- Email: [email protected]
Gm and Km Typing in Forensic Science—A Methods Monograph
3. Forens. Sci. Sac. (1979), 19, 27
and
Received 18 January I979
Forensic ~ e t - h o dMonograph s
ANN E. KIPPS* Home Ofice Central Research Establishment, Aldermaston, Reading, Berkshira, England, RG7 4PN
Blood Grouping in Forensic Science I t is the concern of the forensic biologist to characterise samples of blood and other body fluids in order to determine whether two samples could have come from the same source. This characterisation makes use of inherited properties of the body fluids, such as the ABO blood groups, which remain constant in one person during his whole life and which are inherited, according to wellunderstood principles, from the person's parents. Without exception these grouping systems involve proteins which exist in two or more forms in body fluids and which can either be separated from each other by electrophoresis (e.g. phosphoglucomutase (PGM), haptoglobin) or can be identified by immunological tcchniqucs such as rcd-ccll agglutination (e.g., ABO, Gm, Km) . I n blood, these proteins can be components of the red blood cell membranes (e.g., ABO, Rhesus), or soluble proteins inside the red cell (e.g., PGM, adenylate kinase, (AK)): or soluble proteins in the serum (haptoglobin, Gm, Km). Many of these soluble proteins can be detected in body fluids other than blood. PGM, for example, can be used to group semen samples. I n many individuals, termed 'secretors', the ABO blood group substances exist also in a soluble form in semen, saliva and other body fluids. I n all, more than twenty different grouping systems have been reported as suitable for forensic use, that is as suitable to deal with the dried, aged and contaminated samples frequently encountered in forensic investigations. These grouping systems differ from each other in complexity and reliability, in the amount of sample that they consume in the test and also in the amount of information which they are potentially able to provide. A grouping system which divides the sample population into several commonly found types has a higher probability of discrimination between two different samples than does a grouping system in which most of the samples are of one type. This probability of discrimination between two samples may be expressed as a number called the discriminating power (DP) and may have values between 0 and 1 (Jones, 1972). For example the ABO grouping system divides the British population into four main classes, two of which are found with similar frcqucncy (A:42%, B:9%, AB:30/,, 0:460/,), and has a D P of 0.60. I n the same population the AK grouping system divides the population into two common types, one of which is much more frequent than the other (AK1:91%, AK2-1:9%) and the system has a D P of 0.16 (see Appendix 1). The forensic scientist is generally restricted to a very few tests by the small size of the sample he can obtain (not to mention time and manpower problems) and so it is important that he obtains the maximum amount of information possible in the minimum number of tests. I n this respect the choice of grouping systems to be used can be critical. Blood grouping systems commonly used by British forensic biologists have been reviewed by Culliford (1971). They include ABO, Rhesus, phosphoglucomutase (PGM), erythrocyte acid phosphatase (EAP), haptoglobin and, more recently, Gm and Km, the immunoglobulin *Present address: Max-Planck-Institut fur Erniihrungsphysiologie, Rheinlanddamm 201, 4600 Dortmund 1, W. Germany.
antigens (Khalap, Pereira and Rand, 1976). All of these systems have DP values greater than 0.55 for the British population. They differ, however, in the amount of material required for the test and the stability of the grouping factors involved. I n sensitivity and durability, the ABO system is superior to any other in current use for stain grouping. I n overall terms the Gm and K m system must be considered as the second choice (Khalap, Pereira and Rand, 1976).
Immunoglobulin Structure and Function Gm and K m antigens are located on immunoglobulin molecules and so an understanding of immunoglobulin structure is necessary for an appreciation of the inheritance pattern of these antigens. Immunoglobulin structure is basically simple and pleasingly symmetric and its elucidation in the early 1960s provided an illuminating insight into immunology as well as into many other unrelated aspects of molecular biology. Immunoglobulins are proteins consisting of more than one polypeptide chain linked together by disulphide bonds into a single structural unit. The basic unit consists of four polypeptide chains (Figure I), two of which are called heavy chains (mol. wt. 50,000-70,000) and two, light chains (mol. wt. 20,000). I n any one immunoglobulin molecule the heavy chains are identical, as also are the light chains. There are five different classes of immunoglobulins (Ig) which are currently recognised. These are IgG, IgA, IgM, IgD and IgE, all of which are found in the sera of all normal individuals. The molecular types have, at various times, had various other names and these are listed in Table 1. TABLE 1 NOMENCLATURE OF T H E IMMUNOGLOBULINS Present IgG IgM IgA IgD IgE
Previous yG, 76y, ~ 1 Y,Z , 6.6Sy, Y S S Y M , 19SY9 Yi, Y1 M, Yz M, pz iota, y-macroglobul~n Y A , Y I , BZA, B x Y D , YsJ Y E , Reagin, I g N D
MI
These five different classes of immunoglobulins are distinguished by the chemical nature of their heavy chains which are called gamma (y), alpha ( a ) , mu (p), delta (8) and epsilon (E) for IgG, IgA, IgM, IgD and IgE respectively. Light chains also come in two types, kappa (K) and lamda (A). Since both types of light chain are found in association with all types of heavy chain, they are both found in all immunoglobulin classes. IgG, IgD and IgE exist as monomers of the basic four-chain structural unit. IgG appears to exist in solution as three approximately globular regions joined together by a flexible region to form a Y or T shape. Early work on IgG structure used proteolytic enzymes to break the molecule into fragments. Such enzymes attack the IgG molecule in the flexible region; e.g., papain splits IgG into three major fragments, two of which are identical and can bind antigens (Fab) and a third, quite different, fragment which cannot (Fc) (Figure 1) (Porter, 1959). By such studies it was demonstrated as early as 1962 that IgG molecules have two separate but identical antigen-combining sites which are located at the Fab end of the molecule and which involve both the light and the heavy chains (Edelman and Benacerraf, 1962). The structure of other immunoglobulins has been thoroughly reviewed by Gally (1973). IgA exists mostly as the four-chain monomeric form in serum, but in secretions (saliva, semen, etc.) it is found largely as a dimer with the two four-chain units linked to two other short polypeptide chains, the J-chain and the secretory component (S.C.). These IgA dimers frequently appear under
L
Fc
Fab
Figure 1. Basic 4 chain structure of the immunoglobulin molecule.
1 [.---MIL Figure 2.
I g A dimers
Shapes of IgA and IgM molecules (see Gally, 1973 for review).
the electron microscope as two Y-shaped molecules joined at the base of the Y (Figure 2). IgM exists as a pentamer of the basic unit together with a J-chain. Under the electron microscope IgM molecules appear as five Y-shaped subunits radiating from a central core. The 'arms' appear to be flexible giving the molecule a spider-like appearance (Figure 2). This diverse family of proteins, the immunoglobulins, does, of course, include antibodies which are biologically characterised by their ability to combine specifically with foreign substances. The structural diversity of immunoglobulins implied by the observation that antibodies can be formed against almost any foreign macromolecule and are then specific for that macromolecule is so enormous that it defies both measurement and estimation. This biological diversity is made possible by variation in the amino acid sequences of just one
end of the polypeptide chains. This variable (V) region comprises approximately half of a light chain and about 20% of a heavy chain. The remainder of the chain, in both cases, is referred to as the constant (C) region. The variable regions of the four chains in an IgG molecule are found all together at the Fab end of the molecule (Figure 1) and are involved in the formation of antigenantibody combining sites. The variable regions of the two heavy chains in a single molecule seem to be identical, as are those of the two light chains, so that the two antibody-combining sites are structurally alike and of identical specificity. Gm and Km antigens are found on the constant regions of immunoglobulin polypeptide chains. Km antigens are found on kappa chains (Km = kappa marker) and so are found on all classes of immunoglobulins. Gm antigens are associated with gamma chains (Gm = gamma marker) and so are found only on IgG molecules.
The Gm and K m Blood Group Systems History The first Gm antigen was inadvertently discovered by Grubb (1956) during gamma-globulin determinations in sera from patients suffering from hypogamma-globulinaemia. He was making use of the fact that Rhesus positive red blood cells coated with an incomplete anti-Rh antiserum are agglutinated by the addition of an anti-immunoglobulin antiserum. I n 1956 several workers (reviewed by Prokop and Uhlenbruck, 1969) had shown that certain normal sera and sera from patients suffering from rheumatoid arthritis would also agglutinate red cells coated with anti-Rh antiserum and it had been postulated that rheumatoid serum also contains an anti-immunoglobulin (Grubb, 1956). Then in the same year, Grubb and Laurel1 (1956) reported that this ability of rheumatoid serum to agglutinate anti-Rh-coated red cells could be inhibited by certain human sera. Furthermore, the population could be divided into two groups depending on whether their sera were inhibitory or not. The inhibitory substance was shown to be a gamma-globulin and so was given the symbol Gm(a). Gm(a) proved to be an inherited antigen, inherited independently of other blood groups known at that time including ABO, MNS, Rhesus and haptoglobin. It has since been shown (Khalap, personal communication) to be inherited independently of the phosphoglucomutase isoenzymes as well. The discovery of'Gm(a) was the beginning of the characterisation of a whole series of closely linked Gm antigens. At least twenty-three such antigens have been described but not all of these have proved to be separate entities. Gm nomenclature was revised in 1965 by a World Health Organisation (W.H.O.) committee and the antigens given numbers instead of letters so that Gm(a) is now called Gm(1). A list of the original names and the current Gm terminology is given in Table 2. In 1961, Ropartz, 1,enoir and Rivat found that the serum of a healthy normal donor, Virm, possessed an aeglutinating activity against certain antiRh-sensitised red cells which could be inhibited by the serum of lgO/,of French donors. The inhibitory antigen was shown to be inherited independently of the Gm and, unlike Gm, to be present on all classes of immunoglobulins. The antigen was called InV and in 1974 was renamed K m by a W.H.O. committee at Rouen, France. The K m system does not show the great complexity of the Gm, only three K m antigens having been discovered to date (Table 3). Inheritance The Km antigens are inherited as simple autosomal (not sex-linked) alleles of which there are four known: Kml, Km1'2, Km3 and Km. (Km blank). K m 2 does not appear to occur without Kml. Km. is either an inhibitor of K m expression or a fourth antigen to which no antibody has yet been encountered. These alleles are codominant, and so both alleles present in an individual are
TABLE 2 NOMENCLATURE OF THE Gin SYSTEM
Previous
Current
P ba = b5
b0 Bet
=
25
=
b3 = 10
z Rouen 2 Rouen 3 San Francisco 2 g
Y n c5
Pa = u Ray = v TABLE 3 NOMENCLATURE OF THE Km SYSTEM
Current 1 2 3
Previous 1 a
b
expressed. Thus the K m phenotypes (i.e., observed types) which may be found are K m ( l ) , Km(1,2), Km(1,2,3), Km(1,3), Km(3) and K m blank. 98% of Km(1) individuals are also Km(2) in Caucasians and 91% in other races. 95% of Km(1) individuals are Km(1,3) in the Caucasian population and 82% among Negroes. K m frequencies in various populations are given in Appendix 3. Gm antigens are also inherited as codominant, autosomal alleles and are inherited independently of the K m antigens. The various Gm antigens are not inherited independently of each other but in linked groups called haplotypes, which are almost invariably passed without change from parent to offspring. The phenotype of an individual is thus the sum of two haplotypes, one from each parent. Certain phenotypes are more common than others and the common phenotypes are different for different races. Consider, for example, the Gm antigens Gm (1, 2, 3, 23, 5: 6, 13, 14). I n any phenotype each of these markers could be present or absent. For these eight markers there are thus forty-five different possible phenotypes. However, in the British population only eight of these are common and Gm(-1, -2, 3: 23, 5, -6, 13, 14) is the commonest. This common British phenotype is never found in pure Negroes, none of whom are Gm(-1). Gm(1, -2, -23, 5, -6, 13, 14) is the commonest Negro phenotype. Frequencies of Gm phenotypes in different races are given in Appendix 4.
Suitability for forensic blood grouping Gm and K m antigens are stable to moderate heat and alkali, are unaffected by liaemolysis and non-sterile conditions and may be stored (in solution) at room temperature for many days or at -20°C for many years without apparent loss
of activity. (This system was reviewed by Prokop and Uhlenbruck, 1969). The first reports of the stability of Gm factors in dried blood stains appeared in 1961 (Funfhausen and Sagen; Planques, RuffiC and Ducos). These have since been confirmed and augmented by a long series of publications (Ducos, RuffiC and Varsi, 1962; Nielsen and Henningsen, 1963; Gohler and Hunger, 1963; Brocteur and Moureau, 1964; Sagen and Funfhausen, 1965; Ducos, Ruffit and Varsi, 1964; Ducos, RuffiC and Varsi, 1965; Lenoir and Muller, 1966; Blanc and Gortz, 1971; Prokop and Byrdy, 1965). The very small amount of dried blood required for the Gm typing procedure was emphasised by Blanc, Gortz and Ducos (1973) and also by Khalap, Pereira and Rand ( 1976) who published the results of an extensive investigation into the use of Gm ( l ) , Gm(2) and Km (1) typing for the characterisation of blood stains. This combination of the high stability of the Gm antigens as well as their relatively high concentration in dried blood makes Gm a very attractive typing system for the forensic biologist. When one adds to this the excellent discriminating power of the system (for the British population the D P is 0.70 using Gm(l), Gm(2) and Km(1)) and the simple haemagglutination-inhibition technique used for Gm determination, Gm typing can be seen to approach the ideal for forensic purposes.
