Practical manual book for

Blood Banking and Transfusion

(33051482)

AL Baha University
Faculty of Applied Medical Sciences
Department of Laboratory medicine
Blood Banking and Transfusion lab manual 33051482

Preface
This practical book of blood banking and transfusion contains a series of fifteen
experiments dedicated for the students studying the course of blood banking and
transfusion. The practical experiments presented in this book aims to provide the
students with the basic understanding of Immunohematology procedures and its
techniques. The book is written in simple and easy to understand language with a
variety of figures and illustrations. And ended up with a list of references for those
who need further comprehensive details about a specific topic.

pg. 2 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Table of Contents
Preface……………………………………………………………………………………………..2

Experiment 1: safety guidelines …………………………………………………………………4

Experiment 2: Preparation of Normal Saline and 2‐5% Cell Suspension, Washed RBC Cells

……………………………………………………………….........……………………..…….…5

Experiment 3&4: ABO grouping………………………….......…………………... …………..7

Experiment 5: Rh (D) Typing…………………………………………………………………..10

Experiment 6: Weak D (Du) testing…………………………………………………………….13

Experiment 7&8: Antiglobulin tests………....…………………………………………………14

Experiment 9&10: Cross matching …………….……………………………………………19

Experiment 11: Antibody Screening …….……………………………………………………21

Experiment 12: Antibody Identification ………………………………………………….……25

Experiment 13: Antibody Titration……………………………………………………………30

Experiment 14: transfusion reaction workup……………………………….………………….32

Experiment 15: Gel method……………………………………………………………………35

References……..……………………………………………………………………………….36

pg. 3 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 1
Safety Guidelines
ALWAYS KEEP IN MIND THAT THE SPECIMENS ARE FROM REAL PATIENTS AND
DONORS, AND MOST REAGENTS ARE MADE FROM HUMAN SOURCES.
CONSEQUENTLY, THERE IS AN ASSOCIATED RISK OF DISEASE TRANSMISSION.
OBSERVE UNIVERSAL PRECAUTIONS AT ALL TIMES.

1. Be neat and orderly - the work area should have only specimens, equipment, reagents, lab
manual and other necessary worksheets to complete your work. Coats, purses and other
books must be stored in your locker or in appropriate places away from the work area.
2. ALWAYS wear gloves when handling patient and donor samples, or tubes containing
patient or donor samples.
3. Wear protective eyewear when handling specimen tubes containing patient or donor
samples, when centrifuging, or when performing cell washing techniques.
4. Wash hands frequently and NEVER leave the lab without washing your hands.
5. Always wear a buttoned lab coat when working in the lab.
6. If a spill occurs, soak up the excess fluid with paper towels; then wipe the area clean with
a dilute bleach solution (made fresh daily). Discard paper towels in the isolation trash.
7. Discard materials grossly contaminated with human body fluids in the isolation trash.
Slides, applicator sticks, glass pipets, and lancets go in the sharps container. Gloves can
generally go in the regular trash, unless grossly contaminated with blood.
8. Wipe the work area with a dilute cleaning solution at the end of the day.
9. Wipe off the inside of the serofuge and outside of the saline wash bottles with cleaning
agent at the end of the day.
10. Let the serofuge come to a stop by itself - never open it while it is still rotating, and never
use your finger to stop it.
11. If a reagent or sample tube breaks in the serofuge, leave the lid unopened for 30 minutes
before decontaminating.
12. Reagents are shared, therefore take special precautions to prevent contamination. Never
lay droppers, tops, or corks on the counter top. Never touch the tip of a dropper or saline
wash bottle into a tube; never touch the tip of a dropper with your hands. Close all vials
tightly after use.
13. Keep agglutination viewers, serofuge heads, microscopes and other pieces of equipment
clean at all times. This is the responsibility of each student using the equipment.

pg. 4 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 2
Washing of red cells and making red cell suspension
Background
Suspension of washed red cells is needed for all haem agglutination tests. The cells must
be washed at least once in order to remove plasma, weak cell suspensions are used in
haemagglutination tests since the ratio of serum to cells affects the sensitivity of most
tests – a minimum number of antibodies must bound to RBC's in order to bring about
agglutination.

Principle:
This experiment is based on the A red cell suspension is a common reagent used for many
serologic procedures. Red cell suspensions provide the appropriate serum to cell ratio to allow
for grading and interpretation of tests results.

Aims and objectives:

Aim:
A 3-5% red cell suspension is used for the following tube examination procedures:
• ABO and Rh typing
• Direct antiglobulin test and auto control
• Donor unit compatibility (crossmatch)
• Red cell phenotyping
A 0.8% red cell suspension is used for the following MTS™ test methods:
• Direct Antiglobulin Test
• Donor unit compatibility (crossmatch)
• Indirect Antiglobulin Test
• Identification Panel Test Cells
For best results red cell suspensions should be used for testing on the day of preparation.

Objectives: by the end of this session, the student would be able to do washing of red cell and
prepare red cell suspension of different concentration.

Materials:
Specimen:
EDTA anticoagulated whole blood
Cord blood collected in a plain tube
Reagents:
Isotonic saline
MTS™ Diluent 2

pg. 5 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Supplies:
Test tubes (10x75mm) (12x75)
Transfer pipettes
Test tube rack
Equipment:
Serological centrifuge
MTS™ Pipettor (s)
MTS™ Diluent Dispenser
Procedure:
Time scale expected to perform the experiments is : 20 minutes

1- Place 0.2 -0.5 ml of blood into the tube (2-3 drops).

2- Fill the tube with the saline.
3- Centrifuge at 200 G for 1-2 minutes until the RBC's are packed. (G=relative
centrifugal force)
4- Decant the supernatant.
5- Tap the tube to resuspend the RBC's in the residual fluid. This constitutes one wash.
Repeat step 2-5 at least twice. The last wash should always have a clear supernatant
with no signs of haemolysis.
6- To make a 5% cell suspension add 1 volume of the packed RBC's to 19 volumes of
saline.
7- To make a 3% suspension, add 1 volume packed RBC's to 32 volumes of saline.

pg. 6 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 3&4
ABO grouping
Background
It was in 1901, that Austrian-American immunologist and pathologist Karl
Landsteiner discovered human blood groups. Karl Landsteiner's work helps to determine blood
groups and thus opened a way for blood transfusions which can be carried out safely. He was
awarded the Nobel Prize in Physiology or Medicine in 1930 for this discovery.
ABO typing is usually performed by saline techniques in tubes or micro-plates by testing the
patients or donor's red cells with anti-A, anti-B and the serum or plasma with A cells, B cells and
O cells.

