A simple method for purification of anti-A and anti-B antibodies using glutaraldehyde-fixed human red blood cells

A simple method for purification of anti-A and anti-B antibodies using glutaraldehyde-fixed human erythrocytes is described. Specific antibodies were first absorbed with the corresponding cells, then eluted by heating at 53 degrees-55 degrees C for 15 min. The method is simple and highly efficient with a fair recovery of 15.6%-34.4%.     

A microplate method for reverse ABO typing of bloodstains

A sensitive and reliable hemagglutination assay, using V-bottom microplates, is described for the detection of the ABO blood group alloantibodies in bloodstained material. When used in conjunction with an absorption-elution procedure, the microplate assay resulted in a 300% increase in the number of conclusive grouping results when compared to the Lattes crust test. The use of the microplate reverse grouping assay permits 24 specimens to be assayed conveniently on a single plate and eliminates the tedious and time-consuming microscopic examination required for the Lattes crust test.     

Validation of STR typing by capillary electrophoresis

With the use of capillary electrophoresis (CE), high-resolution electrophoretic separation of short tandem repeat (STR) loci can be achieved in a semiautomated fashion. Laser-induced detection of fluorescently labeled PCR products and multicolor analysis enable the rapid generation of multilocus DNA profiles. In this study, conditions for typing PCR-amplified STR loci by capillary electrophoresis were investigated using the ABI Prism 310 Genetic Analyzer (Applied Biosystems). An internal size standard was used with each run to effectively normalize mobility differences among injections. Alleles were designated by comparison to allelic ladders that were run with each sample set. Multiple runs of allelic ladders and of amplified samples demonstrate that allele sizes were reproducible, with standard deviations typically less than 0.12 bases for fragments up to 317 bases in length (largest allele analyzed) separated in a 47 cm capillary. Therefore, 99.7% of all alleles that are the same length should fall within the measurement error window of +/- 0.36 bases. Microvariants of the tetranucleotide repeats were also accurately typed by the analytical software. Alleles differing in size by one base could be resolved in two-donor DNA mixtures in which the minor component comprised > or = 5% of the total DNA. Furthermore, the quantitative data format (i.e., peak amplitude) can in some instances assist in determining individual STR profiles in mixed samples. DNA samples from previously typed cases (typed for RFLP, AmpliType PM+DQA1, and/or D1S80) were amplified using AmpFlSTR Profiler Plus and COfiler and were evaluated using the ABI Prism 310. Most samples yielded typable results. Compared with previously determined results for other loci, there were no discrepancies as to the inclusion or exclusion of suspects or victims. CE thus provides efficient separation, resolution, sensitivity and precision, and the analytical software provides reliable genotyping of STR loci. The analytical conditions described are suitable for typing samples such as reference and evidentiary samples from forensic casework.     

Blood typing of blood stains on sweat stains by the iso-agglutinins detection method

When a blood typing is made for mixed stains of sweat and blood, erroneous results may be obtained. The reason is that the blood group substance in the sweat is detected at the same time as that in the blood. In this paper the typing of the blood stain on the sweat stain is carried out by the detection of isoagglutinins which may give additional information to the forensic serologist.     

Reliability of SE33 typing by capillary electrophoresis

A ladder of 24 ACTBP2 (SE33) alleles was separated 175 times by denaturing capillary electrophoresis on an ABI Prism 310 Genetic Analyzer using polymer POP-4. The mean standard deviation of fragment size determination was 0.083 bp. Fragments in the whole allelic range of ACTBP2 could be typed with high precision and reproducibility if adjacent fragments differed by at least two nucleotides. The capacity of resolving 1 bp differences was tested by repeatedly running a ACTBP2*14.2/14.3/31.2/31.3 allelic mixture. The 14.2/14.3 fragment pair could be separated in 98%, the 31.2/31.3 fragment pair only in 65% of all runs. Reliable separation of this difficult fragment mixture could exclusively achieved by using POP-6.     

Rapid direct PCR for ABO blood typing

Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.     