Principles of Gm and Km Grouping Cm and K m types are still determined in the manner by which they were discovered. The components of the typing system are: (1) Red cells sensitised with an appropriate incomplete anti-Rh antiserum. (2) Antiserum specific for the Gm antigen being determined. (3) The sample to be typed. The incomplete anti-Rh antiserum is usually an anti-D, but not all anti-D antisera are suitable for use in this test. The anti-D must be selected to be positive for the Gm antigen being determined and such reagents are readily available from a number of commercial sources (Appendix 2). The red cells to be sensitised must be Rhesus type D ( + ) so that they will become coated with the anti-D antibodies and thereby become positive for the Gm antigen, that is, become sensitised. The red cells must also be of ABO type 0 so that they are not agglutinated by any anti-A or anti-B present in the serum to be tested. The specific anti-Gm antisera are commercially available from the sources listed in Appendix 2. The procedure has three stages. Firstly, sensitised cells are prepared by mixing washed red blood cells with the appropriate anti-D, for example, an anti-D for Gm(1) typing. Secondly, the sample to be tested is mixed with anti-Gm(1) antiserum and then, thirdly, the sensitised cells are added. A Gm(1)-positive sample reacts with the anti-Gm(1) leaving insufficient antibody to agglutinate the sensitised cells. That is, a Gm(1)-positive sample inhibits the red cell agglutination. A Gm(1)-negative sample contains no Gm(1) antigen and so the anti-Gm(1) remains unreacted and can agglutinate the sensitised cells. The reaction is illustrated in Figure 3. The choice of Gm and K m tests to be used depends on the population from which the samples have been taken, but for a European population tests for K m ( l ) , G m ( l ) , Gm(2) and Gm(l0) provide a grouping system with a DP of 0.79. This has been calculated from the population frequencies for the British population which are given in Table 4. Frequencies for other populations are given in more detail in Appendices 3 and 4. Reagents for K m ( l ) , G m ( l ) , Gm(2) and Gm(l0) are currently readily available from the sources listed in Appendix 2. Detailed Methods for Gm and Km Grouping The procedures described below are those in use at the Metropolitan Police Forensic Science Laboratory, London, and have been described by Khalap,
Gm(+) Serum
Gm(-)
Anti Gm
Anti G m
Gm(+)RBC
Gm(+)RBC Figure 3.
No Agglutination
Agglutination
Principles of Gm Grouping.
TABLE 4 IMMUNOGLOBULIN PHENOTYPES IN THE BRITISH POPULATION Gm(2) -
+-
++ +-
Gm(l0)
+ + + + +-
+
-
-
Frequency 34% 25% 14%
7%
5%
5% 4%
3% 1% 1% 0%
Pereira and Rand (1976). The procedures are all described for Gm typing but apply equally, of course, to K m typing where anti-D for the K m factor concerned and the relevant anti-Km antisera must be used. The anti-D and the specific anti-Gm antiserum should, for any one marker in any one test, come from the same supplier. The use of an anti-D from one supplier with an anti-Gm from another may, on occasions, lead to erroneous results. Body fluids other than blood do contain Gm antigens and so it is important to avoid fingering samples. The techniques used are very sensitive and great care must be taken to ensure that instruments and glassware are scrupulously clean. A. Sensitisation of Red Cells with an Anti-D of Unknown Titre Each new batch of anti-D must be titrated by the procedure described below in order to obtain the best sensitisation of the red cells. When the titre for that batch of anti-D is known procedure B should be used for cell sensitisation. (1) Take about 15 drops of whole blood from a finger prick, venepuncture, or from a transfusion blood pack. The blood must be group 0, D +
(2) (3) (4) (5)
(6) .
(7) (8) (9) (10)
(1 1) (12)
(see procedure sections F and G). I t is convenient to collect the blood from a finger prick or venepuncture into a tube containing anticoagulant (heparin, EDTA or citrate) . Transfer the blood to a centrifuge tube (volume 5-15cm3) and wash the red cells three times with saline (0.9% w/v sodium chloride in water). Place 1 drop of the washed packed cells in each of six centrifuge tubes. Add 1 drop of saline to each tube. Select the anti-D for the Gm antigen to be determined. Add 1 drop of the anti-D to the first tube, 2 drops to the second, 3 drops to the third and so on to 6 drops in the sixth tube. Stopper the tubes, mix the contents well and incubate them a t 37OC for 90 minutes. During this time the anti-D binds to the red cells. Wash the cells three times with saline to remove any unabsorbed anti-D. Test each tube of sensitised cells with anti-human globulin to establish whether the anti-D has coated the cells (Procedure section H). Prepare a pale cell suspension (fi0.2%) from each of the six tubes of sensitised cells. Test each of these suspensions with the specific anti-Gm antiserum using the following procedure : Prepare doubling dilutions of anti-Gm antiserum in saline from neat antiserum in ten tubes using 7 drops per tube. Set up six rows of ten small tubes. Disposable plastic tubes 2-3cm long 0.5cm internal diameter are convenient. Place 1 drop of each anti-Gm antiserum dilution into each of the six tubes in a column of tubes (see Table 5). TABLE 5 TITRATION OF ANTI-D FOR Gm TYPING
Anti-Gm dilution Red cells 1/64 11128 11256 11512 1/1024 with anti-D 112 1/32 1/1G 118 114 0 0 0 0 0 0 2 0 1 :1 4 3 0 0 0 0 1 0 2 4 3 1 :2 4 0 0 0 0 1 2 3 3 1 :3 4 4 0 0 3 0 4 4 2 1 :4 4 4 0 0 0 0 2 1 1 :5 4 3 4 4 0 0 0 0 1 :6 4 4 3 3 2 The numbers in the body of the table are agglutination scores (see Procedure A(16)).
+
+
(13) (14) (15) (16)
Add 1 drop of each dilute sensitised cell suspension to each tube in a row of tubes (see Table 5). Mix and leave at 4OC for 1-1& hours. Centrifuge the tubes at 1000 rpm for 1 minute in a bench-top centrifuge. This should be timed carefully so that it can be reproduced in all tests. Transfer the cells to microscope slides using a Pasteur pipette and being careful not to break up the pellet. Examine for agglutination both macroscopically and microscopically. Score the agglutination on the scale: 4, 3, 2, 1, 0 as follows: 4 : no free cells; all cells agglutinated. 3 : 0-10% cells free. 2 : 10-50% cells free. 1 : 50-90% cells free. : very few, very small agglutinates. 0 : no agglutination; all cells free. Select the sensitised cells giving maximum titre for that antiserum and always use that cell : anti-D ratio for sensitisation with that batch of anti-D, e.g., in Table 5 the maximum anti-Gm titre is obtained with a red cell : anti-D ratio of 1 : 4. Thus, with that batch of anti-D, 4 drops
+,
+ (17)
(18) (19) (20)
of anti-D should be added to 1 drop of packed red cells for sensitisation by procedure B. Store the selected sensitised cells in the minimum volume of saline at 4°C and wash once in saline before use. Stored in this manner, sensitised cells keep satisfactorily for 24 hours. Discard the other five batches of sensitised cells. For use in the Gm typing procedure (D) prepare a pale pink suspension (+0.2%) of sensitised cells in saline.
B. Sensitisation of Red Cells using an Anti-D o f Known Tbre (1) Place 1 drop of washed, packed red cells of 'blood type 0, D + (see procedure A) in a centrifuge tube and add 1 dnop of saline. (2) Add the appropriate number of drops of anti-D as determined in procedure A for that batch of anti-D. (3) Mix and incubate at 37OC for 1; hours. (4) Wash the sensitised cells three times with saline. (5) Test the sensitised cells with anti-human globulin (procedure section H). (6) Store the sensitised cells in the minimum volume of saline at 4OC for not more than 24 hours before use. (7) For use in Gm typing, stored cells should be washed once in saline and a 0.2% suspension made. C. Titration of Anti-Gm Antisera In procedure A, for determining the titre of a new batch of anti-D, the anti-Gm antiserum titre is determined simultaneously; e.g., in the example shown in Table 5 we chose a 1 : 4 red cell : anti-D ratio. With these sensitised cells complete agglutination occurred down to an anti-Gm dilution of 1/16. At 1/32 only 90% of cells were agglutinated. The anti-Gm titre for routine use would be between these two - 1/24. New batches of anti-Gm antisera which have not been titred during procedure A should be evaluated in the following way: (1) Make doubling dilutions of anti-Gm antiserum in saline from neat antiserum through ten tubes,. preparing 2 drops of each dilution. (2) Transfer 1 drop of each dilutlon to one of a second row of ten tubes and discard the original ten tubes. (2) Add 1 drop of pale pink sensitised cell suspension to each tube. (4) Mix and leave at 4OC for 1-19 hours. (5) Centrifuge at 1000 rpm for 1 minute. This should be timed carefully so that it can be reproduced in all tests. (6) Transfer the cells to a microscope slide and examine for agglutination as before (procedure A( 16)) . (7) Select the antiserum dilution to be used in the inhibition test; e.g., in Table 6 the agglutination results for an anti-Gm antiserum titration are shown. The selected dilution must be between the last dilution giving 100% agglutination and the next, which gives about 90% agglutination. Thus, this antiserum must be used at a dilution of 116. TABLE 6 TITRATION OF AN ANTI-Gm ANTISERUM Antiserum dilution Agglutination score
: :
neat 4
112 4
1$4
1&8
1/16 2
1/32 1
1/64 11128 11256 11512 + 0 0 0
D. Gm Typing of Serum Positive and negative control sera as well as a saline control must be set up with each batch of unknown sera. Control sera may be purchased from the suppliers of Gm reagents (Appendix 2) or serum from laboratory staff of known Gm type may be used.
(1) (2)
(3) (4)
(5) (6) (7) (8) (9) (10)
Dilute all samples and controls ten times in saline. For each sample or control set up two small tubes and place 1 drop of the diluted serum in each. Place 1 drop of saline in each of another pair of tubes for the saline control. T o the first tube of each pair add 1 drop of appropriately diluted anti-Gm antiserum. T o the second tube of each pair add 1 drop of saline. These second tubes are the agglutination controls. Mix the contents of all tubes and leave at 4OC for 1-1& hours for the inhibition reaction to take place. Add 1 drop of the appropriately sensitised cell suspension to all tubes. Mix, and leave the tubes at 4OC for 1-14 hours. Spin the tubes at 1000 rpm for 1 minute in the same centrifuge as used for antiserum titration. This should be timed carefully so that it can be reproduced in all tests. Transfer the cells to a microscope slide and examine for agglutination as before (Procedure A(16)). Record the G m type of the sample. Table 7 shows some typical results. The unknown serum '4' has caused agglutination of the sensitised red cells in the agglutination control, i.e., in the absence of added anti-Gm antibody. The test should be repeated and if the same result is obtained then the sample cannot be typed by this procedure. I t is likely that the sample itself contains an antibody which is agglutinating the sensitised cells. TABLE 7 Gm TYPING OF SERUM
Sample
Agglutination Score Anti-Gm Saline Positive Control Serum 0 0 Negative Control Serum 4 0 4 0 Saline Control Unknown Serum 1 0 0 2 4 0 3 3 0 4 3 3 *See text, procedure D(10).
Result Gm (+I Gm (-1 Gm (-1 Gm (+I Gm (-1 Gm (-1 No result*
E. Gm Typing of Bloodstains I t has been established in a large trial that when bloodstains are G m typed there is no need for agglutination controls. (Khalap, Pereira and Rand, 1976). Any antibodies which may have been present in the original blood sample do not interfere in stain typing when the following methods are used. (a) Direct Inhibition Testing (1) Positive and negative control stains as well as a control (unstained) sample of each cloth involved must be set up in parallel with each batch of unknown stains. (2) Take a length of stained fibre (for Gm(1) : 0.5cm, for Km(1) and Gm(2) : l.cm, for Gm(l0) : 2cm) and cut it into three or four pieces. Take equivalent lengths of unstained fibres for controls. (3) Place the fragments from each length of fibre in the bottom of a small tube and add 1 drop of appropriately diluted anti-Gm antiserum. Ensure that the threads are submerged in the antiserum. (4) Leave the tubes at 4OC overnight (16 hours or more) for the inhibition reaction to take place. ( 5 ) Remove the threads from the tubes, squeezing out any antiserum with clean forceps. (For an alternative method of removing the threads see procedure section J).
(6)
(7) (8)
(9) (10)
(b) (1)
(2) (3)
Add 1 drop of a dilute suspension of sensitised cells in saline containing 0.3% bovine serum albumin. Mix, and leave the tubes at 4OC for 1-la hours. Centrifuge the tubes at 1000 rpm for 1 minute, timing this carefully. Transfer the cells to a microscope slide and examine for agglutination as before (procedure A(16)). Record the Gm types of the stains. If the cloth controls inhibit the agglutination then the test must be repeated, preferably using method (b) below. Extraction Techniquefor Stain Typing Take 0.5cm2 of stained fabric and extract it into 3-4 drops of saline by soaking for 3-4 hours at room temperature. Remove the fabric with forceps or, preferably, by procedure J. Simultaneously, extract similar amounts of positive and negative control stains and of unstained areas of all cloths. Use 1 drop of extract for each test without further dilution, using procedure D as for serum grouping. No agglutination controls are . required.
F. A B O Typing of Liquid Blood I t is very important that the red cells sensitised for use in the Gm typing procedure are blood group 0. Cells of group A or B might be agglutinated by anti-A or anti-B present in antisera or in samples. The ABO group of the cells to be sensitised may be checked by the following procedure: (1) Collect the whole blood (a few drops) in a tube containing anticoagulant, mix well with saline and transfer to a centrifuge tube. A graduated centrifuge tube is convenient. (2) Centrifuge to sediment the cells and discard the supernatant saline. (3) Resuspend the cells in saline at a cell concentration of about 5% v/v. (4) Prepare cells from known A, B and 0 blood in a similar manner. (5) Grouping is carried out on a flat glass plate or tile marked, with a wax pencil, into squares of about 3cm side. For each blood to be tested and for the control bloods place 1 drop of cell suspension in each of three squares. (6) T o the first square add 1 drop of anti-A antiserum, tolthe second 1 drop of anti-B antiserum and to the third 1 d r o of ~ anti-H (a lectin v r e ~ a r e d from the seeds of Ulex europaeus). (7) Mix the antisera and cell suspensions by gently rocking the tile in the hands and then allow it to stand on the bench for 5 minutes. (8) Rock the tile again and read the agglutination macroscopically scoring as described in procedure A(16). Some sample results are shown in Table 8. A
.
