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Human chimerism is the presence of ≥ 2 cell populations in one person that contain genetic material from more than one zygote. Chimerism may be either acquired by transfusion or transplantation of donor cells, or congenital arising from embryo fusion or dizygotic twin-twin transfusion. We encountered a 4-year-old boy with developmental hip dysplasia whose preoperative (serologic) blood group was AB, but whose red cell agglutination was atypical ("mixed field") and caused us to study the patient's parents' ABO blood groups. Parental blood groups (AB and O) suggested possible nonparentage. An alternative explanation of the findings was that the child was chimeric or mosaic. Molecular cloning and genotyping of his ABO locus in leukocytes revealed two heterozygous genotypes: A102/O01 and B101/O01. Other loci, each of which possessed three distinct alleles, unambiguously showed transmission of two alleles from either the child's mother (e.g., HLA-A) or two alleles from the child's father (e.g., D8S1179). Findings indicate that the child is a tetragametic chimera.

Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.

ABO genotyping is often performed to identify the blood type of decomposed samples, which is difficult to be determined by a serological test. In this study, we developed a simple method for ABO genotyping using a DNA chip. In this method, polymerase chain reaction-amplified and fluorescent-labeled fragments in the ABO gene and primate-specific D17Z1 were hybridized with DNA probes on a chip designed to detect single nucleotide polymorphisms (SNPs) in the ABO gene and part of the D17Z1 sequence. Using blood samples from 42 volunteers and 10 animal species, we investigated whether the chip could be used to detect SNPs in the ABO gene and the D17Z1 sequence. This method was then applied to various forensic samples, and it was confirmed that this method was suitable for the simultaneous analyses of ABO genotyping and species identification. This method fulfills the recent need for the development of rapid and convenient methods for criminal investigations.

The short tandem repeat system HumCD4 was amplified by the polymerase chain reaction (PCR) on blood samples from 304 unrelated Austrian Caucasians and analyzed by horizontal, non-denaturing polyacrylamide electrophoresis. The mean exclusion chance was 0.417, the discriminating power 0.850 and the heterozygosity rate 0.628. The observed phenotype distribution is in Hardy-Weinberg equilibrium. In 100 families (200 meioses) no mutations were found. Sufficient amplification could be achieved with as little as 80 pg of high molecular weight cell-line DNA. In a degradation experiment DNA extracted from bloodstains stored for up to 28 days in a moist chamber and DNA boiled for up to 18 min could be amplified. A duplex PCR with TH01 is proposed.

Allele and genotype frequencies were determined for the HLA-DQA1 and Amplitype® Polymarker loci (low density lipoprotein receptor (LDLR), glycophorin A (GYPA), hemoglobin G gammaglobin (HBGG), D7S8, and group-specific component (Gc)) in Hasidic and non-Hasidic Ashkenazi New York City Jewish subpopulations. For all loci tested, except HBGG, the 2 subpopulations meet the assumption of Hardy-Weinberg equilibrium. Comparison of various allele and genotype frequencies for the Hasidic and the non-Hasidic groups showed no significant differences. Comparison of the various allele frequencies in the two subpopulations to another Caucasian group revealed significant differences at the HLA-DQA1 and D7S8 loci in the Hasidic group. These frequency data can be used for comparison to other populations and for frequency estimates in DNA profiling.

A unique sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of ABH antigens in bloodstains has been developed. Human anti-A and -B antisera and Ulex europaeus anti-H lectin were coated on the inner surface of microplate wells. The sample antigens from bloodstains, solubilized with n-octyl-beta-D-glucopyranoside which was then removed by passing through a Sephadex G-25 (G-25) mini-column, were placed in the wells. After washing the wells repeatedly, peroxidase-conjugated Ulex europaeus lectin I was added and incubated. Antigen activities were determined by the development of colors using o-phenylenediamine/H2O2. This technique permitted clear detection of all ABH antigens corresponding to the antisera and lectin with high sensitivities. The A and B antigens were solubilized as aggregates with H antigen from the erythrocyte membrane. Excess detergent remaining in the sample reduced the sensitivity and accuracy of this ELISA, probably due to the removal of antibody from the wells by the effect of the surfactant. The treatment of solubilized antigens with G-25, an indispensable step, eliminated the adverse effect of the detergent on the ELISA. The ELISA method reported here was proved to be easy, economical and sensitive, and this technique should be useful in the forensic practice.

