Essentials of ABO–Rh Grouping and Compatibility Testing. Theoretical Aspects and Practical Application

146 Pages·1982·7.874 MB·English

by  W. John Lockyer (Auth.)

Essentials of ABO-Rh
Grouping and
Compatibility Testing
Theoretical Aspects
and Practical Application
W. John Lockyer PhD BSc FIMLS
Assistant Director, South- West Regional Blood Transfusion Service, Bristol;
Recognized Teacher, University of Bristol
With a Foreword by
H. H. Gunson DSc MD MRCP FRCPath
Medical Director, North-Western Regional Blood Transfusion Centre;
Adviser in Blood Transfusion to the Department of Health and Social
Security
WRIGHT PSG
BRISTOL LONDON BOSTON
1982
© Dr W. J. Lockyer, South-West Regional Blood Transfusion Centre,
Southmead Road, Bristol, Avon, BS10 5ND. 1982.
All Rights Reserved. No part of this publication may be reproduced,
stored in a retrieval system, or transmitted, in any form or by any
means, electronic, mechanical, photocopying, recording or otherwise,
without the prior permission of the Copyright owner.
Published by:
John Wright & Sons Ltd, 42-44 Triangle West, Bristol BS8 1EX,
England
John Wright PSG Inc, 545 Great Road, Littleton, Massachusetts 01460,
U.S.A.
British Library Cataloguing in Publication Data
Lockyer, W. John
Essentials of ABO-Rh grouping and compatibility testing.
1. Compatibility testing (Hematology)
I. Title
612'. 118225 RB45.5
ISBN 0 7236 0635 8
Library of Congress Catalog Card Number: 82-70099
Printed in Great Britain by
John Wright & Sons (Printing) Ltd, at The Stonebridge Press, Bristol
BS4 5NU
Preface
Overall safety in blood transfusion requires the correct application of a
number of different procedures, the most important of which is the
accurate ABO and Rh-D grouping of both patient and donor, followed
by a reliable and well-applied compatibility (cross-matching) test.
Medical, scientific and technical staff carrying out the duties of blood
grouping should be well trained in both the science and technology of
blood groups and should fully understand the major responsibility
which their work involves.
Every year fatalities occur as a result of incompatible blood
transfusion and it can be said that few pathological tests carry a heavier
responsibility than blood grouping and compatibility testing for in no
other test can an error lead so directly to the death of a patient.
Since the introduction of blood transfusion into clinical medicine the
number of units of donor blood matched and transfused has rapidly
and continually increased. Each year within the United Kingdom alone
well over two million blood donors attend sessions organized by the
National Blood Transfusion Service. The practice of providing ade-
quately trained staff for grouping and matching these bloods during
routine working hours does not, under normal circumstances, create
any major difficulty. However, during out-of-routine hours, it is often
left to members of laboratory staff whose prime interest might well be a
branch of pathology other than blood group serology. Whilst such
people need not require a detailed knowledge of the science and
technology of blood grouping, it is essential that they understand the
subject sufficiently well to carry out safely not only blood grouping
and compatibility testing but to cope with any minor difficulties and
follow-up studies that might arise when performing these tests.
The information within this book is directed primarily at such
members of staff and is therefore orientated towards the practical
aspects of the subject. It is designed to provide a sound understanding
V
vi PREFACE
of the essentials of blood grouping together with sufficient technical
instruction to permit the safe carrying out of both the straightforward
and the more complicated compatibility tests. It is also hoped that the
information provided will assist those medical, scientific and technical
staff preparing themselves for primary examinations in pathology and
medical laboratory sciences. It is not intended to satisfy the budding
Landsteiners. Those people wishing to know more about the science of
blood group immunology are advised to consult one of the more
detailed books on the subject, such as Blood Groups in Man by Race and
Sanger, or Applied Blood Group Serology by Issitt and Issitt.
W. J. L.
Practical Procedures
Technique
No.