TABLE 8 ABO TYPING OF L I Q U I D BLOOD Sample Control A Control B Control 0 Unknown 1
2 3
Agglutination Scores Anti-A Anti-B Anti-H 4 0 3 0 4 4 0 0 4 0 0 4 4 0 2 0 4 3
Blood T y p e
A B 0 0 A B
G. Rhesus Typing of Liquid Blood The cells used for Gm typing must be of Rhesus type D + so that the sensitising anti-D antiserum will bind to the cells. The Rhesus type of the blood may be
checked using a complete anti-D antiserum (usually described as 'suitable for saline agglutination tests in tubes or on tiles') and following the procedure described for ABO typing in section F. Control D-positive and D-negative bloods must always be tested in parallel with the unknown samples. There are many antigens other than D in the Rhesus system and more than one nomenclature system. A list of Rhesus types in three nomenclature systems showing the D-positive types is given in Table 9. TABLE 9 SIMPLIFIED NOMENCLATURE OF T H E RHESUS SYSTEM
Fisher Notation CDe cDE cDe CDE Cde cdE CdE cde
Common Notation
RI R2 Ro
Rz R Rl1
Wiener Notation R1 R2 R" R r1 rl1
R~
ry
r
r
D
Positive Positive Positive Positive Negative Negative Negative Negative
H . The Anti-Human Globulin Test The simplest way of checking that the red cells for the Gm test have become coated with anti-D during the sensitisation procedure is to react the washed sensitised cells with an anti-human globulin antiserum. Since the anti-D is a human globulin it reacts with the anti-human globulin and the red cells are agglutinated. All glassware used must be scrupulously clean as the slightest trace of contaminating human protein may neutralise the antiserum giving false negative results. Dilute the anti-human globulin antiserum according to the manu(1) facturer's instructions and use the diluted antiserum immediately. Do not attempt to store diluted antiserum. (2) Place 1 drop of the washed, sensitised red cells on a flat glass plate or tile and add 1 drop of diluted anti-human globulin antiserum. (3) Set up a saline control by adding 1 drop of saline to 1 drop of the sensitised cells. (4) Mix the contents of each square on the tile using the corner of a microscope slide, a clean corner for each square. ( 5 ) Leave the tile undisturbed for 1 minute and then rock it very gently by hand. At no time must the tile be shaken. After 5-7 minutes (or the time recommended by the anti-globulin manufacturer) examine the tile for macroscopic agglutination. (6) If the drops are allowed to dry up false agglutinations may appear in the saline control as well as in other samples.
J. Extraction Technique for Stained Fabric (Baxter and Rees, 1974)
This is a convenient and efficient means of stain extraction. I t may be used to extract the Gm antigens from the stained fabric for procedure E(b) or for removing the threads from the antiserum in procedure E(a). Small plastic disposable test tubes, conveniently 1-2cm long and 0.5-0.6cm internal diameter, are required. A small hole is made in the bottom of one tube with a hot needle. This hole must be as small as possible so that liquids placed in the tube do not run out. A second, unpunctured tube is sellotaped to the first so that the hole in the bottom of the upper tube is over the top of the lower tube. The fabric to be extracted or reacted with antiserum is placed in the upper tube and saline or antiserum is added. The extraction or inhibition time is allowed to elapse and the double tube is centrifuged at low speed, 1000-2000
rpm, for a few minutes. The liquid in the upper tube is forced through the hole and collects in the lower tube. The system may also be used for multiple extractions provided that the lower tube is large enough to contain the accumulated extraction volume. The extract in the lower tube is reached by snapping the sellotape between the two tubes. This extract must always be thoroughly mixed before use. The sellotape may be replaced by a tightly fitting sleeve of plastic or rubber tubing which may then be washed and re-used. The tubes must be discarded.
Choice of Reagents I n practice the biggest single factor limiting the choice of Gm allotypes to be tested is the availability of antisera. Most anti-Gm antisera occur 'naturally' in human serum and are discovered during testing of serum for use by transfusion laboratories. Not all antisera are alike and not all antisera are equally suitable for routine Gm and K m typing. The first anti-Grrl antibodies were found in the sera of patients suffering from rheumatoid arthritis (Grubb and Laurell, 1956) but, very shortly after similar antibodies were discovered in the sera of healthy individuals (Grubb, 1959). The former were called Ragg sera (Rheumatoid agglutinators) and the latter, Snagg (Serum normal agglutinators). Ragg sera have relatively higher anti-Gm titres than do Snagg but with frequent changes in this titre with time in the one individual. Snagg sera have lower but fairly constant titres. Frequently a distinct prozone eflect is observed with Ragg sera such that their agglutinating ability increases with dilution, at first, before decreasing at higher dilution in the normal manner. Such a prozone is not observed with Snagg sera. The anti-Gm antibodies of Ragg sera may be readily inhibited non-specifically by certain levels of globulins. Snagg sera are only inhibited by the specific Gm factor. Ragg antisera often contain antibodies of several different specificities in the one serum whereas Snagg sera are monospecific. Snagg antisera only arise in individuals whose serum is negative for the factor against which the antibody is directed. With Ragg sera there may be simultaneous presence of the anti-Gm antibody and the corresponding G m factor. Thus with Ragg antisera errors can occur in the determination of Gm types and for routine work Snagg antisera must always be used. Much valuable work has been done with Ragg sera but their usefulness is generally restricted to specialist structural and quantitative studies. Snagg antisera are not very coirlmon. For example, in 1,200 non-rheumatoid sera Ropartz et al. (1960) reported nine anti-Gm(1) antisera. I n 1965 were found eighteen anti-Gm(l), nine anti-Gm(6) and two anti-Gm(2) antisera among 13,000 healthy sera. In all instances the antiserum donor is assumed to have been immunized against the Gm or K m allotype. This may be due to 'autoimmunisation' in rheumatoid patients, alloimmunisation by plasma immunoglobulins in patients receiving multiple transfusions (Allen and Kunkel, 1963) and alloimmunisation of the foetus by the mother or of the mother by the foetus during pregnancy, (Steinberg and Wilson, 1963; Fudenberg and Fudenberg, 1964). Antisera have also been raised by immunising primates or rabbits but such sera are not readily available. (Litwin and Kunkel, 1966; Hess and Butler, 1962). The anti-D antiserum used to sensitise the red cell in the Gm test must also be carefully selected. This procedure is carried out by the antiserum retailers so that, provided an anti-D is always used with the specific anti-Gm antiserum marketed by the same manufacturer, the reagent pair will give reliable results in serum typing. For bloodstain typing reagents must be evaluated locally using stains of known type. The red cell coating antisera does not have to be anti-D. Any incomplete antiserum (i.e., one which will sensitise the red cells rather than agglutinate them) which is positive for the specific Gm or K m factor may
be used. Anti-Duffy antisera have been shown to be quite satisfactory, but anti-D are the commonest and the only ones which are widely available. T o choose the optimum groups for testing from the reagents which are available (for suppliers see Appendix 2) requires more knowledge of the structure and inheritance of these immunoglobulin antigens.
Further Structural Considerations Immunoglobulins exist as a number of types: IgG, IgA, IgM, IgD and IgE which differ in the structure of their heavy chains called gamma (y), alpha (a), mu (p), delta (8) and epsilon (E) respectively. This has already been discussed under Immunoglobulin Structure and Function. Each of the different immunoglobulin types can be further subdivided, by their immunological properties, into subclasses. IgG, for example, exists as four subclasses : IgG1, IgG2, IgG3 and IgG4 which differ in the antigenic properties of their heavy chains called yGl? yG2, yG3 and yG4, respectively. Since each of these y chains is found in association with both K and A chains there are eight types of IgG molecule which are present in the serum of all normal individuals. The relative amounts of the various heavy chain subclasses is approximately 71% yG1, 18% yG2, 8% yG3 and 3% yG4. The ratio of IgG molecules with K chains to those with A chains is approximately 2 : 1. The different heavy chains have different amino acid sequences and thus it is not surprising that the disulphide bonds linking the polypeptide chains are in different positions in the different IgG subclasses. The constant regions of the heavy chains are made up of several linearly arranged regions whose amino acid sequences are sufficiently similar as to suggest that they all evolved from a single short polypeptide chain. In y heavy chains there are three such regions, called homology regions (Figure 4) so that we can imagine y chains to have been formed in an evolutionary sense from four shorter polypeptide chains joined end to end. Three of these are similar (the homology regions) and form the constant region and the fourth is the variable region. The three homology regions are called CH1, C H 2 and CH3 counting from the amino terminus of the polypeptide chain (Figure 4). Each homology region is folded into a loop held in place by a disulphide bond across its base (Figure 4). In the intact IgG molecule the homology regions of the different chains appear to interact closely with their counterparts on other chains, thus stabilising the structure. Gm and K m antigens are found on the constant regions of immunoglobulin polypeptide chains. Km antigens are associated with K chains (Km = Kappa marker) and so are found on all classes of immunoglobulins. Gm antigens are associated with y chains (Gm = gamma marker) and so are found only on IgG molecules. Each of the Gm markers is confined to one of the IgG subclasses so that, for example, Gm(1) is found only on IgGl molecules, Gm(23) on IgG2 molecules and Gm(5) on IgG3 (Table 10). In some cases the amino acid sequence associated with a particular antigen has been identified. For example, if amino acid 191 on human kappa chains is leucine the molecule has Km(1) specificity but if amino acid 191 is valine the molecule has Km(3) specificity. Similarly, on yG1 chains of IgGl a certain sequence of amino acids at positions 355-358 is associated with Gm(1) activity. Arg-Asp-Glu-Leu is Gm(1) positive while the only known alternative sequence at this position, Arg-Glu-Glu-Met, is Gm(1) negative. This alternative sequence is also found in the corresponding position on yG2 and yG3 heavy chains and is called the Gm (non-1) marker. Thus IgGl molecules can be Gm(1) or Gm (non-1) but IgG2 and IgG3 molecules can only be Gm (non-1). yG4 chains have a different non-marker sequence in this position and this is called Gm (4 non-1). The non-markers are not useful for genetic studies or for discrimination purposes as they are always present in all normal individuals. Gm(1) and Gm (non-1) on yG1 chains are called homoalleles. Not all pairs of homoalleles are com-
Figure 4. Homology regions of an IgG molecule. Key: V = Variable region C = Constant region L = Light chain H = Heavy chain
posed of a marker and a non-marker. Grn(3) on yG1 chains, for example, has Gm(17) as its homoallele and neither of these markers are found on other IgG subclasses. The homoalleles of all markers are not yet known. For example, an alternative allele to Gm(2) has not yet been discovered. The Gm terminology currently recommended by the W.H.O. Committees on Immunoglobulin Allotypes lists the antigens in order of heavy chain subclasses yG1, yG2, yG3 and writes complete phenotypes such that the two haplotypes of the individual are irrimediately obvious. The phenotype G m ( l ) , for example, is the product of an allele coding for a part of the yG1 chain. The allotype is written Glm(1). Thus, a test for the allotype Glm(1) may have the result Gm(1) when the allotype is present or the result Gm(-1) when it is not. Similarly a positive result for Gm(5) indicates the presence of the allotype G3m(5). A Gm haplotype is written simply in order of heavy chain subclasses with the antigens written as superscripts, e.g., Gm13293823*5913914 or Gm-1-2s A phenotype is written in subclass and haplotype order, e.g., Gm(1,2,17,3,23,6,15,21,24,5,10,11,13,14,26,27) where Gm1z2.l7and Gm3 are the two haplotypes of the yG1 subclass. A partial phenotype can be expressed as Gm(1,2,3,-14) if one only tested for Glm(1,2,3) and G3m(14). I t is, of course, possible on theoretical grounds to predict which combination of Gm markers will be of most use to a forensic laboratory, giving the maximum chance of discrimination between samples for the minimum number of tests. 332395p13914.
TABLE 1 0 STRUCTURAL RELATIONSHIP OF THE Gm ALLOTYPES
Current Gm Nomenclature 1 2 3 5 6
7 8 9 10 11
Heauy Chain Subgroup
YG1 YG1 YG1 YG3 YG3 771 yG1
yG3 vG3
$'I ?
Heauy Chain Fragment Fc Fc Fa b Fc Fc Fc
Homology Region CH3 CH3 CH 1 CH2 CHY
?
Fc E'c Fc
Fab ? ?
CHI ? ?
A consideration of the commonly found Gm phenotypes in the population from which the samples were drawn will indicate the optimum antigens for study. I t is evident from Appendix 4 that the British population can be differentiated into eight common phenotypes by testing for four Gm allotypes Glm(1,2,3) and G2m(23). Where a group of antigens are almost invariably linked, tests for more than one of the group are unlikely to provide any extra information. I n the British population Glm(3) and G3m(5,10,11,13,14) are linked in the haplotype Gm3.5916911913,14and so, in samples from a British population, only one of these markers need be tested. Since yG1 markers are generally present in serum at higher concentrations than yG3 markers, Glm(3) is the obvious choice. Future Prospects The biggest single problem facing forensic biologists wishing to do Km and G m typing on blood stains is the availability of suitable antisera. This is likely to remain a problem unchanged even with the discovery of new Gm and K m groups, until production of antisera by immunisation of laboratory animals is undertaken on a commercial scale. Although Gm and K m factors in blood have been studied for over twenty years, in other body fluids they have received very little attention probably because of the problems encountered by early workers (Klose and Schraven, 1962; Kramer 1963). Theoretically, any body fluid which contains immunoglobulin must contain Gm and K m antigens. The currently used detection techniques were all devised for use with blood serum and body secretions have much lower immunoglobulin levels than does serum. The levels of immunoglobulins found in some body fluids are given in Table 11. Km(1) typing, which depends on the total immunoglobulin level, can be carried out on blood diluted at least 100 and sometimes 1000 times. I t is, therefore, to be expected that one could Km(1) type most body fluids and perhaps even sweat. Km(1) typing of saliva and semen have been reported (Davie and Kipps, 1976; Jorch and Oepen, 1977). Gm typing however,
TABLE 1 1 IMMUNOGLOBULIN LEVELS IN BODY FLUIDS
Body Fluid
Immunoglobulin level mg/100 ml 1.G ZgA ZgM 1200 260 90 2 20 1 15 150 1 20 2 ? 14 21 44 3 1 ?
