immobilized mixture of anti-HIV-1 antibody,
HIV-1/2 antigens, and HIV-1 p24 recombinant antigen
and anti-HIV-1 p24 monoclonal antibody in the Control
Area.
Materials
• 1. Aluminum ziplock pouch containing Determine HIV-
1/2 Ag/Ab Combo Cards. Each card consists of 5 or 10
test units which can be separated from each other
by tearing along the perforated lines. Each test unit has
a cover that is to be removed for sample application
and visualization of test results.
• 2. Desiccant package
• 3. Chase bufer: Containing sodium chloride, disodium
hydrogen phosphate, and Nipasept as a preservative.
• 4. Quick reference card
• 5. Package insert
• 6. Subject information notices
• 7. Customer letter
• 8. Disposable capillary tubes: For collection and
transfer of fingerstick samples.
• 9. Disposable workstations:
TEST PROCEDURE AND INTERPRETATION OF
RESULTS
• Kit Component Preparation
• 1. Open the aluminum pouch containing the
Determine HIV-1/2 Ag/Ab Combo Cards.
• 2. Remove the desired numbers of test units from the
5 or 10-test unit card by bending and tearing at the
perforation.
• 3. Return the unused test units to the aluminum
pouch and close the pouch with the Ziplock.
• 4. Remove the protective foil cover from each test
unit. Lay the test unit fat in the workstation.
• The test should be initiated within 2 hours after
removing the protective foil cover from each test unit.
Do NOT touch the sample pad with your fingers.
• NOTE: Determine HIV-1/2 Ag/Ab Combo must ONLY
be used with capillary (fingerstick) or venous
(venipuncture) whole blood, serum or EDTA plasma.
Using other types of samples or testing of venipuncture
whole blood and plasma samples collected using a tube
containing an anticoagulant other than EDTA may not
yield accurate results. For serum samples, collect blood
without anticoagulant.
• For serum or plasma samples:
• 1. Apply 50 μL of sample (precision pipette) by
touching the tip of the pipette to the sample pad
(marked by the arrow symbol). Do not add chase buffer
when using serum or plasma specimens.
• 2. Read the test result between 20 and 30 minutes
after the addition of the sample. Do not read test
results after 30 minutes.
• For whole blood (venipuncture) samples:
• 1. Using a precision pipette with a disposable tip,
apply 50 μL of sample by touching the tip of the pipette
to the sample pad (marked by the arrow symbol).
• 2. When all of the blood is transferred to the sample
pad, wait one minute to ensure the chase buffer does
not overflow the sample pad.
• 3. Add one drop of chase buffer to the sample pad.
• 4. Read the test result between 20 and 30 minutes
after the addition of chase buffer. Do not read test
results after 30 minutes.
SD BIOLINE HBsAg
• SD BIOLINE HBsAg WB is an in vitro
immunochromatographic, rapid assay designed for
the qualitative detection of Hepatitis B surface antigen,
in human serum, plasma (heparin, EDTA and sodium
citrate) or venous whole blood (heparin, EDTA and
sodium citrate).
• Reactive specimens should be reflexed for additional
testing, either by enzyme immunoassay (EIA) to identify
current HBV infection.
• The membrane is pre-coated with mouse monoclonal
anti-HBsAg pool on the test line region and Mouse
monoclonal anti-chicken on the control line region.
• During testing, the specimen is allowed to react with
the colored conjugate (mouse monoclonal anti-HBsAg
conjugated gold colloid) which was pre-coated on the
test strip. The mixture (mouse monoclonal anti-HBsAg +
HBsAg in specimen) then moves upward on the
membrane chromatographically by capillary action.
• For a reactive result, a purple-colored line with the
mouse monoclonal anti-HBsAg
pool colored conjugate complex will form in the test
line region of the result window.
Dengue NS1
Ag STRIP
• Dengue NS1 Ag STRIP is a disposable test using lateral
flow immunochromatography.
• The test device contains a strip membrane formed
with:
• • a sample pad for sample loading.
• • a conjugate pad containing gold colloidal particles
coated with anti-NS1 monoclonal antibodies and gold
colloidal particles coated with streptavidin.
• • a membrane where occurs the immunological
reaction with two lines corresponding respectively to
the Test Line (anti-NS1 monoclonal antibodies) and the
Control Line (biotin).
• The Dengue NS1 Ag STRIP is placed vertically in a
sample tube containing 50 μl of the specimen to be
tested and one drop of the migration buffer.
• The sample is drawn along the membrane strip.
Results are read after 15 minutes of migration.
• Each Dengue NS1 Ag STRIP kit contains:
• • One (1) strip container with vial stopper including a
desiccant.
• • Twenty-five (25) strips for individual testing.
• • One (1) migration buffer dropper (3 ml).
• • One (1) instruction leaflet.
Sample distribution (50 μL)
• In a single glass or plastic tube, distribute 50 μl of
serum or plasma to be tested. Check for clarity of the
specimen and absence of particles in suspension.
Migration Buffer distribution (1 drop)
• Using the dropper supplied with the kit, add to each
tube one (1) drop of the migration buffer.
Gently shake the tube to ensure adequate mixing.
Add one strip in the specimen tube
• Place vertically one strip in each tube. Ensure
that the strip is placed in the right position with the
arrows directed to the bottom of the tube. Check that
the strip correctly dips in the diluted specimen.
KEEP THE STRIP IN THE TUBE FOR 15 MINUTES
• Maintain the strip in the tube and read the result 15
minutes after adding the strip to the tube. Read the
results from your normal reading distance in well lit
conditions.
The appearance of a blue or purple line at the Test
Line (T) and the Control Line (C) indicates a POSITIVE
RESULT (a)
The appearance of a blue or purple line only at
the Control Line (C) indicates a NEGATIVE RESULT (b)
If the Control Line (C) is not present, the test is
considered INVALID and should be repeated using
fresh sample and a new strip (c)