A preliminary evaluation of typing commonly occurring human erythrocyte acid phosphatase (ACP1) phenotypes in bloodstains, using the LKB Immobiline system

Abstract

A preliminary study has been made of the use of immobilised pH gradients (Immobiline gels) for the separation of the six commonly occurring erythrocyte acid phosphatase (ACPl) phenotypes in blood lysates and stains. These pH gradients are immobilised in the matrix of the polyacrylamide gel and possess different properties from conventional isoelectric focusing (IEF) gels. A comparison was made between the profile of the ACP 1 isoenzymes apparent on conventional IEF gels with that seen on the Immobiline gels. I IntroductionHopkinson et al. [ Although IEF has proved to be a more sensitive method oftyping ACPl activity than the starch gel technique, particularly in bloodstains, there are still problems in interpretation, paricularly where A activity is low and where one has to discriminate between the B and CB phenotypes. Recently immobilised pH gradients have been described [7,81 which are prepared with a set of commercially available acrylamide derivatives (Immobiline). Both basic and acidic Immobilines are mixed separately with a number of reagents normally used in the preparation of polyacrylamide gels in a gradient mixer to form a gel which varies in pH. The Immobilines finally used will depend upon the pIrange ofthe system to be investigated, which, for the common ACPl variants, is pH 5.77-7.58 [91. With these considerations in mind a study was set up to evaluate the Immobiline technique with particular regard to the problems associated with ACP 1 typing in bloodstains. This preliminary report follows the separation of six common phenotypes of ACPl from blood lysates and stains using the Immobiline gel system and discusses the advantages of this technique over conventional IEF. Materials and methods Preparation of red cell lysates and bloodstainsBlood was collected in heparinized tubes by finger pricks from known ACPl donors. After centrifuging, the serum was removed and the erythrocytes were washed twice with 0.85 % wlv saline between further centrifugations. The resultant pellet of erythrocytes were then lysed by freezing and thawing. Bloodstains were made from finger pricks on cotton cloth from donors with known and unknown ACP 1 phenotypes, and were air-dried before analysis. Immobiline gel preparationGeneral details of gel preparation are given in LKB Application Note 32 1 [71. The gels were 245 x 100 x 0.5 mm and, for sample application, they contained slots of the size 10 x 2 x 0.2 mm, made by applying Dymo Tape to the surface of the glass plate. A pH gradient of pH 5.5-7.6 was selected which would give the optimal separation ofthe commonly occurring ACPl phenotypes which have PI values in the range pH 5.77-7.58 191. Stock solutionsAcrylamide 29.1 % w/v containing bisacrylamide 0.9 % w/v; glycerol 87 % w/v; Immobilines 0.2 M solutions of pK 7.0, pK 6.2 and pK 3.6; ammonium persulphate 10 % w/v; NNN'N'-tetramethylethylenediamine (TEMED) 10 % v/v (Table 1).

Forensic science

“…Isoelectric focusing (IEF) has become a valuable technique in the field of forensic serology, and the use of ultrathin gel is making it even more powerful (335). IEF has been used to type esterase D (ESD) (336)(337)(338), group specific component (Gc) (339-343, 335), erythrocyte acid phosphatase (ACPi) (344), transferrin (Tf) (345), and phosphoglucomutase (PGM) (346)(347)(348)(349)(350)(351)(352)335). The value of ultrathin polyacrylamide gels for the simultaneous separation of PGM and EAP by isoelectric focusing of human red cell lysates and bloodstain extracts has been investigated (353).…”

Polymorphism of ACP1 by isoelectric focusing : Population study in Japanese and phenotyping in bloodstains.

“…Recently, isoelectric focusing has been introduced for the analysis of ACP1 isoenzymes because of the high resolution capacity (Burdett and Whitehead 1977;Randall et al 1980;Divall 1981;Carracedo and Concheiro 1982;Dorrill and Sutton 1983;Budowle 1984;Westwood and Sutton 1984;Finney et al 1985;Yuasa et al 1985;Frank and Stolorow 1986). Using this technique some of the above workers phenotyped ACP1 from bloodstains and these ranged in age from 2 weeks to 1 year (Randall et al 1980;Dorrill and Sutton 1983;Westwood and Sutton 1984;Frank and Stolorow 1986).…”

“…Using this technique some of the above workers phenotyped ACP1 from bloodstains and these ranged in age from 2 weeks to 1 year (Randall et al 1980;Dorrill and Sutton 1983;Westwood and Sutton 1984;Frank and Stolorow 1986). However, no systematic stability experiments were made on the time limit of determination of this system.…”