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Studies on the Blood Group Substances in Saliva
3. Forens. Sci. Soc.
(1979), 19, 301
Received 30 November 1979
Studies on the Blood Group Substances in Saliva T . J. ROTHWELL Honze Ofice Forensic Science Laboratory, Usk Road, Chepstow, G'went, Wales, N P 6 6J7E.
Following fractionation of saliva samples on Sephadex columns attempts have been made to detect the variousfractio~zsof bloodgroup activip previously reported by Fiori et al. Only a s i ~ g l email2 fraction of activity has been fozizd in salivas from secretor individuals zlsing both absorption/inhibition and elution techniques. Most non-secretor salivas exhibited activity detectable by absorptionlelution but not by inhibition. ( T h i s paper should be read in conjunction with those by Kind et al., Lang et al., p. 293 in this issue).
p. 287 and
Introduction Traditional blood grouping serology has long recognised the existence of two types of reactions in saliva and other body fluids-"secretor" and "nonsecretor"-depending on the presence or otherwise of blood group substances in the material. These substances havc been detected by the absorption/ inhibition test; more recently the absorption/elution test has also been used. Fiori and his co-workers (Fiori et al., 1969, 1971 and 1973) have studied the blood group substances in saliva using gel filtration chromatography on Sephadex columns. The main blood group active fraction possessed a molecular weight above the exclusion limit of the gel, and thus passcd straight through the column. This main fraction 1 was not found in non-secretor salivas. I n addition to this fraction 1 Fiori et al. reported two sub-fractions: fraction 2 having a molecular weight of about 10,000, and fraction 3 with a molecular weight of about 1,500 (Fiori, Giusti and Panari, 1969). These two fractions were eluted after the main active fraction. Either or both of these sub-fractions were found in saliva from secretors and non-secretors, with some samples containing neither fractions 2 or 3. I t has been suggested that the presence of these latter fractions is genetically controlled (Fiori et al., 1972) and that they are found in semen (Panari, Rossi and Fiori, 1976). The existence of such easily determined sub-groups of the ABO system would be of great significance in the investigation of sexual offences if the sub-groups were found to be present in semen. Until very recently only the ABO system has been available for the typing of semen stains, and any extension of this would improve the evidential value of such grouping tests. Work by Rutter and Whitehead (1976) failed to detect fractions 2 or 3 in saliva, although a fairly large number of samples was examined and a sensitive inhibition technique using automated analysis employed for the detection of blood group substances. I n view of these findings it was decided to repeat this work using a manual absorption/inhibition grouping technique; a t the same time the opportunity was taken to examine the products of gel filtration by the absorption/elution technique. Materials and Methods The method closely followed that of Fiori. A column of 25cm x 1.2cm (bed volume about 30ml) was used, being filled with pre-swollen Sephadex GI00 Superfine, and eluted with 0.05M tris-saline buffer a t p H 7.3. Most of the saliva samples were used immediately after collection, although one or two had been stored for some time a t -20°C. The saliva. samples were centrifuged and
~neasuredvolumes (0.5ml) of the supernatant liquid applied directly to the top of the column. The saliva was eluted using the 0.05M tris-saline buffer, and 40-50 fractions each of about 1.5ml collected. The fractions were tested by both absorption/inhibition (AII) and e1utio:l (A/E) tzchniques. For the A/I test 1 drop of each fraction was mixed with 1 drop of the relevant antiserum and allowed to absorb for 30 minutes a t room temperature. The seracommercial anti-A and B, and anti-H prepared in the laboratory-were titred prior to use and utilised a t the appropriate dilution. Follo~ringabsorption, 1 drop of a red cell suspension (approximately 176) of A, B, or 0 cells was added to the antisera as appropriate and the tests gently agitated on a rotatingshaker for 15 minutes. After t!lis time the degree of agglutination was scored on a n arbitrary scale from 0-4. For the absorption/elution tests clean cotton threads were dipped into each column fraction and allowed to dry at room temperat~re.Each thread was then tested using the Howard and Martin (1969) modification of the Nickolls and Pereira (1962) elution technique, and the degree of agglutination again scored on a scale from 0-4. Between each application of saliva to the column, it was washed with a t least 10 column volumes of buffer. The column characteristics were checked by eluting solutions of Blue Dextran (Sigma) (approx. mol. wt. 2,000,000) and also cytochrome C (mol. wt. 12,400).