The proficiency testing of crime laboratories began in the mid-1970s and presently assumes an important role in quality assurance programs within most forensic laboratories. This article reviews the origins and early results of this testing program and also examines the progress of proficiency testing in allied scientific fields. Beginning in 1978, a fee-based crime laboratory proficiency testing program was launched and has grown to its present level involving almost 400 laboratories worldwide. This is the first of two articles that review the objectives, limitations and results of this testing from 1978 through 1991. Part I reviews the success of laboratories in the identification and classification of common evidence types: controlled substances, flammables, explosives, fibers, bloodstains, and hairs. Laboratories enjoy a high degree of success in identifying drugs and classifying (typing) bloodstains. They are moderately successful in identifying flammables, explosives, and fibers. Animal hair identification and human hair body location results are troublesome. The second paper will review the proficiency of crime laboratories in determining if two or more evidentiary samples shared a common origin.

If a female has sexual intercourse with two males at short intervals within the same ovulatory period, superfecundation may occur. This article reports two cases of paternity identification in twins. The results showed that each twin had come from a different father. Thus, great attention should be paid to such a situation when the twin paternity identification is asked for.

PCR analysis of the VNTR locus D17S30 was assessed for its potential use in forensic identification analysis. "Allelic drop-out," the inefficient amplification of some alleles, complicates the interpretation of DNA typing at this locus. PCR conditions were varied in an effort to improve amplification of the alleles at this locus. Such changes included the use of denaturants, formamide and DMSO, to overcome any incomplete denaturation of template strands due to GC content or allele size. Lowering the annealing temperature during the PCR cycle enhanced the amplification of a larger fragment, but this was not related to the D17S30 locus. It appears that the structure of the genome of some individuals rendered PCR amplification inefficient at this locus.

This report concerns the rape of a woman by a stranger. The complainant stated that, during the course of the rape, penetration took place without ejaculation: these details were later confirmed by a suspect. This suspect, who was identified by the complainant, confessed and was convicted. Laboratory findings, however, indicated that the suspect could not have been the source of the semen found on the complainant's underpants. Further, a vaginal swab taken from the complainant contained intact sperm cells, an indication of a recent sexual contact. In spite of the presence of semen on the underwear which could not have come from the suspect, and the nonoccurrence of an ejaculation during the rape, the complainant maintained that she had not had a sexual contact for a month prior to the rape. This apparent contradiction and its implications are discussed.

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.

Six red blood cell (RBC) antigen systems, coupled with human lymphocyte antigen (HLA) phenotyping, were used to establish paternity on 28 mother/child/alleged-father trios. Samples were subsequently examined using the deoxyribonucleic acid (DNA) fingerprinting test with the multilocus Jeffreys DNA probes 33.6 and 33.15. In 27 of 28 paternity cases, the DNA fingerprinting test results supported and enhanced the results of RBC and HLA typing by resolving disputed paternity cases conclusively. One discrepancy between conventional serological methods and DNA analysis is discussed.

Seven genetic marker systems were analyzed from liquid blood and dried bloodstain specimens submitted to the Nebraska State Patrol Crime Laboratory from various law enforcement agencies throughout Nebraska. The phenotypic and genotypic frequencies for the ABO, Lewis, esterase D (ESD), phosphoglucomutase (PGM), adenylate kinase (AK), adenosine deaminase (ADA), and haptoglobin (HP) systems were calculated. The results indicate that the phenotypic frequencies are generally in agreement with frequencies reported in other populations in the United States.

Urine samples from 28 donors with known blood group and secretor status were concentrated by three different procedures, and ABO typing on the concentrated samples was successfully performed after 12 weeks of storage. The effects of storage with or without sodium azide on ABO typing and on the pH values at several different temperatures were also studied.

Using ABH enzyme-labeled monoclonal antibodies, the authors could rapidly detect the ABO group from body fluids and body fluid stains by the dot enzyme-linked immunosorbent assay (dot-ELISA). In this test, the antigen was immobilized on nitrocellulose paper; the entire piece of paper was coated with an appropriate dilution of enzyme-labeled McAb directly against the antigen of interest; and, finally, 3,3'-diaminobenzidine (DAB) substrate solution was added. The site of a positive reaction is clearly visible as a brown spot. We analyzed 521 samples and got satisfactory results. We also analyzed 99 practical case samples by this method and achieved the same results as those obtained by other researchers using other methods. This method is accurate, simple, direct, rapid, and sensitive; it also produces easily observed results, requires no equipment, and can be completed in 30 min. The test proved to be clearly more sensitive for the detection of the ABO blood group in secretor saliva than the conventional hemagglutination inhibition test. Also saliva diluted 10(-4) to 10(-5) and the ABO group of nonsecretor saliva and urine could be easily detected by this method.

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.

