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Journal of Criminal Law and Criminology
Volume 53 Article 13
Issue 3 September
Fall 1962
The ABO Grouping of Blood Stains
Stuart S. Kind
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Recommended Citation
Stuart S. Kind, The ABO Grouping of Blood Stains, 53 J. Crim. L. Criminology & Police Sci. 367 (1962)
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POLICE SCIENCE
THE ABO GROUPING OF BLOOD STAINS
STUART S. KIND
Stuart S. Kind is Principal Scientific Officer of the Department of Biology at the Home Office
Forensic Science Laboratory, Harrogate, England. He is a
graduate of the University of Nottingham,
and Editor of the Journal of the Forensic Science Society. We are pleased to again be able to publish
a paper of Mr.
Kind. This paper is an ampified version of a lecture delivered to the
course on Some
Aspects of Blood Grouping for Forensic Purposes at the Regional Transfusion Centre, Sheffield,
England, on February 17, 1961.--EiroR.
The grouping of bloodstains falls naturally into know this phenomenon has not been noted in the
two parts, the detection of antigens and the detec- literature, but I have experienced it myself and I
tion of antibodies. Dealing with the detection of suspect that if extraction procedures were less
the antibodies first I have little to say except that popular then it would be noted as a common
I consider it absolutely necessary before a blood- occurrence.
stain is reported on as being fully grouped in the I also suspect that the reason one frequently
ABO system that both antigens and antibodies fails to find agglutinips in bloodstains is because
should be of absorption
proved. rather than deterioration but since
one is subject to a continuous lecture on the
TmB DETECTION OF AGGLUTTINS lability of agglutinins in the literature
of bloodstain
The system we use at Harrogate is of the sim- grouping then the usual reason assumed for lack
plest, and merely entails placing small fragments of success is destruction rather than absorption.
of bloodstained fabric at the bottom of a small It follows that if a bloodstain contains agglutinins
bore tube (about 5 x 0.5 cm) and dispensing onto in an absorbed condition then it should be theo-
it a small amount of 0.5% red cell suspension. retically possible to elute these from the fabric by
The amount used is the smallest possible commen- raising the temperature. I have succeeded in doing
surate with recovering enough cell suspension to this experimentally with dean cloth soaked in
give a reading. In practice no extraction procedure various quantities of saliva, but unfortunately in
can equal this simple process because quite apart all the instances I have attempted to do this on
from the extra manipulation required there are actual case material then that old enemy of the
two factors mitigating against success in any bloodstain grouper-haemolysis-has intervened.
system where extraction and reaction do not If one could find a mode of treatment which
proceed simultaneously. The first of these is that would render red cells immune to haemolysis but
as a bloodstain ages it becomes more insoluble and still fully agglutinable then I would predict that
secondly, and this does not seem to have been the number of successes in agglutinin detection
would go up
explicitly recognised in the literature, that the sharply.
dissolving of dried agglutinins in saline provides Both the absorption and the deterioration of
conditions where they can react with absorbing agglutinins on bloodstained articles can be greatly
substances present in the fabric. It is quite reduced by correct conditions of storage. In our
obvious experience moisture causes
that should a fabric absorb strongly on the A side more deterioration than
then a stain of Group 0 blood present on it will temperature, and indeed it may be that a blood-
lose at least some of its anti A agglutinin. Drying stained object removed from a warm room to a
of the bloodstain on the surface of the fabric will, refrigerator will suffer more reduction in agglutinin
however, frequently prevent this process going content than it would otherwise have done due to
to the concomitant increase in humidity when
completion, and this is shown in practice by some lowering the temperature. At the present time we
bloodstains showing an anti A on testing with red favour the preservation of bloodstained artides by
cells and yet showing A absorption. As far as I storage over P 0r or CaC1.
