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The use of sperm-specific lactate dehydrogenase isoenzyme for the identification of semen in dried stains
Forensic
Science,
0 Elsevier
8 (1976)
Sequoia
269
- 215
S.A., Lausanne
-Printed
269
in the Netherlands
THE USE OF SPERM-SPECIFIC LACTATE DEHYDROGENASE ISOENZYME FOR THE IDENTIFICATION OF SEMEN IN DRIED STAINS
R. H. MOKASHI
and M. S. MADIWALE
Forensic
Laboratory,
Science
(Received 30,1976)
September
State of Maharashtra,
16, 1975;
in revised
Bombay
form February
400
008
(India)
23, 1976; accepted
September
SUMMARY The sperm-specific lactate dehydrogenase (LDH) isoenzyme (Blanc0 and Zinkham, 1963; Goldberg, 1963) separated from other LDH isoenzymes of semen by polyacrylamide gel electrophoresis has been found to be suitable for specific differentiation of human semen from other human body fluids and semen of commonly encountered animals. Seminal isoenzymes were found to be stable even 4 weeks after storage in tropical conditions. The method gave substantially more positive results than microscopic identification of spermatozoa when applied to a large number of relatively old stains on actual crime articles. It is therefore valuable in a large number of cases involving normal males and also in interpreting results of immunological and enzymological individualisation of semen stains.
INTRODUCTION
The reliability of acid phosphatase method [ 1, 21 which was widely used until recently for the detection of semen in stains was questioned by Willott [3] who demonstrated that 1-tartarate inhibitable acid phosphatase was also present in vaginal fluid. Adams and Wraxall [4] then improved the method and showed that differential migration of acid phosphatase isoenzymes obtained by electrophoresis could be used for specific detection of semen in stains even in the presence of blood and vaginal fluid. They have however stated, that although in their laboratory this method is routinely employed in place of chemical methods, microscopic detection of spermatozoa in stains is still used in all cases except those in which azoospermic or vasectomized males are involved. Spermatozoa [5, 61 which have thus retained their original significance for the specific detection of semen are not however, detectable in relatively old semen stains due to their disintegration particularly under tropical conditions. It was therefore thought desirable to develop a biochemical method for the specific detection of semen, based on the presence of sperm-specific LDH isoenzyme reported by Blanc0 and Zinkham [7] and Goldberg [S] , so that semen could be detected even in those stains in which morphology of spermatozoa is not. available.
A preliminary work reported from this laboratory [9] has indicated that LDH isoenzyme pattern obtained by polyacrylamide gel electrophoresis of semen is suitable for its specific identification in the presence of blood and vaginal fluid. This communication reports successful application of the LDH isoenzyme method for the specific detection of semen in actual crimearticles in the presence of commonly encountered human body fluids and animal semen. METHODS AND MATERIALS
Human blood, semen and swabs of human vaginal fluid were collected from a local hospital. Samples of dog, bullock and buffalo semen were obtained from Bombay Veterinary College and Aarey Milk Colony, Bombay. Specimens of saliva, nasal secretion and urine were collected from donors from among members of the laboratory staff. In all, 100 specimens each of blood, seminal fluid and vaginal fluid (taken on swabs) and 10 each of other fluids were collected for the study. Experimental seminal stains were prepared by placing 0.2 ml of fresh seminal fluid on clean cotton cloth pieces (approximately 9 cm X 5 cm). Vaginal swabs which were treated as stains however, contained varying quantities of vaginal fluid. Mixed stains were obtained by placing 0.2 ml of seminal fluid on blood-stains and vaginal fluid stains prepared as given above. These stains were first dried in air and transferred to dry and wet chambers for storage at room temperature for 4 weeks as described earlier for bloodstains [IO]. For verifying the applicability of the method to routine work, articles were randomly chosen from sexual offence cases referred to this laboratory and which had given positive results for the acid phosphatase test ]21* All chemicals used were B.D.H. Analar or equivalent grade. An aliquot of 0.05 ml of human body fluids such as blood/saliva/semen/ nasal secretion and animal semen was mixed with 0.45 ml of a 40% w/v solution of sucrose in distilled water. In the case of urine however, 0.25 ml were mixed with 0.25 ml of the sucrose solution as smaller quantities were found to be inadequate. All experimental stains and stains from crimearticles were extracted with 1.0 ml of distilled water and extracts centrifuged, if necessary, at 3000 rev/minute for 10 minutes. 0.25 ml of these clear extracts was mixed with 0.25 ml of the sucrose solution. Aliquots of these mixtures in sucrose solution (0.05 ml and 0.1 ml in the case of body fluids and aqueous stain-extracts respectively) were used in a vertical polyacrylamide gel electrophoresis method essentially based on that described by was 8% and electroDeitz and Lubrano [ll].The monomer concentration phoresis was carried out in a refrigerator for 150 minutes using 5mA/tube. Gels were then removed and stained in darkness for 60 minutes as described by us earlier [ 121. Smears were prepared from stain-extracts on microscope slides, stained with Haematoxylin and eosin and examined as described by Nickolls [13].
