BMS3136 Transplantation, Transfusion and Specialist Biochemistry

Practicals for Blood grouping

Aim

To allow students to experience practical blood grouping

Learning objectives

After doing this practical class and researching the discussion points, students should be
able to:

 Safely handle blood, and take suitable precautions against infection hazards from the
blood of others.

 Describe the principles of ABO and Rhesus typing of blood.

 Perform ABO and Rhesus typing of blood.

 Explain the terms ‘forward blood grouping’ and ‘reverse blood grouping’

 Describe and manually perform a common automated technique for blood grouping
used in UK hospital laboratories

 Describe how antibody screening on blood samples is performed in the UK

Introduction

ABO blood grouping is the most important serological test performed in blood compatibility
testing – this is because the antibodies of the system (anti-A and anti-B) are preformed
and can cause a rapid transfusion reaction if the wrong group is given. The Rhesus system
is also important, but as the antibodies are not present unless there has been a previous
exposure to the Rhesus antigen in a Rhesus negative individual this is less critical than
ABO grouping.

‘Forward’ ABO blood grouping is now done with monoclonal, separate anti-A and anti-B
reagents used against the patient’s red cells (historically, polyclonal anti-A, anti-B and
anti-A, B together reagents were used). A and B red cells are also used for ‘reverse
grouping’, i.e. the patient’s serum is tested against known A and B group red blood cells
to check that it agglutinates them appropriately. Any discrepancies between the forward and
reverse group need to be investigated using the patient’s original blood sample rather than
any cell suspensions created from it to prevent accumulating errors.

Reminder about safe handling of human blood

For the purposes of this practical, we will supply you with screened donor blood. Although
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the blood has been screened for HIV and hepatitis, you must still take all safety
precautions – there are undoubtedly new viruses as yet undiscovered! Please observe all
of the following safety points.

 Ensure that any cuts or broken skin on your hands is covered with a plaster.

 All the usual lab rules apply – no eating or drinking is particularly important.

 Wear a lab coat, gloves and safety glasses at all times.

 Keep sources of blood sealed as much as you possibly can, i.e. replace the stoppers
or caps on containers of blood as soon as you can, and put lids on test tubes with
blood or solutions of blood in.

 Never put an uncovered tube of blood into a centrifuge – it must have a lid.

 Never mix a tube containing blood on a vortex mixer unless the tube has a lid on.

 If you spill blood on the bench or floor, ask for help to clean it up safely.

 If you break a glass slide that has blood on it, do not touch it – call for help to clear it up
with a dustpan and brush. Sharp fragments and foreign blood are potentially
dangerous.

 Do not run the tap fast and pour blood (or solutions containing blood) into the water
stream – you risk creating aerosols of blood vapour! Pour solutions carefully down
the sink, then run the tap slowly afterwards.

 Ensure that all disposable materials that have touched blood during the practical are
disposed of in an appropriate hazardous waste bin (ask a demonstrator or technician if
you are uncertain of this). Non-disposable materials (e.g. haemocytometer) should be
soaked in Milton sterilising fluid solution.

 If you are unsure of how to proceed safely at any stage of these practicals, ask for
help.

 If you injure yourself in any way with something potentially contaminated with blood, you
must immediately inform one of the staff.

1. ABO and Rhesus grouping

Materials

 Anti-A, Anti-B and Anti-D sera

 Red blood cell suspensions from 4 patients– CAUTION, INFECTION HAZARD
 Tiles
 Eppendorf tubes
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 Method

 Set up the tile by using a marker pen to draw an appropriate number of squares on it
(e.g. have a row for each sample, and columns for each antibody). Draw a diagram
to plot where you have put each reagent on the plate.

 Add one drop of anti-A to squares in one column, one drop of anti-B to squares in
a second column, and one drop of anti-D to squares in the third columns.

 Add 40µl of your red blood cell suspension (from patient Tom) across one row to each of
the anti-A, anti-B and anti-D containing columns.

 Stir the mixture of blood and antibody briefly with a pipette tip (use a separate tip to
avoid cross-contamination).

 Repeat on new rows for each blood sample suspension from other three patients
Meena, Ali and Sara.

 Leave the plate untouched for 20 minutes, then look for agglutination in the wells.

 Read the results in the wells carefully and note them down, using ‘+’ for agglutination
and ‘–’ for no agglutination.

 Continue with part 2 of the protocol (Page 6) – blood grouping using the Diamed ID
cards.