2 2
STUART S. KIND • Vol. 53
A suitable technique for agglutinin detection is from the fabric absorption) is that the bloodstain
shown in Appendix A. shows weak or no absorption and one is left
wondering if this is a result of (a) the absence of
THE DETECTION op AGGLUTINOGENS the antigen tested for or (b) because the antigen is
The detection of antigens in bloodstains, at the present in too small amounts or (c) because we
moment, rests on the demonstration of the specific have used an antiserum which is too strong.
absorption of antibodies. Simple one step processes Absorption of agglutinins by the control is also, in
are not possible because of the destruction of the practice, the rule rather than the exception espe-
red cells by drying. Most of the problems of the cially where anti A reactions are concerned. I
bloodstain grouper would disappear if someone should mention here in passing that it is absolutely
would discover a way of recovering cells from blood- necessary that the control cloth used in bloodstain
stains in an agglutinable form. All the processes of grouping should be taken from immediately adja-
liquid blood group serology would then be appli- cent to the stain under test. One still sees control
cable to these cells. taken from areas remote from the bloodstain
We are then left with the necessity of carrying under test and indeed when I was first shown how
out absorption tests, and these tests fall into two to group bloodstains I was told to take my controls
classes, the first in which the absorption is demon- from a remote area because (a) it nullified the
strated by the reduction of the agglutinating power chance of being contaminated with blood from the
of the antiserum. This is called the Absorption stain, (b) one could then choose an area which did
Inhibition process, a name which is in my opinion not render the garment unusable and (c) one could
not a particularly good one since it suggests the pick a clean area in which the chance of interfering
inhibition of the action of an unchanged antiserum substances was remote!
rather than the removal or neutralisation of an If equivocal results are obtained from the simple
actual chemical entity. However it seems to be in procedure which I have just outlined then it
such common usage that it would be difficult to becomes necessary to employ some form of titra-
change it. tion. This is commonly carried out by making
The second method of demonstrating absorption serial doubling dilutions of the absorbed and
is by proving not what is left in the antiserum but unabsorbed antiserum and comparing any change
by proving what is combined with the bloodstain in titre. A better way of carrying out titrations in
directly. This is the Absorption-Elution technique. cases such as this is to use several dilutions of
Its main virtues are its simplicity compared with antiserum absorbed with constant amounts of
classical techniques and the fact that it takes only a stain either as an extract or in a powdered form.
fraction of the time of inhibition methods, is less This system is more satisfactory but still time
prone to error and is one which the inexperienced consuming. For a discussion of these techniques
worker can master quickly. It also proves actual see Kind (1955): A method used in the Harrogate
absorption of antibodies which, strictly speaking, laboratories is outlined in Appendix C.
the Absorption-Inhibition test does not. All these techniques are dependent on a number
Absorplion-Inhibition of variables which may nullify any results obtained
Tests. The basis of these or make necessary a redetermination. In order to
tests is the ability of a bloodstain to reduce the make bloodstain grouping procedures more simple
agglutinating activity of an antiserum (or to and accurate I embarked on a study which had as
'inhibit' it). The simplest way to operate this test its initial objective the determination whether
is to place a fragment of bloodstain in an aliquot fresh cells could be attached to cells fixed on a
of antiserum where it is left to absorb the agglu- slide and sensitised with an antiserum. I had no
tinins. The bloodstain is subsequently removed success with this (except occasionally with potent
and known cells are added to the 'absorbed' normal anti A sera) although it seems that Ogata
antiserum. This process is described in Appendix (1960) has since then had some success with this
B and in certain cases may be entirely satisfactory method. The present technique is the result of
where, for example, the stain totally absorbs the this initial study, and it is put forward as a sub-
activity of the antiserum and the control has no stitute for classical bloodstain grouping methods
absorbing power at all. Unfortunately, in practice, as being more efficient. It is not advocated as a
straight-forward reactions are the exception rather panacea; but we have made several hundred
than the rule and what frequently happens (apart determinations by this method, and we are con-
BLOOD STAINS
ABO GROUPING OF
vinced that unit amount of information can be out as quickly as possible since elevation of the
from refrigerator tempera-
extracted from a stain in about a fifth of the time temperature of the slide
taken by absorption inhibition methods. ture to room temperature will have some eluting
Absorption Elution Tests. We have tried several action. Next the indicator cells are pipetted into
modifications of this test both in tubes and on the cavities of the slides which are removed to
slides. A simple demonstration of the method moist chambers at 500 to 550 Centigrade for ten
may be made as follows. minutes (the antisera used must, of course, contain
Fresh blood is diluted with approximately ten no haemolysins). Next the slides are removed to
times its own volume of distilled water and room temperature where they are read after five
approximately 50 jlitre lots are dispensed into the and fifteen minutes. The
concentration of indicator
wells of cavity slides. This should be done for all used in liquid blood-
cells used is higher than that
the blood groups in the ABO system, and it is stain grouping since we find it only necessary to
convenient to use for this purpose twin-cavity read the results with the naked eye.