Results and Discussion Secretor and non-secretor salivas of groups A, B, and 0 were testcd (Table 1) and the results are shown in Figures 1-4. O n these diagrams is plotted the degree of egglutination observed in the inhibition and elution tests on the various column fractions. The agglutination score in the elution tests was plotted directly. For the A/I tests the degree of inhibition was subtracted from the score of the unabsorbed serum; the sole purpose of this was to enable both A/I and A/E results to be shown as positive peaks and so be directly comparable. Typical results for a group 0 secretor saliva are shown in Figure 1. A single s!larp peak of blood group activity was observed by both series of serological tests. The peak of activity was located in the same fractions whether detected by the A/I or A/E techniques, and this pea!< corresponded exactly with the exclusion volume indicating that the blood group substance was of a high molecular weight. The results for a group B secretor are shown in Figure 2, and this exhibits similar sharp peaks of activity by both inhibition and elution. I n most samples examined the "H" activity formed a sharper peak than that of the "A" or "B" activity, and this is shown in the sample represented in Figure 2. The results from a group A secretor are shown in Figure 3, and the considerable tailing of the peak in this sample indicates the very high level of blood group substance found in some salivas. This tailing is particularly noticeable in the "A" activity as detected by the A/E technique. T h e apparent peaks of activity, e.g. in column fractions 36-38, were not found to be reproducible in this or in
NUMBER OF SAI.I\'A SARlPLES EXASIINED Group 0 Secretor 3 0 non-srrrelor -9 secretor 4 A non-secretor 7 B secretor 1 R non-secretor 3 No group An salivas were examined in this series of srlmple~.
.\
Agg. S c o r e
A- l
0
5
0
10
15
20
Fraction Figure 1.
SEC
25
30
35
40
No.
Aggl~~tination score of column J'rzctions ohtained from a Group 0 secretor saliva.
Agg. Score
A- i
0
Figure 2.
5
B
10
SEC
20
25
Fraction
!'do.
15
30
35
40
.4galutination scores of colunin f'ractionb obtained from a Group I3 srcrctor saliva.
Agg. Score
A,
A- l
Fraction Figure 3.
SEC
NO.
Agglutination scores of column fractions obtained from a Group A secretor saliva.
other samples. This effect is probably due to the column being overloaded with material. The results from the A/I tests exactly parallel those of Rutter and Whitehead (1976) to which reference has already been made, and similar patterns were obtained using the A/E technique. Only a single pcak of blood group activity (corresponding to the Fiori fraction 1) was detected in secretor salivas; no trace of the Fiori fractions 2 or 3 was recorded. Ever since the absorption/elution technique has been used for the examination of body fluids it has been recognised that the non-secretor may in fact secrrte relatively high concentrations of blood group substance detectable by this technique (Pereira, 1971). Grouping results (Table 2) on two samples of group A non-secretor salivas illustrate the extremes of results which may be obtained when grouping such samples by absorption/elution. Results from a group A secretor saliva are included for comparison. I t will be seen that although there is a clear distinction between the secretor and non-secretor salivas on the basis of their results by the inhibition technique, using the elution technique nonsecretor salivas can yield results which correspond closely to those obtained from secretor samples. I t would thus appear that the inhibition and elution techniques might be detecting different types of blood group substance. Several non-secretor salivas were examined by Sephadex fractionation. Most gave results similar to those shown in Figure 4. This shows that while no activity was recorded using the inhibition technique, a sharp peak of activity corresponding to the exclusion volume was detected by elution. Accordingly, although such salivas may contain a different type of blood group substance it appears to be of similar molecular weight to that present in secretors. Although Fiori has used the presence or absence of blood group activity in fraction 1 to distinguish secretor status, in this study activity has been detected in this fraction in samples from both secretors and non-secretors. These results present no evidence to suggest the activity can be correlated in any way with Fiori's fractions 2 or 3. A small number of non-secretor salivas yielded no detectable blood group substance by either elution or inhibition techniques following fractionation on the Sephadex column. These salivas were of the type which yielded no results when the neat sample was tested for blood group activity using the elution technique, i.e. resembling sample 1 in Table 2, and accordingly it seems likely that the level of blood group substance in these samples was below the limit of detection of the system.
