The use of ultrathin-layer agarose gels for phenotyping erythrocyte acid phosphatase by isoelectric focusing

A method is described for the use of ultrathin-layer agarose gels in phenotyping erythrocyte acid phosphatase (EAP) by isoelectric focusing (IEF). The results obtained using ultrathin-layer agarose gels are shown to be equally reliable and reproducible in comparison to established ultrathin-layer polyacrylamide gels. IEF of EAP on 0.168-mm agarose gels took place in 90 min using the LKB Multiphor system. The technique described allows for both time and cost efficient phenotyping of EAP.     

Studies on the use of isoelectric focusing as a method of phenotyping erythrocyte acid phosphatase

The technique of isoelectric focusing (IEF) in ultra-thin polyacrylamide gels as a method of phenotyping erythrocyte acid phosphatase (EAP) has been applied to a large number of red cell lysates and dried bloodstains. This paper presents the results of this study and discusses some features of the IEF patterns and problems with their interpretation. The IEF patterns of several rare EAP phenotypes are also described. These studies have confirmed that IEF is more sensitive than starch gel electrophoresis as a method of phenotyping EAP in dried bloodstains.     

The use of isoelectric focusing (IEF) as a method for the combined phenotyping of erythrocyte acid phosphatase (EAP) and esterase D (EsD)

The simultaneous isoelectric focusing (IEF) in polyacrylamide gels (PAG) of erythrocyte acid phosphatase (EAP) and esterase D (EsD) allows the poor discriminating power (DP) of EsD to be usefully combined with a highly discriminating system EAP, such that a joint DP of 0.766 was achieved compared with PGM IEF DP 0.756. Focusing was carried out in a centrally flattened gradient containing ampholines (pH 4-6 and 6-8) and the chemical spacer 3-(N-morpholino) propanesulphinic acid (MOPS). It enabled the identification of six EsD phenotypes including the recently discovered EsD5 isozymes. The application of this method to casework bloodstains is discussed.     

Simultaneous electrophoretic determination of phosphoglucomutase subtypes, adenosine deaminase, erythrocyte acid phosphatase, and adenylate kinase enzyme phenotypes

Many of the conventional agarose phosphoglucomutase (PGM) subtyping systems presently in use fail to provide a good separation between the 1 + and 2- bands as well as the 2+ band and the more anodic moving bands. Use of a 1-mm-thick gel composed of 1% ISO GEL (FMC Corp.) and phosphate-citric acid gel and tank buffers with a pH of 5.3 provided exceptionally good separation between all four of the major subtyping bands. The additional criteria for this procedure is a voltage of 21 V/cm and a run time of 4 h. Utilization of this procedure using case samples of varied ages proved the reliability of the procedure. Also examined were the effects of several reducing agents on the enzyme band patterns and the use of this system for the simultaneous determinations of the adenosine deaminase (ADA), erythrocyte acid phosphatase (EAP), and adenylate kinase (AK) enzyme phenotypes.     

Evaluation of a nonequilibrium isoelectric focusing (IEF) method for the simultaneous typing of esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA)

A nonequilibrium isoelectric focusing method incorporating the chemical spacers MOPS and HEPES was developed and subsequently evaluated for its ability to reliably discriminate common and rare phenotypes in the esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA) isoenzyme systems. The validation procedures used were blind testing, comparison of results to conventional methods, and evaluation of known rare variant phenotypes. This method proved to be a quick and reliable method for typing all five isoenzyme systems, while providing an excellent probability of discrimination (PD = 0.96).     

Determination of phosphoglucomutase (PGM1), acid phosphatase (ACP), and esterase D (ESD) in human bloodstains by hybrid isoelectric focusing (HIEF)

PGM1, ESD, and ACP were determined in bloodstain extracts by isoelectric focusing (IEF) with carrier ampholytes (CA) and HIEF. HIEF yields superior results in PGM typing from bloodstain extracts, whereas for ESD and ACP typing isoelectric focusing with carrier ampholytes seems to be the method of choice.     

The simultaneous separation of the enzymes glyoxalase I, esterase D, and phosphoglucomutase

A procedure for the multisystem analysis of bloodstains using the simultaneous separation of the enzymes glyoxalase I, esterase D, and phosphoglucomutase has been developed. The amount of bloodstain required has therefore been reduced threefold without any loss in resolution and sensitivity. Bloodstains at least seven weeks old have been correctly phenotyped in all three systems.     

Esterase D phenotyping of bloodstains and hair roots by low voltage isoelectric focusing

A new isoelectric focusing method is described for phenotyping of esterase D in blood stains and hair roots. It permitted easy and rapid discrimination of six phenotypes determined by ESD*1, ESD*2 and ESD*7. Experiments showed it to be practicable in forensic stain work. In addition, this technique was also usable in phenotyping of ESD 5.     

The esterase D polymorphism as revealed by isoelectric focusing in ultra-thin polyacrylamide gels

The polymorphism of human red cell esterase D (EsD) was studied using isoelectric focusing (pH 4-6) in ultra-thin polyacrylamide gels. Typing was possible without the EsD isozymes attaining true equilibrium focusing conditions. Using this single method, six phenotypes (EsD 1, 2-1, 2, 5-1, 5-2 and 5) could be recognized in the White population of south-east England. Family studies showed these to be controlled by three co-dominant alleles and the gene frequencies were calculated to be EsD1 0.8856; EsD2 0.0946 and EsD5 0.0198. For successful and reliable EsD typing by this method, the electrophoretic system must be carefully optimized with respect to the duration of electrophoresis and the temperature attained in the gel during the electrophoretic run.     

