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Quantitative IgD measurement for discrimination of human bloodstains
Forensic Science International, Elsevier
Scientific
Publishers
31 (1986) Ireland
73-78
73
Ltd.
QUANTITATIVE IgD MEASUREMENT HUMAN BLOODSTAINS
W. KEILa
and MARIE-LOUISE
DISCRIMINATION
OF
GLOWATZKIb
‘Institute for Forensic Medicine, bZnstitute for Forensic Medicine, (Received (Accepted
FOR
Hum bold t University, 1040 Berlin (G. D. R.) and Berne University, 3012 Berne (Switzerland)
August 15, 1985) December 3, 1985)
Summary The IgD concentrations of eluates of artificial bloodstains and of the corresponding sera from 40 subjects of different ages were measured by single radial immunodiffusion. IgD was found in bloodstains stored up to 53 days, i.e. IgD storage stability is sufficient for forensic purposes. Since serum IgD concentrations of individuals, in particular of adults, are almost invariable and serum levels of different individuals can vary by more than a lOOO-fold, the discrimination of bloodstains on the basis of IgD is generally possible. Thus IgD constitutes a further marker in antibody profiling of bloodstains.
Key words:
IgD; Bloodstain
analysis;
Antibody
profile
Introduction
In 1974 King introduced the investigation of antibody profiles into bloodstain analysis [ 11, Since then it has been the IgG and IgE antibodies in particular that have been used against various antigens in practical forensic work [2,3]. Antibodies of the IgD class have, as far as we know, not yet been investigated in bloodstains. However, the serum IgD concentration displays certain properties which could make its application in forensic practice possible: -
The serum IgD level of a particular adult is almost invariable, with exceptional changes occurring only in cases of illness and towards the end of a pregnancy [cf. 41. It has been found in population investigations that the serum IgD level of different individuals can vary by more than a lOOO-fold. The normal adult values range from 0.14 to 400 mg/l and are apparently subject to a trimodal distribution [5,6]. That is, about 10% of adults have very low serum IgD concentrations (<3 mg/l), 10% have high concentrations (>lOO mg/l) and the others have medium concentrations [4,7]. This
0379-0738/86/$03.50 0 1986 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
74
trimodal distribution is obviously genetically influenced, but it has not yet been fully accommodated by genetic theory [6,8,9]. IgD concentrations slowly increase up to adult values during childhood, yet, there may be big differences between individual children even at an early stage [9,10]. The concentrations of newborns are about 0.08 mg,/1[9]. These two phenomena - the almost total invariability of individual serum IgD levels and the large variation of these levels among the population, encouraged us to experiment with quantitative IgD measurements for bloodstain discrimination. Materials and methods Bloodstains
Samples were obtained from 40 subjects (cf. Table 1); the newborns, the adults and five children were apparently healthy while five children suffered from diseases which have, according to the literature, no influence on IgD level. Blood samples were prepared on cotton substrate, air-dried and stored at 22°C until investigation dates. Stain eluates
The stain elution for IgD measurement was carried out with the necessary minimum quantity of distilled water, overnight, at 4°C. Before IgG meausrement, the eluates were further diluted with PBS (1: 5) in order to produce a sufficient quantity of liquid.
Sera
For comparison, sera were sampled along with the stain preparation. Storage was at -18°C. Examination of sera was carried out together with that of stain eluates. Single radial immunodiffusion
IgD partigen plates and IgD standard were purchased from Behring, F.R.G. Almost all tests were carried out according to the directions of the manufacturer. The only exception was when stain eluate samples of 0.03 ml were used at the beginning (after standing for 20 min, the initial 0.02 ml was supplemented by a further 0.01 ml). The IgD concentrations measured in 0.03 ml samples were adjusted so that they could be directly compared with the 0.02 ml sample concentrations. After the first successful IgD determinations in stain eluates, 0.02 ml eluate samples were generally used. Several plates were stained with Amido black after drying. Sensitivity was at 10.0 mg IgD/l. Along with the IgD determination, quantitative IgG were carried out in sera and stain eluates using the same technique. Determining the sensitivity for IgG tests was unnecessary, because the eluates always contained sufficient IgG. Standard deviation in IgD and IgG determination (within one test) was 4.9.