271 RESULTS
LDH isoenzyme profiles obtained with various body fluids are shown in Fig. 1. Patterns of LDH isoenzymes obtained from semen and extracts of fresh and stored semen stains are given in Fig. 2(a).
(a) Fig. 1. From left to right - LDH isoenzyme patterns vaginal secretion, saliva, nasal secretion and urine. Fig. 2(a). From aqueous extracts tions respectively.
given by human
blood,
semen,
left to right - LDH isoenzyme patterns given by human semen and of human semen stains after storage for 4 weeks in dry and moist condi-
Patterns of .LDH isoenzymes given by extracts of neat stains of semen and vaginal fluid and those of mixed stains are shown in Fig. 2(b). A comparison of isoenzyme profiles of semen and vaginal fluid and those representing extracts of various stains from crime-articles is given in Fig. 2(c). LDH isoenzyme patterns obtained with human and animal semen are shown in Fig. 3(a) and their diagrammatic representation is given in Fig. 3(b). Results obtained with stains from actual crime-articles are summarised in Table I. DISCUSSION
The LDH patterns shown in Fig. 1 can be taken to be representative of the various human body fluids because a sufficient number of their specimens
272
(b) Fig. 2(b). From left to right - LDH isoenzyme profiles of experimental stains of semen, vaginal fluid, a mixture mixture of semen and blood.
obtained of semen
with aqueous extra& and vaginal fluid and a
Fig. 2(c). From left to right - LDH isoenzyme profiles obtained with neat semen, neat vaginal fluid and with aqueous extracts of stains from crime-articles showing the presence of semen alone (2 different samples), semen mixed with blood and semen mixed with vaginal fluid.
was studied. It was found that all specimens of all the body fluids except vaginal fluid give the same specific patterns, but as many as 78 out of 100 samples of vaginal swabs gave 5 bands, the remaining 22 giving 4 or less, probably because of lower amounts of vaginal fluid in them. As even 1 ml of seminal fluid was found to give a distinct isoenzyme pattern, the method can be regarded as quite sensitive and specific for the detection of semen in the presence of commonly encountered human body fluids. The results given in Figs 2(a) and 2(b) demonstrate that the method can be successfully applied to stains of neat semen or semen mixed with blood/ vaginal fluid even after their storage for 4 weeks under tropical conditions. This was confirmed by the observation that 2 stains could also give a positive result after more than 12 months after the crimes (Fig. 2(c) and Table I). It was observed that the isoenzyme band specific for vaginal fluid is less stable than the one specific for semen and hence the detection of vaginal fluid may
273
(b)
(a) Fig. 3(a). From left to right bullock and dog.
LDH isoenzyme
patterns
given by semen of man, buffalo,
Fig. 3(b). Diagrammatic representation of LDH isoenzyme man (l), buffalo (2), bullock (3) and dog (4).