Discussion points:

1) Draw your results onto the diagram below. (2 marks)

Anti-sera
Red blood cell 2) What are the blood groups of your 4
samples
Anti-A Anti-B Anti-D samples? What blood groups could
these patients receive during a red
blood cell transfusion (i.e. which
Tom
+ - + group(s) is/are compatible with each of
them?) (2 marks)

Meena
+ + -

Ali
- - +

Sara
- + +
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 Red blood Blood
Compatible blood groups
cell samples groups

Tom A+ A+ , A- , O+ , O-

Meena AB- A- , B-, AB- , O-

Ali O+ O+ , O-

Sara B+ B+ , O+ (in emergency cases also O- , B-)

2) What are the common causes of false positive and false negative reactions in ABO
blood grouping? (4 marks) (Maximum 150 words)

https://onlinelibrary.wiley.com/doi/full/10.1111/j.1751-2824.2008.00186.x

-cross contamination between specimens

- wrong technique used and samples were confused : e.g blood was added onto the
wells first and then reagents.
-reagents and samples at unsuitable temperatures
-expired reagents
-In direct antiglobulin test (DAT), complement binding is likely to cause false positive
results. (Raman, Armstrong and Smart, 2008)

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4) In the diagram below which represents forward grouping, red cell suspensions are
added to each of the wells first, then anti-sera are added. Draw the expected results in
the diagram. (2 marks)

Red blood Anti-A Anti-B Anti-D

cells serum serum serum

Group AB
+ +
Group A
+ -
Group B
- +
Group O
- -
+
Group
RhD+

-
Group
RhD-

5) In the diagram below which shows reverse grouping, patient’s sera are added to each
of the wells first, then reagent red cells are added. Draw the expected results in the
diagram. (2 marks)

Group Group
Antibodies in Group AB Group A Group B Group O RhD+ red RhD- red
serum red cells red cells red cells red cells cells cells

+ + + -
Anti-A and
anti-B

+ + - -
Anti-A

+ - + -
Anti-B

+ -
Anti-D

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2. Column agglutination technology
Most hospital laboratories in the UK used automated systems to group patients’ blood
samples, and column agglutination techniques are in common use. These are based on
the principles of size exclusion chromatography. They use the same system for ABO and
Rhesus grouping that you have used (i.e. anti-A, anti-B and anti-D [the latter in duplicate]
for forward grouping, and known group A and B red cells for reverse grouping), but
reagents are in small tubes containing a gel, within a larger cassette. The patient’s red
cells are added to the small tubes containing anti-A, anti-B or anti-D, and their plasma to
tubes containing group A and group B cells, and once an agglutination reaction has had
time to occur, the cassette is spun in a centrifuge. Where agglutination has occurred, the
large clumps of cells are unable to pass through the small spaces in the gel, and the clump
remains at the top of the column. Where agglutination has not occurred, the small, free
cells are able to pass through the column and can be seen at the base of the tube in the
cassette. The principle of size exclusion chromatography will be demonstrated, and you
will use two samples of patient red blood cells and serum on Diamed blood grouping
cassettes to establish (and cross-check) the ABO and Rhesus groups of these patients.

Materials

 Diamed ABO/D+ reverse grouping ID cards (two cards per group)

 Known blood samples (red cell suspension and sera) (See ABO and Rh grouping above).

 Unknown blood samples (red cell suspension and sera).

Methods

1. Label two Diamed ID cards with your initials and an appropriate label of your known
samples (the first card) and unknown samples (the second card).

2. Hold the Diamed ID card upright and peel back the foil from the wells.
3. To both ID cards: Pipette 10 μl of known blood group A cells into lane 5. Pipette 10 μl of
known blood group B cells into lane 6.
4. To card 1 only: Pipette 10 μl of Tom’s known red cells into lanes 1- 4, and 10 μl of
the correspondent serum of Tom’s (ser-Tom) into lanes 5 and 6.
5. To card 2 only: Pipette 10 μl of the unknown red cell suspension (Un.) into lanes 1- 4,
and 10 μl of the correspondent unknown serum (ser-Un.) into lanes 5 and 6.
6. Centrifuge the cards for 10 minutes in the Diamed centrifuge.

7. Read and record the reactions below.

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Discussion points
6) Draw your results onto the blank cassettes shown below, and identify the blood groups of
the two samples, respectively. (2 marks)

Known sample: Unknown sample:

+ - + - - + - + + - + -

Blood group: A+ Blood group: B+

7) Red cell transfusion in warm-antibody autoimmune haemolytic anaemia presents

difficulty in cross-matching and it is nearly always impossible to find truly sero-compatible
donor blood. Discuss the strategies of red cell transfusion in patients with severe
autoimmune haemolytic anaemia. (8 marks) (maximum 300 words)

Patients with autoimmune haemolytic anaemia (AIHA) may frequently

develop anaemic hypoxia that cannot be relieved by oxygen
administration until therapeutic measures become effective. Apart from
red blood cell (RBC) transfusion, no drug is yet available which can
immediately stop and/or compensate haemolysis in such patients. (Yürek
et al., 2015) Yürek, S., Mayer, B., Almahallawi, M., Pruss, A. and Salama, A. (2015).
Precautions surrounding blood transfusion in autoimmune haemolytic anaemias are
overestimated. Blood Transfusion, 13(4), pp.616–621.