75 TABLE
1
QUANTITATIVE BLOODSTAINS
Su bjec ts
(n)
DETERMINATION OF IgD IN ELUATES AND IN CORRESPONDING SERA
Age (yeam)
(10)
-
Children
(10)
l-13
Adults
(20)
21-_j7
Storage time of bloodstains (days)
IgD-concentrations
(mg/l)
Serum
Elua te’
10x
Newborns
OF STORED
2x 1x 7x
8X
10x
53-55
2x
20-53
8x
9-14
Results results of the quantitative IgD measurement are summarized in Table 1. Our investigations of the bloodstains of seven children and five adults demonstrated that IgD is generally measurable in such material. A section of an original plate is shown in Fig. lb. In the material investigated IgD could be measured after a storage period of 53 days. When this period was doubled, the IgD content of the same stains was no longer measurable, i.e. was lower than 10 mg/l. The serum IgD concentration values fall, as expected, into very different ranges. Due to this variation in concentration and the sensitivity of the investigation technique used (10 mg/l), the subjects can be classified in three groups (cf. also Figs. la and lb): The
(1) clearly measurable serum IgD concentrations (seven children, five
adults) so that the stain IgD content could also be easily demonstrated; (2) serum IgD concentrations little above the sensitivity of the investigation technique used (one child, seven adults), thus the stain IgD content could not be demonstrated because of the relative dilution of the eluates; (3) serum IgD concentrations below the sensitivity of the investigation technique (ten newborns, two children, eight adults) so that IgD measurement in the stains was impossible. The dilution ratio between serum and eluate was usually 1: 3, and only in rare cases was an even lesser dilution of the eluates achieved. Thus it was not possible to measure the IgD contents of stains originating from subjects whose serum IgD concentration was below 30 mg/l, because sensitivity was not reached. When the IgD concentration was measurable both in serum and
76
Fig. 1. Single radial immunodiffusion for IgD concentration determination. Adult samples stained with Amido black. (a) sera (sample volume of 20 ~1) (b) corresponding stain eluates (sample volume of 30 ~1, storage period of 7-12 days). 1 and 2: IgD level of (a) and (b) clearly measurable. 3: IgD level of (a) little above sensitivity; IgD of (b) not measurable. 4: IgD level of (a) below sensitivity; IgD of (b) not measurable.
eluate the values obtained served to determine the dilution ratio between serum and stain. In all other cases the dilution ratio was determined with the help of the IgG values. Discussion Several authors have claimed that IgD, like IgE, is a comparatively unstable immunoglobulin and tends towards “spontaneous” proteolysis in serum
77
samples, but this claim has not remained unchallenged [4-6]. Our results obtained from stains would suggest that IgD is not particularly prone to disintegration: IgD could be measured in bloodstains after periods of several weeks without noticeable loss of activity. This also corresponds with results obtained from IgE tests in bloodstains: IgE could be easily measured after storage periods of 2-3 weeks [ 111; it is not until after 6 weeks that half of the original IgE concentration in artificial bloodstains may have disintegrated [12]. Although a systematic investigation into IgD disintegration rates in bloodstains has yet to come, storage stability is, according to our findings, sufficient for practical forensic purposes. In numerous cases, the almost total invariability of serum IgD concentrations of individuals, particularly of adults, and the more than lOOO-fold variation of these concentrations among the population provide a basis for discriminating bloodstains.The best conditions for discriminating stains of two individuals occur when one individual has a clearly measurable serum IgD concentration and the other has an IgD level below the sensitivity of the investigation technique used (
247-253. H.L.
Spiegelberg,
The structure
and biology
of human
3-24. H. Mauch,
Immunglobulin
D. DMW 106
(1981)
686-689.
IgD. Zmmunol.
Rev., 37 (1977)
6 S.L.
7 8 9 10 11
12 I3
Dunnette, G.J. Gleich, R.D. Miller and R.A. Kyle, Measurement of IgD by a double antibody radioimmunoassay: demonstration of an apparent trimodal distribution of IgD levels in normal human sera. J. Immunol., 119 (1977) 1727-1731. D.S. Rowe and J.L. Fahey, A new class of human immunoglobulins II. Normal serum IgD. J. Exp. Med., 121 (1965) 185-199. S.L. Dunnette, G.J. Gleich and R.M. Weinshilboum, Inheritance of low serum immunoglobulin D. J. Clin. Invest., 62 (1978) 248-255. S.K. Lee, J.D. Metrakos, K.R. Tanaka and D.C. Heiner, Genetic influence on serum IgD levels. Pediatr. Res., 14 (1980) 60-63. T. Berg and S.G.O. Johansson, Immunoglobulin levels during childhood, with special regard to IgE. Acta Paediat. Stand., 58 (1969) 513-524. D.J. Werrett and L.A. King, Application of allergy diagnosis in forensic serology. J Forensic Sci., 22 (1977) 763-770. D.J. Werrett, L.A. King and P.H. Whitehead, The detection of allergen-associated antibodies in bloodstains. J. Forensic Sci. Sot., 16 (1976) 121-126. M.-L. Glowatzki and W. Keil, IgD concentration: a marker in bloodstain analysis. 1 I th Jnt. Congr. Sot. Forensic Haemogenetics, Copenhagen, 1985, Ref. Vol., in press.