The ridge patterns on the fingernails of corresponding fingers of a pair of twins were compared microscopically and found to be readily distinguishable from one another. Based on blood grouping in six blood group systems (ABO, Rhesus, Ss, Duffy, Kidd, and Kell), the probability that the twins were monozygotic was calculated to be 89.1%.

An improved method has been developed for the reliable classification of different C1R genetic variant forms from human serum or plasma. The method combines the use of neuraminidase-digested samples followed by monodimensional isoelectric focusing in the pH range 5 to 8 followed by immunoblotting. The method yields a simple pattern, with one major band in homozygote and two major bands in heterozygote cases.

Using the highly sensitive immunohistochemical staining method EnVision+, which employs a dextran polymer reagent for the secondary antibody, the detection of the ABH antigens was attempted in the oral squamous epithelium. This new technique uses monoclonal antibody as a primary antibody and it takes about three hours for staining. The time is much shorter than conventional absorption-elution testing or absorption-inhibition testing for the determination of ABO blood grouping. Secretor saliva samples were stained at strong intensity by the antibody, which corresponded to its blood group and anti-H. On the one hand, nonsecretor saliva samples were stained at strong intensity only by the antibody that corresponded to its blood group, and at weak intensity only by anti-H. Since human oral squamous epithelium antigens were stained specifically by this method, we can examine the ABO blood group of saliva samples and perform cytodiagnosis at the same time. Our research suggested that the EnVision+ Method is a useful technique for ABO blood grouping of saliva in forensic cases.

A blood group substance (BGS), a protein with ABH antigenic activity, was isolated from human seminal plasma and designated as p 84 (Sato, 1995). We have developed a method for determining the ABO blood type of semen by performing a sandwich enzyme-linked immunosorbent assay (ELISA) in which p 84 is captured with an anti-p 84 monoclonal antibody, and evaluated the specificity and sensitivity of this method. Although BGS activity was detected in semen sensitively by this method, it was not detected in saliva, urine, breast milk, blood or vaginal secretions. Since the concentration of p 84 in semen was independent of the secretion status, the status can be determined as non-secretor when p 84 but not BGS activity was detected. To determine the stability of BGS activity on p 84, dried stains of semen on filter paper were kept at 4, 26, and 37 degrees C for 8 months, 2 years and 1 month, respectively, and their BGS activities were examined. After 8 months at 4 degrees C, over 60% of the original BGS activity was recovered from the stain. The activity could be detected even from a square as small as 0.25 by 0.25 cm. After 1 month at 37 degrees C and 2 years at 26 degrees C, 31 and 20% of the BGS activity, respectively, still remained. It could be detected from the pieces of 1.0 by 1.0 cm and 0.5 by 0.5 cm squares, kept for 1 month at 37 degrees C and 2 years at 26 degrees C, respectively. Finally, semen was mixed with saliva or blood at varying volumetric ratios and used for the sources of dried stains. The BGS activity of p 84 could be detected in the stains until the ratio between semen and saliva or blood reached 1:4. We conclude that this sandwich ELISA offers a more sensitive and specific method for determining the ABO blood type of semen samples obtained from sexual assault victims than existing methods, such as the conventional absorption-elution and classical hemagglutination-inhibition tests.

Twenty-five permanent teeth, including eight carious ones whose pulp cavities had been exposed, were used for this research 3-5 weeks after extraction. Phosphate-buffered saline (PBS, at pH 7.2) was employed to extract ABO blood group substance from tooth powder. ABO grouping was performed on blood-stained compresses from the extraction wound (as controls), tooth fragment, tooth powder, and cotton fibers immersed in PBS extract by absorption-elution (AE) technique and on the PBS extracts by the two-dimensional absorption-inhibition (2-D AI) technique. It was found that blood grouping in PBS extracts by 2-D AI yielded reliable results: no false positive results, and a high rate of correct grouping, (24/25), while blood grouping on other dental materials, such as tooth fragments, tooth powders, immersed fibers, by AE gave an unacceptable rate of false positive/negative results.

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.

Two recent paternity cases are reported. In the first case of paternity exclusion, deoxyribonucleic acid (DNA) restriction fragment length polymorphisms (RFLPs) on variable number of tandem repeat (VNTR) loci with multiple alleles were informative, as well as established systems of red blood antigens, red cell enzymes, serum proteins, and human leukocyte antigens. In the second case, in which both the alleged father and the first wife were deceased, the paternal genotype was determined by using genetic markers from the second wife and four children, which then were compared with the paternal alleles of the child in question, the plaintiff in this case. The high probability of paternity (0.999,998,7) made us conclude that the man probably was the actual father. The DNA analysis by VNTR probes appears to be quite valuable in the study of paternity cases.