TABLE 2 TYPICAL .AGGLUTINATION SCORES IN BLOOD GROUPING TESTS O N SECRETOR AND WON-SECRETOR SAI.I\'AS Antzser~rnz A
u Group A non-secretor Sample 1 Group A non-secrctor Sample 2
H A R tI
Groupzng test results Absorpt*on/tnhrbztzon ~lbsotptz~n/elutzon Neat Saltva dzlntzon~: Neat Salzva dz1:rtlons : sallva 1/10 11100 1'1000 salrva 1/10 1/100 1/1000 0 0 0 0 0 3 7 2 3 3 3 4 o o o n o o n o 7 4 3 o 2 7 4 -1 0 0 0 0 3 4 4 4 0 0 0 0 4 4 4 4 0 0 0 0
Agg. Score
A-l
A
NON-SEC
(14)
4a
3-
2
-
1
-
0
I
I
A4
-
3
-
21
-
I
I
1
1
I
15
20
25
Fraction
No.
I
I
I
I
i
35
40
E
. r, I I I I I I
I I I I I
1
I
I
-
I I
00
Figure 4.
I I L-1
5
10
30
Agglutination scores of colun~nfractions obtained from a non-secretor saliva.
Conclusions This work has extended the attempts made by Rutter and Whitehead to confirm the experiments of Fiori, without success. Only a single peak of blood group activity has been detected in fractions eluted from a Sephadex GlOO column. This activity has been confirmed in secretor salivas by both inhibition and elution techniques. Most non-secretor salivas were found to contain peaks of blood group activity detectable by absorption/elution only. These peaks occurred in the same column fractions as the activity detected in secretor salivas, and thus were high molecular weight substances, and not the low molecular weight compounds postulated by Fiori as fractions 2 or 3. I t would be interesting to ascertain whether the nature of the blood group substances differs in secretor and nonsecretor salivas. References FIORI,A., GIUSTI,G. V. and PANARI,G., 1969, Med. Leg. et dommage corp., 2, 364-366. G., 1971, J. Chromatog., 55, 337-349. FIORI,A., GIUSTI,G. V., and PANARI, FIORI,A., GIUSTI,G. V. and PANARI, G., 1971, J. Chromatog., 55, 351-363. FIORI,A., SERRA,A., PANARI,G. and GIUSTI,G. V., 1972, Atti. Assoc. Genet. Ital., 17, 38-42. G., 1973, J . Chromatog. 84, FIORI,A., PANARI,G., GIUSTI,G. V. and BRANDI, 335-346. HOWARD, H. D. and MARTIN,P. D., 1969, J. Forens. Sci. Soc., 9, 28-30. M., 1962, Med. Sci. Law, 2, 172-179. NICKOLLS, L. C. and PEREIRA, PANARI,G., ROSSI,G. and FIORI,A., 1976, Forensic Science, 7, 55-60. PEREIRA,hf., 1971, I n The Examination and Typing of Bloodstains in the Crime Laboratory, by B. J. Culliford, National Institute of Law Enforcement and Criminal Justice, Washington D.C., p. 98. RUTTER,E. R. and WHITEHEAD, P. H., 1976, J. Forens. Sci. Soc., 16, 241-246.
