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A stability study on Gc subtyping in bloodstains: Comparison by two different techniques
Forensic Elaevier
Science International, Scientific
26 (1984) Ireland Ltd.
Publishers
39-43
39
A STABILITY STUDY ON Gc SUBTYPING IN BLOODSTAINS: COMPARISON BY TWO DIFFERENT TECHNIQUES
A. KIDO,
M. OYA,
Department ken 409-38 (Received (Accepted
N. KOMATSU
of Legal Medicine, (Japan)
and R. SHIBATA
Yamanashi
Medical
University,
Tamaho-mura,
Yamanashi-
March 26, 1984) May 4, 1984)
Summary The limits of determination of Gc subtypes in bloodstains were compared between the immunofixation method and the sulfosalicylic acid precipitation method using isoelectric focusing on polyacrylamide gel. By the immunofixation method Gc subtyping in bloodstains was successfully made at 37°C after 7 weeks, at room temperature after 17 weeks and at 4°C even after 25 weeks storage. By the sulfosalicylic method Gc subtypes were no longer able to be determined a few weeks after stain formation. The superiority of the results obtained by the immunofixation method makes it the recommended method for the Gc subtyping from bloodstains in medicolegal practice.
Key
words:
Stability
study;
Gc subtypes;
Grouping
of bloodstains
Introduction
The grouping of human bloodstains is of practical importante in medicolegal and criminal investigations. Since Hirschfeld [l] first discovered the polymorphism of group-specific component (Gc) in human sera, various techniques such as immunoelectrophoresis [ 21 and immunofixation electrophoresis on agarose [3] or cellulose acetate [4] have been utilized for grouping Gc from bloodstains. Recently, Constans and Viau [5] showed, using isoelectric focusing followed by immunofixation, that the common Gc phenotypes can be divided into six subtypes which are controlled by three autosomal codominant alleles, GclF, GclS and Gc’. With this new technique Baxter et al. [6] demonstrated Gc bands from bloodstains of up to 12-14 weeks old, however, no description was given of the subtype system. On the other hand, Kühnl et al. [7] developed an altemative economie way for detection of Gc subtype bands by sulfosalicylic acid precipitation 0379-0738/84/$03.00 o 1984 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
40
following electrofocusing which required no anti-Gc serum. Hoste attempted Gc subtyping from bloodstains by this non-immunologie cedure, but the results seemed unsatisfactory. In the present stability study the limits of the determination of Gc types in bloodstains were compared between the above-mentioned methods, immunofixation method and sulfosalicylic method.
[8] prosubtwo
Materials and methods Venous bloods from 20 donors with known phenotypes were dropped on filter paper (Toyoroshi No. 2, Tokyo, Japan) and allowed to dry for a few hours at room temperature. The bloodstains thus made were stored in a thermostatic chamber at 37°C at room temperature and in a refrigerator at 4”C, and examined every week over a period of 25 weeks. Bloodstains with a minimal amount of were cut in 5 X 6 mm pieces, moistened physiologic saline and directly applied to gel plates. Isoelectric focusing was performed with an LKB 2117 Multiphor apparatus (Bromma, Sweden) on polyacrylamide gel plates of 230 X 110 X 0.5 mm in dimensions. The gel plate was composed of 20 ml of stock solution (6.25% acrylamide/0.25% N,N’-methylenebisacrylamide), 1 ml of Ampholine pH range 4-6 (LKB), 0.3 ml of 0.01% riboflavin and 2.5 g of sucrose. The electrode paper strips were soaked @th 1 M phosphoric acid for anode and with 1 M sodium hydroxide for cathode. After a prefocusing at a voltage between 300 V and 1000 V for 60 min, the specimens were applied onto the gel surface 2 cm from the cathode. Electrofocusing was conducted at a constant voltage of 1000 V for 300 min. During focusing the gel plate was cooled by circulating water at 4°C. The immunofixation method was carried out as described by Constans et al. [9] with the use of a specific anti-Gc serum (DAKO, Denmark). A piece of cellulose acetate strip (Separax, F’uji, Tokyo, Japan) was soaked with the anti-Gc serum diluted 1: 3 with physiologic saline and placed on the gel surface for a few minutes. The cellulose acetate strip was washed with physiologic saline ovemight and stained for 5 min with 0.5% Amido Black 10 B dissolved in a mixture of methanol and acetic acid (9 : 1). Excess stain was removed by washing in 3% acetic acid. The sulfosalicylic acid precipitation method was performed according to the method of Hoste [ 81. The gel was immersed in 33% methanol containing 3% sulfosalicylic acid and 0.1% Coomassie Brilliant Blue R-250. The Gc bands appeared immediately as sharp white precipitates on a black background with light from the side. Results and discussion Table 1 summarizes the results for the determination limit of Gc subtypes in 20 bloodstains stored at 37°C room temperature and 4°C obtained by the
