1. Wash the donor red blood cells to be tested at least
three times in isotonic saline.
2. Prepare 2 to 3% red blood cell (donor) suspension in
3. To an appropriately labeled test tube, add two drops
of recipient serum to be tested and two drops of donor
red blood cell suspension. Mix the contents thoroughly
4. Immediately add two drops of Liquipap reagent.
5. Incubate at 37°C for 15 to 30 minutes.
6. Centrifuge at 1000 rpm for 2 minutes,
7. Gently resuspend and observe for agglutination and/
or hemolysis macroscopically and microscopically.
1. Wash the reagent red blood cells to be tested at least
three times in isotonic saline.
2. Prepare 2 to 3% reagent red blood cell suspension to
3. To an appropriately labeled test tube, add two drops of
serum to be tested and two drops of reagent red blood
4. Mix the contents and immediately add two drops of
5. Incubate at 37°C for 15 to 30 minutes.
6. Centrifuge at 1000 rpm for 2 minutes.
7. Gently resuspend and observe for agglutination and/
or hemolysis macroscopically and microscopically.
Alternatively, a two-stage test using Liquipap reagent
can also be performed as follows:
1. Wash the donor red blood cells three times in isotonic
2. To an appropriately labeled test tube, add one drop of
washed packed cells (donor) and one drop of Liquipap
3. Incubate the test tube at 37°C for 15 to 30 minutes.
4. Wash the Liquipap-treated donor red blood cells at
least three times with isotonic saline.
5. Prepare Liquipap treated 2 to 3% red blood cell
suspension of donor in isotonic saline.
6. To an appropriately labeled test tube, add one drop
of Liquipap treated 2 to 3% donor red blood cell
7. Add two drops of recipient serum to be tested.
8. Mix well and incubate at 37°C for 30 minutes.
9. Centrifuge at 1000 rpm for 2 minutes.
10. Gently resuspend and observe for agglutination and/
or hemolysis macroscopically and microscopically.
1. Wash the reagent red blood cells to be tested three
2. To an appropriately labeled test tube, add one drop of
washed packed reagent red blood cells under test and
3. Incubate the test tube at 37°C for 15 to 30 minutes.
4. Wash the Liquipap treated reagent red blood cells
under test at least three times with isotonic saline.
5. Prepare Liquipap treated 2 to 3% reagent red blood
cell suspension under test in isotonic saline.
6. To an appropriately labeled test tube, add one drop
of Liquipap treated 2 to 3% reagent red blood cell
7. Add two drops of serum to be tested.
8. Mix well and incubate at 37°C for 30 minutes.
9. Centrifuge at 1000 rpm for 2 minutes.
10. Gently resuspend and observe for agglutination and/
or hemolysis macroscopically and microscopically.
Agglutination and/or hemolysis indicate an antibody
directed against the antigen present on the red blood cell
No agglutination and/or hemolysis indicate absence of
enzyme reactive antibodies directed against the antigen
present on the red blood cell under test.
1. As undercentrifugation or over-centrifugation could
lead to erroneous results, it is recommended that each
Blood Banking (Immunohematology) 363
laboratory calibrate its own equipment and determine
the time required for achieving the desired results.
2. Erroneous results may also occur due to improper red
blood cell concentration, improper incubation time or
temperature while performing the test.
3. All enzyme preparations are subject to some loss of
potency, it is therefore, recommended to check the
reagent performance with known negative control
(neutral AB serum) and positive control (Coomb’s
control cells) on a regular basis.
4. The ability of papain to denature IgG molecule
renders the one-stage technique less sensitive though
it is a convenient method for use in cross-match
during the proteolytic action of papain enzyme.
6. Liquipap reagent is a colorless to pale yellow clear
solution. Repeated exposure to elevated temperatures
may impart a dark color to the reagent. In such cases,
the reagent performance must be assessed closely
7. Usage of isotonic buffered saline while performing the
test ensures in maintaining the optimum pH of the
reaction milieu for antigen antibody reaction.
8. Alternatively, LISS (Low ionic strength solution) can
be used while performing the test. LISS lowers the
ionic concentration of the reaction milieu thereby
potentiating the rate of antibody uptake by the antigen
present on the red blood cell membrane.
9. It is recommended to run a control with each assay
The procedures described earlier in this chapter are done
mainly for providing appropriate blood for transfusion, i.e.
the donor’s blood should be compatible in all ways with
Most individuals can give blood without any ill effects, as
a matter of fact, without any symptoms at all. The donors
may be volunteers or paid donors. Blood transfusion
should always be a safe, harmless procedure, therefore,
the donors must be screened. Two basic principles govern
1. The donor should not be harmed in any way.
2. The recipient should be equally safe, i.e. the transfusion
should be absolutely safe for the recipient too.
1. Identification: Identify the donor name, address and
other pertinent information. No donor should donate
twice in 3 months’ period. Preferably, the donors
should be between the ages 18 and 60 years.