(1979), 19, 301
Received 30 November 1979
Studies on the Blood Group Substances in Saliva T . J. ROTHWELL Honze Ofice Forensic Science Laboratory, Usk Road, Chepstow, G'went, Wales, N P 6 6J7E.
Following fractionation of saliva samples on Sephadex columns attempts have been made to detect the variousfractio~zsof bloodgroup activip previously reported by Fiori et al. Only a s i ~ g l email2 fraction of activity has been fozizd in salivas from secretor individuals zlsing both absorption/inhibition and elution techniques. Most non-secretor salivas exhibited activity detectable by absorptionlelution but not by inhibition. ( T h i s paper should be read in conjunction with those by Kind et al., Lang et al., p. 293 in this issue).
p. 287 and
Introduction Traditional blood grouping serology has long recognised the existence of two types of reactions in saliva and other body fluids-"secretor" and "nonsecretor"-depending on the presence or otherwise of blood group substances in the material. These substances havc been detected by the absorption/ inhibition test; more recently the absorption/elution test has also been used. Fiori and his co-workers (Fiori et al., 1969, 1971 and 1973) have studied the blood group substances in saliva using gel filtration chromatography on Sephadex columns. The main blood group active fraction possessed a molecular weight above the exclusion limit of the gel, and thus passcd straight through the column. This main fraction 1 was not found in non-secretor salivas. I n addition to this fraction 1 Fiori et al. reported two sub-fractions: fraction 2 having a molecular weight of about 10,000, and fraction 3 with a molecular weight of about 1,500 (Fiori, Giusti and Panari, 1969). These two fractions were eluted after the main active fraction. Either or both of these sub-fractions were found in saliva from secretors and non-secretors, with some samples containing neither fractions 2 or 3. I t has been suggested that the presence of these latter fractions is genetically controlled (Fiori et al., 1972) and that they are found in semen (Panari, Rossi and Fiori, 1976). The existence of such easily determined sub-groups of the ABO system would be of great significance in the investigation of sexual offences if the sub-groups were found to be present in semen. Until very recently only the ABO system has been available for the typing of semen stains, and any extension of this would improve the evidential value of such grouping tests. Work by Rutter and Whitehead (1976) failed to detect fractions 2 or 3 in saliva, although a fairly large number of samples was examined and a sensitive inhibition technique using automated analysis employed for the detection of blood group substances. I n view of these findings it was decided to repeat this work using a manual absorption/inhibition grouping technique; a t the same time the opportunity was taken to examine the products of gel filtration by the absorption/elution technique. Materials and Methods The method closely followed that of Fiori. A column of 25cm x 1.2cm (bed volume about 30ml) was used, being filled with pre-swollen Sephadex GI00 Superfine, and eluted with 0.05M tris-saline buffer a t p H 7.3. Most of the saliva samples were used immediately after collection, although one or two had been stored for some time a t -20°C. The saliva. samples were centrifuged and
~neasuredvolumes (0.5ml) of the supernatant liquid applied directly to the top of the column. The saliva was eluted using the 0.05M tris-saline buffer, and 40-50 fractions each of about 1.5ml collected. The fractions were tested by both absorption/inhibition (AII) and e1utio:l (A/E) tzchniques. For the A/I test 1 drop of each fraction was mixed with 1 drop of the relevant antiserum and allowed to absorb for 30 minutes a t room temperature. The seracommercial anti-A and B, and anti-H prepared in the laboratory-were titred prior to use and utilised a t the appropriate dilution. Follo~ringabsorption, 1 drop of a red cell suspension (approximately 176) of A, B, or 0 cells was added to the antisera as appropriate and the tests gently agitated on a rotatingshaker for 15 minutes. After t!lis time the degree of agglutination was scored on a n arbitrary scale from 0-4. For the absorption/elution tests clean cotton threads were dipped into each column fraction and allowed to dry at room temperat~re.Each thread was then tested using the Howard and Martin (1969) modification of the Nickolls and Pereira (1962) elution technique, and the degree of agglutination again scored on a scale from 0-4. Between each application of saliva to the column, it was washed with a t least 10 column volumes of buffer. The column characteristics were checked by eluting solutions of Blue Dextran (Sigma) (approx. mol. wt. 2,000,000) and also cytochrome C (mol. wt. 12,400).