A technique was developed for Gm/Km typing of bloodstains and sera in U-bottom microtiter plates. Gm/Km typing of sweat and urine samples was also attempted with limited success.

The use of the principles of genetic inheritance to link a bloodstain to a missing victim is discussed. Genetic marker typing can determine whether the parents of the missing victim are possible parents of the bloodstain source. Given a parental inclusion, it is possible to calculate the probability that a randomly selected couple would possess the necessary genetic combinations to be the parents of the person who bled. The applications of this concept using several genetic marker systems are examined. General formulas have been developed for the probability calculation using phenotype distribution frequencies and gene frequencies. This approach was applied to a homicide case in which it was shown that the victim's parents were among only 0.8% of couples from the general population who would be able to bequeath the genetic marker types found in the bloodstain. This evidence was helpful in producing a conviction of first degree murder.

A conventional agarose gel electrophoretic method was described for typing phosphoglucomutase-1, esterase D, or glyoxalase I as single systems. Bloodstain extracts were absorbed into 1-mm-thick agarose gels via an application mask. The electrode wick distance was 12 cm and electrophoresis was carried out at 400 V at 6 degrees C. The electrophoretic run times were 30 min for glyoxalase and 1 h for esterase D or phosphoglucomutase. This method is reliable and produces highly resolved band patterns. Additionally, the shorter separation times as a result of the increased voltage gradient permitted typing of more samples in a given time period compared with presently used methods. This technique requires little technical expertise and can be incorporated into the laboratory at a minimal cost.

A simple and inexpensive capillary tube procedure, which can be applied in the forensic science laboratory, is described for the detection of the Lewisa (Lea) and Lewisb (Leb) antigens on red blood cells. This procedure will permit approximately 4000 tests to be performed from a single 2-mL bottle of Lewis antiserum.

An absorption inhibition method for the detection of gamma marker Gm(11) in dried bloodstains is described. Particular reference is made to the association of Gm(11) with Gm(-1, -2). When a dried bloodstain fails to inhibit anti-Gm(1) and anti-Gm(2), this may represent a true Gm(-1, -2) result or there may be insufficient material to inhibit either antibody. The detection of Gm(11) in a bloodstain extract provides an objective means of confirming the apparent absence of Gm(1) and Gm(2) as representing a true Gm(-1, -2) result. This antigen compares very well with other blood group systems with regard to the amount of bloodstain required for analysis and its stability. No evidence is available for preferential loss of Gm(1) and Gm(2) relative to Gm(11) in dried bloodstains.

The proteolytic enzyme, proteinase K, has been found to destroy all vaginal cells though it does not have the same effect on spermatozoa. In cases of sexual offenses, in which a swab has been used to wipe out the vagina, the female cells and their nuclei on that swab may also contain the heads of spermatozoa. After as short a time as 30 min of proteinase K treatment, the spermatozoa that had separated from the enzymatically destroyed vaginal cells were recovered. This proteinase destruction furnishes some spermatozoa with deformed heads and a somewhat greater number of isolated tails though a sufficient number of spermatozoan heads still remain for a reliable diagnosis. For detection of spermatozoa from a vaginal swab after proteinase K pretreatment, the heads of the spermatozoa are distinctly stained by Oppitz's method. Further, on prior treatment with proteinase K, the ABO blood grouping of the spermatozoa could also be determined on the vaginal swab by using the absorption-elution technique. The resistance of the spermatozoa to proteinase K is the basis for this method.

Analysis of bloodstains subjected to heat or improper collection techniques may yield expressions of erythrocyte acid phosphatase (EAP) phenotypes other than those originally coded. This report illustrates a case in which the deterioration of an EAP Type CB sample caused it to appear as an EAP Type C. Suggestions are offered to minimize such problems and to make the analyst aware of possible misinterpretations.

ABO typing was successfully performed on 46 urine samples whose ABO group and secretor status had been determined previously from blood and saliva. Twenty-four urine samples were collected on which blind studies, time studies, and storage studies were performed. Multiple urines from several individuals were collected to evaluate the duplicity of the test. Also, urines were collected from pregnant and menstruating females to determine if ABO typing was affected under these conditions. Results of these studies are discussed.

The Lewis blood grouping of human dried bloodstains could be determined by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies with an avidin-biotin complex (ABC). The bloodstains aged 1 year were used as samples, and approximately 1 mg of the stains was enough to type each Lewis antigen reliably by this method. The Lewis substances of 106 individual stains were correctly typed regardless of their ABO blood group system.

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.