patterns
given by semen
of
not be possible in some old stains. However, semen could be specifically detected in aged stains even in the presence of blood and vaginaI fluid. Figures 3(a) and 3(b) indicate the possibility of the simultaneous use of LDH isoenzyme patterns for differentiation of human seminal stains from those of common domestic animals like buffalo, bullock and dog. The value of these results should be taken as enhanced in the light of an earlier observation by Iosifov [14] that LDH isoenzyme profiles of semen from bull, ram, boar and stallion are also species-specific. The interference by vaginal fluid in the use of the acid phosphatase method [ 21 for detection of semen which was pointed out by Willott [3 ] is confirmed by this study employing LDH method as the basis. The significance of this interference is evident from distinct positive response given by residual LDH activity of vaginal fluid in 4 stains even after 5 months (Table I). It can be seen that, if only the acid phosphatase method [ 21 was relied upon for detection, results for at least 4 stains containing only vaginal fluid would have been falsely reported as positive for semen. It is obvious therefore, that to avoid such misleading results isoenzyme methods which are based on resolution of enzyme components are essential in routine detection of semen in stains. The presence or absence of vaginal fluid alone or admixed with semen as indicated by this method may also be helpful in
274 TABLE Detection
I of spermatozoa
in stains by LDH isoenzyme
and microscopic
methods
--Age of stain
(days)
<30
Total no. tested*
22
LDH isoenzyme -_______ positive for S
v
s+v
16 -
-2 --
1 -
31-60
19
3 -
>60
25
,2@--
Total * ** @ ”
66
36
method
results** negative
S+B
S and V
-
-
9
3
-2-
4 -
-
4” -
4 9 ___-
for
Microscopic detection of spermatozoa (positive results)
-
-
-
-
5
1 -
-
-
-
-
2 4 5
9 1
16
21 .___
All stains examined were positive for I-tartrate inhibitable acid phosphatase. S = semen; V = vaginal secretion; B = blood. 5 stains were more than 4 months old of which 2 were more than a year old. All stains were more than 5 months old.
some cases for attempting the detection of human blood group antigens/ isoenzymes or interpretation of its results with stains. Results given in Table I are strongly indicative of the superiority of the LDH isoenzyme method to microscopic detection of spermatozoa. Instead of 46 stains giving positive results for semen, out of 66 attempted, only 21 would have been positive by microscopy. Gradual loss of morphology of spermatozoa is shown by a sharp decline of positive microscopic results with increasing age of stains. It may therefore be inferred that the intrinsic high specificity of spermatozoa is much better utilised by the LDH isoenzyme method, giving a distinct advantage in routine examination of seminal stains in a majority of sexual offence cases in which normal males (neither azoospermic nor vasectomized) are involved. ACKNOWLEDGEMENT
The authors wish to express their sincere thanks to Dr. B. N. Mattoo, Director, Forensic Science Laboratories, State of Maharashtra for giving
275
constant encouragement and taking keen interest in the progress of this work. They are grateful to Dr. D. P. Velhankar and Dr. K. S. Deshpande from Bombay Veterinary College and Dr. R. N. Samant and Dr. A. D. Sohoni from Aarey Milk Colony for kind supply of specimens of semen of various animals.
REFERENCES
4 5 6 7 8 9 10 11 12 13 14
S. S. Kind, The Acid Phosphatase Test, in A. S. Curry (ed.), Methods of Forensic Science, Vol. 3, Interscience, New York, 1964, pp. 267--287. S. Sivaram and H. L. Bami, Identification of seminal stains by the inhibition of acid phosphatase by L (+) tartrate, J. Forensic Sci. Sot., 11 (1971) 187-194. G. M. Willott, L-tartrate inhibitable acid phosphatase in semen and vaginal secretions, J. Forensic Sci. Sot., 12 (1972) 363-366. E. C. Adams and B. G. Wraxall, Phosphatase in body fluids: The differentiation of semen and vaginal secretion, Forensic Sci., 3 (1974) 57-62. L. C. Nickolls, The Scientific Investigation of Crime, Butterworth, London, 1966, pp. 198. J. Glaister, Medical Jurisprudence and Toxicology, Livingstone, Edinburgh and London, i957, pp. 415. A. Blanc0 and W. H. Zinkham, Lactate debydrogenases in human testis, Science, 139 (1963) 601. E. Goldberg, Lactic and malic dehydrogenase in human spermatozoa, Science, 139 (1963) 602. R. H. Mokashi, A. G. Malwankar and M. S. Madiwale, Detection of semen in the presence of blood and vaginal secretion, J. Indian Acad. Forensic Sci., 14 (1975) l-3. M. S. Madiwale, H. S. Mahal and R. W. P. Master, Der Nachweis der Blutart im Blutspuren durch Isoenzymbestimmung, Arch. Kriminol., 150 (1972) 160.. 166. A. A. Dietz and T. Lubrano, Separation and quantitation of LDH isoenzymes by disc electrophoresis, Anal. Biochem., 20 (1967) 246 257. R. H. Mokashi and M. S. Madiwale, Isozym-methoden zur Unterscheidung von Menschen-und Affenblut, Arch. Kriminol., 153 (1974) 48 ~53. L. C. Nickolls, The Scientific Investigation of Crime, Butterworth, London, 1956, pp. 200. K. Iosifov, Isoenzyme composition of the lactate dehydrogenase in ram, bull, boar and stallion seminal plasma, spermatozoa and blood serum, Vet. Med. Nauki, 8 (1971) 43m 50.