Results and Discussion Secretor and non-secretor salivas of groups A, B, and 0 were testcd (Table 1) and the results are shown in Figures 1-4. O n these diagrams is plotted the degree of egglutination observed in the inhibition and elution tests on the various column fractions. The agglutination score in the elution tests was plotted directly. For the A/I tests the degree of inhibition was subtracted from the score of the unabsorbed serum; the sole purpose of this was to enable both A/I and A/E results to be shown as positive peaks and so be directly comparable. Typical results for a group 0 secretor saliva are shown in Figure 1. A single s!larp peak of blood group activity was observed by both series of serological tests. The peak of activity was located in the same fractions whether detected by the A/I or A/E techniques, and this pea!< corresponded exactly with the exclusion volume indicating that the blood group substance was of a high molecular weight. The results for a group B secretor are shown in Figure 2, and this exhibits similar sharp peaks of activity by both inhibition and elution. I n most samples examined the "H" activity formed a sharper peak than that of the "A" or "B" activity, and this is shown in the sample represented in Figure 2. The results from a group A secretor are shown in Figure 3, and the considerable tailing of the peak in this sample indicates the very high level of blood group substance found in some salivas. This tailing is particularly noticeable in the "A" activity as detected by the A/E technique. T h e apparent peaks of activity, e.g. in column fractions 36-38, were not found to be reproducible in this or in
NUMBER OF SAI.I\'A SARlPLES EXASIINED Group 0 Secretor 3 0 non-srrrelor -9 secretor 4 A non-secretor 7 B secretor 1 R non-secretor 3 No group An salivas were examined in this series of srlmple~.
.\
Agg. S c o r e
A- l
0
5
0
10
15
20
Fraction Figure 1.
SEC
25
30
35
40
No.
Aggl~~tination score of column J'rzctions ohtained from a Group 0 secretor saliva.
Agg. Score
A- i
0
Figure 2.
5
B
10
SEC
20
25
Fraction
!'do.
15
30
35
40
.4galutination scores of colunin f'ractionb obtained from a Group I3 srcrctor saliva.
Agg. Score
A,
A- l
Fraction Figure 3.
SEC
NO.
Agglutination scores of column fractions obtained from a Group A secretor saliva.
other samples. This effect is probably due to the column being overloaded with material. The results from the A/I tests exactly parallel those of Rutter and Whitehead (1976) to which reference has already been made, and similar patterns were obtained using the A/E technique. Only a single pcak of blood group activity (corresponding to the Fiori fraction 1) was detected in secretor salivas; no trace of the Fiori fractions 2 or 3 was recorded. Ever since the absorption/elution technique has been used for the examination of body fluids it has been recognised that the non-secretor may in fact secrrte relatively high concentrations of blood group substance detectable by this technique (Pereira, 1971). Grouping results (Table 2) on two samples of group A non-secretor salivas illustrate the extremes of results which may be obtained when grouping such samples by absorption/elution. Results from a group A secretor saliva are included for comparison. I t will be seen that although there is a clear distinction between the secretor and non-secretor salivas on the basis of their results by the inhibition technique, using the elution technique nonsecretor salivas can yield results which correspond closely to those obtained from secretor samples. I t would thus appear that the inhibition and elution techniques might be detecting different types of blood group substance. Several non-secretor salivas were examined by Sephadex fractionation. Most gave results similar to those shown in Figure 4. This shows that while no activity was recorded using the inhibition technique, a sharp peak of activity corresponding to the exclusion volume was detected by elution. Accordingly, although such salivas may contain a different type of blood group substance it appears to be of similar molecular weight to that present in secretors. Although Fiori has used the presence or absence of blood group activity in fraction 1 to distinguish secretor status, in this study activity has been detected in this fraction in samples from both secretors and non-secretors. These results present no evidence to suggest the activity can be correlated in any way with Fiori's fractions 2 or 3. A small number of non-secretor salivas yielded no detectable blood group substance by either elution or inhibition techniques following fractionation on the Sephadex column. These salivas were of the type which yielded no results when the neat sample was tested for blood group activity using the elution technique, i.e. resembling sample 1 in Table 2, and accordingly it seems likely that the level of blood group substance in these samples was below the limit of detection of the system.