Scientific
Publishers
31 (1986) Ireland
73-78
73
Ltd.
QUANTITATIVE IgD MEASUREMENT HUMAN BLOODSTAINS
W. KEILa
and MARIE-LOUISE
DISCRIMINATION
OF
GLOWATZKIb
‘Institute for Forensic Medicine, bZnstitute for Forensic Medicine, (Received (Accepted
FOR
Hum bold t University, 1040 Berlin (G. D. R.) and Berne University, 3012 Berne (Switzerland)
August 15, 1985) December 3, 1985)
Summary The IgD concentrations of eluates of artificial bloodstains and of the corresponding sera from 40 subjects of different ages were measured by single radial immunodiffusion. IgD was found in bloodstains stored up to 53 days, i.e. IgD storage stability is sufficient for forensic purposes. Since serum IgD concentrations of individuals, in particular of adults, are almost invariable and serum levels of different individuals can vary by more than a lOOO-fold, the discrimination of bloodstains on the basis of IgD is generally possible. Thus IgD constitutes a further marker in antibody profiling of bloodstains.
Key words:
IgD; Bloodstain
analysis;
Antibody
profile
Introduction
In 1974 King introduced the investigation of antibody profiles into bloodstain analysis [ 11, Since then it has been the IgG and IgE antibodies in particular that have been used against various antigens in practical forensic work [2,3]. Antibodies of the IgD class have, as far as we know, not yet been investigated in bloodstains. However, the serum IgD concentration displays certain properties which could make its application in forensic practice possible: -
The serum IgD level of a particular adult is almost invariable, with exceptional changes occurring only in cases of illness and towards the end of a pregnancy [cf. 41. It has been found in population investigations that the serum IgD level of different individuals can vary by more than a lOOO-fold. The normal adult values range from 0.14 to 400 mg/l and are apparently subject to a trimodal distribution [5,6]. That is, about 10% of adults have very low serum IgD concentrations (<3 mg/l), 10% have high concentrations (>lOO mg/l) and the others have medium concentrations [4,7]. This
0379-0738/86/$03.50 0 1986 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
74
trimodal distribution is obviously genetically influenced, but it has not yet been fully accommodated by genetic theory [6,8,9]. IgD concentrations slowly increase up to adult values during childhood, yet, there may be big differences between individual children even at an early stage [9,10]. The concentrations of newborns are about 0.08 mg,/1[9]. These two phenomena - the almost total invariability of individual serum IgD levels and the large variation of these levels among the population, encouraged us to experiment with quantitative IgD measurements for bloodstain discrimination. Materials and methods Bloodstains
Samples were obtained from 40 subjects (cf. Table 1); the newborns, the adults and five children were apparently healthy while five children suffered from diseases which have, according to the literature, no influence on IgD level. Blood samples were prepared on cotton substrate, air-dried and stored at 22°C until investigation dates. Stain eluates
The stain elution for IgD measurement was carried out with the necessary minimum quantity of distilled water, overnight, at 4°C. Before IgG meausrement, the eluates were further diluted with PBS (1: 5) in order to produce a sufficient quantity of liquid.
Sera
For comparison, sera were sampled along with the stain preparation. Storage was at -18°C. Examination of sera was carried out together with that of stain eluates. Single radial immunodiffusion
IgD partigen plates and IgD standard were purchased from Behring, F.R.G. Almost all tests were carried out according to the directions of the manufacturer. The only exception was when stain eluate samples of 0.03 ml were used at the beginning (after standing for 20 min, the initial 0.02 ml was supplemented by a further 0.01 ml). The IgD concentrations measured in 0.03 ml samples were adjusted so that they could be directly compared with the 0.02 ml sample concentrations. After the first successful IgD determinations in stain eluates, 0.02 ml eluate samples were generally used. Several plates were stained with Amido black after drying. Sensitivity was at 10.0 mg IgD/l. Along with the IgD determination, quantitative IgG were carried out in sera and stain eluates using the same technique. Determining the sensitivity for IgG tests was unnecessary, because the eluates always contained sufficient IgG. Standard deviation in IgD and IgG determination (within one test) was 4.9.