Science,
0 Elsevier
8 (1976)
Sequoia
269
- 215
S.A., Lausanne
-Printed
269
in the Netherlands
THE USE OF SPERM-SPECIFIC LACTATE DEHYDROGENASE ISOENZYME FOR THE IDENTIFICATION OF SEMEN IN DRIED STAINS
R. H. MOKASHI
and M. S. MADIWALE
Forensic
Laboratory,
Science
(Received 30,1976)
September
State of Maharashtra,
16, 1975;
in revised
Bombay
form February
400
008
(India)
23, 1976; accepted
September
SUMMARY The sperm-specific lactate dehydrogenase (LDH) isoenzyme (Blanc0 and Zinkham, 1963; Goldberg, 1963) separated from other LDH isoenzymes of semen by polyacrylamide gel electrophoresis has been found to be suitable for specific differentiation of human semen from other human body fluids and semen of commonly encountered animals. Seminal isoenzymes were found to be stable even 4 weeks after storage in tropical conditions. The method gave substantially more positive results than microscopic identification of spermatozoa when applied to a large number of relatively old stains on actual crime articles. It is therefore valuable in a large number of cases involving normal males and also in interpreting results of immunological and enzymological individualisation of semen stains.
INTRODUCTION
The reliability of acid phosphatase method [ 1, 21 which was widely used until recently for the detection of semen in stains was questioned by Willott [3] who demonstrated that 1-tartarate inhibitable acid phosphatase was also present in vaginal fluid. Adams and Wraxall [4] then improved the method and showed that differential migration of acid phosphatase isoenzymes obtained by electrophoresis could be used for specific detection of semen in stains even in the presence of blood and vaginal fluid. They have however stated, that although in their laboratory this method is routinely employed in place of chemical methods, microscopic detection of spermatozoa in stains is still used in all cases except those in which azoospermic or vasectomized males are involved. Spermatozoa [5, 61 which have thus retained their original significance for the specific detection of semen are not however, detectable in relatively old semen stains due to their disintegration particularly under tropical conditions. It was therefore thought desirable to develop a biochemical method for the specific detection of semen, based on the presence of sperm-specific LDH isoenzyme reported by Blanc0 and Zinkham [7] and Goldberg [S] , so that semen could be detected even in those stains in which morphology of spermatozoa is not. available.
A preliminary work reported from this laboratory [9] has indicated that LDH isoenzyme pattern obtained by polyacrylamide gel electrophoresis of semen is suitable for its specific identification in the presence of blood and vaginal fluid. This communication reports successful application of the LDH isoenzyme method for the specific detection of semen in actual crimearticles in the presence of commonly encountered human body fluids and animal semen. METHODS AND MATERIALS
Human blood, semen and swabs of human vaginal fluid were collected from a local hospital. Samples of dog, bullock and buffalo semen were obtained from Bombay Veterinary College and Aarey Milk Colony, Bombay. Specimens of saliva, nasal secretion and urine were collected from donors from among members of the laboratory staff. In all, 100 specimens each of blood, seminal fluid and vaginal fluid (taken on swabs) and 10 each of other fluids were collected for the study. Experimental seminal stains were prepared by placing 0.2 ml of fresh seminal fluid on clean cotton cloth pieces (approximately 9 cm X 5 cm). Vaginal swabs which were treated as stains however, contained varying quantities of vaginal fluid. Mixed stains were obtained by placing 0.2 ml of seminal fluid on blood-stains and vaginal fluid stains prepared as given above. These stains were first dried in air and transferred to dry and wet chambers for storage at room temperature for 4 weeks as described earlier for bloodstains [IO]. For verifying the applicability of the method to routine work, articles were randomly chosen from sexual offence cases referred to this laboratory and which had given positive results for the acid phosphatase test ]21* All chemicals used were B.D.H. Analar or equivalent grade. An aliquot of 0.05 ml of human body fluids such as blood/saliva/semen/ nasal secretion and animal semen was mixed with 0.45 ml of a 40% w/v solution of sucrose in distilled water. In the case of urine however, 0.25 ml were mixed with 0.25 ml of the sucrose solution as smaller quantities were found to be inadequate. All experimental stains and stains from crimearticles were extracted with 1.0 ml of distilled water and extracts centrifuged, if necessary, at 3000 rev/minute for 10 minutes. 0.25 ml of these clear extracts was mixed with 0.25 ml of the sucrose solution. Aliquots of these mixtures in sucrose solution (0.05 ml and 0.1 ml in the case of body fluids and aqueous stain-extracts respectively) were used in a vertical polyacrylamide gel electrophoresis method essentially based on that described by was 8% and electroDeitz and Lubrano [ll].The monomer concentration phoresis was carried out in a refrigerator for 150 minutes using 5mA/tube. Gels were then removed and stained in darkness for 60 minutes as described by us earlier [ 121. Smears were prepared from stain-extracts on microscope slides, stained with Haematoxylin and eosin and examined as described by Nickolls [13].
