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A Simplified Mixed Agglutination Technique for ABO Grouping of Dried Bloodstains Using Cellulose Acetate Sheets
3. Forens.
Sci. SOC.(1977), 17, 143
A Simplified Mixed Agglutination Technique for ABO Grouping of Dried Bloodstains Using Cellulose Acetate Sheets -
P. K. CHATTERJI Central Forensic Science Lzboratory, New Delhi, India.
A quick and sensitive method.fir the determination of the ABO groups of dried bloodstains is described. The dtjiculties experienced with the earlier methods of mixed agglutination using cavity slides have been overcome by using cellulose acetate sheets instead. Results of extensive trials have demonstrated that the test is as sensitive and reliable as the absorption-elution test currently in use. Introduction A number of methods for grouping of dried bloodstains have been developed from time to time: absorption-inhibition (Boyd and Boyd, 1937), mixed agglutination (Coombs and Dodd, 1961) and absorption-elution (Kind, 1960). All the techniques described so far have their own advantages and handicaps. Howard and Martin (1969), however, developed a modified and simpler method for grouping dried bloodstains which was based on the absorptionelution technique outlined by Nickolls and Pereira (1962) and Outteridge (1963). This absorption-elution technique is currently in use in many laboratories. With the increase in number of cases there is a need for a quicker method having the same sensitivity as the methods currently in use. I n this communication, a simplified mixed agglutination technique is described, which is both quick and sensitive with a time saving of a t least 40%, compared with the Howard and Martin technique. The most advantageous part of this method is that during the normal working day, two batches of bloodstains can be processed by the same worker. Materials and Method Standard blood grouping antisera, anti-A and anti-B each having a titre of 1 :256 were obtained commercially while Anti-H serum with a titre of 1:128 was prepared from the seeds of Ulex europeus. Reference bloodstains A, B, AB and 0 were prepared from the members of staff. A few drops of blood were soaked on a clean white cotton cloth piece, dried in air and kept in paper envelopes in a dessicator at room temperature. White cellulose acetate sheets (0.4mm thickness) were cut to size and divided into small squares. Three threads (each 2-3mm long) were taken out from the stained area and glued at one end to the acetate sheet. Unstained control threads were similarly fixed. One drop each of anti-A, anti-B and anti-H serum was added to the respective squares and the free ends of the fibres were teased with a fine needle. The sheets were then transferred to moist chambers for the absorption stage in the refrigerator (4°C). They were then washed 2-3 times with a jet of chilled saline using a polythene wash bottle. The sheets were blotted dry and one drop of indicator cells (0.4-1.0% in physiological saline) were added to respective squares. The sheets were then put on a rotary shaker for a period of 15-20 minutes after which readings were taken using a stereo-
microscope. Bead-like cell cluster attachments on the fibres were interpreted as a positive result. Slight or doubtful cell attachments were not considered.
Results and Discussion The accuracy of the method was first established using the known stains. Subsequently, results of grouping nearly 150 dried bloodstains of various ages (up to 5 years old) by this method were found to be in complete agreement with the results previously obtained by the Howard and Martin method. The two methods were thus compared on the basis of sensitivity, reliability and time involved in the laboratory. O n no occasion did unstained control threads give a positive reaction but even for older stains, the degree of agglutination was quite conspicuous. In this present method, the absorption period is reduced to one hour instead of the minimum period of 3 hours. This present method was also tried with a 3 hour absorption period as well as overnight absorption periods at 4OC but the results showed no improvement over the 1 hour period. Furthermore, a cumbersome washing procedure has been minimised while the elution step is eliminated altogether. With these modifications, there is not only considerable time saving but the sensitivity and reliability of the test is maintained. The degree of agglutination on the threads was sufficient for scoring. Occasionally, there was also some agglutination of cells in the medium around the thread which could be possibly due to some elution having taken place during the shaking procedure as this was done at room temperature in a tropical area. These agglutinations were ignored for scoring purposes. Absorption at room temperature as suggested by Coombs and Dodd (1961) and Roy Chowdhary (1963) was found to be unsuitable. Fiori et al., (1963) also found better results by absorbing the antisera at 4OC. Indicator cells of higher dilutions have been recommended by various workers (Roy Chowdhary, 1963; Mitra and Ganguly, 1973) but in the present method results were satisfactory with cells in a 0-5-1.0% suspension. Recently, Mitra and Ganguly (1973) described a method of mixed agglutination carried out on glass cavity slides. However, difficulties are experienced when teasing the fibres which are lying free on the slide cavities and because the washing procedure has been completely dispensed with free agglutinates due to the presence of unabsorbed antiserum can lead to confusing results. In the present method both these difficulties have been eliminated. Acknowledgement The author is thankful to Dr. H. L. Bami, Director, Central Forensic Science Laboratory, New Delhi, for constructive suggestions. Thanks are also due to Shri Katar Singh for technical assistance. References BOYD,W. C. and BOYD,L. G., 1937,J. Immunol., 33, 159. COOMBS, R. R. A. and DODD,Barbara, 1961, Med. Sci. Law, 1, 354. P., 1963,J. For. Sci., 8,419 and 535. FIORI,A. M. M. and BENCIOLINI, HOWARD, H. D. and MARTIN,P. D., 1969,J. For. Sci. Soc., 9, 28. KIND,S. S., 1960, Nature, 185, 397. MITRA,A. K. and GANGULY, H. N., 1973, Ind. 3. Expt. Biol., 11, 433. NICKOLLS, L. D. and PEREIRA, M., 1962, Med. Sci. Law, 2, 172. OUTTERIDGE, R. A., 1963, Nature, 198, 698. A. B., 1963, Annals of Biochemistry and Experimental Medicine, ROYCHOWDHARY, 23, 271.