There must be a careful clinical evaluation and, in addition, the laboratory

must perform some specialized compatibility tests to determine the
presence or absence of RBC alloantibodies that have the potential to
cause a hemolytic transfusion reaction. Patients with AIHA relates to the
detection of red cell alloantibodies in patients with a broadly reactive
autoantibody. These alloantibodies are developed as a result of previous
transfusions or pregnancies, and are capable of causing hemolytic
transfusion reactions. Undetected alloantibodies may be the cause of
increased hemolysis following transfusion, which may be falsely attributed
to an increase in the severity of AIHA. In warm antibody AIHA, the
autoantibody in the patient’s serum will generally react with all RBCs

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tested, thus masking the presence of potentially hemolytic alloantibodies.
adsorption tests which remove autoantibody from the patient’s serum and
allow for detection and identification of alloantibodies in the adsorbed
serum. An alternative approach, which may be about as effective in
avoiding the effects of alloantibodies, but which is not widely implemented
in transfusion services, is to perform extensive RBC phenotyping of the
patient and the donor units. Other simple tests which provide a modicum
of safety include routine testing of the patient’s serum against a red cell
panel and diluting the patient’s serum before doing compatibility testing.
Warm Autoadsorption. The optimal adsorption technique for detecting
alloantibodies in the presence of a broadly reactive autoantibody is the
warm autoadsorption procedure.1,4,15 In this technique, some of the
autoantibody is eluted from the patient’s RBCs, as with “ZZAP” reagent16
(a mixture of 0.1M dithiothreitol plus 0.1% cysteine-activated papain or
0.1% ficin), and then these cells are used to adsorb the autoantibody
from the patient's serum at 37o C. The adsorbed serum can then be
tested for alloantibodies, since alloa (Petz, 2005) Petz, L.D. (2005). Emergency
Transfusion Guidelines for Autoimmune Hemolytic Anemia. Laboratory Medicine, 36(1),
pp.45–48.

8) Describe the consequences of receiving ABO-mismatched red blood transfusions.

Explain the mechanisms by which the consequences may arise and summarise the
clinical features associated with the consequences. (10 marks) (maximum 400 words)

9) Discuss the laboratory investigations for patients with suspected acute haemolytic

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 transfusion reactions. (8.5 marks) (maximum 350 words)

3. Antibody screening

Antibody screening is usually undertaken at the same time as blood grouping, and is
important in antenatal patients (who may have or develop antibodies that will affect the
foetus/baby), and prior to transfusion. It works by taking some of the patient’s serum and
mixing it with at least two different preparations of group O red blood cells (i.e. eliminating
the potential effects of anti-A and anti-B antibodies that the patient has anyway – these
cannot react against group O cells). These group O cells will have been tested and are
known to express a minimum set of antigens. In the UK, the minimum set of antigens is:

C, c, D, E, K, k, Fya, Fyb, Jka, Jkb, S, s, M, N, and Lea

Ideally, red cells should be homozygous for antigens (e.g. one set of Group O cells should
be SS and the other ss) to get the maximum agglutination reaction from any antibodies
present in the patient’s serum, so multiple sets of Group O cells are needed.

Like ABO and Rhesus grouping, most hospital labs now use automated systems for
antibody screening, and column agglutination is a common method which is demonstrated
here. Small volumes of the patient’s serum are added to each tube within a cassette –
each tube contains Group O red blood cells which express known antigens, and an anti-
human immunoglobulin. If the patient’s serum contains antibodies against any of the
antigens on the red blood cells, binding of the antibody to the antigen occurs. The anti-
human globulin present in the tube then causes the antibody-coated red blood cells to
cross-link and agglutinate into clumps. After a suitable incubation time, the cassette is
centrifuged. If agglutination has occurred, the large clump of red cells is unable to move
through the pores of the gel and remains at the top of the gel. In the normal sample
where no agglutination has occurred, the small single cells move easily through the gel
and are seen at the bottom of the tube.

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Discussion points
10) A 30-year-old female at 39 weeks of gestation was admitted to the Obstetric unit for a
Caesarean section (C-section). She had a history of C-section 4 years before this visit.
A sample (ethylenediaminetetraacetic acid [EDTA] anticoagulant) was submitted to the
blood bank for type and screen along with an order for two units of red blood cells. The
patient had no history of prior transfusion.

The blood group typing results were shown as below.

Reverse typing (patient

Forward typing (patient red cells)
plasma)

Anti-A Anti-B Anti-D A1 cells B cells

4+ 0 3+ 0 4+

Antibody screening and identification showed the presence of anti-Le a antibodies.

What is the patient’s ABO/Rh blood group type? Discuss if the anti-Le a antibodies are
clinically significant. Is there a risk of haemolytic disease of the foetus/newborn
(HDFN)? Why or why not? (7 marks) (maximum 300 words)

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 References

Raman, L., Armstrong, B. and Smart, E. (2008). Principles of laboratory

techniques. ISBT Science Series, 3(2), pp.33–60.

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