The simultaneous identification of seminal acid phosphatase and phosphoglucomutase by starch gel electrophoresis

Although elevated acid phosphatase (AP) activity in vaginal fluid is a consistent indicator for semen, differentiation between vaginal AP and seminal AP provides a more meaningful result. Detection of seminal AP in mixtures of vaginal AP, feces, and blood is accomplished by starch gel electrophoresis, employing the substrate thymolphthalein monophosphate as a selective visualization agent. Genetic phosphoglucomutase isoenzymes are simultaneously separated by this method and allow differentiation in some semen/vaginal fluid mixtures.     

Behavior of animal blood in blood typing systems. Isoelectric focusing of erythrocyte acid phosphatase and phosphoglucomutase

Isoenzyme band patterns of animal blood erythrocyte acid phosphatase (EAP) and phosphoglucomutase-1 (PGM) were studied by isoelectric focusing on ultrathin polyacrylamide gels. For blood from all animals tested (dog, cat, cow, sheep, and goat), the overall band patterns for both isoenzymes were different from those of the most common human types of these enzymes, although some animal EAP and PGM bands appeared in the human band areas. When mixtures of human and animal red blood cells were studied, it was found that misinterpretation of human types was possible only if the overall band pattern of the mixtures was ignored. For the animal blood tested, the strong PGM bands appearing outside the human band areas could be used as "markers" for the possible presence of animal blood in the samples tested.     

Haptoglobin phenotyping by polyacrylamide gel isoelectric focusing and its application to simultaneous typing of serum proteins

A simple isoelectric focusing method for haptoglobin (HP) typing is described. Serum was pretreated first with C. perfringens neuraminidase (CPN) and then with dithiothreitol (DTT). The treated serum was subjected to polyacrylamide gel isoelectric focusing (PAGIF), and the band patterns were detected by immunoblotting. The method could be successfully applied to HP typing of bloodstains as old as 2 months. A slight modification of it enabled HP, complement component C81, and factor I (IF) to be typed simultaneously. The immunoblotting facilitated preservation of HP patterns. Thus, the PAGIF method for HP typing is suitable for routine use in the forensic laboratory.     

The forensic identification of semen by isoelectric focusing of seminal acid phosphatase.

An isoelectric focusing technique for the separation of acid phosphatases is described. The focusing patterns for semen and vaginal secretion are examined in detail and the technique is evaluated as a means of identifying and distinguishing between these body fluids and other materials of potential forensic interest. The method can reliably be used to diagnose the presence of semen in stains and in the post-coital vagina.     

Further alleles of phosphoglucomutase in human semen detected by isoelectric focusing

A technique for identifying further alleles of PGM1 in human semen detected by isoelectric focusing has been described. A survey of 100 semen samples has shown that there was close agreement between the observed and expected gene frequencies of these new alleles on the basis that there were four common alleles determined by the PGM1 locus and not two as originally proposed [1,2]. The use of these new genetic variatns of PGM will considerably enhance the investigation of semen stains in forensic science.     

Usefulness of two methods of isoelectric focusing for the erythrocyte acid phosphatase phenotypes determination in human bloodstains

The authors tried to compare the usefulness of the isoelectric focusing of EAP in bloodstains on 0.2 mm polyacrylamide gel with their method of determination of the enzyme on 1 mm polyacrylamide gel. Both methods turned out to be useful but better results were obtained on 0.2 mm gel. Isoelectric focusing on the ultra-thin gel is more sensitive; it gives clear enzyme strips, takes less time (30 min) and demands about half the amount of material.     

DIA3 phenotyping in human semen and seminal stains by isoelectric focusing

The polymorphism of DIA3 was investigated by isoelectric focusing in semen samples from 235 unrelated Japanese volunteers and patients. Besides the three common phenotypes seven samples of the type 3-1 were observed. However, readable isoenzyme patterns were not demonstrated in semen samples of oligospermia under about 10 X 10(6)/ml sperm cells. The allele frequencies were DIA3*1 = 0.821, DIA3*2 = 0.164, and DIA3*3 = 0.015. The DIA3*1 frequency in oligospermia (0.765) was lower than that in normospermia (0.836). The isoelectric focusing method was successfully applied to phenotyping DIA3 in seminal stains; each phenotype was demonstrated at 37 degrees C for up to 4 weeks, at room temperature for up to 8 weeks, and at 4 degrees C for over 12 weeks after stain formation. In vaginal swabs the isoenzyme bands were very faint and not identifiable.     

C6 phenotyping in sera and bloodstains by isoelectric focusing followed by electroimmunoblotting

The genetic polymorphism of C6 was investigated in 329 unrelated Japanese individuals using isoelectric focusing in polyacrylamide gels followed by an electroimmunoblotting technique. Besides six common phenotypes C6 A, AB, B, AB2, BB2 and B2, six rare variants were observed. The allele frequencies were: C6*A = 0.4422, C6*B = 0.4757, C6*B2 = 0.0714, C6*A3 = 0.0015, C6*M1 = 0.0046 and C6*B3 = 0.0046. The population data confirmed that the C6*B2 allele is the third common allele characterizing Japanese. The present electroimmunoblotting technique was applied to demonstrate C6 types in dried bloodstains. The C6 types were determined from bloodstains stored at 4 degrees C for up to 10 weeks, at room temperature for up to 2 weeks and at 37 degrees C for up to 4 days. The results show that this component system offers a new powerful means for the medico-legal grouping of bloodstains.