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.     

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.     

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.     

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%.     

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.     

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.     

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.     

Phenotyping of carbonic anhydrase II in fresh blood and bloodstains on cellulose acetate membrane

A simple and rapid procedure is presented for the identification of carbonic anhydrase II in fresh blood and bloodstains using cellulose acetate membranes. Identification of the phenotypes is simplified by the migration of the isozyme bands to one side and away from the origin.     

Determination of the age of bloodstains using immunoelectrophoresis.

The technique of immunoelectrophoresis was used to determine the age of bloodstains. The immunoelectrophoretic patterns (IEP) of bloodstains ranging from 15 days to one year old were obtained by the use of high titer anti-whole human serum. The IEPs revealed gradual disappearance of beta-globulins and gamma-globulin with increase in the age of bloodstains. A comparative study of the IEP of normal human serum with those of the experimental bloodstains showed the absence of some of the corresponding proteins. The absence of a particular serum protein in the IEP of a given bloodstain will indicate the age of that bloodstain.     

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.     

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.     

Development of a radioimmunoassay technique for the detection of human hemoglobin in dried bloodstains

A sensitive radioimmunoassay for the detection of human hemoglobin in dried bloodstains for the purpose of forensic science species identification has been developed. Bloodstains from 13 animal species were tested and found to be negative for human blood. A minimum volume of 0.8 microL of fresh blood is required to produce sufficient stain for successful testing. Bloodstains prepared from newborn and sickle-cell bloods were determined to be human. Bloodstains ranging in age from 1 month to 6 years which had been maintained desiccated at 20 to 25 degrees C were also successfully tested. Positive results were obtained on human bloodstains stored at 24 degrees C with relative humidity ranging from 0 to 98% for a period of 3 weeks. Absolute counts per minute (CPM) decreased with increased humidity. Human bloodstains exposed to bacterial contamination (gram positive or negative species) under humid conditions for 2 weeks also tested positive. Bacterial contamination caused a decrease in CPM, but insufficient to result in an erroneous conclusion as to species of origin. Positive results were also obtained on human bloodstains stored for 6 weeks at various temperatures ranging from -16 to 37 degrees C. No significant decreases in CPM were noted for any of the temperature conditions described.