Elaine M. Pagliaro

A PCR-based strategy for ABO genotype determination.

The ABO blood group system has been widely used in forensic serology. Several techniques have been developed which detect ABH antigens. To overcome the problems associated with conventional methods such as bacterial contamination, extreme environmental conditions, antigen activity, non-secretor issues, and non-specific absorption, several new strategies have been employed to detect ABO genotypes by PCR. We have developed improved amplimers for the glycosyl transferase locus on chromosome 9 and examined the suitability of PCR-based ABO genotyping for forensic identification. We show that the ABO system is primate specific and that DNA extracted from various tissues commonly encountered in criminal cases can be quickly and reliably typed by ABO-PCR. The results indicate that ABO genotyping by PCR and restriction enzyme digestion of the amplified product is a useful procedure for forensic analysis that can provide additional discriminating power compared to conventional immunological methods.

ABH Antigen Typing in Bone Tissue

Results obtained from the ABH grouping of bone tissues by the absorption-elution procedure and by a recently described two-dimensional absorption-inhibition procedure are reported. Neither the elution nor the inhibition procedure alone yielded uniformly correct results. A combination procedure consisting of the use of both absorption-elution and two-dimensional absorption-inhibition is proposed for bone ABH grouping. When elution and inhibition were used in combination, specimens yielding concordant results with both techniques were correctly grouped.

Two-Dimensional Absorption-Inhibition

A novel inhibition procedure, called two-dimensional absorption-inhibition, is described. The theory underlying this technique is developed based on a review of and comparison with existing inhibition methods. Two-dimensional inhibition takes advantage of the best features of inhibition-titration and titration-inhibition, and is shown to be more sensitive than either of them. Results obtained using all the inhibition methods on secretor saliva, semen, urine, urine stain, and perspiration stain specimens show that the new technique is especially powerful in correctly determining the ABH antigens in secretor body fluids having lower concentrations of soluble blood group antigens. A two-stage version of the two-dimensional procedure that makes it a practical casework method is described as well.

Evaluation of antisera for bloodstain grouping. II. Ss, Kell, Duffy, Kidd, and Gm/Km.

Thirty-one different examples of commercially available blood grouping antisera specific for the S, s, K, k, Fya, Fyb, Jka, and Jkb antigens and anti-human globulin sera were serologically evaluated with red cells and in absorption-elution tests to determine their applicability to bloodstain antigen determinations. Nineteen examples of commercially available antisera specific for various Gm and Km antigens and their corresponding anti-D reagents were likewise evaluated in inhibition tests with sera and bloodstains. Elution tests with the blood grouping antisera and inhibition tests with the Gm/Km antisera on a series of aging bloodstains on cotton cloth, and on bloodstains on a number of different substrata, demonstrated that properly evaluated commercial antisera are useful reagents for bloodstain grouping in forensic serology.

Evaluation of Antisera for Bloodstain Grouping I. ABH, MN, and Rh

Sixty-eight different commercially available blood grouping antisera and lectins with ABH, MN, and Rh D, C, E, c, and e specificities were serologically evaluated for their applicability to bloodstain antigen determination. The characteristics of the antisera were determined with red cells, with fresh bloodstains, and with series of aging bloodstains. The Rh antisera were tested under a variety of serological conditions and with bloodstains on various substrata. Additionally, studies on optimization of absorption-elution procedure variables were carried out, and some data on the storage characteristics of red cells and blood grouping antisera were gathered.

Genetic Markers in Human Bone: II. Studies on ABO (and IGH) Grouping

A combination absorption-elution, two-dimensional absorption-inhibition procedure was used to determine the ABH antigen composition of a series of human bone specimens of known ABO type that had been aged up to nine months under dry and humid conditions at ambient temperature, 37 degrees C, and 56 degrees C; at ambient temperature in dry and wet soil; and buried in soil outdoors. Grouping data for the separate elution and inhibition testing, as well as for the combination procedure, are given. The combination method was found to be a highly reliable procedure for bone tissue ABH typing. Some data on microbial contaminants of human bone specimens aging in soil, and their effects on ABH typing results, are presented. No direct correlation between the properties of microbial contaminants and specific changes in the ABH antigenic composition of aging bone tissue specimens could be ascertained. Data on IGH antigen determination and on the quantitation of immunoglobulin G (IgG) in human bone tissue extracts indicated that immunoglobulin levels were typically too low to expect routinely successful Gm antigen testing results. However, these factors can sometimes be determined in fresh bone tissue extracts, particularly if the extracts are concentrated.

Enzyme Assays for the Identification of Gastric Fluid

Simple, reliable procedures for the assay of pepsin and rennin-like enzyme activities are described as a means of identifying gastric fluid-containing samples in forensic science laboratories. These samples are usually vomitus, or stomach contents originating from wounds that perforate the stomach. They may be encountered at scenes or on articles submitted for examination, in fresh form or as dried stains. The pepsin activity assay is based on proteolytic activity with bovine albumin as substrate and the rennin-like activity assay is based on the coagulation of milk protein.

Studies on DNA polymorphisms in human bone and soft tissues

Abstract The determination of genetic characteristics in human bone and soft tissues is interest both to forensic scientists and to physical anthropologists, although for different purposes. The application of genetic typing from human remains to anthropological and forensic identification problems is briefly reviewed. DNA analyses of ancient and modern human remains by Southern blot (RFLP) and amplification (PCR) techniques are reviewed. The results of studies on the effects of exposing bone and soft tissues to different environmental conditions for defined time periods on subsequent DNA typing are presented. Bone specimens were temperatures and soil under dry and humid conditions, and a complete series of soft tissues were exposed to dry and moist air and to salt water, for periods of 1–9 months.

DNA analysis in human bone and other specimens of forensic interest: PCR typing and testing

Polymerase chain reaction (PCR) procedures that enable the faithful replication of millions of copies of a specific DNA sequence in vitro have been described and refined in recent years [I]. PCR techniques have been used in a variety of genetic and clinical diagnostic applications, including evolutionary studies, cloning and sequencing, detection of sickle cell anaemia genes, detection of human papilloma virus, HIV and other retroviruses. Because many specimens submitted for forensic DNA analysis contain limited quantities of DNA and the DNA in these specimen may be degraded, RFLP analysis is not always possible. This limitation has prompted the interest of forensic scientists in the applicability of PCR typing procedures that may be applicable to small and/or degraded specimens. PCR techniques may be applied to forensic identification problems using specific primers for the amplification of coding loci, variable number of tandem repeats (VNTR) loci, or sequences specific for X and/or Y chromosomes that can serve as sex markers. The most refined PCR procedure applicable to forensic identification thus far utilized specific primers and allele-specific oligonucleotide probes to detect genotypes at the HLA-DQ, locus [2-41. HLA-DQ, typing techniques have been applied to blood and seminal stains [5] and to hair roots [6].

Simultaneous identification and determination of species origin, ABH antigens and isoenzyme markers in the same bloodstain

A reliable procedure for the simultaneous identification of blood, determination of species origin and ABH antigens, and typing of isoenzyme markers from a sample of three 1-cm threads is described. Results obtained from known control as well as from casework bloodstains using this procedure were consistent with those obtained in parallel, conventional, individual tests under blind trial conditions.