Scrum Saliva Nasal Secretion Semen Vaginal Secretion Sweat
References: Waissbluth and Langman, 197 1 . Brandtzaeg, Fjellanger, Gzeruldsen, 1970. Blenk et al., 1974. TABLE 12 Gm AND Km ANTIGENS IN BODY FLUIDS
Body Fluid Km(1)
+ + +
Gm(1)
+ + + +
Gm(2) 0
Antigen Gm(3) G m ( 5 ) G m ( l 0 ) Gm(14) Gm(21) 0 0 0 0 0 0 0
Saliva Nasal Secretion Semen Vaginal Secretion 0 Sweat 0 0 0 0 0 0 0 + : detected in that body fluid but not yet reliably enough for routine typing. 0: either not yet detected or not yet tested for.
+
+ + +
+
$
+
1
+
+ + 0 0
depends only on the IgG fraction and although (from the data in Table 12) one might expect to be able to Gm type nasal secretion, semen and vaginal secretion, saliva and sweat would appear to be too dilute. Jorch and Oepen (1977) have reported Gm factors in semen and nasal secretion, and work a t the Home Office Central Research Establishment, Aldermaston, England, has shown that Gm(1) may be unreliably detected in both saliva and vaginal secretion (unpublished results). Table 12 shows the various Gm and K m antigens which have been detected in body secretions, but in no case has the detection system been shown to be reliable. For Gm(1) in saliva, for example, this unreliability may well be due to insufficiently sensitive detection systems but for Gm and K m markers in semen this is an unlikely explanation. Agglutination systems are very sensitive to the protein and ionic concentration of their suspension media (Pollack 1965). Body secretions particularly when undiluted, are thus far from ideal fluids in which to measure degree of agglutination. False agglutinations and false negative results are very common features of Gm and K m typing of body fluids and much more basic methodological research is necessary before this extremely valuable and flexible typing system can be recommended for routine typing of fluids other than blood.
References ALLEN,J. C. and KIJNKEL, H. G., 1963, Antibodies to genetic types of gamma globulin after mu1tiple transfusions. Science, 139, 4 18. BAXTER,S. J. and REES,B., 1974, Simultaneous haptoglobin and haemoglobin typing of blood and bloodstains using gradient polyacrylamide gel electrophoresis, Med. Sci. Law, 14, 231-236. BLANC,M. and GORTZ,R., 1971, Identification of a new factor Gm 'Bet' in blood stains. Application in forensic medicine, Vox Sang, 20, 263-266. BLANC,M., GORTZ,R. and Ducos, J., 1973, Identification d'un tache de sang minime g r k e B la mise en tvidence des facteurs Inv (1) et Inv (2) au cours d'une expertise mtdico-1Cgale. C. R. Skances Soc. Biol. Ses. Fil., 167, 777-780 43
BLENK,H., HOFSTETTER, R., BOWERING, R., BUTTLER, R., HARTMANN, M. and MARX,F., 1974, Immunelektrophorese des Ejakulats, Miinch. med. Wschr. 116, 35-38. BRANDTZAEG, P., FJELLANGER, I. and GZERULDSEN, S., 1970, Human secretory immunoglobulins. I : Salivary secretions from individuals with normal or low levels of serum immunoglobulins, Scand. 3.Haemat., Supkl., 12, 1-83. BRAZIER, D. M. and GOLDSMITH, K . L. G., 1968, Frequency of certain Gm and Inv factors in the United Kingdom, Nature, 219, 193. BROCTEUR, J. and MOUREAU,P., 1964, U n cas d'application pratique des l'identification i de taches de sang, Ann. mid. lbg., groupes plasmatiques Gm ? 44, 315-321. CIJLLIFORD, B. J., 1971, The examination and typing of bloodstains in the crime laboratory, U.S. Dept. of Justice, Washington D.C. 20402. DAVIE,M. J. and KIPPS,A. E., 1976, Km(1) (Inv (1)) typing of saliva and semen, Vox Sang., 31, 363-367. Drrcos, J., R U F F I ~J., and VARSI,M., 1962, Mise en Cvidence des antigtnes Gma, Gmb, Gmx dans les taches de sang sec, Vox Sang., 7, 722-731. Ducos, J., R U F F I ~J ,. and VARSI,M., 1964, Une nouvelle application du systtme Gm : l'identification des taches de sang sec., Proc. 9th Congr. int. Soc., Blood Transf. Mexico, 1962, pp. 478-482. Ducos, J . , R U F F IJ. ~ , and VARSI,M., 1965, Efficiency of searching Gm antigens for identification of bloodstains in forensic medicine, Nature (Lond.), 205, 1332-1333. EDELMAN, G. M. and BENACERRAF, B., 1962, O n structural and functional relations between antibodies and proteins of the gamma system, Proc. Nut. Acad. Sci. U.S., 48, 1035-1042. FIIDENBERG, H. H. and FUDENBERG, B. R., 1964, Antibody to hereditary human gamma globulin (Gm) factor resulting from maternal-fetal incompatibility, Science, 145, 170-1 7 1. FUNFHAUSEN, G. and SAGEN, Z., 1961, Uber die Moglichkeit des Nachweises der Gruppeneigenschaft Gm in Blutspuren, Deutsch. Gesch. Wes., 16, 52, 2468-9. GALLY, J. A., 1973, Structure of immunoglobulins p. 161-298 in 'The Antigens' Vol. I, Ed M. Sela Academic Press. GOHLER,W. and HUNGER,H., 1963, Der Nachweis von Gm und InvEigenschaften in Blutspuren., Zschr. Aerztl. Fortbild, 58, 794-795. GRITBB, R., (1956), Agglutination of erythrocytes coated with 'incomplete' anti-Rh by certain rheumatoid arthritic sera and some other sera, Acta Pathol. Microbiol. Scand., 39, 195-197. G R ~ B BR., , 1959, Ciba Foundation Symposium 264. GRIJBB,R. and LAIJRELL, A. B., 1956, Hereditary sero\ogical human serum groups. Acta Pathol. Microbiol. Scand., 39, 390-398. HESS,M. and BUTLER, R., 1962, Anti-Gm specificities in sera of rhesus monkeys immunised with human gamma globulin, Vox Sang., 7, 93. JONES,D. A., 1972, Blood samples: probability of discrimination, 3.Forens. Sci. soc., 12, 355-359. JORCH,G. and OEPEN,I., 1977, Nachweis der Faktoren Gm (1,2,4,5,21) und Inv (1) in menschlichen Sekreten: Speichel, Schweiss, Nasensekret und Sperma, Z. Rechtsmedizin, 79, 1-6. KHALAP, S., PEREIRA, M. and RAND,S., 1976, Gm and Inv grouping of bloodstains, Med. Sci. Law., 16, 40-43. KLOSE,I . and SCHRAVEN, J., 1962, Die Eigenschaft Gma im menschlichen Sperma und ihre Beziehung zur ABO Ausscheidereigenschaft, Dt. 5.ges gericht. Med., 52, 610. KRAMER, K., 1963, Untersuchungen iiber das Vorkommen von Gma Substanz in menschlichen Geweben und extravasalen Korperfliissigkeiten, 3. gericht. Med., 53, 131-141. 44
LENOIR,L. and MULLER,P. N., 1966, Determination des groupes Gm sur taches de sang, Ann. mid. lig., 46, 191-193. LITWIN,S. D. and KUNKEL, N. G., 1966, Genetic factors of human gamma globulin detected by rabbit antisera, Transfusion, 6, 140. K., 1963, Experimental studies on the NIELSEN,J. C. and HENNINGSEN, determination of the Gm groups in bloodstains, Med. Sci. Law, 3,49-58. PLANQUES, J., R U F F I ~ J., and Ducos, J., 1961, Le systkme Gm en mtdecine ltgale. Toulouse Med., 62, 685-699. POLLACK, W., 1965, Ann. N.Y. Acad. Sc ., 127, 892. PORTER,R. P., 1959, Biochem. J., 73, 119. PROKOP, 0.and BYRDY, M., 1965, 'Aktuelle Fragen der geritchlichen Medizin', Ursg. M. Varmosi. 0 . and UHLENBRUCK, G., 1969, 'Human Blood and Serum Groups', PROKOP, Maclaren and Sons Ltd., London. ROPARTZ,C., LENOIR, J., HEMET,Y. and RIVAT,L., 1960, Possible origins of the anti-Gm sera, Nature, 188, 1120-1 121. ROPARTZ, C., LENOIR, J. and RIVAT,L., 1961, A new inheritable property of human sera: the InV factor, Nature (Lond.), 189, 586. SAGEN, A. and FUNFHAUSEN, G , 1965, Gm group characteristics in bloodstains, Arch. Irnrnunol. Ther. Exp., 13, 149-156. R. and WAINWRIGHT, P., 1978, Gm and Kni polymorphism in man, STEDMAN, Biotest Bulletin, 4, 3-14. STEINBERG, A. G. and WILSON, J. A., 1963, Hereditary globulin factors and immune tolerance in man, Science, 100, 303-304. J. G. and LANGMAN, M. J. S., 1971, ABO blood groups, secretor WAISSBLUTH, status, salivary protein and serum and salivary immunoglobulin concentrations, Gut, 12, 646-649. Acknowledgements This monograph was inspired by and based on a symposium on Gm and K m typing of body fluids held in July, 1976, at the Home Office Central Research Establishment, Aldermaston, England. The credit for its contents must therefore go to those who organised and took part in the symposium - in particular to Mr. P. Martin, Dr. S. Khalap and Mrs. M. Brady of the Metropolitan Police Forensic Science Laboratory and to Dr. P. H . Whitehead of the Home Office Central Research Establishment. Thanks for assistance and advice are also due to Mr. S. S. Kind, who sugiested the writing of the monograph, and helped at every stage that followed, to Miss M. Pereira and Dr. L. King of the Home Office Central Research Establishment, to Miss D. Brazier of the World Health Organisation Blood Group Reference Laboratory, London, and to the Information Division of the Home Office Central Research Establishment who tirelessly sought references as required. APPENDIX 1 DISCRIMINATING POWER Discriminating Power (Jones, 1972) is defined by the expression: DP = 1-P where P is the probability of two random samples matching. P is calculated from the expression: P = ~ , ~ + p ~ ~ + p , ~ + . .pn2 . - . . . where p,, p,, p, . . . . . . p, are the proportions of the population being studied in each class 1, 2, 3 . . . . . . n. Consider the following examples: 1. 111 the Metropolitan Police area the ABO blood grouping system divides the population as fbllows: Class ABO type P 1 A 0.42 2 BL 0.09 3 A'B 0.03
The probability of a match, using this system, is given by: 0.462 P = 0.4Z2 + 0.092 + 0.03' = 0.1764 + 0.0081 + 0.0009 + 0.2116 = 0.3970 Thus the probability of being able to distinguish between two samples using this test alone is DP = 0.603.
+
2.
In the adenylate kinase (AK) grouping system the class frequencies are as follows: Class Type P 1 AK I 0.912 2 AK2-1 0.087 3 AK2 0.001 P = 0.912' + 0.0872 + 0.0012 = 0.84 DP = 0.16 APPENDIX 2 COMMERCIAL SOURCES O F Gm AND I
Behring Hoechst Pharmaceuticals, Hoechst House, Salisbury Road, Hounslow, Middlesex, England. Telephone: 01-570-77 12 Biotest Folex Limited, 1649 Pershore Road, Birmingham B30 3DR, England. Telephone: 02 1459-2 112 Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, Postbus 9 190, Amsterdam. Dade, A. R. Horwell Limited, 2 Grangeway, Kilburn High Road, London NW6 2BP. England. Telephone: 01-328-1551 Dr Molter GmbH, 69 Heidelberg 1, Postfach 104049, West Germany. APPENDIX 3 I
Number of Samples 43
Percentage of popu!atiou Km(1) Km(2) Km(3) 58
African negroes (Mauritania) Amcricans (White) 295 American negroes 189 British2 1000 Chinere 507 Czechs 190 Dutch 354 Filipinos 126 Finns 162 French 339 Germans 968 Greeks 297 Iranians 297 Japanese 68 1 Jugoslavs 157 Swiss 600 'Prokop and Uhlenbruck. 1969. 2Brazier and Goldsmith, 1968.
18
58 17 13
18
46
14 61 16 56 12 16 51 11 16 12 17 12 48 15
100 83
APPENDIX 4 G m POPULATION FREQUENCIES* Phenotype 1
12
1,13 1,2,13 1,5,6 1,2,5,6 1,5,6,13 1,5,14 1,2,5,14 1,5,6,14 1,2,5,6,14 1,5,6,13,14 1,5,13,14 1,2,5,13,14 3,5,13,14
Greek
Indian
American Negro
3 7 -
-
3 1
14 16 6 3
5
3 1 2 i 1 8 1 2 1 <1 1 <1 24 27 3 2
8 13
1 9
<1
-
20 5
<1
4
-
-
-
-
-
-
-
-
<1
<1 <1 <1 A
7
-
-
A
1
<1
African Negro (South) <1
Afrian Negro (West) i 1
British
A
-
5 2 3 1
<1
6 -
1 -
32 48
35 52
-
10 A
<1
1
-
-
-
-
Chinese (Hong Kong) 2 1 1
<1
-
A
-
63 3,23,5,13,14 1,3,5,13,14
35 8
29
1,3,23,5,13,14 1,2,3,5,13,14
22 4
-
90
-
3
1,2,3,23,5,13,14 12 8 1 1,3,5,6,13,14 7 1,3,23,5,6,13,14 1 3 Frequency (%) o f phenotypes in various races based on G3m(5,6,13,14). ( T h e generic term G m has been omitted). *Stedman and Wainwright, 1978.