1. SCOPE & APPLICATION:

To determine the correct ABO group of an individual and ensure the reliability of the
result. This procedure describes the method of detection of ABO antigens on the red cell
and the reciprocal antibodies in the serum (Landsteiner’s Law). It provides guidance for
the use of blood grouping reagents (antisera & standard red cells) in order to detect weak
variants, acquired antigens, Bombay (Oh) blood group and irregular red cell antibodies.
2. Material Required:
Equipment:

• Refrigerator to store samples and reagents at 2- 6 C.

• Table top centrifuge.
• Microscope.
Specimen:

• Clotted and anticoagulated blood samples of donors.

• Clotted blood sample of patients.
• Test red cells suspended in native serum/plasma or saline.
Reagents:

• Anti A, Anti-B, Anti-AB antisera.

• Group A,B and O pooled cells.
• 0.9% saline.
• Distilled water.
Glassware:
• Serum tubes.
• Micro tubes.
• Pasteur pipettes.
• Glass slides.
3. PROCEDURE:

pg. 7 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Principle: ABO system is the only system in which there is a reciprocal relationship
between the antigen on the red cells and the naturally occurring antibodies in the serum.
Routine grouping of donors and patients must therefore include both RBC and serum tests,
each serving as check on the other.
The procedure is based on the principle of agglutination of antigen positive red cells in the
presence of antibody directed towards the antigen.
Forward Grouping (Cell Grouping):
1. Label tubes with donor/patient and test identification.
2. Prepare cell suspension for cells being tested (Refer SP 015)
3. Place two drops of anti-A, anti-B and anti-AB reagent in the appropriately labelled
tubes.
4. Add to each tube one drop of a 2 – 5% cell suspension (in normal saline, serum or
plasma) of the red cells to be tested.
5. Mix the contents of the tubes gently and incubate at room temperature for 15
minutes.
6. Centrifuge at 1000 rpm for 1 minute.
(Note: Always follow manufacturer’s instructions from package insert)
Reverse Grouping (Serum Grouping):
1. Label tubes with donor-patient and test identification.
2. Add 2 drops of test serum in all tubes in the corresponding column.
3. Prepare cells for testing of A, B and O groups by pooling 3 samples of each group.
4. Add 1 drop of 2% pooled A red cell suspension in tube labelled Ac.
5. Add 1 drop of 2% pooled B red cell suspension in tube labelled Bc.
6. Add 1 drop of 2% pooled O red cell suspension in tube labelled Oc
7. Mix the contents of the tubes gently and incubate the test for minimum 15 minutes at
room temperature.
8. Centrifuge all tubes at 1000 rpm for 1 minute.
9. Gently resuspend the red cell button & examine for agglutination.
RESULTS:
• Depending on presence (+) or absence (-) of agglutination, the position of the tubes is
changed or remains unaltered as shown below:
• Confirm the cell grouping results with those obtained in serum grouping and vice
versa.
Grading:
• The following system can be used as a guideline:
• Table.1. Grading system
C or ++++ Complete agglutination; one clump of agglutinated
cells. Score: 12

V or +++ Visual agglutination; several large clumps of cells. Score: 10

++ A few large clumps seen with the naked eye. Score: 8

+ Small evenly distributed clumps seen more easily

pg. 8 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

under the microscope. Score: 5

± Microscopic clumps of 5-10 cells and mainly non-

agglutinated cells. Score: 3

W Weak; 3-5 cells per clump. Score: 2

0, neg or –ve Negative; no agglutination observed. It is pre-

preferable not to use the minus sign '-' to record a
negative result but 0 or neg.

L Haemolysis; means a positive reaction. Total

haemolysis should be scored as 12 and partial
haemolysis as 10.

MF Mixed field reaction; a mixture of agglutination and non-

agglutinated cells.

INTERPRETATION:
1. Agglutination in any tube of RBC tests and agglutination or haemolysis in serum test
constitutes a positive test result. The expected agglutination reaction for positive tests
are 3+ to 4+.
2. A smooth suspension of RBCs after resuspension of RBC button is a negative test
result. All negative results must be verified under microscope. Cells should be
separate without any clumping.
3. The interpretation of ABO group is as follows:

Cell Typing Serum Typing Interception of

ABO group
Anti-A Anti-B Anti-AB AC BC OS
C - C - C/L - A
- C C C/L - - B
C C C - - - AB
- - - + + - O
C=Clumps, L=Lysis

4. Resolve any discrepancies between cell and serum typing tests before the
patient’s or donor’s ABO group is interpreted.

pg. 9 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 5
Rh (D) Typing
1. SCOPE & APPLICATION:
This Standard Operating Procedure (SOP) provides the method to be followed to determine
the Rh D type of an individual and ensure the reliability of the result. This procedure
describes the method for detection of D antigen on the red Cells. It provides guidance for
the use of anti D blood grouping reagent.
2. MATERIAL REQUIRED:
Equipment:
§ Refrigerator to store samples and reagents at 2 – 60C
§ Table top centrifuge.
§ Microscope
§ Incubator/dri bath.
Specimen:
§ Clotted or anti-coagulated blood samples of donors.
§ Clotted blood sample of patients.
§ Test red cells suspended in native serum/plasma or saline.
Reagents:
§ Anti D bioclone.
§ Anti D monoclonal (IgM/IgG blend)
§ 0.9% saline.
§ Distilled water.
Glassware:
§ Serum tubes.
§ Micro tubes.
§ Pasteur pipettes.
§ Glass slides.

pg. 10 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

3. Procedure:
Principle: After ABO next clinically significant blood group system is Rhesus. Though no
naturally occurring antibodies are there typing is done, because antigens are very
immunogenic and Transfusion into Rh negative person can lead to promotion of clinically
significant antibodies which can cause problems of hemolytic disease of newborn and
homolytic transfusion reactions, of all Rh antigens. D is most immunogenic, Hence
routinely only Rh D Typing is done. ABO & Rh (D) Typing is done simultaneously.
1. Label 2 test tubes with Pt./Donor name/D1, Pt./Donor name/D2.
2. Add 1 drop of respective anti D in the test tubes.
3. Add 2-5% washed test cell suspension.
4. Mix well, centrifuge at 1000 rpm x 1 min.
4. Resuspend cell button & look for agglutination
5. Confirm all negative reactions under microscope.
6. Control: +ve control: Known Rh-D +sample & Anti D
7. Negative control; Known Rh -D Neg. sample & Anti D
8. RESULTS:
1. Centrifuge all tubes at 1000 rpm for 1 minute (or as specified by manufacturer).
2. Gently resuspend the red cell button & examine for agglutination.
3. Grade and record test results.
9. Interpretation:
1. Agglutination of the red blood cells in the presence of reagent is a positive test result
and indicates the presence of the D antigen.
2. A smooth suspension of RBCs after resuspension of RBC button is a negative test
result. All negative results must be verified under microscope. Cells should appear
separate without ant agglutination.
3. The interpretation of Rh D type is as follows:

Cell Typing Anti – D Cell Typing Anti – D Interpretation of Rh D

Bioclone Monoclonal type
V V Positive

pg. 11 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

- - Negative*
V - ?*
- V ?*
V= Visible Clumps

4. Proceed with weak D (Du) TYPING using indirect anti-globulin technique in case
of blood donor sample.
N.B.:
1. Invalid test results may be obtained with this reagent if the blood tested is from a
person with autoantibodies or abnormal serum proteins. Concurrent testing for the
ABO blood group serves as a routine simultaneous control. The simultaneous use of
an Rh – hr control is required only when the cells under test are found to be reactive
with anti-A, anti-B and anti-D. If the use of an additional control is necessary,
isotonic saline or 6% to 8% albumin in isotonic saline or a patient auto control may
be used. If the control gives a positive result, a valid interpretation cannot be made.
2. Cord red blood cells heavily sensitised with anti-D may demonstrate a false negative
test result.

pg. 12 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 6
Weak D (Du) testing
1. SCOPE & APPLICATION:
Some red cells possess the D antigen but it is expressed so weakly that the cells are not
agglutinated directly by anti-D sera. An indirect antiglobulin test is necessary to identify
patients with the Weak D (formerly known as Du) phenotype. Weak D testing is done on
all prenatal patients and candidates for Rh immune globulin. Weak D testing is also done
on Rh negative donors to ensure they are truly D negative. It may or may not be done
routinely on Rh negative candidates for transfusion, depending on the policy of the
transfusing institution. If routine weak D testing is done, weak D positive patients should
receive Rh positive blood.
2. MATERIAL REQUIRED:
• 37oC incubator
• Wash bottle with physiologic saline
• Coombs serum - either polyspecific or anti-IgG
• Coombs control cells
• All reagents, equipment, and supplies used in the Rh TESTING procedure.

3. PROCEDURE:
1. Prepare a washed, 3% suspension of patient cells.
2. Take one 1 drop of anti D (Ig G) in a cleaned labelled test tube.
3. Add 1 drop of 2-5% labelled test RBCs suspension.
4. Mix & incubate at 370 C x 45 min.
5. If still negative, add 1 drop of AHG reagent and centrifuge at 1000 rpm x I min.
6. Record results in the appropriate column on the worksheet
7. Record results. Confirm all negative reactions microscopically.
8. Confirm all negative results by adding one drop Coombs control cells to all tubes
showing no agglutination and centrifuge 15-30 seconds at high speed in the serofuge.
9. Gently resuspend and examine for agglutination. Agglutination should be present in
this step or the test is invalid.
4. Interpretation:
No agglutination- DU negative
Agglutination - DU positive
A true weak D should give at least a 2+ positive result. Weaker results may be due to
mixed field agglutination in an Rh negative individual who received Rh positive blood, or
vice-versa. Obtain a recent transfusion history on patients who give inconclusive weak D
results.

pg. 13 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 7&8
Antiglobulin Test
PRINCIPLE
The DAT detects red cells coated in vivo with immunoglobulin or complement.
Washed red cells from a patient or donor are tested directly with antihuman globulin reagents.
The test is used to detect hemolytic disease of the newborn, autoimmune hemolytic anemia,
sensitization of red cells due to drugs, investigation of transfusion reactions, and hemolysis due
to ABO-incompatible platelet transfusions or ABO- mismatched organ transplants.
The DAT may be set up with only the polyspecific tube first, and then repeated with the
monospecific reagents of the polyspecific test is positive. This is generally done when you have
no indication what the results of the test will be. However, if you are expecting the DAT to be
positive, it is permissible to set up tubes for the polyspecific and monospecific reagents at the
same time and test them all together
SAMPLE

A 4 or 7 ml EDTA (lavender top) tube is the sample of choice. A sample from a red top tube may
be tested, but is less desirable, because complement may become attached to red cells in vitro
when they are exposed to their own serum, especially at lower temperatures. If you get a positive
result due to complement on a sample from a red top tube, obtain a lavender top tube and repeat
the test.

REAGENTS, EQUIPMENT, AND SUPPLIES

• 12 x 75 mm tubes
• Polyspecific AHG (Coombs)
• Dispo pipettes
• Anti-IgG
• Indelible marking pen
• Anti-C3
• Isotonic saline
• Coombs Control Cells
• Serofuge
• Lighted agglutination viewer
• Microscope

PROCEDURE – POLYSPECIFIC TEST

1. Add 1-2 drops of patient cells to a tube labelled with the patient’s initials. These are cells
directly from the EDTA tube - they do not have to be diluted at this point.
2. Wash this tube three times with isotonic saline.

pg. 14 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

3. After the third wash, prepare a 3% suspension from the washed cells.
4. Label 2 tubes – POLY IS and POLY 5” (plus the patient’s initials)
5. Add one drop of the washed 3% suspension to each tube.
6. Wash these tubes one more time. When decanting, position the tubes so that the cell
button is on top. This will prevent too many cells from being lost in the washing process.
7. Drain well, and blot dry with a biowipe.
8. Immediately add one drop Polyspecific Antihuman Globulin (also known as AHG or
Coombs serum) to both tubes, and shake to mix.
9. Allow the 5” tube to incubate at room temperature 5 minutes.
10. Centrifuge the POLY IS tube for the time calibrated for the Coombs spin on the serofuge.
11. Immediately resuspend gently and examine for agglutination using the lighted
agglutination viewer.
12. Grade and record results in the POLYSPECIFIC I.S. column of the worksheet.
13. If positive, continue with MONOSPECIFIC TESTING below. It is not necessary to read
the 5” tube if the IS tube is positive, and it is not necessary to examine it microscopically.
14. If the POLY IS tube is negative, examine for agglutination under the microscope. Record
results under the I.S. – MICR. column of the worksheet. If positive, continue with
MONOSPECIFIC TESTING below.
15. If still negative microscopically, add one drop of IgG-coated Coombs Control Cells
(Check Cells) to the tube and centrifuge.
16. Examine for agglutination. Agglutination should be present in this step, or the test is
invalid. See NOTES and PRECAUTIONS, below.
17. Record the check cell results on the worksheet in the I.S. – CCC column. A checkmark (
) is sufficient if agglutination of any degree is present – no need to grade.
18. If the POLY-IS tube was negative through the microscopic reading, centrifuge the
POLY-5” tube after its incubation period and repeat steps 11-17 above, recording results
on the worksheet in the POLY-5” column.
19. If there is no agglutination in any of the steps BEFORE addition of the check cells
(CCC), the test is interpreted as NEGATIVE and no further work needs to be done.
20. If there is agglutination in any of the above steps BEFORE addition of the check cells
(CCC), continue with MONOSPECIFIC TESTING below.

