A new method for typing haptoglobin in bloodstains using immobilized allo A lectin

Allo A lectin from the beetle, which is beta-D-galactose specific, reacts to haptoglobin but not to hemoglobin. The use of allo A-Sepharose for typing haptoglobin in bloodstains helped eliminate hemoglobin from the bloodstain extract and presented highly resolved haptoglobin patterns by disc gel electrophoresis. This method is simple and rapid for typing haptoglobin in bloodstains and can be easily used in forensic science laboratories.     

The application of immunoblotting to the phenotyping of haptoglobin.

An immunoblotting method for phenotyping haptoglobin in serum and bloodstains has been developed. Haptoglobin isoproteins were separated by polyacrylamide gradient gel electrophoresis and then transferred to nitrocellulose by electroblotting. The use of 1 mm gels facilitated more rapid and effective transfer than conventional 3 mm thick gels. Nitrocellulose blots were developed by double antibody enzyme immunoassay. The detection limit for serum and bloodstains was improved 16 times compared to conventional staining using O-tolidine. The method could detect haptoglobin phenotypes from 0.001 microliter of whole blood. This detection limit is approximately 8 times lower than that of group specific-component analysis by immunoblotting.     

Haptoglobin typing in canine bloods

Haptoglobin typing by vertical electrophoresis in a discontinuous polyacrylamide gel was conducted on 47 dog blood samples, of which 19 were from Doberman pinschers, 20 from German shepherds, and 8 from pit bullterriers. Two phenotypes were common in the three breeds and could not be used to differentiate between them. Canine haptoglobin phenotypes were, however, sufficiently different from those of humans to warrant using haptoglobin typing as a method for determining the origin of bloodstains.     

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.     

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.     

Subtyping phosphoglucomutase-1 in semen stains and bloodstains: a report on the method

A method is described for obtaining nondistorted, reproducible phosphoglucomutase-1 subtyping patterns from semen stains and bloodstains. Isoelectric focusing of phosphoglucomutase-1 was accomplished in 80 min in a 0.2-mm-thick polyacrylamide gel with an interelectrode wick distance of 8.0 cm. The gel contained 1.2% (w/v) N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid (EPPS) and pH 5 to 7 ampholytes (4% w/v). When maintained at room temperature, laboratory-prepared bloodstains and semen stains could be typed for phosphoglucomutase-1 up to four months and three weeks, respectively. An evaluation of phosphoglucomutase-1 typing by isoelectric focusing and the Group I system was performed on casework samples submitted to the FBI Laboratory. In addition to the increased discriminating probability of phosphoglucomutase-1 when subtyped, isoelectric focusing yielded an increase in positive calls on questioned bloodstains (65.6 versus 36.2%) and dried seminal stains (16.4 versus 13.1%) compared with the Group I system.     

The application of immunoblotting to the phenotyping of group-specific component

A sensitive immunoblotting method for the routine detection of group-specific component (GC) from fresh serum, and from control and casework bloodstains has been developed. GC phenotypes were separated in a thin layer polyacrylamide gel by isoelectric focusing, transferred to nitrocellulose by a rapid capillary blotting procedure, and detected using a double antibody enzyme immunoassay. This method is capable of phenotyping 8 ng of GC extracted from bloodstains, a four-fold increase in sensitivity when compared to immunofixation and silverstaining. A total of 2424 casework bloodstains have been analysed and GC phenotypes identified in 78% of samples. The method is suitable for use in routine laboratories and is more sensitive than other methods for GC phenotyping of casework bloodstains.     

The typing of α1-antitrypsin in human bloodstains by isoelectric focusing

A polyacrylamide gel isoelectric focusing (PAGIF) technique is described for the determination of α₁-antitrypsin (Pi) phenotypes in bloodstains. The time limits for Pi type determination of bloodstains kept under different storage conditions are given. The resolution of PAGIF in the typing of Pi phenotypes in human bloodstains in investigated.     

Transferrin subtyping of human bloodstains

A method was described for subtyping transferrin derived from human bloodstains. Bloodstain cuttings were extracted in 0.5% ferrous ammonium sulfate. The extracts were subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. After isoelectric focusing, transferrin was detected by silver staining. This method permitted the successful typing of Tf in 6-month-old blood stains maintained at -20 degrees C and room temperature and 3-month-old bloodstains maintained at 37 degrees C.     

Haptoglobin phenotyping from older bloodstains by enzyme immunoassay and haptoglobin phenotypes within a Nebraska Caucasian population

Enzyme immunoassay and Western blotting (electrophoretic) techniques were used to determine haptoglobin (HP) phenotypes from older bloodstains. Serum was collected from liquid blood and the HP phenotypes were determined. Bloodstains were prepared from these specimens and stored at various temperatures for several months. The stains were extracted and applied to gradient polyacrylamide gels. The Western blotting technique was used to achieve the transfer of HP bands from the gels to the nitrocellulose membranes. Enzyme immunoassay with goat anti-HP antiserum and rabbit anti-goat immunoglobulin peroxidase were used to identify the HP bands from the extracted samples. Enzyme immunoassay was found to be clearly more sensitive than o-dianisidine or o-tolidine in detecting HP bands from diluted serum samples. The haptoglobin frequency in a Caucasian population in Nebraska was calculated. The frequencies of Phenotypes 1, 2-1, and 2 were found to be 15.8, 48.4, and 35.8%, respectively.     