1 Saline Agglutination Test 80
2 Albumin Agglutination Test 81
3 Enzyme Agglutination Test 82
4 Direct Anti-human Globulin Test 85
5 Indirect Anti-human Globulin Test 85
6 Controlling Anti-human Globulin Serum 87
7 Low Ionic Strength Saline Anti-human Globulin Test
(LISS/AHG) 89
8 Enzyme/Anti-human Globulin Test 90
9 ABO Grouping Test—Tube Technique 91
10 ABO Grouping Test—Slide or Tile Technique 93
11 ABO Sub-grouping using Anti-A (aj Reagent 93
t
12 Rh-D Grouping—Saline Technique 94
13 Rh-D Grouping—Albumin Layering Technique 95
14 Rh-D Grouping by Enzyme Technique 96
15 Direct Compatibility Test 97
16 Minor Compatibility Test 102
17 Rh Genotyping 103
18 Irregular Antibody Screening 104
19 Irregular Antibody Identification 106
20 Preparation of Low Ionic Strength Saline 108
21 Antibody Elution 108
22 Titration of Anti-A and Anti-B Agglutinins 109
23 Avidity Testing of Anti-A and Anti-B Sera 110
24 Specificity Testing of Anti-A and Anti-B Sera 111
25 Preparation of Anti-A (a ) Reagent 111
x x
26 Testing for A-B-H Blood Group Substance 112
ix
x PRACTICAL PROCEDURES
27 Quantitative Analysis of Blood Group Specific
Substance (Inhibition Index) 113
28 Method for the Complement Coating of Red Cells 114
29 Preparation of Leucocyte- and Platelet-poor Blood by
Dextran Sedimentation 116
30 Testing for Serum Protein Antibodies using Chromic
Chloride Treated Red Cells 118
31 Schumm's Test for Haemochromogen 119
32 Laboratory Investigation of Transfusion Reactions 120
33 Preparation of Platelet Concentrates 122
34 Preparation of Granulocyte Concentrates (Manual
Method) 123
35 Testing for HBsAg by Reversed Passive
Haemagglutination 124
36 Testing for IgG Anti-A and Anti-B using the Partial
Neutralization Method of Witebsky 127
37 Testing for IgG Anti-A and Anti-B using the Gel
Separation Technique of Webb 128
38 Testing for IgG Anti-A and Anti-B using Dithio-
threitol (Pirofsky and Rosner) 129
39 Testing for Alpha-beta Haemolysins 129
40 The Kleihauer-Betke Acid Elution Technique for
demonstrating Fetal Haemoglobin 130
Foreword
H. H. Gunson DSc MD MRCP FRCPath
Medical Director, North-Western Regional Blood Transfusion Centre;
Adviser in Blood Transfusion to the Department of Health and Social
Security
Modern blood transfusion practice has evolved from the fundamental
discoveries, at the turn of the twentieth century, of antigenic differences
between red blood cells of human beings. Since that time the extreme
complexity of antigen-antibody interactions relating to red cells,
leucocytes and platelets has become apparent. Knowledge of these,
together with an understanding of the physicochemical properties of
the cellular and plasma components of blood has enabled maximum
use to be made of many blood products in the clinical situation.
Research workers have many avenues to explore in the ever-
widening field of blood transfusion and doubtless their efforts will
continue to provide more effective and safer transfusions of blood and
its products. However, it must be remembered that in the majority of
transfusions in clinical practice the patient requires red cells in the form
of whole blood or as a red cell concentrate. The principal efforts of
Regional Transfusion Centres and Hospital Laboratories are, there-
fore, directed towards the safe transfusion of these preparations. One
must not lose sight of the fact, whilst investigating the intricate
complexities in immunohaematology, that two of the early blood
groups discovered, viz. the ABO and Rh, are of paramount importance
in preparing blood for transfusion.
It is difficult to prevent a sense of bemusement in those who are
entering the field of blood transfusion as a career, and in this book they
will find an easily readable account of basic techniques which are used
in everyday practice of blood transfusion. Dr Lockyer is well qualified
to write this book and has drawn extensively on his many years of
experience as a scientist in this field and as a teacher of the subject. The
theoretical aspects of the subject are, of necessity, restricted and there
will be some who may favour variations in some of the techniques
described, and the author has recognized this fact. However, those who
follow the methods detailed in this book will be assured of safe and
reproducible results.
