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.     

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.     

Forensic determination of pregnancy hormones in human bloodstains.

When different bloodstains are encountered at the scene of crime, it is possible to discriminate those from a pregnant woman from others. Human chorionic gonadotrophin, human placental lactogen, total oestriol and progesterone in the stains may be determined with radioimmunoassay techniques using commercial kits. Only 1 cm2 of bloodstain is needed for the determination of all four parameters, which gives information about the state of pregnancy. More than 100 stains of blood from women in all stages of pregnancy, normal menstruating women, menopausal and post-menopausal women and male subjects, and of menstrual blood were analysed. Bloodstains from pregnant women were easy to evaluate with the four determinations, only very early pregnancies being undetected. Stains from non-pregnant women were negative or below the cut-off level. Two case examples are also described.     

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.     

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.     

The determination of glyoxalase I phenotypes in human bloodstains

A method is described for the determination of glyoxalase I phenotypes in liquid blood and dry bloodstains. The frequencies of glyoxalase I phenotypes in various populations of S.E. England are included.     

Glucose phosphate isomerase variants in human bloodstains and seminal stains

Glucose phosphate isomerase (GPI) variants occurring in human red cells were also demonstrated in human semen. Phenotyping was possible from bloodstains of 6 weeks storage and seminal stains of 12 weeks storage. The GPI system may be a supplemental tool for medicolegal individualization of seminal stains.     

Quantitative IgD measurement for discrimination of human bloodstains

The IgD concentrations of eluates of artificial bloodstains and of the corresponding sera from 40 subjects of different ages were measured by single radial immunodiffusion. IgD was found in bloodstains stored up to 53 days, i.e. IgD storage stability is sufficient for forensic purposes. Since serum IgD concentrations of individuals, in particular of adults, are almost invariable and serum levels of different individuals can vary by more than a 1000-fold, the discrimination of bloodstains on the basis of IgD is generally possible. Thus IgD constitutes a further marker in antibody profiling of bloodstains.     

Gc in bloodstains

Gc-subtyping was carried out on blood stains that had been made on cotton and glass and stored under a variety of conditions ranging from -20 degrees to +56 degrees C. The limits of detection ranged from 2 weeks at 56 degrees C up to 92 weeks at +4 degrees C and greater than 116 weeks at -20 degrees C. Additional bands that have been reported in other studies could not be detected during this study, and this difference is thought to be due to storage of the samples in the liquid state.     

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.     

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.     

An improved method for subtyping Pi in old bloodstains.

Immunofixation procedures were used for detecting alpha-1 antitrypsin protease inhibitor (Pi) phenotypes in bloodstains. Neuraminidase elution of bloodstains, together with isoelectric focusing, immunofixation, and silver staining techniques, makes possible Pi subtyping in old bloodstains. No extra bands appear when the storage time is no longer than three months.     

Identification of fetal bloodstains by enzyme-linked immunosorbent assay for human alpha-fetoprotein

A rapid and highly sensitive enzyme-linked immunosorbent assay (ELISA) for determination of human alpha-fetoprotein (AFP) using commercially available reagents was devised and applied to identification of fetal bloodstains. When experimentally prepared bloodstains, 1 by 2 mm in area, were submitted to analysis, only fetal bloodstains showed positive reactions in the present ELISA. The reactions did not change significantly when these bloodstains were stored at room temperature for one week. The present ELISA seems to be suitable for forensic science practice.