A routine method is described for obtaining reproducible haptoglobin patterns from bloodstains by discontinuous polyacrylamide gel electrophoresis. By employing a stacking gel, proteins from bloodstain extracts are concentrated into narrow zones, before entering the resolving gel. This effect yields highly resolved haptoglobin patterns. Therefore, laboratories without the specialized equipment and expertise for polyacrylamide gradient gel preparation can still obtain highly resolved haptoglobin patterns from bloodstains.

A rapid, reliable method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II by agarose gel electrophoresis is presented. This method uses a double origin sample application system. Unreduced sample extracts for adenylate kinase analysis are applied 13.0 cm from the anode. Reduced sample extracts for the remaining proteins of interest are applied 7.0 cm from the anode. The use of applicator foils and an increased voltage gradient result in superior resolution, linearity, and band sharpness of the allozyme patterns. Further, there is no masking of the adenylate kinase 2 band as a result of the use of a reducing agent, and carbonic anhydrase II is resolved without interference from hemoglobin as has been observed with other multisystem methods.

A paternity test is presented in which a father and his two children possessed an extremely rare amorphic gene R−29( r = , –––). One of the children was determined to be illegitimate at the first trial as her Rh phenotype was R2R2 (ccDEE) and the father's phenotype was R1R1 (CCDee). At the Court of Appeal, however, the rare Rh gene ( r = –––) was shown to be inherited from the father to the appellant child through extended tests including her brother whose phenotype was also R2R2 (ccDEE). She was acknowledged to be legitimate.

This paper constitutes a feasibility report on the use of the human leukocyte antigen (HLA) system for the typing of dried bloodstains. Antigens tested include the HLA-A2, A3, A10, B7, B8, and B14 alleles. An aging study conducted on A3 positive bloodstains showed that HLA-A3 could be reliably detected on bloodstains stored up to 30 days at 22 degrees C. Unlike most earlier reports on HLA typing of bloodstains, no cross-reactivity problems were detected with the antisera used in this study. In addition to the successful typing of bloodstains, we were also able to type fresh, neat seminal and saliva stains in the A2 and A10 antigenic systems.

A case of kidnapping involving the concept of paternity testing is described. The couple arrested and charged with kidnapping stated the child in their possession was their biological daughter. Serological tests were performed on liquid blood samples in an effort to determine if the couple could have been the biological parents of the child. Two genetic inconsistencies were discovered in the 15 blood marker systems analyzed.

A sensitive and reliable hemagglutination assay, using V-bottom microplates, is described for the detection of the ABO blood group alloantibodies in bloodstained material. When used in conjunction with an absorption-elution procedure, the microplate assay resulted in a 300% increase in the number of conclusive grouping results when compared to the Lattes crust test. The use of the microplate reverse grouping assay permits 24 specimens to be assayed conveniently on a single plate and eliminates the tedious and time-consuming microscopic examination required for the Lattes crust test.

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.

Vaginally inserted plastic tampon applicators were obtained from 42 female volunteers. The applicators were examined for the presence of ABH blood group substances, phosphoglucomutase (PGM), amylase, acid phosphatase, P30, and intact spermatozoa. Each applicator was accompanied by a control blood sample, a saliva specimen, a brief sexual and menstrual history, and method of birth control of the donor. Eight of the male sexual partners of the donors submitted blood and saliva samples. One male sexual partner submitted only a saliva sample. ABH blood group substances corresponding to the donor were recovered from 36 of the 42 applicators. The remaining 6 applicators revealed a combination of the donor's and sexual partner's ABH substances. The female's PGM type was recovered from 34 of the applicators. The remaining 8 applicators failed to show PGM activity. Of the applicators, 15 indicated evidence of prior sexual intercourse by the detection of ABH substances not consistent with the applicator donor (6 samples), high levels of acid phosphatase (11 samples), or recovery of spermatozoa (8 samples) or some combination of these. All applicator samples failed to show the presence of either P30 activity or PGM factors foreign to the female.

A sensitive method for the detection of anti-A and anti-B hemagglutinins in fresh saliva has been developed. The method utilizes a bromelin treated erythrocyte suspension as indicator cells and includes a simple procedure to concentrate these hemagglutinins. Antiserum directed against immunoglobulin A enhances the hemagglutination assay. We find that these salivary hemagglutinins are present in over 90% of the population and that their titer remains stable over a period of two months. These hemagglutinins can be used to blood type the donor of a saliva sample and can be used in a confirmatory test that complements the commonly used absorption-inhibition test which is used to detect salivary blood group agglutinogens. In preliminary studies we have determined that hemagglutinins can be successfully isolated and analyzed from dried saliva stains.