TABLE 2 TYPICAL .AGGLUTINATION SCORES IN BLOOD GROUPING TESTS O N SECRETOR AND WON-SECRETOR SAI.I\'AS Antzser~rnz A
u Group A non-secretor Sample 1 Group A non-secrctor Sample 2
H A R tI
Groupzng test results Absorpt*on/tnhrbztzon ~lbsotptz~n/elutzon Neat Saltva dzlntzon~: Neat Salzva dz1:rtlons : sallva 1/10 11100 1'1000 salrva 1/10 1/100 1/1000 0 0 0 0 0 3 7 2 3 3 3 4 o o o n o o n o 7 4 3 o 2 7 4 -1 0 0 0 0 3 4 4 4 0 0 0 0 4 4 4 4 0 0 0 0
Agg. Score
A-l
A
NON-SEC
(14)
4a
3-
2
-
1
-
0
I
I
A4
-
3
-
21
-
I
I
1
1
I
15
20
25
Fraction
No.
I
I
I
I
i
35
40
E
. r, I I I I I I
I I I I I
1
I
I
-
I I
00
Figure 4.
I I L-1
5
10
30
Agglutination scores of colun~nfractions obtained from a non-secretor saliva.
Conclusions This work has extended the attempts made by Rutter and Whitehead to confirm the experiments of Fiori, without success. Only a single peak of blood group activity has been detected in fractions eluted from a Sephadex GlOO column. This activity has been confirmed in secretor salivas by both inhibition and elution techniques. Most non-secretor salivas were found to contain peaks of blood group activity detectable by absorption/elution only. These peaks occurred in the same column fractions as the activity detected in secretor salivas, and thus were high molecular weight substances, and not the low molecular weight compounds postulated by Fiori as fractions 2 or 3. I t would be interesting to ascertain whether the nature of the blood group substances differs in secretor and nonsecretor salivas. References FIORI,A., GIUSTI,G. V. and PANARI,G., 1969, Med. Leg. et dommage corp., 2, 364-366. G., 1971, J. Chromatog., 55, 337-349. FIORI,A., GIUSTI,G. V., and PANARI, FIORI,A., GIUSTI,G. V. and PANARI, G., 1971, J. Chromatog., 55, 351-363. FIORI,A., SERRA,A., PANARI,G. and GIUSTI,G. V., 1972, Atti. Assoc. Genet. Ital., 17, 38-42. G., 1973, J . Chromatog. 84, FIORI,A., PANARI,G., GIUSTI,G. V. and BRANDI, 335-346. HOWARD, H. D. and MARTIN,P. D., 1969, J. Forens. Sci. Soc., 9, 28-30. M., 1962, Med. Sci. Law, 2, 172-179. NICKOLLS, L. C. and PEREIRA, PANARI,G., ROSSI,G. and FIORI,A., 1976, Forensic Science, 7, 55-60. PEREIRA,hf., 1971, I n The Examination and Typing of Bloodstains in the Crime Laboratory, by B. J. Culliford, National Institute of Law Enforcement and Criminal Justice, Washington D.C., p. 98. RUTTER,E. R. and WHITEHEAD, P. H., 1976, J. Forens. Sci. Soc., 16, 241-246.