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.     

Detectability of group-specific component (Gc) in aged bloodstains

An improved method of group-specific component (Gc) typing was conducted electrophoretically on agarose gel. Individual bloodstains randomly collected from different individual donors over a five-year period at intervals of approximately one month were checked for Gc activity. Group-specific component was typed accurately in dried bloodstains stored at room temperature up to 43 months in age. From 100 different donors, bloodstains ranging in age from 38 to 43 months were tested by the methods described and 73% of the samples were interpretable for Gc.     

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.     

Effects of luminol on the subsequent analysis of bloodstains.

The effects of luminol upon additional presumptive chemical tests, subsequent confirmatory blood tests, species determination by immunoelectrophoresis, ABO typing by absorption elution, and genetic marker analysis by multienzyme system electrophoresis were examined. Results indicate that luminol does not affect additional presumptive chemical tests, confirmatory tests, species determination, or ABO typing, but does affect certain genetic marker systems.     

Detection of low levels of flunitrazepam and its metabolites in blood and bloodstains.

Detection of low levels of flunitrazepam and its metabolites was developed using solid-phase extraction to isolate the drugs from whole liquid blood and dried bloodstains, with subsequent derivatization with pentafluoropropionic anhydride (PFPA) followed by N-(tert-butyldimethyl-silyl)-N-methyl-trifluoroacetamide (MTBSTFA) with 1% TBDMSCI. Analysis was confirmed by gas chromatography/mass spectroscopy using select ion monitoring (sim) in electron impact mode. The limit of detection of this procedure using 1 ml of blood was determined to be 0.1 microgram/dl.     

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.     

The simultaneous electrophoretic analysis of esterase D and phosphoglucomutase subtyping in fresh blood and in dried bloodstains

Phosphoglucomutase1 (PGM) subtyping and esterase D phenotyping were simultaneously performed by electrophoresis of bloodstained fibers using agarose and a Tris-maleic acid buffer system , pH 5.4. This method reduces anodal gel shrinkage and shortens development time when compared to the conventional electrophoretic technique for PGM subtyping which is performed at pH 7.4 using an agarose-starch substrate.     

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.     

Destruction of intracerebrally applied red blood cells in cervical lymph nodes. Experimental investigations

In rabbits, intracerebrally applied erythrocytes can move with the cerebrospinal fluid along connecting pathways between the subarachnoid space and the cervical lymph nodes. This study compares the time dependency for the degradation of intracerebrally injected erythrocytes in the brain as well as in the cervical lymph nodes. Rabbits were killed at various predetermined intervals after the intracerebral erythrocyte injection. Microscopic and histologic examination of the brain and the cervical lymph nodes revealed the following findings: (1) Erythrophages first appeared in the brain 24 h after the injection; siderophages, 4 days after the injection. Siderophages were still demonstrable at the conclusion of the study, i.e. 240 days after the injection. (2) In the cervical lymph nodes erythrophages were first observed 1 h after the injection; siderophages, 9 h after the injection. Only isolated erythrophages and siderophages were found in the lymph nodes 12 days after intracerebral injection of red blood cells. Later on no erythrocytes or siderophages were observed in the lymph nodes. The findings indicate that non-phagocytized red blood cells arriving at the lymph nodes were ingested by local macrophages. The extremely rapid ingestion and digestion of the red blood cells by lymph node macrophages as well as the possible reasons were discussed.     

Simultaneous screening and detection of drugs in small blood samples and bloodstains

A gas chromatography-mass spectrometry (GC-MS) method is described for the screening and detection of morphine, codeine, cocaine, benzoylecgonine, methylecgonine, cocaethylene, delta-9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THC-COOH), 11-hydroxy-THC (11-OH-THC), amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymetamphetamine (MDMA) and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in small blood samples and bloodstains using solid phase SPE columns and a pipetting robot (Gilson Aspec XL). The detection limits are in the order of 1.62-4.10 ng/50 microl spot (amphetamines), 0.15-0.82 ng/50 microl spot (cannabinoids), 1.67-4.70 ng/50 microl spot (cocaine and derivatives) and 4.53-4.91 ng/50 microl spot (opiates) and the correlation factors are between 0.9957 and 0.9999. The method has proven useful in forensic cases with only small sample volumes or bloodstains.     