PROCEDURE – MONOSPECIFIC TESTING

If the patient test shows agglutination in step 11, 14, or 18, repeat the test using monospecific
reagents and a saline patient control:

1. Add 1-2 drops of patient cells from the EDTA tube to a new labelled tube. You cannot
continue using the previously-washed cells at this point. See NOTES AND
PRECAUTIONS below.
2. Wash this tube 3 times.
3. While this tube is washing, label three more tubes with the patient’s initials and: IgG, C3,
Saline
4. After the third wash, prepare a 3% suspension from the washed cells.
5. Add one drop of the 3% washed cells to each of the tubes labelled in step C above.
6. Wash these three tubes one more time, decant well and blot the last drop of saline with a
biowipe. Remember to position the cell button UP when decanting.

pg. 15 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

7. Add one drop Anti-IgG to the IgG tube, 1 drop Anti-C3 to the C3 tube, and 1 drop saline
to the saline tube.
8. Centrifuge and read as before, recording results in the appropriate columns on the
worksheet.
9. If the IgG tube is negative, examine it microscopically. Record all results in the
appropriate place on the worksheet.
10. After the 5" incubation, read the C3tube and record the results on the appropriate place
on the worksheet.
11. Confirm any negative IgG reactions with the IgG-coated Coombs Control cells, and
confirm any negative C3 reactions with C3-coated Coombs Control cells. No need to
confirm negative saline reactions.

PROCEDURE – POLYSPECIFIC AND MONOSPECIFIC TESTING DONE AT THE SAME

TIME

NOTE THAT THIS PROCEDURE IS DONE WHEN THE RESULTS OF THE DAT ARE
EXPECTED TO BE POSITIVE.

1. Add 1-2 drops of patient cells to a tube labelled with the patient’s initials. These are cells
directly from the EDTA tube - they do not have to be diluted at this point.
2. Wash this tube three times with isotonic saline.
3. After the third wash, prepare a 3% suspension from the washed cells.
4. Label 4 tubes – POLY, IgG, C3, and SALINE (along with the patient’s initials)
5. Add one drop of the washed 3% suspension to each tube.
6. Wash these tubes one more time. When decanting, position the tubes so that the cell
button is on top.
7. Drain well, and blot dry with a Kimwipe.
8. Immediately add one drop Polyspecific Antihuman Globulin to the Poly tube, one drop
anti-IgG to the IgG tube, one drop anti-C3 to the C3 tube, and one drop saline to the
saline tube. Shake all tubes gently to mix.
9. Centrifuge all tubes for the time calibrated for the Coombs spin on the serofuge.
10. Immediately resuspend gently and examine for agglutination using the lighted
agglutination viewer.
11. Read, grade and record results in the appropriate columns of the worksheet.
12. If the IgG tube is negative, examine it immediately microscopically and record the result
on the worksheet.
13. If the Polyspecific or C3 tube is negative, incubate 5 minutes at room temperature,
centrifuge, and read again. Record results.
14. If the Polyspecific or C3 tube is still negative after the 5 minute incubation, examine
microscopically. Record results on the worksheet.
15. Confirm all negative results with the appropriate Coombs Control Cells. Remember to
use complement-coated cells for the C3 tube. Record the results of the check cells on the
worksheet.

INTERPRETATION

1. No agglutination indicates a negative DAT - nothing is coating the patient's cells in vivo.

pg. 16 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

2. Agglutination with Polyspecific AHG indicates a positive DAT, due to either IgG
antibodies or complement coating the patient's cells. To determine which it is, the
monspecific testing must be done:

§ Agglutination with Anti-IgG indicates antibody (immunoglobulin) is coating the cells.

§ Agglutination with Anti-C3 indicates complement is coating the cells.
§ Both IgG and complement may be present.

3. If the polyspecific tube is positive but all the monospecific tubes are negative, repeat the
entire test with the polyspecific and monospecific reagents.
4. Agglutination with saline indicates either spontaneous agglutination due to cells heavily
coated with antibody, (frequently IgM), spontaneous agglutination due to Wharton's Jelly
coating cord cells, or polyagglutinable cells due to T activation. The test is inconclusive
and the results cannot be reported.
5. If the patient has a positive DAT, obtain the medications list, diagnosis and transfusion
history. In some circumstances it may be necessary to perform an eluate to identify the
antibody coating the patient's cells.

NOTES AND PRECAUTIONS

1. The washing step must proceed uninterrupted. A delay in washing may cause antibodies
to be eluted off the cell and washed away.
2. AHG must be added to the cells immediately following washing. Antibodies may elute
from the cells if they are allowed to sit in saline without the addition of AHG.
3. The Coombs Control Cells must be positive in the Polyspecific and IgG tubes at the end
of the procedure. Reasons for negative check cells include:

• Inadequate washing of the cells. Residual serum neutralizes the AHG so that it cannot
react with the antibody-coated Coombs Control Cells at the end of the procedure. Be sure
the cells are thoroughly resuspended between each wash.
• A delay in adding AHG. Antibodies that were attached to the red cells may elute off into
the saline, and neutralize the AHG when it is added. Add the AHG immediately after the
final wash.
• A small fibrin clot has formed in the tube, neutralizing the AHG. Be sure the sample has
thoroughly clotted, if using a red-top tube.
• AHG was omitted, is inactive, or was diluted out by too much residual saline in the tube.
Drain the last wash well and blot before adding AHG.