slides using one slide for each blood to be grouped. It is probably better to err on the side of too
This enables the grouping to be conveniently concentrated a cell suspension rather than too
carried out in duplicate. It is next necessary to dilute since the process is self-compensating and
fix this bloodstain onto the slide by such a method the higher the cell concentration the more com-
that will render the bloodstain insoluble and yet petitive removal of agglutinin from the stain
have no effect on the agglutinogens. ABO agglu- occurs. (Taking 60 pl of 3% cells and assuming
tinogens are thermostable to boiling water, and that 45% cells contain 5,000,000 cells per yl then
so we fix merely by plunging the dried slides into this volume of 3% cells will contain 20,000,000
boiling Mcllvaine buffer for 30 secs. at pH 7.4. cells each of an average surface area of 0.00013
It may possibly be that it would be equally satis- 2
mm which will give a total surface area of 26
factory to use distilled water for the fixation, but Cn2.)
we use buffer since in practice one is liable to meet The actual size of a blood smear grouped by this
with acid or alkaline substances much more than method is about 1 cm2. The actual surface area
in the antiseptic precints of a blood grouping will be somewhat greater than this due to micro-
laboratory. It may be .that there is some method scopic irregularities. On the other hand some of
of fixing other than boiling which would be equally the receptor groups on the surface of the blood-
satisfactory, but we have had no opportunity of stain will be occluded by denatured serum proteins.
experimenting in this direction. If the method is Diffusion of antibody molecules may occur over a
extended out of the ABO system, then obviously short distance through this denatured protein,
some other method of fixation must be found, but it is safe to say that the effective area of the
but all I have to say here refers to tests made in bloodstain is certainly smaller than the surface of
the ABO system. the cells onto which the antibody is being eluted.
The slides are next sensitised with the necessary It would be interesting to elute for a second time
antisera and allowed to absorb for a period of two with a fresh batch of cells without re-absorbing
to three hours. Since the reaction of ABO anti- and see how much antibody comes off. I would
bodies and antigens is reversible and the equilib- suspect that the amount would be very small
rium lies further towards the combined side at since convection currents caused on eluting by
lower temperatures then we find it advantageous heating the slide from room 0
temperature to 50 C
to carry out the final 30 minutes of absorption at would carry the antibody molecules into the body
about 4°C. This, however, is not absolutely of the liquid and disperse them where they would
necessary, and good results are usually obtained be preferentially absorbed by the indicator cells.
without it. The well of the slide is in each case One must not be led into using very dilute cells in
filled with antiserum. The amount used is not the hope that thereby the technique will be ren-
critical, and the only criterion to observe is that dered more sensitive. Although this may work
excess should be used so that antigen is saturated quite well in experimental smears, in actual case
with antibody under the conditions of the test. examples of bloodstain grouping the fabric nearly
Next, after the period of absorption the anti- always causes some interfering absorption on the
serum is washed off the slides with cold saline for a control side and it may be that the small amount of
period of at least a minute. The slides are then antibody eluted from this will be enough to agglu-
quickly blotted dry. This process should be carried tinate a small amount of dilute cell suspension.
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