XI
Section One
The ABO Blood Group
System
When the history of human blood groups and blood transfusion is
studied it can be seen that as far back as the twelfth century people were
experimenting with the possible transfusion of blood from one animal
to another. Mainly these experiments involved the transfusion of
human beings with blood taken from dog, sheep or goat. Needless
to say they almost always resulted in complete failure and often
caused the death of the recipient. Efforts to transfuse blood from one
human to another met with a greater degree of success, although
workers at that time could not account for the reason why in some
cases the patient's condition improved, while in others a severe and
often fatal reaction would occur.
The main answer to this did not manifest itself until very early in the
twentieth century when an immunologist by the name of Karl Land-
steiner, working in his Viennese laboratory, made what must have been
at that time one of the most important findings in the science of blood
transfusion since Harvey (1611) first showed that blood did not stay
still but moved rapidly around the body through a network of arteries
and veins. By cross-testing one blood sample against another, Land-
steiner was able to show that while some would mix successfully with
no visual signs of reaction, others would react strongly, causing
massive clumping (agglutination) of the red cells. This Landsteiner
attributed to the presence of two factors, one located on the red cells
and referred to as an antigen, the other found in the plasma and
referred to as an antibody. It was postulated that two different antigens
existed and these were called A and B, and that human red cells would
possess either or neither of these. Those having the A were referred to
as group A, while those possessing the B became known as group B.
Those lacking both the A and B were referred to as group O. The
plasma antibody present was shown to differ according to the A or B
antigens present on the red cells. Those grouping as A would have anti-
l
2 ESSENTIALS OF ABO-Rh GROUPING
B (P), those grouping as B would have anti-A (a) while those of group
O would have both anti-A and anti-B (a(3). These antibodies were
rarely present at birth and appeared in the blood stream around the
twelfth week of life, hence they became known as 'naturally-occurring'
antibodies (see p.8).
About a year after Landsteiner's discovery of the three basic ABO
groups, Decastello and Sturli showed the presence of a fourth group
when they tested a blood specimen which had both A and B antigens
present on the red cell; this was called group AB and was shown to be in
keeping with Landsteiner's earlier work in that the plasma lacked both
the oc and |3 antibodies. As a result of the work of Landsteiner and
Decastello and Sturli the ABO blood group system was born and this
laid the foundation of a new and important biological science. Table 1
Table 1. The basic blood groups
Group Red cell antigen Plasma antibody
O Nil oc + p
A A P
B B a
AB A + B Nil
shows the basic ABO red cell antigen and plasma antibody content of
the four basic ABO blood groups.
To the blood group immunologist, the greatest significance concern-
ing the fundamental difference between A and B blood group antigens
is the knowledge that the transfusion of red cells possessing either
antigen to someone lacking the same may rapidly bring about the death
of that person. To the immunochemist, while appreciating the clinical
significance of ABO incompatible transfusion, the difference between
the two antigens is seen primarily as a difference between two very
simple sugars, the sugar specificity of the blood group A antigen being
N-acetyl-D-galactosamine and group B a simple oe-linked D-galactose.
The blood group O receives its specificity indirectly from an oc-linked
fucose sugar. The term 'group O' can be somewhat confusing when
talking of the red cell antigens sugar specificity, as group O, unlike A
and B, has no specific gene and therefore no specific sugar for its
identification. The term 'group O' simply implies a total lack of A and
B. There is, however, a further blood group gene with the specificity H.
This gene shows its specificity through a fucose sugar which is to be
found in varying amounts in each of the four basic blood groups. It is
therefore common practice when talking of the sugar structure on red
cells to talk of ABH rather than ABO. Each of these A-B-H specific
sugars are referred to as the terminal sugar because they are linked on
ABO BLOOD GROUP SYSTEM 3
Note that five sugars remain constant with each group
Five Constant Sugars
1 = Galactose
2 = N-acetyl-D-glucosamine
3 = Galactose
4 = N-acetyl-D-glucosamine
5 = Fucose
Blood group 0(H) 0.—© 0 -o
Blood group A
_ © © ©
N-acetyl-D-
galactosamine
(A specificity)
Blood group B 0 0 © 0
° '" ©
Galactose
(B specificity)
Blood group AB (mixture of A and B specific sugars)
^ . -0 0 - 0 0
N-acetyl-D-
galactosammii ne
(A specificity)
^ ..-'-0 0 © -0
°" A
Galactose ( 5 )
(B specificity)
Fig. 1. A simplified explanation of the basic sugar structures responsible for giving
the four blood groups their specificity.