4. If the test is positive for complement and the sample was drawn into a red top tube, repeat
the entire test on a lavender top tube. If the C3 is now negative, this indicates the
previous positive result was due to in vitro binding of complement, and is a false positive.

pg. 17 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 9&10
Cross matching
1. SCOPE & APPLICABILITY:
This procedure is applied for compatibility testing of all patients requiring transfusion.
2. MATERIAL REQUIRED:
Equipment:
§ Refrigerator to store samples & reagents at 2 – 60C.
§ Deep Freezer to store enzyme papine – cystein in frozen state.
§ Tabletop centrifuge.
§ Automated cell washer (for patient pre-transfusion and prenatal testing).
§ Microscope.
§ Dry bath.
Specimen:
Clotted blood sample of donors/patients.

Reagents:
§ Group O polled cells/Antibody-screening reagent red blood cells (two or three
cells).
§ Papain –cystein.
§ 22% Bovine albumin.
§ Antihuman globulin reagent(anti-IgG+anti-C3d)
§ IgG – sensitised control cells.
§ 0.9% saline.
§ Distilled water
Glassware:
§ Glass test tubes.
§ Coombs’ tubes (for patient pre-transfusion & prenatal testing).
§ Micro tubes.
§ Pasteur pipettes.
§ Glass slides.

pg. 18 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

3. PROCEDURE:
Principle: The major cross-match is used to detect unexpected blood group antibodies in
patient’s serum against antigens on donor cells. Positive reaction in any test indicates
incompatibility.
Cross-match:
1. Label 3 tubes with patient/donor test identification.
2. Add 2 drops of patient’s serum to each tube.
3. Prepare 5% cell suspension in 0.9% saline from each donor unit segment.
4. Add 1 drop 5% donor red cell suspension to the tubes containing patient’s serum.
5. Add 1 drop pap-cysteine to tubes labelled enzyme.
6. Add 1 drop of 22% albumin to the tubes labelled albumin.
7. Mix the contents of tubes gently and incubate for minimum 15 minutes. (Saline
tubes at room temperature and Enzyme / Album at 370C)
8. Centrifuge the tubes at 1000 rpm for 1 minute.
9. Examine for hemolysis.
10. Gently resuspend red cell button and examine for agglutination.
11. Examine all visually negative reactions under microscope.
12. Grade and record test results immediately.
13. Let a second technician check the results.
Interpretation:
1. Hemolysis or agglutination in any test indicates incompatibility.
2. Absence of hemolysis / agglutination in all tests indicates compatibility.
Limitations:

Th saline / enzyme cross match will not:

1. Detect error in Rh typing

2. Prevent isoimmunisation of the recipient
3. Ensure normal red blood cell survival
4. Detect some weakly reactive antibodies

pg. 19 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 11
Antibody Screening
10. SCOPE & APPLICATION:
Detection of Unexpected Blood group Antibodies. This procedure applies to all testing
that requires antibody screening, including donor units, patient's pre-transfusion blood
grouping and prenatal specimens.

11. MATERIAL REQUIRED:

Equipment:

• Refrigerator to store samples & reagents at 2-6 degree Centigrade.

• Deep Freezer to store enzyme papaine cystein in frozen state.
• Tabletop centrifuge.
• Automated cell washer (for patient pre-transfusion and prenatal testing).
• Microscope.
• Incubator
Specimen:

Clotted blood sample of donors /patients.

Reagents:

• Group O polled cells/Antibody-screening reagent red blood cells (two or three cells).
• Papain cystein.
• 22% Bovine albumin.
• Antihuman globulin reagent(anti-IgG+anti-C3d)
• IgG sensitised control cells.
• 0.9% saline
• Distilled water
Glassware:

• Serum tubes.

pg. 20 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

• Coombs' tubes(for patient pre-transfusion & prenatal testing).

• Micro tubes.
• Pasteur pipettes.
• Glass slides.
12. PROCEDURE
Principle: The antibody screen test is used in the detection of unexpected blood group
antibodies. In this test, pooled O cells or the antibody-screening reagent red blood cells are
combined with serum under investigation. The addition of a potentiating medium enzyme /
albumin helps to promote the interaction of red cells and antibodies allowing
antibody/antigen reactions to occur. Positive reactions (haemolysis or agglutination) in any
tests indicate the presence of allo antibody or auto antibody in the serum.

Antibody Screen:

1. Label tubes with donor/patient and test identification.

2. Add two drops of test serum to each tube.
3. Add 1 drop of papain cystein to all tubes labelled 'enzyme' (if enzyme method is
being followed).*
4. To each of the tubes labelled 'saline' or 'enzyme/albumin', add 1 drop of 2% pooled O
red cell suspension (or 2% suspension of the antibody-screening reagent red cells).
5. Add 1 drop of 22% abovine albumin to tubes labelled 'albumin' (if albumin method is
being followed).*
6. Add 1 drop of 5% pooled O red cell suspension (or 5% suspension of antibody-
screening reagent red cells) to tubes labelled 'IAT', followed by 2 drops of 22%
bovine albumin.
7. Mix the contents of the tubes gently and incubate for minimum 15 minutes.
* Either enzyme or albumin method may be followed for detection of incomplete
antibodies.

Results:

1. Centrifuge saline, enzyme and albumin tests at 1000rpm for 1 minute.

2. Examine for haemolysis.

pg. 21 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

3. Gently resuspend the red cell button and examine for agglutination.
4. Examine all visually negative tests microscopically.
5. Grade and record test results immediately.
6. Proceed to perform antiglobulin phase of the indirect antiglobulin test on tubes labelled
'IAT'.
7. Wash the cells 3 times with saline. Decant completely after last wash. (washing can be
done manually or using automated cell washer).
8. Add 2 drops antihuman globulin reagent to the dry cell button.
9. Mix well and centrifuge at 1000 rpm for 1 minute.
10. Read and record results.
11. Add drop IgG sensitised cells to all negative results. This shows a positive
agglutination.
Interpretation:

1. Hemolysis or agglutination in any test may indicate the presence of an unexpected

antibody.
2. The absence of agglutination and hemolysis in all tests is a negative test result.
3. After addition of IgG-sensitized cells to a negative test, the presence of agglutination
indicates that the AHG serum added was capable of reacting and that the negative
antiglobulin test is valid.

TEST INCUBATION IDEAL INCUBATION

TEMPERATURE TIME
Saline Room Temperature 1 hour
Enzyme 370 C 45 minutes
Albumin 370 C 45 minutes
IAT 370 C 1 hour

• Follow manufacturer’s directions when using commercial reagents.