41 TABLE 1 POSITIVE RESULTS IN Gc SUBTYPING FROM 20 BLOODSTAINS BY THE IMMUNOFIXATION METHOD AND THE SULFOSALICYLIC ACID PRECIPITATION METHOD &?e of bloods tains (Weeks) ~-
Immunofixation method 37°C Room 4°C temperature
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
20 20 20 20 20 20 20 19 17 15 9 5 0
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 19 18 18 18 16 16 12 11
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
Sulfosalicylic acid precipitation 37°C
Room temperature
4°C
6 4 0
13 6 1 0
19 17 16 14 9 8 0
method
immunofixation method and the sulfosalicylic acid precipitation method. Our samples included five Gc lF-lF, one Gc lS-lS, four Gc lF-lS, five Gc 2-lF, three Gc 2-1s and two Gc 2-2. The typical Gc subtype pattems in bloodstains stored at room temperature for 1 week are shown in Fig. 1. By the immunofixation method all the bloodstains examined were successfully subtyped for Gc at 37°C for periods of up to 7 weeks, at room temperature for periods of up to 17 weeks and at 4°C even for periods in excess of 25 weeks. However, the bands became fainter and more indistinct with increasing time of storage, particularly in the anodal bands of Gc F and Gc S. NO significant differences on the determination limits were seen among the six subtypes. The sulfosalicylic method yielded much poorer results than the immunofixation method, and it was difficult to identify the accurate Gc phenotypes from bloodstains of 1 week old. The Gc subtyping was no longer possible a few weeks after stain formation.
42
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
Fig. 1. Isoelectric focusing patterns of Gc subtypes in bloodstains stored at room temperature for 1 week. The anode is at the top. A: immunofixation method; B: sulfosalicylic acid precipitation method. 1: Gc 2-1F; 2: Gc 2-1s; 3: Gc lF-1F; 4: Gc lS8: Gc 2-2; 9: Gc 2-1F; 10: Gc lF-1s. 6: Gc 2-1s; 7: Gc lF-1s; 1s; 5: Gc lF-1F;
The present stability study clearly shows that the results obtained by the immunofixation method is far superior to those obtained by the sulfosalicylic acid precipitation method. Immunofixation combined with isoelectric focusing permits Gc subtyping from bloodstains after fairly long storage periods (about 4 months at room temperature) and is therefore recommended for the grouping of bloodstains in medicolegal practice. Although the determination limit largely depends on the storage conditions, this method is useful also in tropical countries. The sulfosahcylic method is not fit for this purpose. References J. Hirschfeld, Immuno-electrophoretic demonstration of qualitative differences in human sera and their relation to the haptoglobina Acta Pathol. Microbiol. Stand., 47 (1959) 160-168. B. Nerstrbm and J. Skafte Jensen, Immunoelectrophoretic anaiysis of blood stains with special reference to Gc grouping. Acts Pathol. Microbiol. Stand., 58 (1963) 257-263. S. Berg und S. Kijewski, Beiträge zur Blutgruppenbestimmung in gelagerten Biutproben, Leichenblut und Blutspuren. Anwendung der Immunfixationsmethode zur Gc-Darsteiiung. Arch. Kriminol., 161 (1978) 95-104. P.L. Zajac and B.W. Grunbaum, Determination of group specific component phenotypes in dried bloodstains by immunofixation on cellulose acetate. J. Foreneic Sci., 23 (1978) 353-355. J. Constans and M. Viau, Group-specific component: Evidente for two subtypes of the Gc’ gene. Science, 198 (1977) 1070-1071.
43 6 M. Baxter, T.W. Randall and J.W. Thorpe, A method of phenotyping group specific component protein from dried bloodstains by immunofixation thin-layer polyacrylamide gel isoelectric focusing. J. Forensic Sci. Sec., 22 (1982) 367-371. 7 P. Kühnl, W. Spielmann and M. Loa, An improved method for the identification of Gc’ subtypes (group-specific component) by isoelectric focusing. Vox Sang., 35 (1978) 401-404. 8 B. Hoste, Group-specific component (Gc) and transferrin (Tf) subtypes ascertained by isoelectric focusing. Hum. Genet., 50 (1979) 75-79. 9 J. Constans, M. Viau, H. Cleve, G. Jaeger, J.C. Quilici and M.J. Palisson, Analysis of the Gc polymorphism in human populations by isoelectrofocusing on polyacrylamide gels. Demonstration of subtypes of the Gc’ allele and of additional Gc variants. Hum. Genet., 41 (1978) 53-60.