Sci. SOC.(1977), 17, 143
A Simplified Mixed Agglutination Technique for ABO Grouping of Dried Bloodstains Using Cellulose Acetate Sheets -
P. K. CHATTERJI Central Forensic Science Lzboratory, New Delhi, India.
A quick and sensitive method.fir the determination of the ABO groups of dried bloodstains is described. The dtjiculties experienced with the earlier methods of mixed agglutination using cavity slides have been overcome by using cellulose acetate sheets instead. Results of extensive trials have demonstrated that the test is as sensitive and reliable as the absorption-elution test currently in use. Introduction A number of methods for grouping of dried bloodstains have been developed from time to time: absorption-inhibition (Boyd and Boyd, 1937), mixed agglutination (Coombs and Dodd, 1961) and absorption-elution (Kind, 1960). All the techniques described so far have their own advantages and handicaps. Howard and Martin (1969), however, developed a modified and simpler method for grouping dried bloodstains which was based on the absorptionelution technique outlined by Nickolls and Pereira (1962) and Outteridge (1963). This absorption-elution technique is currently in use in many laboratories. With the increase in number of cases there is a need for a quicker method having the same sensitivity as the methods currently in use. I n this communication, a simplified mixed agglutination technique is described, which is both quick and sensitive with a time saving of a t least 40%, compared with the Howard and Martin technique. The most advantageous part of this method is that during the normal working day, two batches of bloodstains can be processed by the same worker. Materials and Method Standard blood grouping antisera, anti-A and anti-B each having a titre of 1 :256 were obtained commercially while Anti-H serum with a titre of 1:128 was prepared from the seeds of Ulex europeus. Reference bloodstains A, B, AB and 0 were prepared from the members of staff. A few drops of blood were soaked on a clean white cotton cloth piece, dried in air and kept in paper envelopes in a dessicator at room temperature. White cellulose acetate sheets (0.4mm thickness) were cut to size and divided into small squares. Three threads (each 2-3mm long) were taken out from the stained area and glued at one end to the acetate sheet. Unstained control threads were similarly fixed. One drop each of anti-A, anti-B and anti-H serum was added to the respective squares and the free ends of the fibres were teased with a fine needle. The sheets were then transferred to moist chambers for the absorption stage in the refrigerator (4°C). They were then washed 2-3 times with a jet of chilled saline using a polythene wash bottle. The sheets were blotted dry and one drop of indicator cells (0.4-1.0% in physiological saline) were added to respective squares. The sheets were then put on a rotary shaker for a period of 15-20 minutes after which readings were taken using a stereo-
microscope. Bead-like cell cluster attachments on the fibres were interpreted as a positive result. Slight or doubtful cell attachments were not considered.
Results and Discussion The accuracy of the method was first established using the known stains. Subsequently, results of grouping nearly 150 dried bloodstains of various ages (up to 5 years old) by this method were found to be in complete agreement with the results previously obtained by the Howard and Martin method. The two methods were thus compared on the basis of sensitivity, reliability and time involved in the laboratory. O n no occasion did unstained control threads give a positive reaction but even for older stains, the degree of agglutination was quite conspicuous. In this present method, the absorption period is reduced to one hour instead of the minimum period of 3 hours. This present method was also tried with a 3 hour absorption period as well as overnight absorption periods at 4OC but the results showed no improvement over the 1 hour period. Furthermore, a cumbersome washing procedure has been minimised while the elution step is eliminated altogether. With these modifications, there is not only considerable time saving but the sensitivity and reliability of the test is maintained. The degree of agglutination on the threads was sufficient for scoring. Occasionally, there was also some agglutination of cells in the medium around the thread which could be possibly due to some elution having taken place during the shaking procedure as this was done at room temperature in a tropical area. These agglutinations were ignored for scoring purposes. Absorption at room temperature as suggested by Coombs and Dodd (1961) and Roy Chowdhary (1963) was found to be unsuitable. Fiori et al., (1963) also found better results by absorbing the antisera at 4OC. Indicator cells of higher dilutions have been recommended by various workers (Roy Chowdhary, 1963; Mitra and Ganguly, 1973) but in the present method results were satisfactory with cells in a 0-5-1.0% suspension. Recently, Mitra and Ganguly (1973) described a method of mixed agglutination carried out on glass cavity slides. However, difficulties are experienced when teasing the fibres which are lying free on the slide cavities and because the washing procedure has been completely dispensed with free agglutinates due to the presence of unabsorbed antiserum can lead to confusing results. In the present method both these difficulties have been eliminated. Acknowledgement The author is thankful to Dr. H. L. Bami, Director, Central Forensic Science Laboratory, New Delhi, for constructive suggestions. Thanks are also due to Shri Katar Singh for technical assistance. References BOYD,W. C. and BOYD,L. G., 1937,J. Immunol., 33, 159. COOMBS, R. R. A. and DODD,Barbara, 1961, Med. Sci. Law, 1, 354. P., 1963,J. For. Sci., 8,419 and 535. FIORI,A. M. M. and BENCIOLINI, HOWARD, H. D. and MARTIN,P. D., 1969,J. For. Sci. Soc., 9, 28. KIND,S. S., 1960, Nature, 185, 397. MITRA,A. K. and GANGULY, H. N., 1973, Ind. 3. Expt. Biol., 11, 433. NICKOLLS, L. D. and PEREIRA, M., 1962, Med. Sci. Law, 2, 172. OUTTERIDGE, R. A., 1963, Nature, 198, 698. A. B., 1963, Annals of Biochemistry and Experimental Medicine, ROYCHOWDHARY, 23, 271.