271 RESULTS
LDH isoenzyme profiles obtained with various body fluids are shown in Fig. 1. Patterns of LDH isoenzymes obtained from semen and extracts of fresh and stored semen stains are given in Fig. 2(a).
(a) Fig. 1. From left to right - LDH isoenzyme patterns vaginal secretion, saliva, nasal secretion and urine. Fig. 2(a). From aqueous extracts tions respectively.
given by human
blood,
semen,
left to right - LDH isoenzyme patterns given by human semen and of human semen stains after storage for 4 weeks in dry and moist condi-
Patterns of .LDH isoenzymes given by extracts of neat stains of semen and vaginal fluid and those of mixed stains are shown in Fig. 2(b). A comparison of isoenzyme profiles of semen and vaginal fluid and those representing extracts of various stains from crime-articles is given in Fig. 2(c). LDH isoenzyme patterns obtained with human and animal semen are shown in Fig. 3(a) and their diagrammatic representation is given in Fig. 3(b). Results obtained with stains from actual crime-articles are summarised in Table I. DISCUSSION
The LDH patterns shown in Fig. 1 can be taken to be representative of the various human body fluids because a sufficient number of their specimens
272
(b) Fig. 2(b). From left to right - LDH isoenzyme profiles of experimental stains of semen, vaginal fluid, a mixture mixture of semen and blood.
obtained of semen
with aqueous extra& and vaginal fluid and a
Fig. 2(c). From left to right - LDH isoenzyme profiles obtained with neat semen, neat vaginal fluid and with aqueous extracts of stains from crime-articles showing the presence of semen alone (2 different samples), semen mixed with blood and semen mixed with vaginal fluid.
was studied. It was found that all specimens of all the body fluids except vaginal fluid give the same specific patterns, but as many as 78 out of 100 samples of vaginal swabs gave 5 bands, the remaining 22 giving 4 or less, probably because of lower amounts of vaginal fluid in them. As even 1 ml of seminal fluid was found to give a distinct isoenzyme pattern, the method can be regarded as quite sensitive and specific for the detection of semen in the presence of commonly encountered human body fluids. The results given in Figs 2(a) and 2(b) demonstrate that the method can be successfully applied to stains of neat semen or semen mixed with blood/ vaginal fluid even after their storage for 4 weeks under tropical conditions. This was confirmed by the observation that 2 stains could also give a positive result after more than 12 months after the crimes (Fig. 2(c) and Table I). It was observed that the isoenzyme band specific for vaginal fluid is less stable than the one specific for semen and hence the detection of vaginal fluid may
273
(b)
(a) Fig. 3(a). From left to right bullock and dog.
LDH isoenzyme
patterns
given by semen of man, buffalo,
Fig. 3(b). Diagrammatic representation of LDH isoenzyme man (l), buffalo (2), bullock (3) and dog (4).
patterns
given by semen
of
not be possible in some old stains. However, semen could be specifically detected in aged stains even in the presence of blood and vaginaI fluid. Figures 3(a) and 3(b) indicate the possibility of the simultaneous use of LDH isoenzyme patterns for differentiation of human seminal stains from those of common domestic animals like buffalo, bullock and dog. The value of these results should be taken as enhanced in the light of an earlier observation by Iosifov [14] that LDH isoenzyme profiles of semen from bull, ram, boar and stallion are also species-specific. The interference by vaginal fluid in the use of the acid phosphatase method [ 21 for detection of semen which was pointed out by Willott [3 ] is confirmed by this study employing LDH method as the basis. The significance of this interference is evident from distinct positive response given by residual LDH activity of vaginal fluid in 4 stains even after 5 months (Table I). It can be seen that, if only the acid phosphatase method [ 21 was relied upon for detection, results for at least 4 stains containing only vaginal fluid would have been falsely reported as positive for semen. It is obvious therefore, that to avoid such misleading results isoenzyme methods which are based on resolution of enzyme components are essential in routine detection of semen in stains. The presence or absence of vaginal fluid alone or admixed with semen as indicated by this method may also be helpful in
274 TABLE Detection
I of spermatozoa
in stains by LDH isoenzyme
and microscopic
methods
--Age of stain
(days)
<30
Total no. tested*
22
LDH isoenzyme -_______ positive for S
v
s+v
16 -
-2 --
1 -
31-60
19
3 -
>60
25
,2@--
Total * ** @ ”
66
36
method
results** negative
S+B
S and V
-
-
9
3
-2-
4 -
-
4” -
4 9 ___-
for
Microscopic detection of spermatozoa (positive results)
-
-
-
-
5
1 -
-
-
-
-
2 4 5
9 1
16
21 .___
All stains examined were positive for I-tartrate inhibitable acid phosphatase. S = semen; V = vaginal secretion; B = blood. 5 stains were more than 4 months old of which 2 were more than a year old. All stains were more than 5 months old.