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.     

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%.     

Isotachophoretic analysis of bloodstains: differentiation of human, menstrual, bovine, and ovine bloods

Isotachophoresis, a technique to separate components by constant current electrophoresis, was used to differentiate between bloodstains of male, female, menstrual, bovine, and ovine bloods on cotton cloth and filter paper. Bloodstain analysis by isotachophoresis of stains from male and female subjects showed identical cationic patterns, but gave different profiles in the anionic system. Plasma had one extra peak in the anionic system when compared to the profile of serum. This extra peak is due to the presence of fibrinogen in plasma. Some hemoglobin peaks overlapped with serum protein peaks, but these could be identified by comparisons at lower concentrations. Menstrual blood had a much different pattern than normal human blood as was expected since many more compounds are found in menstrual blood than in normally circulating blood. Human, bovine, and ovine bloodstains showed different profiles both in the cationic and anionic systems. These results indicate that isotachophoresis can be used for the rapid and simple analysis of bloodstains to differentiate reliably human male, female, and menstrual blood and also to distinguish human bloodstains from those of cattle or sheep.     

Autolytic changes in blood cells of human cadavers. II. Morphological studies

The morphology of various types of cells in the peripheral blood of human cadavers was investigated. The material comprised 123 medicolegal autopsy cases with post-mortem (p.m.) times ranging from 1.7 to 270.4 hours. The corpses were kept at +4 °C. The haematocrit values of the blood increased rapidly after death. The haematocrit-corrected red cell count, and the total white cell and platelet counts remained quite stable during the whole p.m. time range. Red cells were quite rapidly transformed from a discoid configuration to crumbled discs, echinocytes and spherocytes, but no debris or burst cell configurations were seen. Rapid deterioration of the staining properties and marked morphological changes in many leucocytes occurred quite rapidly after death. Lymphocytes seemed to be the most resistant and basophils the least resistant to the effects of autolysis. Morphologically altered platelets and aggregates of them were seen in each cadaver.The present morphological observations and the quantitative results suggest that various cellular elements of the blood seem to be quite resistant to autolytic effects, and many cells apparently retain their viability for longer periods of time in the blood of cadavers kept at reduced temperature.     

Lewis blood group determination in bloodstains by planimetric measurement of eluted monoclonal antibodies

Planimetric measurements were employed for reading the results of an elution test to determine Lewis blood groups in dry human bloodstains. In the absorption-elution test, two varieties of indicators were used to detect eluted Lewis antibodies. First, 64 blood-stains aged between 2 to 8 months were tested with glutaraldehyde (GLA)-treated erythrocytes (planimetric hemagglutination assay, PMHA). This method demonstrated that dry stains weighing approximately 0.4 mg (equivalent to 3 microliters of whole blood) were sufficient for detection of Lea or Leb antigen. Results were obtained within 1 h. Then, 37 of these stains were tested with Lewis substance-coated latex particles (planimetric latex agglutination assay, PMLA). The presence of Lea and Leb antigen were detected from dry stains weighing 0.1 mg (equivalent to 1 microL of whole blood) within 3 h. Both these assays are faster and simpler with accuracy than the enzyme-linked immunosorbent assay (ELISA). Latex particles coated with Lewis substance are, in particular, strongly agglutinated and show agglutination patterns more clearly than erythrocytes. The blind tests using these two methods properly classified 7 Le(a + b-) and 23 Le(a-b + ) bloodstains; whereas, 5 Le(a-b-) stains were undetermined by the criteria for these tests. These results indicate the usefulness of the PMHA and PMLA for typing Lewis blood groups from small bloodstains.