A stability study on Gc subtyping in bloodstains: comparison by two different techniques

The limits of determination of Gc subtypes in bloodstains were compared between the immunofixation method and the sulfosalicylic acid precipitation method using isoelectric focusing on polyacrylamide gel. By the immunofixation method Gc subtyping in bloodstains was successfully made at 37 degrees C after 7 weeks, at room temperature after 17 weeks and at 4 degrees C even after 25 weeks storage. By the sulfosalicylic method Gc subtypes were no longer able to be determined a few weeks after stain formation. The superiority of the results obtained by the immunofixation method makes it the recommended method for the Gc subtyping from bloodstains in medicolegal practice.     

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.     

血痕中PGM_1亚型的检出及酶谱的保存方法

本文报告用等电聚焦方法,采用拉丁方设计,对血痕保存的温度、布质及含量,以及血痕中 PGM_1亚型检出时间进行了研究。保存在0℃(6个月)、4℃(2个月)、18℃(1个月)及30℃(3周)的6μL 血痕,PGM_1亚型均可检出。血痕的总量对 PGM_1亚型的检出时间也有一定的影响。不同布质对血痕中 PGM_1亚型的检出时间,无明显差异。另外,利用聚脂膜具有亲水膜面的特点,将 PGM_1原始酶谱贴附在聚酯膜上,可长期保存酶谱。     

A method for subtyping group-specific component in bloodstains

A method is described for subtyping group-specific component (Gc) derived from human bloodstains. Bloodstained cuttings were extracted in 6 M urea. The extracts were subjected to ultrathin-layer polyacrylamide gel isoelectric focusing in the pH 4.5-5.4 range. After isoelectric focusing, Gc was detected by immunofixation in cellulose acetate membranes. This method permitted the successful typing of Gc in at least four-month-old bloodstains maintained at room temperature. Bloodstains from 266 liquid blood samples of known origin were subjected to both this method and immunofixation conventional agarose gel electrophoresis with no phenotypic discrepancies observed. The Gc population data for Whites from Baltimore, Maryland, were homogeneous with white sample populations from other geographical locations within the U.S.A.; while Gc data from northern U.S.A. black sample populations appeared to be heterogeneous compared with a southern United States black sample population.     

A hybrid ampholyte focusing technique for esterase D subtyping of evidentiary material

An ultrathin-layer polyacrylamide gel isoelectric focusing technique that uses a composite of ampholytes from three commercial sources is described for subtyping esterase D. All common allelic products of esterase D were separated clearly. The technique described in this paper provides a higher conclusive call rate on known blood specimens (95.8%) and questioned bloodstains (69.7%) compared with continuous zone electrophoresis in agarose gels (89.9 and 37.6%, respectively).     

Esterase D typing of bloodstains by non-equilibrium focusing

Non-equilibrium focusing in a pH 4-6 gradient in ultra-thin polyacrylamide gels has been shown to be a reliable and reproducible method for detecting the six common esterase D phenotypes (EsD 1,2-1,2,5-1,5-2 and 5) in dried bloodstains. Successful typing is dependent on both the age and phenotype of the stain in question. The effects of age on the isozyme pattern of each phenotype are described and illustrated. In a comparative trial using 100 simulated and 300 authentic casework bloodstains, non-equilibrium focusing was shown to be more efficient than thin-layer starch gel electrophoresis for the typing of esterase D.     

An efficient method to eliminate streaking in the electrophoretic analysis of haptoglobin in bloodstains

A bloodstain extraction procedure that improves the analysis of haptoglobin in dried bloodstains has been developed. The streaking of electrophoresis gels caused by deteriorated hemoglobin can be eliminated by incorporating chloroform in the bloodstain extraction procedure. The method is easier to execute than previously published techniques for eliminating the adverse effects of deteriorated hemoglobin on the analysis of haptoglobin. Bloodstains up to two years old were correctly phenotyped in haptoglobin by this method.     

Evaluation of four deoxyribonucleic acid (DNA) extraction protocols for DNA yield and variation in restriction fragment length polymorphism (RFLP) sizes under varying gel conditions.

This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.     

Haptoglobin phenotyping of bloodstains by horizontal electrophoresis on a compact polyacrylamide gradient gel

A method is described for phenotyping haptoglobin by horizontal electrophoresis on a small polyacrylamide gradient gel. This method employs the same apparatus used in the separation of many red cell enzyme phenotypes and thereby eliminates the necessity for specialized vertical electrophoresis equipment.     

Haptoglobin typing of human bloodstains using a specific DNA probe

The stability of DNA in human bloodstains and various post mortem tissues has been investigated. High molecular weight (HMW) DNA was usually recovered from dried bloodstains, even those up to a few years old, but very rapid degradation was found to occur post mortem in the liver, pancreas, spleen and kidney. Other tissues such as the heart, thyroid and skeletal muscle were found to give a reasonable yield of HMW DNA during the first few days after death. The feasibility of using DNA extracted from forensic bloodstain specimens for the detection of DNA polymorphisms was explored using a human haptoglobin (Hp) alpha chain specific probe. Using HindIII and XbaI digests the Hp genotypes Hp2, Hp1F and Hp1S were distinguished by Southern blot analysis in DNA prepared from 1 cm2 bloodstains up to 15-18 months old.