some cases for attempting the detection of human blood group antigens/ isoenzymes or interpretation of its results with stains. Results given in Table I are strongly indicative of the superiority of the LDH isoenzyme method to microscopic detection of spermatozoa. Instead of 46 stains giving positive results for semen, out of 66 attempted, only 21 would have been positive by microscopy. Gradual loss of morphology of spermatozoa is shown by a sharp decline of positive microscopic results with increasing age of stains. It may therefore be inferred that the intrinsic high specificity of spermatozoa is much better utilised by the LDH isoenzyme method, giving a distinct advantage in routine examination of seminal stains in a majority of sexual offence cases in which normal males (neither azoospermic nor vasectomized) are involved. ACKNOWLEDGEMENT
The authors wish to express their sincere thanks to Dr. B. N. Mattoo, Director, Forensic Science Laboratories, State of Maharashtra for giving
275
constant encouragement and taking keen interest in the progress of this work. They are grateful to Dr. D. P. Velhankar and Dr. K. S. Deshpande from Bombay Veterinary College and Dr. R. N. Samant and Dr. A. D. Sohoni from Aarey Milk Colony for kind supply of specimens of semen of various animals.
REFERENCES
4 5 6 7 8 9 10 11 12 13 14
S. S. Kind, The Acid Phosphatase Test, in A. S. Curry (ed.), Methods of Forensic Science, Vol. 3, Interscience, New York, 1964, pp. 267--287. S. Sivaram and H. L. Bami, Identification of seminal stains by the inhibition of acid phosphatase by L (+) tartrate, J. Forensic Sci. Sot., 11 (1971) 187-194. G. M. Willott, L-tartrate inhibitable acid phosphatase in semen and vaginal secretions, J. Forensic Sci. Sot., 12 (1972) 363-366. E. C. Adams and B. G. Wraxall, Phosphatase in body fluids: The differentiation of semen and vaginal secretion, Forensic Sci., 3 (1974) 57-62. L. C. Nickolls, The Scientific Investigation of Crime, Butterworth, London, 1966, pp. 198. J. Glaister, Medical Jurisprudence and Toxicology, Livingstone, Edinburgh and London, i957, pp. 415. A. Blanc0 and W. H. Zinkham, Lactate debydrogenases in human testis, Science, 139 (1963) 601. E. Goldberg, Lactic and malic dehydrogenase in human spermatozoa, Science, 139 (1963) 602. R. H. Mokashi, A. G. Malwankar and M. S. Madiwale, Detection of semen in the presence of blood and vaginal secretion, J. Indian Acad. Forensic Sci., 14 (1975) l-3. M. S. Madiwale, H. S. Mahal and R. W. P. Master, Der Nachweis der Blutart im Blutspuren durch Isoenzymbestimmung, Arch. Kriminol., 150 (1972) 160.. 166. A. A. Dietz and T. Lubrano, Separation and quantitation of LDH isoenzymes by disc electrophoresis, Anal. Biochem., 20 (1967) 246 257. R. H. Mokashi and M. S. Madiwale, Isozym-methoden zur Unterscheidung von Menschen-und Affenblut, Arch. Kriminol., 153 (1974) 48 ~53. L. C. Nickolls, The Scientific Investigation of Crime, Butterworth, London, 1956, pp. 200. K. Iosifov, Isoenzyme composition of the lactate dehydrogenase in ram, bull, boar and stallion seminal plasma, spermatozoa and blood serum, Vet. Med. Nauki, 8 (1971) 43m 50.