• Either enzyme or albumin method may be followed for detection of incomplete
Antibodies

pg. 22 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Limitations:

If tests with all reagent red cells are reactive, the possibility of spontaneous agglutination
should be considered. A control of cells washed three to four times added to two drops of
saline must be non-reactive.

pg. 23 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 12
Antibody Identification
PRINCIPLE

Determining the specificity of an unexpected alloantibody is important in pre-transfusion and

prenatal testing. If the antibody specificity is known, it is possible to test donor blood for the
absence of the corresponding antigen. Antibodies should also be identified in donor blood so that
this blood is not transfused to antigen-positive recipients.

In prenatal testing, knowledge of the specificity of the antibody helps predict the likelihood of
hemolytic disease of the newborn.

SAMPLE

One 10 ml clotted tube (red top) is generally sufficient; however, if you need to request that extra
tubes be drawn so you can identify the antibody, request two 10-ml red tops and one 7 ml
lavender top tube. This will provide sufficient sample to test, and a sample for the DAT and
eluate if necessary. The sample should be as fresh as possible when tested; this assures adequate
complement activity.

REAGENTS, EQUIPMENT, AND SUPPLIES

• 12 x 75 mm test tubes
• Reagent panel cells
• Indelible marking pen
• Coombs control cells
• Controlled drop dispo pipettes
• Physiologic saline
• PEG or 30% bovine albumin
• 37oC incubator
• Polyspecific Anti-human Globulin
• Agglutination viewer
• (Coombs Serum)
• Serofuge

PROCEDURE

The patient's sample should first be tested with Screening Cells I and II. If one or both of these
show agglutination, proceed with the antibody identification.

1. Select a cell panel and the corresponding antigen matrix. Be sure the lot number on the
cell panel matches the lot number on the antigen matrix.
2. Fill in all known patient information on the cell panel worksheet.

pg. 24 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

3. Number as many tubes as there are cells in the panel. Include the patient's initials on each
tube.
4. Using a controlled drop dispo pipette held at a consistent angle, add 2 drops of patient
serum to each tube.
5. Gently invert the panel cells several times to resuspend.
6. Add one drop of the appropriate panel cells to each corresponding numbered tube.
7. Set up an autologous control, if none was run with the antibody screen:
8. To a tube labeled with the patient's initials and "auto", add 2 drops patient serum and one
drop of a washed 3% suspension of patient cells.
9. If the Screening Cells were negative at the Immediate Spin phase, proceed directly to step
11. If they were positive at Immediate Spin, centrifuge the tubes the calibrated time for
saline and continue with step 10.
10. Gently resuspend and examine for agglutination using the lighted agglutination viewer.
11. Record all reactions under a column headed IS (for Immediate Spin) on the panel
worksheet.
12. Add 2 drops PEG to all tubes, shake to mix, and incubate 10 - 30 minutes at 37oC. Note
that you must incubate at least 15 minutes if using a dry heat block.
13. Wash all tubes three or four times with physiologic saline and add one drop Polyspecific
Anti-Human Globulin to each tube.
14. Shake to mix and centrifuge the time appropriate to the Coombs spin calibration in the
serofuge.
15. Gently resuspend and examine for agglutination using the lighted agglutination viewer.
16. Record all reactions under a column headed AHG on the panel worksheet.
17. The auto control may be positive or negative. If it is positive, do a DAT and get a recent
transfusion history and medications list.
18. Confirm all negative reactions with Coombs Control Cells. No agglutination after
centrifugation following the addition of Coombs Control Cells invalidates the cell panel
results.

INTERPRETATION

To determine antibody specificity, use the following protocol:

1. Look at each negative cell and cross off all antigens that are present (positive) on that
cell. Use X for homozygous cells, and / for heterozygous cells.
2. Eliminate antigens along the top of the cell panel by crossing off all that have been
crossed off at least three times in the antigen matrix, ideally with at least one of them
homozygous for the antigen. Do not eliminate antigens based on only one or two
heterozygous cells crossed off, especially if you are getting different strength reactions on
different cells.
3. Exceptions to the above policy include:

• you may rule out Kell based on 3 heterozygous cells - no need for a homozygous cross-
off
• you may rule out low frequency antigens (Cw, V, VS, Kpa, Jsa, Lua) based on only one
cross-off, whether homozygous or heterozygous

pg. 25 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

• you may also rule out low-frequency antigens if there are no cells positive for them on
the panel
• if anti-D is present, you may rule out anti-C or anti-E based on three heterozygous cells
(r'r for C and r"r for E)

4. From the antigens not crossed off, look for a pattern of agglutination matching the pattern
you got in the test. This should identify the antibody specificity.
5. At this point there still may be one or more low-frequency antigens not crossed off. If the
cell that has the low-frequency antigen also has the antigen that corresponds to the
antibody you believe you have identified, you may now cross off this low frequency
antigen, because the positive reaction on this cell is most likely due to the antibody you
have identified.
6. Often you are unable to rule out the possibility of a second or third antibody because the
corresponding antigens are all present on the same cells. For example, you have
identified Anti-Jka but can't eliminate Anti-Kell because all Kell positive cells are also
Jka positive. Check other cell panels and find 3 other cells that are Kell positive and Jka
negative. A negative result when testing these cells with the patient's serum eliminates
that antibody; a positive result confirms the second antibody.
7. When the antibody(ies) have been identified, be sure there are at least 3 cells that are
possess that antigen and give a positive reaction, and at least 3 cells that are lack that
antigen and give a negative reaction. Always check the results of the screening cells. You
may have to test more cells from other panels.
8. As a final confirmation of the antibody specificity, if the autocontrol is negative or only
weakly positive, type the patient's cells for the antigen. The result should be negative,
unless the patient has been recently transfused. The result will then be a mixed field.
9. If you get different reactions at different phases of testing; if you get different strengths
of reactions on different cells, or if your results don't form a pattern corresponding to one
of the antigens on the matrix, consider multiple antibodies. See (6) above.
10. If you get a pattern of reactivity not matching any of the antigens on the matrix, the
antibody may be showing dosage and reacting only with homozygous cells. This is most
often seen in the MNS and Kidd systems, but can be seen in other systems. Eliminate
only the non-reactive homozygous cells; the pattern of reaction may then match an
antigen present only on the remaining homozygous cells. See (2) above.