<1 tests
6
-
for Glm(1,2,3), G2m(23),
and
Received 18 January I979
Forensic ~ e t - h o dMonograph s
ANN E. KIPPS* Home Ofice Central Research Establishment, Aldermaston, Reading, Berkshira, England, RG7 4PN
Blood Grouping in Forensic Science I t is the concern of the forensic biologist to characterise samples of blood and other body fluids in order to determine whether two samples could have come from the same source. This characterisation makes use of inherited properties of the body fluids, such as the ABO blood groups, which remain constant in one person during his whole life and which are inherited, according to wellunderstood principles, from the person's parents. Without exception these grouping systems involve proteins which exist in two or more forms in body fluids and which can either be separated from each other by electrophoresis (e.g. phosphoglucomutase (PGM), haptoglobin) or can be identified by immunological tcchniqucs such as rcd-ccll agglutination (e.g., ABO, Gm, Km) . I n blood, these proteins can be components of the red blood cell membranes (e.g., ABO, Rhesus), or soluble proteins inside the red cell (e.g., PGM, adenylate kinase, (AK)): or soluble proteins in the serum (haptoglobin, Gm, Km). Many of these soluble proteins can be detected in body fluids other than blood. PGM, for example, can be used to group semen samples. I n many individuals, termed 'secretors', the ABO blood group substances exist also in a soluble form in semen, saliva and other body fluids. I n all, more than twenty different grouping systems have been reported as suitable for forensic use, that is as suitable to deal with the dried, aged and contaminated samples frequently encountered in forensic investigations. These grouping systems differ from each other in complexity and reliability, in the amount of sample that they consume in the test and also in the amount of information which they are potentially able to provide. A grouping system which divides the sample population into several commonly found types has a higher probability of discrimination between two different samples than does a grouping system in which most of the samples are of one type. This probability of discrimination between two samples may be expressed as a number called the discriminating power (DP) and may have values between 0 and 1 (Jones, 1972). For example the ABO grouping system divides the British population into four main classes, two of which are found with similar frcqucncy (A:42%, B:9%, AB:30/,, 0:460/,), and has a D P of 0.60. I n the same population the AK grouping system divides the population into two common types, one of which is much more frequent than the other (AK1:91%, AK2-1:9%) and the system has a D P of 0.16 (see Appendix 1). The forensic scientist is generally restricted to a very few tests by the small size of the sample he can obtain (not to mention time and manpower problems) and so it is important that he obtains the maximum amount of information possible in the minimum number of tests. I n this respect the choice of grouping systems to be used can be critical. Blood grouping systems commonly used by British forensic biologists have been reviewed by Culliford (1971). They include ABO, Rhesus, phosphoglucomutase (PGM), erythrocyte acid phosphatase (EAP), haptoglobin and, more recently, Gm and Km, the immunoglobulin *Present address: Max-Planck-Institut fur Erniihrungsphysiologie, Rheinlanddamm 201, 4600 Dortmund 1, W. Germany.
antigens (Khalap, Pereira and Rand, 1976). All of these systems have DP values greater than 0.55 for the British population. They differ, however, in the amount of material required for the test and the stability of the grouping factors involved. I n sensitivity and durability, the ABO system is superior to any other in current use for stain grouping. I n overall terms the Gm and K m system must be considered as the second choice (Khalap, Pereira and Rand, 1976).
Immunoglobulin Structure and Function Gm and K m antigens are located on immunoglobulin molecules and so an understanding of immunoglobulin structure is necessary for an appreciation of the inheritance pattern of these antigens. Immunoglobulin structure is basically simple and pleasingly symmetric and its elucidation in the early 1960s provided an illuminating insight into immunology as well as into many other unrelated aspects of molecular biology. Immunoglobulins are proteins consisting of more than one polypeptide chain linked together by disulphide bonds into a single structural unit. The basic unit consists of four polypeptide chains (Figure I), two of which are called heavy chains (mol. wt. 50,000-70,000) and two, light chains (mol. wt. 20,000). I n any one immunoglobulin molecule the heavy chains are identical, as also are the light chains. There are five different classes of immunoglobulins (Ig) which are currently recognised. These are IgG, IgA, IgM, IgD and IgE, all of which are found in the sera of all normal individuals. The molecular types have, at various times, had various other names and these are listed in Table 1. TABLE 1 NOMENCLATURE OF T H E IMMUNOGLOBULINS Present IgG IgM IgA IgD IgE
Previous yG, 76y, ~ 1 Y,Z , 6.6Sy, Y S S Y M , 19SY9 Yi, Y1 M, Yz M, pz iota, y-macroglobul~n Y A , Y I , BZA, B x Y D , YsJ Y E , Reagin, I g N D
MI
These five different classes of immunoglobulins are distinguished by the chemical nature of their heavy chains which are called gamma (y), alpha ( a ) , mu (p), delta (8) and epsilon (E) for IgG, IgA, IgM, IgD and IgE respectively. Light chains also come in two types, kappa (K) and lamda (A). Since both types of light chain are found in association with all types of heavy chain, they are both found in all immunoglobulin classes. IgG, IgD and IgE exist as monomers of the basic four-chain structural unit. IgG appears to exist in solution as three approximately globular regions joined together by a flexible region to form a Y or T shape. Early work on IgG structure used proteolytic enzymes to break the molecule into fragments. Such enzymes attack the IgG molecule in the flexible region; e.g., papain splits IgG into three major fragments, two of which are identical and can bind antigens (Fab) and a third, quite different, fragment which cannot (Fc) (Figure 1) (Porter, 1959). By such studies it was demonstrated as early as 1962 that IgG molecules have two separate but identical antigen-combining sites which are located at the Fab end of the molecule and which involve both the light and the heavy chains (Edelman and Benacerraf, 1962). The structure of other immunoglobulins has been thoroughly reviewed by Gally (1973). IgA exists mostly as the four-chain monomeric form in serum, but in secretions (saliva, semen, etc.) it is found largely as a dimer with the two four-chain units linked to two other short polypeptide chains, the J-chain and the secretory component (S.C.). These IgA dimers frequently appear under
L
Fc
Fab
Figure 1. Basic 4 chain structure of the immunoglobulin molecule.
1 [.---MIL Figure 2.
I g A dimers
Shapes of IgA and IgM molecules (see Gally, 1973 for review).
the electron microscope as two Y-shaped molecules joined at the base of the Y (Figure 2). IgM exists as a pentamer of the basic unit together with a J-chain. Under the electron microscope IgM molecules appear as five Y-shaped subunits radiating from a central core. The 'arms' appear to be flexible giving the molecule a spider-like appearance (Figure 2). This diverse family of proteins, the immunoglobulins, does, of course, include antibodies which are biologically characterised by their ability to combine specifically with foreign substances. The structural diversity of immunoglobulins implied by the observation that antibodies can be formed against almost any foreign macromolecule and are then specific for that macromolecule is so enormous that it defies both measurement and estimation. This biological diversity is made possible by variation in the amino acid sequences of just one
end of the polypeptide chains. This variable (V) region comprises approximately half of a light chain and about 20% of a heavy chain. The remainder of the chain, in both cases, is referred to as the constant (C) region. The variable regions of the four chains in an IgG molecule are found all together at the Fab end of the molecule (Figure 1) and are involved in the formation of antigenantibody combining sites. The variable regions of the two heavy chains in a single molecule seem to be identical, as are those of the two light chains, so that the two antibody-combining sites are structurally alike and of identical specificity. Gm and Km antigens are found on the constant regions of immunoglobulin polypeptide chains. Km antigens are found on kappa chains (Km = kappa marker) and so are found on all classes of immunoglobulins. Gm antigens are associated with gamma chains (Gm = gamma marker) and so are found only on IgG molecules.
The Gm and K m Blood Group Systems History The first Gm antigen was inadvertently discovered by Grubb (1956) during gamma-globulin determinations in sera from patients suffering from hypogamma-globulinaemia. He was making use of the fact that Rhesus positive red blood cells coated with an incomplete anti-Rh antiserum are agglutinated by the addition of an anti-immunoglobulin antiserum. I n 1956 several workers (reviewed by Prokop and Uhlenbruck, 1969) had shown that certain normal sera and sera from patients suffering from rheumatoid arthritis would also agglutinate red cells coated with anti-Rh antiserum and it had been postulated that rheumatoid serum also contains an anti-immunoglobulin (Grubb, 1956). Then in the same year, Grubb and Laurel1 (1956) reported that this ability of rheumatoid serum to agglutinate anti-Rh-coated red cells could be inhibited by certain human sera. Furthermore, the population could be divided into two groups depending on whether their sera were inhibitory or not. The inhibitory substance was shown to be a gamma-globulin and so was given the symbol Gm(a). Gm(a) proved to be an inherited antigen, inherited independently of other blood groups known at that time including ABO, MNS, Rhesus and haptoglobin. It has since been shown (Khalap, personal communication) to be inherited independently of the phosphoglucomutase isoenzymes as well. The discovery of'Gm(a) was the beginning of the characterisation of a whole series of closely linked Gm antigens. At least twenty-three such antigens have been described but not all of these have proved to be separate entities. Gm nomenclature was revised in 1965 by a World Health Organisation (W.H.O.) committee and the antigens given numbers instead of letters so that Gm(a) is now called Gm(1). A list of the original names and the current Gm terminology is given in Table 2. In 1961, Ropartz, 1,enoir and Rivat found that the serum of a healthy normal donor, Virm, possessed an aeglutinating activity against certain antiRh-sensitised red cells which could be inhibited by the serum of lgO/,of French donors. The inhibitory antigen was shown to be inherited independently of the Gm and, unlike Gm, to be present on all classes of immunoglobulins. The antigen was called InV and in 1974 was renamed K m by a W.H.O. committee at Rouen, France. The K m system does not show the great complexity of the Gm, only three K m antigens having been discovered to date (Table 3). Inheritance The Km antigens are inherited as simple autosomal (not sex-linked) alleles of which there are four known: Kml, Km1'2, Km3 and Km. (Km blank). K m 2 does not appear to occur without Kml. Km. is either an inhibitor of K m expression or a fourth antigen to which no antibody has yet been encountered. These alleles are codominant, and so both alleles present in an individual are
TABLE 2 NOMENCLATURE OF THE Gin SYSTEM
Previous
Current
P ba = b5
b0 Bet
=
25
=
b3 = 10
z Rouen 2 Rouen 3 San Francisco 2 g
Y n c5
Pa = u Ray = v TABLE 3 NOMENCLATURE OF THE Km SYSTEM
Current 1 2 3
Previous 1 a
b
expressed. Thus the K m phenotypes (i.e., observed types) which may be found are K m ( l ) , Km(1,2), Km(1,2,3), Km(1,3), Km(3) and K m blank. 98% of Km(1) individuals are also Km(2) in Caucasians and 91% in other races. 95% of Km(1) individuals are Km(1,3) in the Caucasian population and 82% among Negroes. K m frequencies in various populations are given in Appendix 3. Gm antigens are also inherited as codominant, autosomal alleles and are inherited independently of the K m antigens. The various Gm antigens are not inherited independently of each other but in linked groups called haplotypes, which are almost invariably passed without change from parent to offspring. The phenotype of an individual is thus the sum of two haplotypes, one from each parent. Certain phenotypes are more common than others and the common phenotypes are different for different races. Consider, for example, the Gm antigens Gm (1, 2, 3, 23, 5: 6, 13, 14). I n any phenotype each of these markers could be present or absent. For these eight markers there are thus forty-five different possible phenotypes. However, in the British population only eight of these are common and Gm(-1, -2, 3: 23, 5, -6, 13, 14) is the commonest. This common British phenotype is never found in pure Negroes, none of whom are Gm(-1). Gm(1, -2, -23, 5, -6, 13, 14) is the commonest Negro phenotype. Frequencies of Gm phenotypes in different races are given in Appendix 4.
Suitability for forensic blood grouping Gm and K m antigens are stable to moderate heat and alkali, are unaffected by liaemolysis and non-sterile conditions and may be stored (in solution) at room temperature for many days or at -20°C for many years without apparent loss
of activity. (This system was reviewed by Prokop and Uhlenbruck, 1969). The first reports of the stability of Gm factors in dried blood stains appeared in 1961 (Funfhausen and Sagen; Planques, RuffiC and Ducos). These have since been confirmed and augmented by a long series of publications (Ducos, RuffiC and Varsi, 1962; Nielsen and Henningsen, 1963; Gohler and Hunger, 1963; Brocteur and Moureau, 1964; Sagen and Funfhausen, 1965; Ducos, Ruffit and Varsi, 1964; Ducos, RuffiC and Varsi, 1965; Lenoir and Muller, 1966; Blanc and Gortz, 1971; Prokop and Byrdy, 1965). The very small amount of dried blood required for the Gm typing procedure was emphasised by Blanc, Gortz and Ducos (1973) and also by Khalap, Pereira and Rand ( 1976) who published the results of an extensive investigation into the use of Gm ( l ) , Gm(2) and Km (1) typing for the characterisation of blood stains. This combination of the high stability of the Gm antigens as well as their relatively high concentration in dried blood makes Gm a very attractive typing system for the forensic biologist. When one adds to this the excellent discriminating power of the system (for the British population the D P is 0.70 using Gm(l), Gm(2) and Km(1)) and the simple haemagglutination-inhibition technique used for Gm determination, Gm typing can be seen to approach the ideal for forensic purposes.