Science International, Scientific
26 (1984) Ireland Ltd.
Publishers
39-43
39
A STABILITY STUDY ON Gc SUBTYPING IN BLOODSTAINS: COMPARISON BY TWO DIFFERENT TECHNIQUES
A. KIDO,
M. OYA,
Department ken 409-38 (Received (Accepted
N. KOMATSU
of Legal Medicine, (Japan)
and R. SHIBATA
Yamanashi
Medical
University,
Tamaho-mura,
Yamanashi-
March 26, 1984) May 4, 1984)
Summary The limits of determination of Gc subtypes in bloodstains were compared between the immunofixation method and the sulfosalicylic acid precipitation method using isoelectric focusing on polyacrylamide gel. By the immunofixation method Gc subtyping in bloodstains was successfully made at 37°C after 7 weeks, at room temperature after 17 weeks and at 4°C even after 25 weeks storage. By the sulfosalicylic method Gc subtypes were no longer able to be determined a few weeks after stain formation. The superiority of the results obtained by the immunofixation method makes it the recommended method for the Gc subtyping from bloodstains in medicolegal practice.
Key
words:
Stability
study;
Gc subtypes;
Grouping
of bloodstains
Introduction
The grouping of human bloodstains is of practical importante in medicolegal and criminal investigations. Since Hirschfeld [l] first discovered the polymorphism of group-specific component (Gc) in human sera, various techniques such as immunoelectrophoresis [ 21 and immunofixation electrophoresis on agarose [3] or cellulose acetate [4] have been utilized for grouping Gc from bloodstains. Recently, Constans and Viau [5] showed, using isoelectric focusing followed by immunofixation, that the common Gc phenotypes can be divided into six subtypes which are controlled by three autosomal codominant alleles, GclF, GclS and Gc’. With this new technique Baxter et al. [6] demonstrated Gc bands from bloodstains of up to 12-14 weeks old, however, no description was given of the subtype system. On the other hand, Kühnl et al. [7] developed an altemative economie way for detection of Gc subtype bands by sulfosalicylic acid precipitation 0379-0738/84/$03.00 o 1984 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
40
following electrofocusing which required no anti-Gc serum. Hoste attempted Gc subtyping from bloodstains by this non-immunologie cedure, but the results seemed unsatisfactory. In the present stability study the limits of the determination of Gc types in bloodstains were compared between the above-mentioned methods, immunofixation method and sulfosalicylic method.
[8] prosubtwo
Materials and methods Venous bloods from 20 donors with known phenotypes were dropped on filter paper (Toyoroshi No. 2, Tokyo, Japan) and allowed to dry for a few hours at room temperature. The bloodstains thus made were stored in a thermostatic chamber at 37°C at room temperature and in a refrigerator at 4”C, and examined every week over a period of 25 weeks. Bloodstains with a minimal amount of were cut in 5 X 6 mm pieces, moistened physiologic saline and directly applied to gel plates. Isoelectric focusing was performed with an LKB 2117 Multiphor apparatus (Bromma, Sweden) on polyacrylamide gel plates of 230 X 110 X 0.5 mm in dimensions. The gel plate was composed of 20 ml of stock solution (6.25% acrylamide/0.25% N,N’-methylenebisacrylamide), 1 ml of Ampholine pH range 4-6 (LKB), 0.3 ml of 0.01% riboflavin and 2.5 g of sucrose. The electrode paper strips were soaked @th 1 M phosphoric acid for anode and with 1 M sodium hydroxide for cathode. After a prefocusing at a voltage between 300 V and 1000 V for 60 min, the specimens were applied onto the gel surface 2 cm from the cathode. Electrofocusing was conducted at a constant voltage of 1000 V for 300 min. During focusing the gel plate was cooled by circulating water at 4°C. The immunofixation method was carried out as described by Constans et al. [9] with the use of a specific anti-Gc serum (DAKO, Denmark). A piece of cellulose acetate strip (Separax, F’uji, Tokyo, Japan) was soaked with the anti-Gc serum diluted 1: 3 with physiologic saline and placed on the gel surface for a few minutes. The cellulose acetate strip was washed with physiologic saline ovemight and stained for 5 min with 0.5% Amido Black 10 B dissolved in a mixture of methanol and acetic acid (9 : 1). Excess stain was removed by washing in 3% acetic acid. The sulfosalicylic acid precipitation method was performed according to the method of Hoste [ 81. The gel was immersed in 33% methanol containing 3% sulfosalicylic acid and 0.1% Coomassie Brilliant Blue R-250. The Gc bands appeared immediately as sharp white precipitates on a black background with light from the side. Results and discussion Table 1 summarizes the results for the determination limit of Gc subtypes in 20 bloodstains stored at 37°C room temperature and 4°C obtained by the