NOTES

1. If the reactions with LISS are weak or inconclusive, it may be necessary to potentiate the
reactions:

a. Use 3-4 drops patient serum, omit the LISS, and incubate 30-60 minutes. Wash and add AHG
as described above.

b. Omit the LISS and add PEG. Incubate at least 15 minutes. Wash and add AHG as described
above. PEG strengthens almost all antibodies without destroying any antigens. Unfortunately it
also enhances some clinically insignificant alloantibodies and low-titered autoantibodies as well.

pg. 26 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

c. Set up a panel using enzyme-treated cells. Enzyme enhances reactions with I, P, Rh and Kidd
antibodies; it destroys reactions with MNS and Duffy antibodies. Follow manufacturer's
directions.

2. Special considerations apply to antibodies reacting at room temperature only:

a. Reactions at room temperature only may not be clinically significant. Do not attempt to
identify these antibodies as part of routine pretransfusion testing unless they are interfering with
the backtyping, are from patients who are candidates for bypass surgery, or an antibody screen
has been specifically requested by the ordering physician.

b. If you do want to enhance cold-reactive antibodies, incubate 15 minutes at room temperature

or 4C, centrifuge and read as usual.

c. Strong-reacting antibodies at room temperature may persist in Coombs even though they may
not be clinically significant. They may also irreversibly bind complement at the room
temperature phase and cause agglutination when polyspecific AHG is used in Coombs.

d. To prevent interference from cold antibodies persisting in Coombs, do a pre-warmed cell

panel:

1. Warm the serum and cells separately, add cells to serum, omit the IS step and PEG,

2. Incubate at 37oC 20-30 minutes,

3. Wash with saline warmed to 37oC

4. Add anti-IgG rather than Polyspecific in the Coombs phase.

3. All clinically significant antibodies must go into the patient's permanent record.

QUICK REFERENCE FOR ANTIBODY IDENTIFICATION

1. Cross off antigens based on negative reactions. Use X for homozygous and / for
heterozygous.
2. Look at the columns for each individual antigen. If there are at least 3 cells in the column
that are crossed off, with at least one of them homozygous, rule out antibody formation to
that antigen. If one of your Screening Cells is negative, you can use it to help rule out.
3. If not enough cells are crossed off in the column to rule out that antibody, place a 1 or 2
above that column. Use H to indicate homozygous cross-off needed.
4. Low frequency antigens need only one cross-off. They can also be ruled out if all the
cells on the panel are negative for that antigen, or if the cell that has the low-frequency
antigen also has the antigen corresponding to the antibody you have identified.
5. Kell does not ever need a homozygous cross-off; C or E do not require a homozygous
cross-off if you have identified an anti-D.

pg. 27 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

6. To determine antibody specificity, look at the column's where nothing is crossed off
(excluding low-frequency antigens). Compare your positive cells with the cells that are
positive for that antigen in those columns.
7. Test additional cells as needed to rule out other antigens not completely ruled out. These
cells should be positive for the antigen you want to rule out, but negative for the one(s)
you have identified.
8. Confirm all antibodies by making sure there are at least three cells positive for that
antigen that give a positive reaction, and at least three cells negative for that antigen that
give a negative reaction. Test more cells from additional panels, if necessary.
9. If there are multiple antibodies, make sure each antibody can be confirmed by 3 cells
positive for each individual antigen but negative for the others.
10. Look at the autocontrol - if it is positive, perform a DAT and obtain the recent transfusion
and medications history. Try to determine if it is an autoantibody or an alloantibody
reacting with antigens on transfused cells.
11. Antigen type the patient - should be negative for the antigen(s) unless recently transfused.
12. If blood is needed, antigen type compatible donor units

pg. 28 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 13
Antibody Titration
PRINCIPLE AND APPLICATIONS:

Antibody titration is a determination of the concentration of a specific antibody in the patient's

serum or to determine the strength of antigen expression on different red cell samples.

If the concentration of the specific antibody is being determined, the cells must contain the
known antigen and the procedure should be performed under the optimal conditions for that
antibody.

The usual applications of titration studies are:

1. estimating the antibody activity of an alloimmunized pregnant female.

2. attempting to determine if there is any specificity to an autoantibody
3. characterizing antibodies that may be high titer, low avidity antibodies

SAMPLE :

Either plasma and serum can be titrated. In the case of a pregnant female, frozen serum or
plasma from previous months should be run along with the fresh specimen to determine if there
is a rise in titer.

REAGENTS, EQUIPMENT, AND SUPPLIES:

• Reagent cells known to have the antigen that will be reacting with the titered antibody. If
titering for anti-A or anti-B levels, A1 or B Cells would be used. If titering for anti-D
levels, O cells that are homozygous for D should be used.
• 12 x 75 mm tubes
• test tube rack
• marking pen
• dispo pipettes
• physiologic saline
• serofuge
• lighted agglutination viewer

PROCEDURE

1. Label 10 tubes according to the serial dilution: 1, 2, 4, 8, 16, 32, 64, 128, 256, 512 and
the patient identification. The first tube will be undiluted serum. Tube 2 will be a 1/2
dilution, 4 will be a 1/4 dilution.
2. Add 0.3 ml of saline to tubes 2 through 512. No saline in tube 1
3. Add 0.3 ml of serum to both tubes 1 and 2.

pg. 29 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

4. Use a clean pipette to mix the 1/2 dilution several times and then transfer 0.3 ml to tube
4.
5. Use a clean pipette to mix the 1/4 dilution several times and then transfer 0.3 ml to tube
8.
6. Continue the process for all dilutions (512). Remove 0.3 ml from tube 10 (512) and
reserve in a clean tube if the titration needs to be continued.
7. Label a new set of 10 tubes with the appropriate dilutions.
8. Using a separate pipettes for each dilution, transfer 2 drops of each tube to the
appropriate tubes.
9. Add one drops of specific red blood cells.
10. Mix well and test by the appropriate technique for the specific antibody.
11. Examine test results macroscopically, grade and record the reactions

NOTES AND PRECAUTIONS

• If titrating anti-A, anti-B, or anti-A,B, the serologic technique is performed by the same
method as ABO Typing
• If titrating Rh, Kell, Duffy, or Kidd antibodies, the serologic technique includes a 37oC
followed by antiglobulin testing.
• If testing a pregnant female, each month serum should be compared to the previous
month.
• Prozone phenomenon may occur so the first tubes may have a weaker reaction than the
more diluted serum. AABB recommends reading the most dilute tubes first and then
shake out the other tubes.
• Careful pipetting is essential.
• Cells with known antigens may have different reactivity and therefore the serum from
each month must use the same cells
• Measurement is more accurate at larger dilution, therefore the larger dilution should be
made before smaller volumes are used to test with the red cells antigen.