Principles of Gm and Km Grouping Cm and K m types are still determined in the manner by which they were discovered. The components of the typing system are: (1) Red cells sensitised with an appropriate incomplete anti-Rh antiserum. (2) Antiserum specific for the Gm antigen being determined. (3) The sample to be typed. The incomplete anti-Rh antiserum is usually an anti-D, but not all anti-D antisera are suitable for use in this test. The anti-D must be selected to be positive for the Gm antigen being determined and such reagents are readily available from a number of commercial sources (Appendix 2). The red cells to be sensitised must be Rhesus type D ( + ) so that they will become coated with the anti-D antibodies and thereby become positive for the Gm antigen, that is, become sensitised. The red cells must also be of ABO type 0 so that they are not agglutinated by any anti-A or anti-B present in the serum to be tested. The specific anti-Gm antisera are commercially available from the sources listed in Appendix 2. The procedure has three stages. Firstly, sensitised cells are prepared by mixing washed red blood cells with the appropriate anti-D, for example, an anti-D for Gm(1) typing. Secondly, the sample to be tested is mixed with anti-Gm(1) antiserum and then, thirdly, the sensitised cells are added. A Gm(1)-positive sample reacts with the anti-Gm(1) leaving insufficient antibody to agglutinate the sensitised cells. That is, a Gm(1)-positive sample inhibits the red cell agglutination. A Gm(1)-negative sample contains no Gm(1) antigen and so the anti-Gm(1) remains unreacted and can agglutinate the sensitised cells. The reaction is illustrated in Figure 3. The choice of Gm and K m tests to be used depends on the population from which the samples have been taken, but for a European population tests for K m ( l ) , G m ( l ) , Gm(2) and Gm(l0) provide a grouping system with a DP of 0.79. This has been calculated from the population frequencies for the British population which are given in Table 4. Frequencies for other populations are given in more detail in Appendices 3 and 4. Reagents for K m ( l ) , G m ( l ) , Gm(2) and Gm(l0) are currently readily available from the sources listed in Appendix 2. Detailed Methods for Gm and Km Grouping The procedures described below are those in use at the Metropolitan Police Forensic Science Laboratory, London, and have been described by Khalap,
Gm(+) Serum
Gm(-)
Anti Gm
Anti G m
Gm(+)RBC
Gm(+)RBC Figure 3.
No Agglutination
Agglutination
Principles of Gm Grouping.
TABLE 4 IMMUNOGLOBULIN PHENOTYPES IN THE BRITISH POPULATION Gm(2) -
+-
++ +-
Gm(l0)
+ + + + +-
+
-
-
Frequency 34% 25% 14%
7%
5%
5% 4%
3% 1% 1% 0%
Pereira and Rand (1976). The procedures are all described for Gm typing but apply equally, of course, to K m typing where anti-D for the K m factor concerned and the relevant anti-Km antisera must be used. The anti-D and the specific anti-Gm antiserum should, for any one marker in any one test, come from the same supplier. The use of an anti-D from one supplier with an anti-Gm from another may, on occasions, lead to erroneous results. Body fluids other than blood do contain Gm antigens and so it is important to avoid fingering samples. The techniques used are very sensitive and great care must be taken to ensure that instruments and glassware are scrupulously clean. A. Sensitisation of Red Cells with an Anti-D of Unknown Titre Each new batch of anti-D must be titrated by the procedure described below in order to obtain the best sensitisation of the red cells. When the titre for that batch of anti-D is known procedure B should be used for cell sensitisation. (1) Take about 15 drops of whole blood from a finger prick, venepuncture, or from a transfusion blood pack. The blood must be group 0, D +
(2) (3) (4) (5)
(6) .
(7) (8) (9) (10)
(1 1) (12)
(see procedure sections F and G). I t is convenient to collect the blood from a finger prick or venepuncture into a tube containing anticoagulant (heparin, EDTA or citrate) . Transfer the blood to a centrifuge tube (volume 5-15cm3) and wash the red cells three times with saline (0.9% w/v sodium chloride in water). Place 1 drop of the washed packed cells in each of six centrifuge tubes. Add 1 drop of saline to each tube. Select the anti-D for the Gm antigen to be determined. Add 1 drop of the anti-D to the first tube, 2 drops to the second, 3 drops to the third and so on to 6 drops in the sixth tube. Stopper the tubes, mix the contents well and incubate them a t 37OC for 90 minutes. During this time the anti-D binds to the red cells. Wash the cells three times with saline to remove any unabsorbed anti-D. Test each tube of sensitised cells with anti-human globulin to establish whether the anti-D has coated the cells (Procedure section H). Prepare a pale cell suspension (fi0.2%) from each of the six tubes of sensitised cells. Test each of these suspensions with the specific anti-Gm antiserum using the following procedure : Prepare doubling dilutions of anti-Gm antiserum in saline from neat antiserum in ten tubes using 7 drops per tube. Set up six rows of ten small tubes. Disposable plastic tubes 2-3cm long 0.5cm internal diameter are convenient. Place 1 drop of each anti-Gm antiserum dilution into each of the six tubes in a column of tubes (see Table 5). TABLE 5 TITRATION OF ANTI-D FOR Gm TYPING
Anti-Gm dilution Red cells 1/64 11128 11256 11512 1/1024 with anti-D 112 1/32 1/1G 118 114 0 0 0 0 0 0 2 0 1 :1 4 3 0 0 0 0 1 0 2 4 3 1 :2 4 0 0 0 0 1 2 3 3 1 :3 4 4 0 0 3 0 4 4 2 1 :4 4 4 0 0 0 0 2 1 1 :5 4 3 4 4 0 0 0 0 1 :6 4 4 3 3 2 The numbers in the body of the table are agglutination scores (see Procedure A(16)).
+
+
(13) (14) (15) (16)
Add 1 drop of each dilute sensitised cell suspension to each tube in a row of tubes (see Table 5). Mix and leave at 4OC for 1-1& hours. Centrifuge the tubes at 1000 rpm for 1 minute in a bench-top centrifuge. This should be timed carefully so that it can be reproduced in all tests. Transfer the cells to microscope slides using a Pasteur pipette and being careful not to break up the pellet. Examine for agglutination both macroscopically and microscopically. Score the agglutination on the scale: 4, 3, 2, 1, 0 as follows: 4 : no free cells; all cells agglutinated. 3 : 0-10% cells free. 2 : 10-50% cells free. 1 : 50-90% cells free. : very few, very small agglutinates. 0 : no agglutination; all cells free. Select the sensitised cells giving maximum titre for that antiserum and always use that cell : anti-D ratio for sensitisation with that batch of anti-D, e.g., in Table 5 the maximum anti-Gm titre is obtained with a red cell : anti-D ratio of 1 : 4. Thus, with that batch of anti-D, 4 drops
+,
+ (17)
(18) (19) (20)
of anti-D should be added to 1 drop of packed red cells for sensitisation by procedure B. Store the selected sensitised cells in the minimum volume of saline at 4°C and wash once in saline before use. Stored in this manner, sensitised cells keep satisfactorily for 24 hours. Discard the other five batches of sensitised cells. For use in the Gm typing procedure (D) prepare a pale pink suspension (+0.2%) of sensitised cells in saline.
B. Sensitisation of Red Cells using an Anti-D o f Known Tbre (1) Place 1 drop of washed, packed red cells of 'blood type 0, D + (see procedure A) in a centrifuge tube and add 1 dnop of saline. (2) Add the appropriate number of drops of anti-D as determined in procedure A for that batch of anti-D. (3) Mix and incubate at 37OC for 1; hours. (4) Wash the sensitised cells three times with saline. (5) Test the sensitised cells with anti-human globulin (procedure section H). (6) Store the sensitised cells in the minimum volume of saline at 4OC for not more than 24 hours before use. (7) For use in Gm typing, stored cells should be washed once in saline and a 0.2% suspension made. C. Titration of Anti-Gm Antisera In procedure A, for determining the titre of a new batch of anti-D, the anti-Gm antiserum titre is determined simultaneously; e.g., in the example shown in Table 5 we chose a 1 : 4 red cell : anti-D ratio. With these sensitised cells complete agglutination occurred down to an anti-Gm dilution of 1/16. At 1/32 only 90% of cells were agglutinated. The anti-Gm titre for routine use would be between these two - 1/24. New batches of anti-Gm antisera which have not been titred during procedure A should be evaluated in the following way: (1) Make doubling dilutions of anti-Gm antiserum in saline from neat antiserum through ten tubes,. preparing 2 drops of each dilution. (2) Transfer 1 drop of each dilutlon to one of a second row of ten tubes and discard the original ten tubes. (2) Add 1 drop of pale pink sensitised cell suspension to each tube. (4) Mix and leave at 4OC for 1-19 hours. (5) Centrifuge at 1000 rpm for 1 minute. This should be timed carefully so that it can be reproduced in all tests. (6) Transfer the cells to a microscope slide and examine for agglutination as before (procedure A( 16)) . (7) Select the antiserum dilution to be used in the inhibition test; e.g., in Table 6 the agglutination results for an anti-Gm antiserum titration are shown. The selected dilution must be between the last dilution giving 100% agglutination and the next, which gives about 90% agglutination. Thus, this antiserum must be used at a dilution of 116. TABLE 6 TITRATION OF AN ANTI-Gm ANTISERUM Antiserum dilution Agglutination score
: :
neat 4
112 4
1$4
1&8
1/16 2
1/32 1
1/64 11128 11256 11512 + 0 0 0
D. Gm Typing of Serum Positive and negative control sera as well as a saline control must be set up with each batch of unknown sera. Control sera may be purchased from the suppliers of Gm reagents (Appendix 2) or serum from laboratory staff of known Gm type may be used.
(1) (2)
(3) (4)
(5) (6) (7) (8) (9) (10)
Dilute all samples and controls ten times in saline. For each sample or control set up two small tubes and place 1 drop of the diluted serum in each. Place 1 drop of saline in each of another pair of tubes for the saline control. T o the first tube of each pair add 1 drop of appropriately diluted anti-Gm antiserum. T o the second tube of each pair add 1 drop of saline. These second tubes are the agglutination controls. Mix the contents of all tubes and leave at 4OC for 1-1& hours for the inhibition reaction to take place. Add 1 drop of the appropriately sensitised cell suspension to all tubes. Mix, and leave the tubes at 4OC for 1-14 hours. Spin the tubes at 1000 rpm for 1 minute in the same centrifuge as used for antiserum titration. This should be timed carefully so that it can be reproduced in all tests. Transfer the cells to a microscope slide and examine for agglutination as before (Procedure A(16)). Record the G m type of the sample. Table 7 shows some typical results. The unknown serum '4' has caused agglutination of the sensitised red cells in the agglutination control, i.e., in the absence of added anti-Gm antibody. The test should be repeated and if the same result is obtained then the sample cannot be typed by this procedure. I t is likely that the sample itself contains an antibody which is agglutinating the sensitised cells. TABLE 7 Gm TYPING OF SERUM
Sample
Agglutination Score Anti-Gm Saline Positive Control Serum 0 0 Negative Control Serum 4 0 4 0 Saline Control Unknown Serum 1 0 0 2 4 0 3 3 0 4 3 3 *See text, procedure D(10).
Result Gm (+I Gm (-1 Gm (-1 Gm (+I Gm (-1 Gm (-1 No result*
E. Gm Typing of Bloodstains I t has been established in a large trial that when bloodstains are G m typed there is no need for agglutination controls. (Khalap, Pereira and Rand, 1976). Any antibodies which may have been present in the original blood sample do not interfere in stain typing when the following methods are used. (a) Direct Inhibition Testing (1) Positive and negative control stains as well as a control (unstained) sample of each cloth involved must be set up in parallel with each batch of unknown stains. (2) Take a length of stained fibre (for Gm(1) : 0.5cm, for Km(1) and Gm(2) : l.cm, for Gm(l0) : 2cm) and cut it into three or four pieces. Take equivalent lengths of unstained fibres for controls. (3) Place the fragments from each length of fibre in the bottom of a small tube and add 1 drop of appropriately diluted anti-Gm antiserum. Ensure that the threads are submerged in the antiserum. (4) Leave the tubes at 4OC overnight (16 hours or more) for the inhibition reaction to take place. ( 5 ) Remove the threads from the tubes, squeezing out any antiserum with clean forceps. (For an alternative method of removing the threads see procedure section J).
(6)
(7) (8)
(9) (10)
(b) (1)
(2) (3)
Add 1 drop of a dilute suspension of sensitised cells in saline containing 0.3% bovine serum albumin. Mix, and leave the tubes at 4OC for 1-la hours. Centrifuge the tubes at 1000 rpm for 1 minute, timing this carefully. Transfer the cells to a microscope slide and examine for agglutination as before (procedure A(16)). Record the Gm types of the stains. If the cloth controls inhibit the agglutination then the test must be repeated, preferably using method (b) below. Extraction Techniquefor Stain Typing Take 0.5cm2 of stained fabric and extract it into 3-4 drops of saline by soaking for 3-4 hours at room temperature. Remove the fabric with forceps or, preferably, by procedure J. Simultaneously, extract similar amounts of positive and negative control stains and of unstained areas of all cloths. Use 1 drop of extract for each test without further dilution, using procedure D as for serum grouping. No agglutination controls are . required.
F. A B O Typing of Liquid Blood I t is very important that the red cells sensitised for use in the Gm typing procedure are blood group 0. Cells of group A or B might be agglutinated by anti-A or anti-B present in antisera or in samples. The ABO group of the cells to be sensitised may be checked by the following procedure: (1) Collect the whole blood (a few drops) in a tube containing anticoagulant, mix well with saline and transfer to a centrifuge tube. A graduated centrifuge tube is convenient. (2) Centrifuge to sediment the cells and discard the supernatant saline. (3) Resuspend the cells in saline at a cell concentration of about 5% v/v. (4) Prepare cells from known A, B and 0 blood in a similar manner. (5) Grouping is carried out on a flat glass plate or tile marked, with a wax pencil, into squares of about 3cm side. For each blood to be tested and for the control bloods place 1 drop of cell suspension in each of three squares. (6) T o the first square add 1 drop of anti-A antiserum, tolthe second 1 drop of anti-B antiserum and to the third 1 d r o of ~ anti-H (a lectin v r e ~ a r e d from the seeds of Ulex europaeus). (7) Mix the antisera and cell suspensions by gently rocking the tile in the hands and then allow it to stand on the bench for 5 minutes. (8) Rock the tile again and read the agglutination macroscopically scoring as described in procedure A(16). Some sample results are shown in Table 8. A
.