基于等位基因特异性PCR原理建立的SNP分型新方法

目的建立一种新方法,对多个单核苷酸多态性(singlenucleotidepolymorphism,SNP)位点进行分型。方法基于等位基因特异性PCR原理,采用荧光标记复合扩增和毛细管电泳技术,根据PCR片段长度差异进行分型。选择SNP位点共11个,每个SNP位点设计两条长度不同、3’末端分别与SNP两个等位基因碱基配对的上游引物,同时为了增加特异性,在两条等位基因上游引物的3’末端第3或第4位碱基人为引入错配。在距离上游引物100～300bp范围内的合适位置,设计下游共用引物,并进行荧光标记。所有位点经过复合扩增后,PCR产物经ABIPrismTM310型遗传分析仪电泳分离,确定每个SNP的基因型。结果每个SNP位点纯合子为单一产物峰,杂合子则为长度不同的两个产物峰。不同的SNP位点扩增产物长度不同,根据产物长度和产物峰的数量进行SNP分型,一次完成11个SNP位点分型,其结果与直接测序完全一致。结论荧光标记复合扩增片段长度差异等位基因特异性PCR法是一种简单快速而有效的SNP分型新方法。     

单克隆抗体双位点一步ELISA方法检测MN血型

目的 :对法医学样品中微量液体血、血痕进行MN分型。方法 :利用抗M、抗N及抗血型糖蛋白A单抗 ,采用双位点一步ELISA方法。结果 :此法可检测的最低全血量约0 065μl,血痕约10～50ng。对455份新鲜血液及200份新鲜血痕的标本均正确检出 ;对58例陈旧血痕的检出率为96 6 %。结论 :此法准确率高且简便、快速 ,具有很大的实用价值。     

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.     

Evaluation of an enzyme linked immunosorbent assay (ELISA) method for ABO and Lewis typing of body fluids in forensic samples.

An ELISA for the detection of the ABO group and secretor status of body fluids and stains other than blood is described, together with the validation procedures employed before its introduction into forensic casework. Criteria for the interpretation of results have been formulated for the method in use in this laboratory. The method was found to be reliable and to have a higher success rate than the haemagglutination techniques previously employed.     

A modification of the microplate method for reverse ABO typing of bloodstains and additional validation studies

The results of additional validation studies of a sensitive microplate hemagglutination assay for ABO reverse grouping of bloodstains are presented. The results of the validation study demonstrate the reliability of the microplate assay for use in routine serological casework. Based on these studies, the microplate assay has now replaced the Lattes crust test for ABO reverse grouping of bloodstains in the FBI Laboratory.     

SEM analysis of red blood cells in aged human bloodstains.

Mammal red blood cells (RBC) in bloodstains have been previously detected by light microscopy on stone tools from as early as 100,000 +/- 25,000 years ago. In order to evaluate the degree of morphological preservation of erythrocytes in bloodstains, an accidental human blood smear on white chert and several experimental bloodstains on hard substrates (the same stone-white chert; another type of stone-graywacke; a non-stone support-stainless steel), were stored in a room, in non-sterile and fluctuating conditions, for lengths of time ranging from 3 to 18 months. Afterwards, the specimens were coated with gold and examined by a Cambridge Stereoscan 120 scanning electron microscope. Results revealed a high preservation of RBC integrity, with the maintenance of several discocytary shapes, a low tendency to echinocytosis and a frequent appearance of a moon-like erythrocytary shape in the thinner areas of the bloodstains.     

Preparation of high-titre anti-P1 sera for blood typing in man

For the last 25 years, antisera for testing P1 in man have been obtained by i.v. injections in goats using hydatid cyst fluid from livers of pigs infected by Echinococcus granulosus. Due to the fact that this antigen has become very rare today, efforts have been made by the authors to substitute it by different antigens and to save antigen by modifying the technique of immunisation as well. By this, very high titres up to 1:4096 could be reached after specification absorption.     

A method for the determination of MN antigens in dried blood

MN phenotypes of experimentally prepared dried blood samples, some as old as six months, were obtained using sodium dodecyl sulfate polyacrylamide gels, electroblotting, and monoclonal antibodies.     

A simple spectrophotometric method for the determination of carboxyhemoglobin in blood

The spectrophotometric method for the determination of carboxyhemoglobin (HbCO) in blood reported by Fretwurst and Meinecke was modified so as to give the same values of percentage HbCO (HbCO%) as those determined by the oxygen electrode method. Values of HbCO% of nine practical samples determined by both the oxygen electrode method and the present method were nearly identical regardless of the presence of methemoglobin (Met-Hb) in blood. The present method is suitable for forensic practice.     

Studies and observations on the use of isoelectric focusing in ultra-thin polyacrylamide gels as a method of typing human red cell phosphoglucomutase

The technique of isoelectric focusing in ultra-thin polyacrylamide gels as a method of typing human red cell phosphoglucomutase (PGM1) has been studied. Typing was possible without the samples attaining true equilibrium focusing conditions. The isozyme patterns so obtained were clearly defined and free from distortion. The importance of assessing relative band intensities when interpreting the isozyme patterns is discussed. Our experience of using the technique to analyse casework material is described.