2. History: The questions should be guided about the
present state of health; recent illness, if any; operations,
if any; if the donor has ever been transfused; (for
woman) if she ever gave birth to children who
developed jaundice shortly after birth; if they have
had malaria, jaundice, syphilis, tuberculosis; if they
have heart disease, diabetes, etc. Donors may give
wrong information on two accounts: (i) They want to
donate blood for want of money. (ii) They do not want
to donate blood for they are afraid of doing so. Hence,
a battery of tests to be done becomes necessary.
• Temperature reading, (exclude those having fever)
• Blood pressure should be normal or only slightly
raised. Disqualify those who have moderate to
• Pulse should be normal without any irregularities
• Auscultate the chest for any respiratory or cardiac
disorder. Discard individuals with respiratory/
• Pregnant women should not donate if they are
• Finding of any venereal disease should outrightly
disqualify the donor until he receives adequate
treatment and gets rid of his problem.
4. Hemoglobin: The donors should not be anemic. It
is best to disqualify anybody having Hb < 12 g% but
keeping in view our health, dietetic, and economic
structure up to 10 g% can, however, be accepted,
weighing the urgency of the demand for blood. As
given in the hematology section specific gravity by
copper sulfate method is adequate for screening
5. Malarial parasite: Malarial blood can cause transfusion
malaria if it is transfused. Screen all donors by making
thick and thin smears of their blood. In some chronic
malaria patient, no parasites may be seen on the
peripheral smear examination but a unit of their blood
would pass on enough parasites to the recipient.
6. Microfilaria: Screening for microfilaria should be done
both by making a wet, coverslip mount of a drop of
fresh blood (looking for the shipping movement of the
microfilaria) and also on the direct and stained films
used for searching malarial parasites. All microfilariaharboring donors are rejected.
and quantitating the amount of pigment (mostly
bilirubin) in the blood. Serum hepatitis is one of the
most dreaded complications of transfusion. If a person
has had jaundice during the last 5 years or is having it—
he should be disqualified as a donor. History might be
difficult to obtain and hence, liver function tests may
be necessary where there is even slightest of doubt.
The simplest and quickest to perform is icteric index,
a measurement of the level of bilirubin, expressed
in units based upon a comparison of the color of the
serum and the color of a standard dilute solution of
potassium dichromate. If the Icterus index is > 6 units,
the donor should not be allowed to donate.
A major complication of transfusing blood from a
hepatitis B/C patient or carrier is the transmission
of the disease to the recipient. Now ELISA, ICT
8. Serology: VDRL/Kahn’s tests should be performed
on blood of all donors. The blood positive for this test
may pass on live spirochetes to the recipient. Weighing
the need of blood even such a positive sample may
be transfused and the recipient given a course of
penicillin. In no case should any drug be put into
the transfusion bottle. It is known that Treponema
pallidum cannot survive for more than 48–72 hours
with the condition of storage of anticoagulated
blood in a refrigerator at 4oC. Within that time they
are all dead. Hence, blood which has been stored
as mentioned above cannot transmit syphilis since
there would be no viable organisms remaining. A
new syphilis patient’s blood may be VDRL/Kahn’s
negative even though his blood would contain the
spirochetes, hence, some believe in storing all blood
for 48–72 hours before allowing it to be transfused. It
is a good practice to indicate to the doctor who has
requisitioned the blood, date and time of procuring
9. Grouping and typing of donor: This has already been
dealt with at great length earlier in this chapter.
10. Antibody Screening: Ideally an indirect Coombs’ test
should be done on every donor to screen for atypical,
11. HIV/AIDS screening is a Must.
(Information regarding kits__refer to Serology chapter).
2. Under aseptic conditions, do a venepuncture with a
fairly large needle on the donor set-15 or 16 gauge
needle, for gravity flow collection method. The bottle
is kept below the level of arm and has an airway so that
air may escape while the bottle gets filled with blood.
3. Where vacuum bottles are available a smaller gauge
needle can be used. Do not puncture such a bottle.
4. Plastic bags containing the preservative fluid are
already collapsed and hence, need no airway.
5. A unit of blood may mean different volumes at
different centers. A standard unit is 420 mL of blood
and 80 mL of ACD or CPD solution, i.e. a total 500 mL.
6. While the blood is being drawn swirl the bottle gently
7. Flow of blood is usually hastened by: (i) having a blood
pressure cuff on the arm above the needle, inflated to
a pressure of 40–50 mm Hg and (ii) asking the patient
to gently flex the fingers or clench and unclench his fist
to assist the flow of the blood into the vein from which
8. When the full amount of donation has been collected,
draw the needle from the bottle and put 5 to 10 mL
samples into 2 or 3 sterile, dry tubes, each with the
same number as that of the large blood transfusion
9. Now the needle from the vein and the blood pressure
10. Immediately apply pressure with cotton at the area,
preferably with the arm held straight up in an extended
position. Have the donor hold it for minimum 3 to 5
11. Close the bottle immediately after sterilizing the
12. Once closed do not open and enter the bottle until the
requisitioning doctor inserts the giving set. Be sure the
bottle is completely and correctly labeledwith number,
name, group, type, date, etc. Double check this.
13. Do not let the donor get up immediately. Let the
donor’s body adjust to the loss of this blood. Offering
a nourishing drink and snacks to the donor is indeed a
very good way of saying thanks to the donor. Encourage
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