TABLE 8 ABO TYPING OF L I Q U I D BLOOD Sample Control A Control B Control 0 Unknown 1
2 3
Agglutination Scores Anti-A Anti-B Anti-H 4 0 3 0 4 4 0 0 4 0 0 4 4 0 2 0 4 3
Blood T y p e
A B 0 0 A B
G. Rhesus Typing of Liquid Blood The cells used for Gm typing must be of Rhesus type D + so that the sensitising anti-D antiserum will bind to the cells. The Rhesus type of the blood may be
checked using a complete anti-D antiserum (usually described as 'suitable for saline agglutination tests in tubes or on tiles') and following the procedure described for ABO typing in section F. Control D-positive and D-negative bloods must always be tested in parallel with the unknown samples. There are many antigens other than D in the Rhesus system and more than one nomenclature system. A list of Rhesus types in three nomenclature systems showing the D-positive types is given in Table 9. TABLE 9 SIMPLIFIED NOMENCLATURE OF T H E RHESUS SYSTEM
Fisher Notation CDe cDE cDe CDE Cde cdE CdE cde
Common Notation
RI R2 Ro
Rz R Rl1
Wiener Notation R1 R2 R" R r1 rl1
R~
ry
r
r
D
Positive Positive Positive Positive Negative Negative Negative Negative
H . The Anti-Human Globulin Test The simplest way of checking that the red cells for the Gm test have become coated with anti-D during the sensitisation procedure is to react the washed sensitised cells with an anti-human globulin antiserum. Since the anti-D is a human globulin it reacts with the anti-human globulin and the red cells are agglutinated. All glassware used must be scrupulously clean as the slightest trace of contaminating human protein may neutralise the antiserum giving false negative results. Dilute the anti-human globulin antiserum according to the manu(1) facturer's instructions and use the diluted antiserum immediately. Do not attempt to store diluted antiserum. (2) Place 1 drop of the washed, sensitised red cells on a flat glass plate or tile and add 1 drop of diluted anti-human globulin antiserum. (3) Set up a saline control by adding 1 drop of saline to 1 drop of the sensitised cells. (4) Mix the contents of each square on the tile using the corner of a microscope slide, a clean corner for each square. ( 5 ) Leave the tile undisturbed for 1 minute and then rock it very gently by hand. At no time must the tile be shaken. After 5-7 minutes (or the time recommended by the anti-globulin manufacturer) examine the tile for macroscopic agglutination. (6) If the drops are allowed to dry up false agglutinations may appear in the saline control as well as in other samples.
J. Extraction Technique for Stained Fabric (Baxter and Rees, 1974)
This is a convenient and efficient means of stain extraction. I t may be used to extract the Gm antigens from the stained fabric for procedure E(b) or for removing the threads from the antiserum in procedure E(a). Small plastic disposable test tubes, conveniently 1-2cm long and 0.5-0.6cm internal diameter, are required. A small hole is made in the bottom of one tube with a hot needle. This hole must be as small as possible so that liquids placed in the tube do not run out. A second, unpunctured tube is sellotaped to the first so that the hole in the bottom of the upper tube is over the top of the lower tube. The fabric to be extracted or reacted with antiserum is placed in the upper tube and saline or antiserum is added. The extraction or inhibition time is allowed to elapse and the double tube is centrifuged at low speed, 1000-2000
rpm, for a few minutes. The liquid in the upper tube is forced through the hole and collects in the lower tube. The system may also be used for multiple extractions provided that the lower tube is large enough to contain the accumulated extraction volume. The extract in the lower tube is reached by snapping the sellotape between the two tubes. This extract must always be thoroughly mixed before use. The sellotape may be replaced by a tightly fitting sleeve of plastic or rubber tubing which may then be washed and re-used. The tubes must be discarded.
Choice of Reagents I n practice the biggest single factor limiting the choice of Gm allotypes to be tested is the availability of antisera. Most anti-Gm antisera occur 'naturally' in human serum and are discovered during testing of serum for use by transfusion laboratories. Not all antisera are alike and not all antisera are equally suitable for routine Gm and K m typing. The first anti-Grrl antibodies were found in the sera of patients suffering from rheumatoid arthritis (Grubb and Laurell, 1956) but, very shortly after similar antibodies were discovered in the sera of healthy individuals (Grubb, 1959). The former were called Ragg sera (Rheumatoid agglutinators) and the latter, Snagg (Serum normal agglutinators). Ragg sera have relatively higher anti-Gm titres than do Snagg but with frequent changes in this titre with time in the one individual. Snagg sera have lower but fairly constant titres. Frequently a distinct prozone eflect is observed with Ragg sera such that their agglutinating ability increases with dilution, at first, before decreasing at higher dilution in the normal manner. Such a prozone is not observed with Snagg sera. The anti-Gm antibodies of Ragg sera may be readily inhibited non-specifically by certain levels of globulins. Snagg sera are only inhibited by the specific Gm factor. Ragg antisera often contain antibodies of several different specificities in the one serum whereas Snagg sera are monospecific. Snagg antisera only arise in individuals whose serum is negative for the factor against which the antibody is directed. With Ragg sera there may be simultaneous presence of the anti-Gm antibody and the corresponding G m factor. Thus with Ragg antisera errors can occur in the determination of Gm types and for routine work Snagg antisera must always be used. Much valuable work has been done with Ragg sera but their usefulness is generally restricted to specialist structural and quantitative studies. Snagg antisera are not very coirlmon. For example, in 1,200 non-rheumatoid sera Ropartz et al. (1960) reported nine anti-Gm(1) antisera. I n 1965 were found eighteen anti-Gm(l), nine anti-Gm(6) and two anti-Gm(2) antisera among 13,000 healthy sera. In all instances the antiserum donor is assumed to have been immunized against the Gm or K m allotype. This may be due to 'autoimmunisation' in rheumatoid patients, alloimmunisation by plasma immunoglobulins in patients receiving multiple transfusions (Allen and Kunkel, 1963) and alloimmunisation of the foetus by the mother or of the mother by the foetus during pregnancy, (Steinberg and Wilson, 1963; Fudenberg and Fudenberg, 1964). Antisera have also been raised by immunising primates or rabbits but such sera are not readily available. (Litwin and Kunkel, 1966; Hess and Butler, 1962). The anti-D antiserum used to sensitise the red cell in the Gm test must also be carefully selected. This procedure is carried out by the antiserum retailers so that, provided an anti-D is always used with the specific anti-Gm antiserum marketed by the same manufacturer, the reagent pair will give reliable results in serum typing. For bloodstain typing reagents must be evaluated locally using stains of known type. The red cell coating antisera does not have to be anti-D. Any incomplete antiserum (i.e., one which will sensitise the red cells rather than agglutinate them) which is positive for the specific Gm or K m factor may
be used. Anti-Duffy antisera have been shown to be quite satisfactory, but anti-D are the commonest and the only ones which are widely available. T o choose the optimum groups for testing from the reagents which are available (for suppliers see Appendix 2) requires more knowledge of the structure and inheritance of these immunoglobulin antigens.
Further Structural Considerations Immunoglobulins exist as a number of types: IgG, IgA, IgM, IgD and IgE which differ in the structure of their heavy chains called gamma (y), alpha (a), mu (p), delta (8) and epsilon (E) respectively. This has already been discussed under Immunoglobulin Structure and Function. Each of the different immunoglobulin types can be further subdivided, by their immunological properties, into subclasses. IgG, for example, exists as four subclasses : IgG1, IgG2, IgG3 and IgG4 which differ in the antigenic properties of their heavy chains called yGl? yG2, yG3 and yG4, respectively. Since each of these y chains is found in association with both K and A chains there are eight types of IgG molecule which are present in the serum of all normal individuals. The relative amounts of the various heavy chain subclasses is approximately 71% yG1, 18% yG2, 8% yG3 and 3% yG4. The ratio of IgG molecules with K chains to those with A chains is approximately 2 : 1. The different heavy chains have different amino acid sequences and thus it is not surprising that the disulphide bonds linking the polypeptide chains are in different positions in the different IgG subclasses. The constant regions of the heavy chains are made up of several linearly arranged regions whose amino acid sequences are sufficiently similar as to suggest that they all evolved from a single short polypeptide chain. In y heavy chains there are three such regions, called homology regions (Figure 4) so that we can imagine y chains to have been formed in an evolutionary sense from four shorter polypeptide chains joined end to end. Three of these are similar (the homology regions) and form the constant region and the fourth is the variable region. The three homology regions are called CH1, C H 2 and CH3 counting from the amino terminus of the polypeptide chain (Figure 4). Each homology region is folded into a loop held in place by a disulphide bond across its base (Figure 4). In the intact IgG molecule the homology regions of the different chains appear to interact closely with their counterparts on other chains, thus stabilising the structure. Gm and K m antigens are found on the constant regions of immunoglobulin polypeptide chains. Km antigens are associated with K chains (Km = Kappa marker) and so are found on all classes of immunoglobulins. Gm antigens are associated with y chains (Gm = gamma marker) and so are found only on IgG molecules. Each of the Gm markers is confined to one of the IgG subclasses so that, for example, Gm(1) is found only on IgGl molecules, Gm(23) on IgG2 molecules and Gm(5) on IgG3 (Table 10). In some cases the amino acid sequence associated with a particular antigen has been identified. For example, if amino acid 191 on human kappa chains is leucine the molecule has Km(1) specificity but if amino acid 191 is valine the molecule has Km(3) specificity. Similarly, on yG1 chains of IgGl a certain sequence of amino acids at positions 355-358 is associated with Gm(1) activity. Arg-Asp-Glu-Leu is Gm(1) positive while the only known alternative sequence at this position, Arg-Glu-Glu-Met, is Gm(1) negative. This alternative sequence is also found in the corresponding position on yG2 and yG3 heavy chains and is called the Gm (non-1) marker. Thus IgGl molecules can be Gm(1) or Gm (non-1) but IgG2 and IgG3 molecules can only be Gm (non-1). yG4 chains have a different non-marker sequence in this position and this is called Gm (4 non-1). The non-markers are not useful for genetic studies or for discrimination purposes as they are always present in all normal individuals. Gm(1) and Gm (non-1) on yG1 chains are called homoalleles. Not all pairs of homoalleles are com-
Figure 4. Homology regions of an IgG molecule. Key: V = Variable region C = Constant region L = Light chain H = Heavy chain
posed of a marker and a non-marker. Grn(3) on yG1 chains, for example, has Gm(17) as its homoallele and neither of these markers are found on other IgG subclasses. The homoalleles of all markers are not yet known. For example, an alternative allele to Gm(2) has not yet been discovered. The Gm terminology currently recommended by the W.H.O. Committees on Immunoglobulin Allotypes lists the antigens in order of heavy chain subclasses yG1, yG2, yG3 and writes complete phenotypes such that the two haplotypes of the individual are irrimediately obvious. The phenotype G m ( l ) , for example, is the product of an allele coding for a part of the yG1 chain. The allotype is written Glm(1). Thus, a test for the allotype Glm(1) may have the result Gm(1) when the allotype is present or the result Gm(-1) when it is not. Similarly a positive result for Gm(5) indicates the presence of the allotype G3m(5). A Gm haplotype is written simply in order of heavy chain subclasses with the antigens written as superscripts, e.g., Gm13293823*5913914 or Gm-1-2s A phenotype is written in subclass and haplotype order, e.g., Gm(1,2,17,3,23,6,15,21,24,5,10,11,13,14,26,27) where Gm1z2.l7and Gm3 are the two haplotypes of the yG1 subclass. A partial phenotype can be expressed as Gm(1,2,3,-14) if one only tested for Glm(1,2,3) and G3m(14). I t is, of course, possible on theoretical grounds to predict which combination of Gm markers will be of most use to a forensic laboratory, giving the maximum chance of discrimination between samples for the minimum number of tests. 332395p13914.
TABLE 1 0 STRUCTURAL RELATIONSHIP OF THE Gm ALLOTYPES
Current Gm Nomenclature 1 2 3 5 6
7 8 9 10 11
Heauy Chain Subgroup
YG1 YG1 YG1 YG3 YG3 771 yG1
yG3 vG3
$'I ?
Heauy Chain Fragment Fc Fc Fa b Fc Fc Fc
Homology Region CH3 CH3 CH 1 CH2 CHY
?
Fc E'c Fc
Fab ? ?
CHI ? ?
A consideration of the commonly found Gm phenotypes in the population from which the samples were drawn will indicate the optimum antigens for study. I t is evident from Appendix 4 that the British population can be differentiated into eight common phenotypes by testing for four Gm allotypes Glm(1,2,3) and G2m(23). Where a group of antigens are almost invariably linked, tests for more than one of the group are unlikely to provide any extra information. I n the British population Glm(3) and G3m(5,10,11,13,14) are linked in the haplotype Gm3.5916911913,14and so, in samples from a British population, only one of these markers need be tested. Since yG1 markers are generally present in serum at higher concentrations than yG3 markers, Glm(3) is the obvious choice. Future Prospects The biggest single problem facing forensic biologists wishing to do Km and G m typing on blood stains is the availability of suitable antisera. This is likely to remain a problem unchanged even with the discovery of new Gm and K m groups, until production of antisera by immunisation of laboratory animals is undertaken on a commercial scale. Although Gm and K m factors in blood have been studied for over twenty years, in other body fluids they have received very little attention probably because of the problems encountered by early workers (Klose and Schraven, 1962; Kramer 1963). Theoretically, any body fluid which contains immunoglobulin must contain Gm and K m antigens. The currently used detection techniques were all devised for use with blood serum and body secretions have much lower immunoglobulin levels than does serum. The levels of immunoglobulins found in some body fluids are given in Table 11. Km(1) typing, which depends on the total immunoglobulin level, can be carried out on blood diluted at least 100 and sometimes 1000 times. I t is, therefore, to be expected that one could Km(1) type most body fluids and perhaps even sweat. Km(1) typing of saliva and semen have been reported (Davie and Kipps, 1976; Jorch and Oepen, 1977). Gm typing however,
TABLE 1 1 IMMUNOGLOBULIN LEVELS IN BODY FLUIDS
Body Fluid
Immunoglobulin level mg/100 ml 1.G ZgA ZgM 1200 260 90 2 20 1 15 150 1 20 2 ? 14 21 44 3 1 ?