75 TABLE
1
QUANTITATIVE BLOODSTAINS
Su bjec ts
(n)
DETERMINATION OF IgD IN ELUATES AND IN CORRESPONDING SERA
Age (yeam)
(10)
-
Children
(10)
l-13
Adults
(20)
21-_j7
Storage time of bloodstains (days)
IgD-concentrations
(mg/l)
Serum
Elua te’
10x
Newborns
OF STORED
2x 1x 7x
8X
10x
53-55
2x
20-53
8x
9-14
Results results of the quantitative IgD measurement are summarized in Table 1. Our investigations of the bloodstains of seven children and five adults demonstrated that IgD is generally measurable in such material. A section of an original plate is shown in Fig. lb. In the material investigated IgD could be measured after a storage period of 53 days. When this period was doubled, the IgD content of the same stains was no longer measurable, i.e. was lower than 10 mg/l. The serum IgD concentration values fall, as expected, into very different ranges. Due to this variation in concentration and the sensitivity of the investigation technique used (10 mg/l), the subjects can be classified in three groups (cf. also Figs. la and lb): The
(1) clearly measurable serum IgD concentrations (seven children, five
adults) so that the stain IgD content could also be easily demonstrated; (2) serum IgD concentrations little above the sensitivity of the investigation technique used (one child, seven adults), thus the stain IgD content could not be demonstrated because of the relative dilution of the eluates; (3) serum IgD concentrations below the sensitivity of the investigation technique (ten newborns, two children, eight adults) so that IgD measurement in the stains was impossible. The dilution ratio between serum and eluate was usually 1: 3, and only in rare cases was an even lesser dilution of the eluates achieved. Thus it was not possible to measure the IgD contents of stains originating from subjects whose serum IgD concentration was below 30 mg/l, because sensitivity was not reached. When the IgD concentration was measurable both in serum and
76
Fig. 1. Single radial immunodiffusion for IgD concentration determination. Adult samples stained with Amido black. (a) sera (sample volume of 20 ~1) (b) corresponding stain eluates (sample volume of 30 ~1, storage period of 7-12 days). 1 and 2: IgD level of (a) and (b) clearly measurable. 3: IgD level of (a) little above sensitivity; IgD of (b) not measurable. 4: IgD level of (a) below sensitivity; IgD of (b) not measurable.
eluate the values obtained served to determine the dilution ratio between serum and stain. In all other cases the dilution ratio was determined with the help of the IgG values. Discussion Several authors have claimed that IgD, like IgE, is a comparatively unstable immunoglobulin and tends towards “spontaneous” proteolysis in serum
77
samples, but this claim has not remained unchallenged [4-6]. Our results obtained from stains would suggest that IgD is not particularly prone to disintegration: IgD could be measured in bloodstains after periods of several weeks without noticeable loss of activity. This also corresponds with results obtained from IgE tests in bloodstains: IgE could be easily measured after storage periods of 2-3 weeks [ 111; it is not until after 6 weeks that half of the original IgE concentration in artificial bloodstains may have disintegrated [12]. Although a systematic investigation into IgD disintegration rates in bloodstains has yet to come, storage stability is, according to our findings, sufficient for practical forensic purposes. In numerous cases, the almost total invariability of serum IgD concentrations of individuals, particularly of adults, and the more than lOOO-fold variation of these concentrations among the population provide a basis for discriminating bloodstains.The best conditions for discriminating stains of two individuals occur when one individual has a clearly measurable serum IgD concentration and the other has an IgD level below the sensitivity of the investigation technique used (
247-253. H.L.
Spiegelberg,
The structure
and biology
of human
3-24. H. Mauch,
Immunglobulin
D. DMW 106
(1981)
686-689.
IgD. Zmmunol.
Rev., 37 (1977)
6 S.L.
7 8 9 10 11
12 I3
Dunnette, G.J. Gleich, R.D. Miller and R.A. Kyle, Measurement of IgD by a double antibody radioimmunoassay: demonstration of an apparent trimodal distribution of IgD levels in normal human sera. J. Immunol., 119 (1977) 1727-1731. D.S. Rowe and J.L. Fahey, A new class of human immunoglobulins II. Normal serum IgD. J. Exp. Med., 121 (1965) 185-199. S.L. Dunnette, G.J. Gleich and R.M. Weinshilboum, Inheritance of low serum immunoglobulin D. J. Clin. Invest., 62 (1978) 248-255. S.K. Lee, J.D. Metrakos, K.R. Tanaka and D.C. Heiner, Genetic influence on serum IgD levels. Pediatr. Res., 14 (1980) 60-63. T. Berg and S.G.O. Johansson, Immunoglobulin levels during childhood, with special regard to IgE. Acta Paediat. Stand., 58 (1969) 513-524. D.J. Werrett and L.A. King, Application of allergy diagnosis in forensic serology. J Forensic Sci., 22 (1977) 763-770. D.J. Werrett, L.A. King and P.H. Whitehead, The detection of allergen-associated antibodies in bloodstains. J. Forensic Sci. Sot., 16 (1976) 121-126. M.-L. Glowatzki and W. Keil, IgD concentration: a marker in bloodstain analysis. 1 I th Jnt. Congr. Sot. Forensic Haemogenetics, Copenhagen, 1985, Ref. Vol., in press.