Report of a European collaborative exercise comparing DNA typing results using a single locus VNTR probe

A collaborative exercise was carried out in 1989 among 12 European forensic laboratories using the single locus VNTR probe pYNH24, the restriction enzyme HinfI, the same set of human genomic DNA samples, and a standardized DNA size marker. The objectives of the exercise were: (1) to study the degree of variation within and between laboratories, (2) to obtain information on requirements for technical standardization allowing the exchange of typing results and (3) to compare different approaches for the identification of allelic DNA fragments of unknown size. Each laboratory carried out up to 10 independent typing experiments using the same DNA samples. The results were analysed independently by two laboratories using three different methods. The results of the exercise demonstrate the correlation of typing that can be achieved within and between laboratories under conditions of minimal standardization.     

A study of sex identification of trace, dried bloodstains using a Y-chromosome-specific deoxyribonucleic acid (DNA) probe

A new method is discussed which examines trace, dried bloodstains by gel in situ hybridization using a Y-chromosome-specific deoxyribonucleic acid (DNA) probe to determine the sex of the bloodstain for forensic medicine application. The complete DNA is transferred directly by electrophoresis onto the gel intact, bypassing the possibilities of impurities contaminating the sample and of DNA degradation. The method has proven accurate for small (2.5-mm-diameter) samples aged up to eight years and is quick, simple, and easily read.     

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.     

Identification of male bloodstains by dot hybridization of human Y chromosome-specific deoxyribonucleic acid (DNA) probe

The sex determination of bloodstains was performed using a human Y chromosome-specific (DNA) fragment of 1.9-kb length as a hybridization probe. The DNA samples were taken from 1- and 4-week-old bloodstains of males and females, respectively. Strong signals with male DNA were observed by Y-probe, while faint signals with female DNA were detected. In addition, clear signals were observed in the extract samples from male bloodstains (16-week-old) on paper. Dot hybridization of the Y-probe would be widely applicable to studies on sex determination of medicolegal materials such as blood, bloodstains, teeth, and cadaverous parts.     

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.     

Successful DNA typing of a urine sample in a doping control case using human mitochondrial DNA analysis

In a doping control case, a urine sample was tested positive for nandrolon. We were asked by the athlete to perform DNA investigations on the questioned urine sample and compare these to a fresh blood sample taken from the athlete in order to detect or rule out manipulation and/or switching of the samples. The urine sample had been collected nine months prior to the investigation and had been stored at 4 degrees C. In a first approach, nuclear DNA systems were investigated that failed with the exception of the Amelogenin system. Due to the high copy number of mitochondrial DNA molecules and the robustness of the mitochondrial genome, we investigated the HVR I and HVR II regions of mitochondrial DNA and obtained reproducible and clear sequencing results for both the blood and the urine samples. Due to the identical sequences, it could not be excluded that the blood sample and the urine sample were from the same individual or an individual having the same maternal lineage.     

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.     

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.     

Lewis typing of human bloodstains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b)

The Lewis blood grouping of human dried bloodstains could be determined by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies with an avidin-biotin complex (ABC). The bloodstains aged 1 year were used as samples, and approximately 1 mg of the stains was enough to type each Lewis antigen reliably by this method. The Lewis substances of 106 individual stains were correctly typed regardless of their ABO blood group system.     

The presumptive reagent fluorescein for detection of dilute bloodstains and subsequent STR typing of recovered DNA

A presumptive reagent for dilute blood detection other than luminol is fluorescein. The sensitivity of fluorescein approaches the sensitivity of detection levels of luminol. The fluorescein detection method offers the advantages of working in a lighted environment, and the reaction persists longer than luminol. A series of diluted bloodstains, ranging from neat to 1:1,000,000, was placed on a variety of substrates. Three sets were made per substrate. One set was exposed to fluorescein, one set was exposed to luminol, and one set served as an uncontaminated control. The fluorescein signal persisted longer than luminol. However, background staining for fluorescein was observed on some substrates within 30 s to 1 min, and no background staining was observed for luminol. Stains on non-absorbent surfaces were detectable at 1:100,000 dilutions, and stains on absorbent surfaces were detectable usually at no more than 1:100. The sensitivity of detection of fluorescein was comparable to that of luminol in this study. In all cases, where sufficient DNA was recovered, typeable results at all 13 core CODIS STR loci were obtained from treated bloodstains and controls. The results from STR typing indicate that there was no evidence of DNA degradation.     

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.     

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.     

Gm/Km typing of bloodstains in U-bottom microtiter plates

A technique was developed for Gm/Km typing of bloodstains and sera in U-bottom microtiter plates. Gm/Km typing of sweat and urine samples was also attempted with limited success.     

Successful DNA typing of urine stains using a DNA purification kit following dialfiltration

To evaluate the utility of DNA polymorphism typing of urine stains in forensic investigations, the amplifiable amount of DNA was estimated in 20 urine specimens obtained from 10 male and 10 female volunteers using a DNA purification kit following dialfiltration. DNA obtained from both urine and urine stains was amplified with the AmpflSTR Profiler PCR Amplification Kit, and was analyzed by capillary electrophoresis using the Genetic Analyzer. The amount of male and female urine necessary for obtaining a complete DNA profile was 0.2 mL and 0.08 mL, respectively. When 0.2 mL of male urine were used to create urine stains, complete DNA profiles could be obtained from just some of the stains. However, when only 0.1 mL of female urine was used, complete profiles could be successfully obtained from all of the stains. DNA on bleached cotton remained amplifiable for 3-6 weeks. This method using a DNA purification kit following dialfiltration can be recommended for the genotyping of urine stains.     

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.     

血痕特异性mRNA标记研究

血痕是案发现场尤其是命案中比较常见的生物物证,而血痕的正确组织来源推断是当前鉴定工作中急需解决的问题。随着法医物证学的不断发展,以mRNA(messenger RNA)为基础的体液斑迹组织来源鉴定技术作为一种不同于传统血痕免疫学检测的新型方法,已经越加显示其独特的优越性。在该技术的基础上,实现共同提取生物物证的RNA与DNA的目标,建立体液斑迹鉴定与DNA分型兼容的方法,有利于现场重建,提高生物物证的证据效力,完善证据链。本研究建立了一个包括血痕总RNA提取、逆转录、荧光特异引物扩增、遗传分析仪电泳检测分析等步骤的血痕来源推断技术平台。实验共采集制备了40份的中国人群(女性)外周血、16份月经血样本,筛选了5个外周血标记:HBA、HBB、GYPA、SPTB、ALAS2,2个月经血标记:MMP7、MMP11,构建了一个囊括外周血、月经血特异标记的荧光复合扩增体系。结果显示mRNA技术为基础的鉴定血痕来源的方法是可行的,并且建立了血痕RNA检验的遗传分析仪结果判读方法。