41 TABLE 1 POSITIVE RESULTS IN Gc SUBTYPING FROM 20 BLOODSTAINS BY THE IMMUNOFIXATION METHOD AND THE SULFOSALICYLIC ACID PRECIPITATION METHOD &?e of bloods tains (Weeks) ~-
Immunofixation method 37°C Room 4°C temperature
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
20 20 20 20 20 20 20 19 17 15 9 5 0
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 19 18 18 18 16 16 12 11
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
Sulfosalicylic acid precipitation 37°C
Room temperature
4°C
6 4 0
13 6 1 0
19 17 16 14 9 8 0
method
immunofixation method and the sulfosalicylic acid precipitation method. Our samples included five Gc lF-lF, one Gc lS-lS, four Gc lF-lS, five Gc 2-lF, three Gc 2-1s and two Gc 2-2. The typical Gc subtype pattems in bloodstains stored at room temperature for 1 week are shown in Fig. 1. By the immunofixation method all the bloodstains examined were successfully subtyped for Gc at 37°C for periods of up to 7 weeks, at room temperature for periods of up to 17 weeks and at 4°C even for periods in excess of 25 weeks. However, the bands became fainter and more indistinct with increasing time of storage, particularly in the anodal bands of Gc F and Gc S. NO significant differences on the determination limits were seen among the six subtypes. The sulfosalicylic method yielded much poorer results than the immunofixation method, and it was difficult to identify the accurate Gc phenotypes from bloodstains of 1 week old. The Gc subtyping was no longer possible a few weeks after stain formation.
42
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
Fig. 1. Isoelectric focusing patterns of Gc subtypes in bloodstains stored at room temperature for 1 week. The anode is at the top. A: immunofixation method; B: sulfosalicylic acid precipitation method. 1: Gc 2-1F; 2: Gc 2-1s; 3: Gc lF-1F; 4: Gc lS8: Gc 2-2; 9: Gc 2-1F; 10: Gc lF-1s. 6: Gc 2-1s; 7: Gc lF-1s; 1s; 5: Gc lF-1F;
The present stability study clearly shows that the results obtained by the immunofixation method is far superior to those obtained by the sulfosalicylic acid precipitation method. Immunofixation combined with isoelectric focusing permits Gc subtyping from bloodstains after fairly long storage periods (about 4 months at room temperature) and is therefore recommended for the grouping of bloodstains in medicolegal practice. Although the determination limit largely depends on the storage conditions, this method is useful also in tropical countries. The sulfosahcylic method is not fit for this purpose. References J. Hirschfeld, Immuno-electrophoretic demonstration of qualitative differences in human sera and their relation to the haptoglobina Acta Pathol. Microbiol. Stand., 47 (1959) 160-168. B. Nerstrbm and J. Skafte Jensen, Immunoelectrophoretic anaiysis of blood stains with special reference to Gc grouping. Acts Pathol. Microbiol. Stand., 58 (1963) 257-263. S. Berg und S. Kijewski, Beiträge zur Blutgruppenbestimmung in gelagerten Biutproben, Leichenblut und Blutspuren. Anwendung der Immunfixationsmethode zur Gc-Darsteiiung. Arch. Kriminol., 161 (1978) 95-104. P.L. Zajac and B.W. Grunbaum, Determination of group specific component phenotypes in dried bloodstains by immunofixation on cellulose acetate. J. Foreneic Sci., 23 (1978) 353-355. J. Constans and M. Viau, Group-specific component: Evidente for two subtypes of the Gc’ gene. Science, 198 (1977) 1070-1071.
43 6 M. Baxter, T.W. Randall and J.W. Thorpe, A method of phenotyping group specific component protein from dried bloodstains by immunofixation thin-layer polyacrylamide gel isoelectric focusing. J. Forensic Sci. Sec., 22 (1982) 367-371. 7 P. Kühnl, W. Spielmann and M. Loa, An improved method for the identification of Gc’ subtypes (group-specific component) by isoelectric focusing. Vox Sang., 35 (1978) 401-404. 8 B. Hoste, Group-specific component (Gc) and transferrin (Tf) subtypes ascertained by isoelectric focusing. Hum. Genet., 50 (1979) 75-79. 9 J. Constans, M. Viau, H. Cleve, G. Jaeger, J.C. Quilici and M.J. Palisson, Analysis of the Gc polymorphism in human populations by isoelectrofocusing on polyacrylamide gels. Demonstration of subtypes of the Gc’ allele and of additional Gc variants. Hum. Genet., 41 (1978) 53-60.