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.     

Acidic and neutral drugs screen in blood with quantitation using microbore high-performance liquid chromatography–diode array detection and capillary gas chromatography–flame ionization detection

Blood previously acidified with aqueous saturated ammonium chloride solution was extracted with ethyl acetate. The dried extract was subjected to acetonitrile–hexane partition. The acetonitrile portion was analysed for the presence of acidic and neutral drugs by HPLC–DAD (200 mm×2.1 mm I.D. microbore ODS-Hypersil column) and GC–FID (25 m narrow-bore×0.25 mm I.D. HP-5 column with 0.33 μm film thickness). The protocol was found to be suitable for both clinical toxicology (including emergency toxicology) and postmortem toxicology. At least 66 drugs of interest were unequivocally identified by RRTs (HPLC) and UV spectra (DAD) match while another 12 were unequivocally identified by double RRTs match (HPLC and GC). Quantitation was facilitated by incorporating calibration blood standards in each assay batch. The five drugs most commonly encountered in clinical blood specimens (1150 cases) were: paracetamol (47.4% of the cases); chlormezanone (6.6%), theophylline (1.74%), naproxen (1.65%) and mefenamic acid (1.56%). The following drugs were detected in toxicologically significant quantities in postmortem blood specimens (245 cases): phenobarbitone (1.22% of the cases), naproxen (0.82%), chlormezanone (0.82%), theophylline (0.82%), carbamazepine (0.41%) and paracetamol (0.41%).     

Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA 'fingerprinting' analysis

A minisatellite probe, MZ 1.3, detecting hypervariable fragment patterns was isolated from a human genomic library. A repetitive sequence of 27 bp length was identified which is contained in the probe approx. 40 times. The MZ 1.3 repeat shows variable homology of 53-73% to the repetitive sequence of the protein III gene of the bacteriophage M13 genome. Polymorphic restriction fragment patterns were found with MZ 1.3 using the enzymes Hinf I, BstN I, Hae III, Mbo I, PstI/Pvu II, and Rsa I. An average of 18 polymorphic fragments was observed using Hinf I as enzyme. The band sharing frequency after Hinf I digestion among unrelated individuals was determined to be 23.8 +/- 7.2%. An example for the application of MZ 1.3 to paternity testing in an incest case is given. The probe can be used with radioactive or non-radioactive detection systems. An approach is presented to compare polymorphic fragment patterns from individuals obtained by independent gel runs on the basis of relative band positions (RBP) and calculated in a computerized analysis.     

Capillary gas-liquid chromatography separation of phenethylamines in amphetamine-positive urine samples

Good gas chromatography (GC) separation of molecules is essential for clean gas chromatography/mass spectrometry (GC/MS) confirmation of compounds. The trifluoro derivatives of ephedrine (E) and methamphetamine (MA) coelute on dimethyl silicone capillary columns, such as DB-1, which are most commonly used by chromatographers. Methods are described to separate E and MA to aid GC/MS confirmations of methamphetamine, ephedrine, or both E and MA together, whichever may be present in Enzyme Immunoassay (EIA)-analyzed amphetamine-positive urine samples. The use of the heptafluoro derivatives of E and MA on a DB-1 column, or the trifluoro derivatives of E and MA on a DB-17 column, is suggested for good gas chromatographic separation.     

Determination of deslanoside in antemortem and postmortem specimens. Unusual case report

One case of the erroneous administration of deslanoside and high level of drug in antemortem plasma and postmortem specimens has been reported owing to the unusual surrounding circumstances. Deslanoside in antemortem plasma was determined by FPIA and the analysis was done by HPLC in the postmortem tissue samples. The analytical results and methods used in the examinations are discussed in the following paper.     

The effect of various stain carriers on the quality and quantity of DNA extracted from dried bloodstains

Bloodstains were made with 200 microliters blood on each of 11 different common substrates to examine the effect of the stain carrier on the amount and quality of DNA recoverable. High-molecular-weight DNA was extracted from all samples after 2 days. The yield of DNA from each sample varied considerably, not only between the different stain carriers but also within a given category. With a DNA yield of up to 10 micrograms, paper, glass, nylon, wood, smooth leather and wool gave the best results, followed by blue denim and wallpaper (up to 6 micrograms), cotton fabric and carpeting (up to 4 micrograms) and suede (up to 2 micrograms). For several stain carriers the DNA-containing solution was contaminated by chemical substances, which in the case of the blue denim, suede, and carpet samples inhibited the digestion of the DNA with restriction enzymes and prevented DNA typing. The different textures of the stain carriers tested and (as for varying yields on the same carrier) the differing degree of loss of DNA during extraction and the physiological variation in the number of leukocytes in human blood are discussed as possible reasons for the wide range of variation in the amounts of DNA it was possible to extract.