Medicolegal assessment of blood transfusion errors--an interdisciplinary challenge

Given a current total incidence of erroneously administered blood transfusions of 1:12,000-1:36,000 (AB0 incompatible 1:38,000), the percentage of lethal outcomes ranges between 2 and 5%; i.e. the sole fact of an erroneous transfusion does not mandatorily result in a causal connection with lethal outcome, which can give rise to problems in the medicolegal assessment. We report on the conception and results of a novel interdisciplinary approach to assess the lethal significance of blood transfusion errors. Besides autopsy, histological investigation and immunohistochemical detection of AB0 incompatible foreign red blood cells in autopsy specimens, transfusion medicine investigations offer the opportunity to assess several immunohaematologic features. We assessed the immunohaematologic gel card ("microcolumn") technique for suitability in the forensic assessment of an AB0 incompatible transfusion incident in a septic patient, who had had no history of previous blood transfusions, with lethal outcome. After such an erroneous transfusion had been simulated in vitro, pre-transfusion and cadaver patient blood samples (p.m. interval: 3 days) were analysed. Amongst other things, IgG-loaded erythrocytes were detected in pre- and post-transfusion samples; the presence of irregular antibodies directed against blood group antigens and anti-A or anti-B isoagglutinins, respectively, especially in the pre-transfusion sample was ruled out. Besides the demonstration of AB0 incompatible red blood cells in the cadaver blood sample, blood group incompatibilities other than AB0 were excluded. With regard to the cause of death, in synopsis with autopsy findings and clinical symptoms, the results did not allow for a final discrimination between the impact of the pre-existing septic inflammatory response syndrome and sepsis, respectively, and potential lethal effects of a (haemolytic) transfusion reaction. Besides pre- and post-transfusion compatibility testing in clinical transfusion medicine as required by German National Guidelines, the reported immunohaematologic investigations offer an important supportive tool for the forensic assessment of lethal erroneous transfusions and investigation of blood samples of survivors of transfusion incidents as well. Besides established morphological techniques, they allow for a certain evaluation of the pathophysiological impact of transfusion incidents as well as a diversified assessment of immunohaematologic features beyond the AB0 system.     

Simplified low-copy-number DNA analysis by post-PCR purification

Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low-copy-number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28-cycle polymerase chain reaction (PCR) was used to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post-PCR purification is described which increases the sensitivity of standard 28-cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post-PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28-cycle amplification.     

A Study of PCR Inhibition Mechanisms Using Real Time PCR*,†

Abstract: In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co‐extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.     

Determining the Source of Equine Bloodstains by Dinucleotide Repeats*

Abstract: A novel multiplex of independent dinucleotide tandem repeat (DTR) loci was previously described that is capable of not only discriminating human and equine DNA, but of identifying a single equine source. We report a case in which a bloodstained syringe and two needles were found during inspection of a barn by inspectors of the Pennsylvania Racing Commissions. Using the multiplex and single‐locus detection, all 21 equine DTR markers were detected in a suspect horse and two evidence samples, indicating the evidence samples came from the suspect animal. Only six markers were detected in the third evidence sample because the volume of blood was limited. Following whole‐genome amplification and single‐locus PCR, the third evidence sample detected a total of 17 markers and the likelihood of identity (probability from suspect horse/probability from a random pacer) was 7.0 × 10⁶. The DTR multiplex has some technical limitations, but it is already practical for casework.     

Investigation of Reproducibility and Error Associated with qPCR Methods using Quantifiler® Duo DNA Quantification Kit*

Abstract: Reproducibility of quantitative PCR results is dependent on the generation of consistent calibration curves via accurate volume transfers and instrument performance. A review of 14 standard curves, using two different QuantDuo^® standard DNA lots, showed variability of cycle threshold values between assays were larger than those of the Internal PCR Control (IPC). This prompted a set of experiments designed to determine the source of variability. Results showed that error introduced during DNA addition to the plate resulted in little variation. A comparison of seven independent series demonstrated cycle threshold variation between dilutions was larger than the variation expected from repeated samples. Modeling the influence of pipette errors on dilution series accuracy indicated that a more rigorous approach to external calibration curve production is required and showed that improvement in calibration curve stability is expected if the pipette conditions are carefully chosen and/or a single validated curve is utilized as the calibrator.     

Multicolor Real-Time PCR Genotyping of ABO System Using Displacing Probes

Abstract:  Rapid and informative ABO genotyping has become increasingly popular in forensic use. We developed a multiplex real-time polymerase chain reaction (PCR) approach to genotype ABO major groups and subgroups. Seven differently fluorophor-labeled displacing probes for O¹(261delG), A(261G), A(796C/803C), B(796A/803C), O² (802G>A), A² (1059delC), and A² (1009A>G) were combined in one or two PCRs to determine either ABO major groups or subgroups. The method correctly detected 13 reference DNA samples. A blind test of 237 samples resulted in complete agreement with their phenotypes, and 110 of these 237 samples as well as with PCR-SSP method. The whole analysis could be finished in less than 100 min at substantially low material cost and the template DNA ranging from 0.16 to 500 ng per reaction could be quantitatively detected. Despite the limited informativeness of ABO genotyping, the developed methods could find application in rapid and inexpensive screening of forensic settings.     

Study: The Lack of Significant Association of the Catechol-O-Methyl Transferase (COMT) Gene Polymorphism in Violent Offenders with Mental Retardation

Abstract:  Little is known about criminality of cognitively impaired people and also there have been no reports on the relationship between catechol- O -methyl transferase (COMT) and committed Mental Retardation (MR) subjects. In the present study, the association between committed (violent offences) MR subjects and genetic variants of COMT were investigated by using polymerase chain reaction and based restriction fragment length polymorphism methods. During 6 years of follow-up, 36 violent offenders with mild MR were investigated. Thirty-six control volunteers were included in the study as a control group. H/L polymorphism of the COMT gene was investigated in these two groups. In conclusion, the COMT gene genotype distribution and allele frequency is not significantly different between the two groups ( p  > 0.05). This result suggests that the H/L polymorphism of the COMT gene does not show an association with the potential of "commits-violent offense" of Turkish subjects with mental retardation, compared with control group.     

Evaluation of Tamm‐Horsfall Protein and Uroplakin III for Forensic Identification of Urine

Abstract: In this study, Tamm‐Horsfall protein (THP), a major component of urinary protein, and uroplakin III (UPIII), a transmembrane protein widely regarded as a urothelium‐specific marker, were evaluated for forensic identification of urine by ELISA and/or immunohistochemistry. THP was detected in urine, but not in plasma, saliva, semen, vaginal fluid, or sweat by the simple ELISA method developed in this study. In addition, most aged urine stains showed positive results. The urine specificity of THP was confirmed by gene expression analysis. Therefore, as reported previously, ELISA detection of THP can be used as a presumptive test for urine identification. UPIII was specific for immunohistochemical staining of cells in centrifuged precipitate of urine. However, ELISA and RT‐PCR for UPIII were not specific for urine. UPIII may be applicable for forensic urine identification by immunohistochemistry.