Scrum Saliva Nasal Secretion Semen Vaginal Secretion Sweat
References: Waissbluth and Langman, 197 1 . Brandtzaeg, Fjellanger, Gzeruldsen, 1970. Blenk et al., 1974. TABLE 12 Gm AND Km ANTIGENS IN BODY FLUIDS
Body Fluid Km(1)
+ + +
Gm(1)
+ + + +
Gm(2) 0
Antigen Gm(3) G m ( 5 ) G m ( l 0 ) Gm(14) Gm(21) 0 0 0 0 0 0 0
Saliva Nasal Secretion Semen Vaginal Secretion 0 Sweat 0 0 0 0 0 0 0 + : detected in that body fluid but not yet reliably enough for routine typing. 0: either not yet detected or not yet tested for.
+
+ + +
+
$
+
1
+
+ + 0 0
depends only on the IgG fraction and although (from the data in Table 12) one might expect to be able to Gm type nasal secretion, semen and vaginal secretion, saliva and sweat would appear to be too dilute. Jorch and Oepen (1977) have reported Gm factors in semen and nasal secretion, and work a t the Home Office Central Research Establishment, Aldermaston, England, has shown that Gm(1) may be unreliably detected in both saliva and vaginal secretion (unpublished results). Table 12 shows the various Gm and K m antigens which have been detected in body secretions, but in no case has the detection system been shown to be reliable. For Gm(1) in saliva, for example, this unreliability may well be due to insufficiently sensitive detection systems but for Gm and K m markers in semen this is an unlikely explanation. Agglutination systems are very sensitive to the protein and ionic concentration of their suspension media (Pollack 1965). Body secretions particularly when undiluted, are thus far from ideal fluids in which to measure degree of agglutination. False agglutinations and false negative results are very common features of Gm and K m typing of body fluids and much more basic methodological research is necessary before this extremely valuable and flexible typing system can be recommended for routine typing of fluids other than blood.
References ALLEN,J. C. and KIJNKEL, H. G., 1963, Antibodies to genetic types of gamma globulin after mu1tiple transfusions. Science, 139, 4 18. BAXTER,S. J. and REES,B., 1974, Simultaneous haptoglobin and haemoglobin typing of blood and bloodstains using gradient polyacrylamide gel electrophoresis, Med. Sci. Law, 14, 231-236. BLANC,M. and GORTZ,R., 1971, Identification of a new factor Gm 'Bet' in blood stains. Application in forensic medicine, Vox Sang, 20, 263-266. BLANC,M., GORTZ,R. and Ducos, J., 1973, Identification d'un tache de sang minime g r k e B la mise en tvidence des facteurs Inv (1) et Inv (2) au cours d'une expertise mtdico-1Cgale. C. R. Skances Soc. Biol. Ses. Fil., 167, 777-780 43
BLENK,H., HOFSTETTER, R., BOWERING, R., BUTTLER, R., HARTMANN, M. and MARX,F., 1974, Immunelektrophorese des Ejakulats, Miinch. med. Wschr. 116, 35-38. BRANDTZAEG, P., FJELLANGER, I. and GZERULDSEN, S., 1970, Human secretory immunoglobulins. I : Salivary secretions from individuals with normal or low levels of serum immunoglobulins, Scand. 3.Haemat., Supkl., 12, 1-83. BRAZIER, D. M. and GOLDSMITH, K . L. G., 1968, Frequency of certain Gm and Inv factors in the United Kingdom, Nature, 219, 193. BROCTEUR, J. and MOUREAU,P., 1964, U n cas d'application pratique des l'identification i de taches de sang, Ann. mid. lbg., groupes plasmatiques Gm ? 44, 315-321. CIJLLIFORD, B. J., 1971, The examination and typing of bloodstains in the crime laboratory, U.S. Dept. of Justice, Washington D.C. 20402. DAVIE,M. J. and KIPPS,A. E., 1976, Km(1) (Inv (1)) typing of saliva and semen, Vox Sang., 31, 363-367. Drrcos, J., R U F F I ~J., and VARSI,M., 1962, Mise en Cvidence des antigtnes Gma, Gmb, Gmx dans les taches de sang sec, Vox Sang., 7, 722-731. Ducos, J., R U F F I ~J ,. and VARSI,M., 1964, Une nouvelle application du systtme Gm : l'identification des taches de sang sec., Proc. 9th Congr. int. Soc., Blood Transf. Mexico, 1962, pp. 478-482. Ducos, J . , R U F F IJ. ~ , and VARSI,M., 1965, Efficiency of searching Gm antigens for identification of bloodstains in forensic medicine, Nature (Lond.), 205, 1332-1333. EDELMAN, G. M. and BENACERRAF, B., 1962, O n structural and functional relations between antibodies and proteins of the gamma system, Proc. Nut. Acad. Sci. U.S., 48, 1035-1042. FIIDENBERG, H. H. and FUDENBERG, B. R., 1964, Antibody to hereditary human gamma globulin (Gm) factor resulting from maternal-fetal incompatibility, Science, 145, 170-1 7 1. FUNFHAUSEN, G. and SAGEN, Z., 1961, Uber die Moglichkeit des Nachweises der Gruppeneigenschaft Gm in Blutspuren, Deutsch. Gesch. Wes., 16, 52, 2468-9. GALLY, J. A., 1973, Structure of immunoglobulins p. 161-298 in 'The Antigens' Vol. I, Ed M. Sela Academic Press. GOHLER,W. and HUNGER,H., 1963, Der Nachweis von Gm und InvEigenschaften in Blutspuren., Zschr. Aerztl. Fortbild, 58, 794-795. GRITBB, R., (1956), Agglutination of erythrocytes coated with 'incomplete' anti-Rh by certain rheumatoid arthritic sera and some other sera, Acta Pathol. Microbiol. Scand., 39, 195-197. G R ~ B BR., , 1959, Ciba Foundation Symposium 264. GRIJBB,R. and LAIJRELL, A. B., 1956, Hereditary sero\ogical human serum groups. Acta Pathol. Microbiol. Scand., 39, 390-398. HESS,M. and BUTLER, R., 1962, Anti-Gm specificities in sera of rhesus monkeys immunised with human gamma globulin, Vox Sang., 7, 93. JONES,D. A., 1972, Blood samples: probability of discrimination, 3.Forens. Sci. soc., 12, 355-359. JORCH,G. and OEPEN,I., 1977, Nachweis der Faktoren Gm (1,2,4,5,21) und Inv (1) in menschlichen Sekreten: Speichel, Schweiss, Nasensekret und Sperma, Z. Rechtsmedizin, 79, 1-6. KHALAP, S., PEREIRA, M. and RAND,S., 1976, Gm and Inv grouping of bloodstains, Med. Sci. Law., 16, 40-43. KLOSE,I . and SCHRAVEN, J., 1962, Die Eigenschaft Gma im menschlichen Sperma und ihre Beziehung zur ABO Ausscheidereigenschaft, Dt. 5.ges gericht. Med., 52, 610. KRAMER, K., 1963, Untersuchungen iiber das Vorkommen von Gma Substanz in menschlichen Geweben und extravasalen Korperfliissigkeiten, 3. gericht. Med., 53, 131-141. 44
LENOIR,L. and MULLER,P. N., 1966, Determination des groupes Gm sur taches de sang, Ann. mid. lig., 46, 191-193. LITWIN,S. D. and KUNKEL, N. G., 1966, Genetic factors of human gamma globulin detected by rabbit antisera, Transfusion, 6, 140. K., 1963, Experimental studies on the NIELSEN,J. C. and HENNINGSEN, determination of the Gm groups in bloodstains, Med. Sci. Law, 3,49-58. PLANQUES, J., R U F F I ~ J., and Ducos, J., 1961, Le systkme Gm en mtdecine ltgale. Toulouse Med., 62, 685-699. POLLACK, W., 1965, Ann. N.Y. Acad. Sc ., 127, 892. PORTER,R. P., 1959, Biochem. J., 73, 119. PROKOP, 0.and BYRDY, M., 1965, 'Aktuelle Fragen der geritchlichen Medizin', Ursg. M. Varmosi. 0 . and UHLENBRUCK, G., 1969, 'Human Blood and Serum Groups', PROKOP, Maclaren and Sons Ltd., London. ROPARTZ,C., LENOIR, J., HEMET,Y. and RIVAT,L., 1960, Possible origins of the anti-Gm sera, Nature, 188, 1120-1 121. ROPARTZ, C., LENOIR, J. and RIVAT,L., 1961, A new inheritable property of human sera: the InV factor, Nature (Lond.), 189, 586. SAGEN, A. and FUNFHAUSEN, G , 1965, Gm group characteristics in bloodstains, Arch. Irnrnunol. Ther. Exp., 13, 149-156. R. and WAINWRIGHT, P., 1978, Gm and Kni polymorphism in man, STEDMAN, Biotest Bulletin, 4, 3-14. STEINBERG, A. G. and WILSON, J. A., 1963, Hereditary globulin factors and immune tolerance in man, Science, 100, 303-304. J. G. and LANGMAN, M. J. S., 1971, ABO blood groups, secretor WAISSBLUTH, status, salivary protein and serum and salivary immunoglobulin concentrations, Gut, 12, 646-649. Acknowledgements This monograph was inspired by and based on a symposium on Gm and K m typing of body fluids held in July, 1976, at the Home Office Central Research Establishment, Aldermaston, England. The credit for its contents must therefore go to those who organised and took part in the symposium - in particular to Mr. P. Martin, Dr. S. Khalap and Mrs. M. Brady of the Metropolitan Police Forensic Science Laboratory and to Dr. P. H . Whitehead of the Home Office Central Research Establishment. Thanks for assistance and advice are also due to Mr. S. S. Kind, who sugiested the writing of the monograph, and helped at every stage that followed, to Miss M. Pereira and Dr. L. King of the Home Office Central Research Establishment, to Miss D. Brazier of the World Health Organisation Blood Group Reference Laboratory, London, and to the Information Division of the Home Office Central Research Establishment who tirelessly sought references as required. APPENDIX 1 DISCRIMINATING POWER Discriminating Power (Jones, 1972) is defined by the expression: DP = 1-P where P is the probability of two random samples matching. P is calculated from the expression: P = ~ , ~ + p ~ ~ + p , ~ + . .pn2 . - . . . where p,, p,, p, . . . . . . p, are the proportions of the population being studied in each class 1, 2, 3 . . . . . . n. Consider the following examples: 1. 111 the Metropolitan Police area the ABO blood grouping system divides the population as fbllows: Class ABO type P 1 A 0.42 2 BL 0.09 3 A'B 0.03
The probability of a match, using this system, is given by: 0.462 P = 0.4Z2 + 0.092 + 0.03' = 0.1764 + 0.0081 + 0.0009 + 0.2116 = 0.3970 Thus the probability of being able to distinguish between two samples using this test alone is DP = 0.603.
+
2.
In the adenylate kinase (AK) grouping system the class frequencies are as follows: Class Type P 1 AK I 0.912 2 AK2-1 0.087 3 AK2 0.001 P = 0.912' + 0.0872 + 0.0012 = 0.84 DP = 0.16 APPENDIX 2 COMMERCIAL SOURCES O F Gm AND I
Behring Hoechst Pharmaceuticals, Hoechst House, Salisbury Road, Hounslow, Middlesex, England. Telephone: 01-570-77 12 Biotest Folex Limited, 1649 Pershore Road, Birmingham B30 3DR, England. Telephone: 02 1459-2 112 Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, Postbus 9 190, Amsterdam. Dade, A. R. Horwell Limited, 2 Grangeway, Kilburn High Road, London NW6 2BP. England. Telephone: 01-328-1551 Dr Molter GmbH, 69 Heidelberg 1, Postfach 104049, West Germany. APPENDIX 3 I
Number of Samples 43
Percentage of popu!atiou Km(1) Km(2) Km(3) 58
African negroes (Mauritania) Amcricans (White) 295 American negroes 189 British2 1000 Chinere 507 Czechs 190 Dutch 354 Filipinos 126 Finns 162 French 339 Germans 968 Greeks 297 Iranians 297 Japanese 68 1 Jugoslavs 157 Swiss 600 'Prokop and Uhlenbruck. 1969. 2Brazier and Goldsmith, 1968.
18
58 17 13
18
46
14 61 16 56 12 16 51 11 16 12 17 12 48 15
100 83
APPENDIX 4 G m POPULATION FREQUENCIES* Phenotype 1
12
1,13 1,2,13 1,5,6 1,2,5,6 1,5,6,13 1,5,14 1,2,5,14 1,5,6,14 1,2,5,6,14 1,5,6,13,14 1,5,13,14 1,2,5,13,14 3,5,13,14
Greek
Indian
American Negro
3 7 -
-
3 1
14 16 6 3
5
3 1 2 i 1 8 1 2 1 <1 1 <1 24 27 3 2
8 13
1 9
<1
-
20 5
<1
4
-
-
-
-
-
-
-
-
<1
<1 <1 <1 A
7
-
-
A
1
<1
African Negro (South) <1
Afrian Negro (West) i 1
British
A
-
5 2 3 1
<1
6 -
1 -
32 48
35 52
-
10 A
<1
1
-
-
-
-
Chinese (Hong Kong) 2 1 1
<1
-
A
-
63 3,23,5,13,14 1,3,5,13,14
35 8
29
1,3,23,5,13,14 1,2,3,5,13,14
22 4
-
90
-
3
1,2,3,23,5,13,14 12 8 1 1,3,5,6,13,14 7 1,3,23,5,6,13,14 1 3 Frequency (%) o f phenotypes in various races based on G3m(5,6,13,14). ( T h e generic term G m has been omitted). *Stedman and Wainwright, 1978.
<1 tests
6
-
for Glm(1,2,3), G2m(23),