INTERPRETATION

• Observe the highest dilution that produces 1+ macroscopic agglutination

• The titer is reported as the reciprocal of the dilution level: 32 not 1/32
• A rise in titer would need to be at least 2 dilution increase between the current specimen
and the previous month.
• For identification of high-titer, low-avidity antibodies would generally have a titer of 64
or greater.

pg. 30 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 14
Transfusion Reaction Workup
PRINCIPLE

A transfusion service must have a system to evaluate and report suspected adverse reactions to
transfusion. Detection of the reaction is the responsibility of the transfusionist, who should
immediately notify a responsible physician and the Blood Bank. Any adverse reaction of the
patient associated with transfusion is a suspected transfusion reaction.

No additional units of blood should be issued to a patient with a suspected transfusion reaction
until the laboratory testing portion of the evaluation has been completed.

A basic workup, consisting of clerical check, visual inspection of the plasma for hemolysis, and
DAT on the post-transfusion sample, should be done on all suspected transfusion reactions. If
any of these tests are abnormal, or if the patient's symptoms are severe, an extended workup
should be performed.

PROCEDURE - BASIC WORKUP

1. When a transfusion reaction is suspected, the nursing unit will send to the Blood Bank:

• A completed transfusion reaction form indicating unit number, time transfusion started
and stopped, and clinical findings.
• Post-Transfusion Sample: A clotted blood sample and an EDTA tube, both properly
labeled, drawn from the patient at the time of the reaction.
• The discontinued unit of blood with the administration set attached.
• The blood issue slip.
• A post-transfusion urine sample may be included.

2. Complete the clerical check:

• Verify that the donor number on the blood bag matches the number on the form.
• Verify that nursing personnel have double-checked patient identity and blood product
information. The appropriate spaces should have been filled in on the blood issue slip.
• Verify that the blood was transfused to the patient for whom it was issued. The name of
the patient on the sample and on the transfusion reaction form should match the name on
the blood issue slip.
• Return the blood issue slip to the nursing unit after verification so that the information
may be recorded on the patient's chart.
• If there is a discrepancy in the identification of the patient or the donor blood,
immediately notify the Blood Bank medical director and the patient's physician. It may
be necessary to check other Blood Bank records to determine if other patients are at risk.

pg. 31 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

3. Retrieve the pre-transfusion sample and label this PRE-TX.

4. Label the post-transfusion sample POST-TX, centrifuge it, and remove the serum to a
labeled tube.
5. Compare the color of the serum from the pre and post samples. If the serum from the post
sample is hemolyzed, call the nursing unit to see if mechanical hemolysis might have
occurred when the sample was drawn. If so, have the sample re-drawn.

NOTE: If the sample was drawn 4 to 10 hours following the reaction, there may be a
yellow or brown color from bilirubin and other hemoglobin breakdown products,
indicating hemolysis.
5a. Perform the ABO/Rh on the post sample.
6. Perform a DAT on the post sample EDTA tube.
7. If the post sample DAT is negative, and there is no visual evidence of hemolysis, no
further serological testing is required and the workup is complete. Report this to the
nursing unit.
8. If the post sample has a positive DAT, or if there is evidence of hemolysis in the post-
transfusion sample with none in the pre-transfusion sample, perform an extended workup.

PROCEDURE - EXTENDED WORKUP

1. Immediately notify the Blood Bank medical director and the patient's physician of any
abnormal findings in the basic workup.
2. Perform a DAT on the pre-transfusion sample (IgG only).
3. Repeat the ABO/Rh on the pre-transfusion sample, and on the donor unit.
4. Repeat the antibody screen on the pre and post samples, including an autocontrol. It may
be desirable to use enhancement techniques such as or PEG.
5. Repeat the crossmatch, through Coombs, on the pre and post samples. For greater
efficiency, steps 4 and 5 may be set up and run together.
6. Perform an eluate on the post transfusion sample, if the DAT is positive.
7. If an antibody is found in the eluate, type the donor unit and pre-transfusion sample for
the corresponding antigen. The post-transfusion sample may also be typed to demonstrate
mixed-field results.
8. Test the post transfusion urine for the presence of free hemoglobin, using a dipstick. If
the urine is grossly bloody, a visual examination of the centrifuged urine for free
hemoglobin will be a more accurate determination.
9. If there is visual evidence of hemolysis, examine the donor unit and IV tubing for
hemolysis.
10. Obtain pre and post hematocrit results, to determine if the transfused unit caused a rise in
hematocrit.
11. Save the donor blood in the refrigerator pending further testing. This may be discarded
after the medical director evaluates the transfusion reaction and determines no further
testing is necessary.
12. Further testing may be required based on evaluation of the patient's symptoms and
clinical findings, review of the accuracy of the records, and results of the laboratory tests.

FURTHER TESTING - ONE OR MORE OF THE FOLLOWING TESTS MAY BE

DONE:

pg. 32 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

1. Complete RBC phenotype on the patient and donor unit.

2. Serum bilirubin, haptoglobin, and LDH levels.
3. Pre-and Post-transfusion hematocrit levels.
4. Urine hemosiderin.
5. Reticulocyte count and peripheral smear.
6. Gram stain and culture on the donor unit and on the patient.
7. Test for HLA antibodies in the patient or donor.
8. Test for anti-IgA in the recipient.
9. In vivo cell survival studies.

pg. 33 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

Experiment 15
Gel Method

The original technique is based on the principle of gel filtration for separation of red blood cells
from human blood. The column consists of special microtubes containing a dextran gel matrix.
Red blood cells and serum or red blood cells alone are dispensed into the microtubes, incubated
if necessary, and then centrifuged under strictly controlled parameters. The gel within the
microtubes acts as a sieve, unagglutinated red blood cells form a pellet at the bottom of the
microtubes, and agglutinated red blood cells are trapped in the gel. Reactions are easily visible
and may be graded.

The aim of this technology is to standardize red blood cell agglutination reactions, and by
trapping the agglutinates, to permit simple and reliable reading.

Advantages:

1. Stability of reactions
2. Easy to use
3. Results can be photocopied
4. Small sample volumes used
5. Decreased false positive reaction

Disadvantages:

1. Cost
2. Loss of "traditional" skills

pg. 34 Prepqred by Dr.Rasha Abd Elgleel Mohammed

Blood Banking and Transfusion lab manual 33051482

References list
-Technical Manual of the American Association of Blood Banks – 17th Edition, 2011.
--Practical Transfusion Medicine, Fifth Edition,2017

pg. 35 Prepqred by Dr.Rasha Abd Elgleel Mohammed