Estimating bloodstain age in the short term based on DNA fragment length using nanopore sequencer.

We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.

A method for determining the origin of crude drugs derived from animals using MinION, a compact next-generation sequencer.

We evaluated whether MinION, an inexpensive portable sequencer, can be used to identify the origin of crude drugs derived from animals. Standard and nonstandard crude drugs with different species of origin were examined. In addition, standards mixed with nonstandard samples were used. As a target gene, cytochrome c oxidase I was amplified and sequenced. The fast mode results had a slightly lower match ratio than high-accuracy mode, but the animals of origin were correctly determined by BLAST for all samples. For antler velvet derived from Rangifer tarandus, even when the sequences were aligned based on Cervus elaphus, the animal of origin was determined correctly. Minor contents could be detected from mixtures of two animals, if the mixtures contained at least 19:1 mtDNA when the coverage allele-fraction threshold was 0.05. By contrast, in fast mode, two sequences could not be separated due to the low accuracy of the base-calling for each read. For fieldwork, the species of origin of crude drugs could be identified with only simple DNA extraction and library preparation. Therefore, MinION appears to be a convenient tool for identifying the origins of crude drugs derived from animals.

Diagnostic significance of the histopathology of bone marrow macrophages in forensic autopsies.

Forensic pathologists often encounter autopsies that require an assessment of antemortem general conditions (e.g., infection, metabolic disorders). To establish evaluation clues for such cases, we quantitatively examined macrophages and the general pathology of bone marrow in samples from 180 forensic autopsy cases of decedents with various conditions. Hematoxylin-eosin staining, Berlin blue staining, and immunostainings for CD163, CD138, and CD61 were performed. We determined the numbers per field (density) of total macrophages, swollen macrophages, macrophages with hemophagocytosis, and hemosiderin-laden macrophages. Each density was standardized by identifying its ratio to the total number of macrophages. The decedents' background data (cause of death, other pathological findings, postmortem interval, antemortem symptoms, and presence of resuscitation) were extracted. No correlations were found between the postmortem interval and the other decedent data, indicating that these data are not affected by postmortem changes. In the group in which inflammatory disease was the cause of death, there were significant elevations in the ratio of the swollen macrophage density to total macrophages. Significantly higher ratios of the density of swollen and hemophagocytic macrophages were observed in the group in which conditions with a prolonged agonal period were the cause of death. The group with a return of spontaneous circulation to resuscitation showed a significantly higher ratio of macrophage density with hemophagocytosis. This study provides the first statistical analysis focused on bone marrow histopathology in forensic autopsies. The results will be useful for elucidating causes of death and agonal-period conditions.

Estimating individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq.

We tried to estimate individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq. The BAM files produced by MiSeq were viewed using Integrative Genomics Viewer (IGV) to verify mixed bases. By sorting the reads according to base type for each mixed base, partial haplotypes were determined. Then, the BAM files produced by MinKNOW were viewed using IGV. To determine haplotypes with IGV, only mixed bases determined by MiSeq were used as target bases. By sorting the reads according to base type for each target base, each contributor's haplotype was estimated. In mixed samples from two contributors, even a haplotype with a minor contribution of 5% could be distinguished from the haplotype of the major contributor. In mixed samples of three contributors (mixture ratios of 1:1:1 and 4:2:1), each haplotype could also be distinguished. Sequences of C-stretches were determined very inaccurately in the MinION analysis. Although the analysis method was simple, each haplotype was correctly detected in all mixed samples with two or three contributors in various mixture ratios by combining MinION and MiSeq. This should be useful for identifying contributors to mixed samples.

Estimating included animal species in mixed crude drugs derived from animals using massively parallel sequencing.

We developed a method that can detect each animal species of origin for crude drugs derived from multiple animal species based on massively parallel sequencing analysis of mitochondrial genes. The crude drugs derived from animals investigated in this study were Cervi Parvum Cornu and Trogopterorum feces, which are derived from a mix of different animal species, two chopped cicada sloughs, and two commercial Kampo drugs. The mitochondrial 12S rRNA, 16S rRNA, and cytochrome oxidase subunit I gene regions were amplified and sequenced using MiSeq. The ratios of haplotype to total number of sequences reads were calculated after sequence extraction and trimming. Haplotypes that exceeded the threshold were defined as positive haplotypes, which were compared with all available sequences using BLAST. In the Cervi Parvum Cornu and Trogopterorum feces samples, the haplotype ratios corresponded roughly to the mixture ratios, although there was a slight difference from mixture ratios depending on the gene examined. This method could also roughly estimate the compositions of chopped cicada sloughs and Kampo drugs. This analysis, whereby the sequences of several genes are elucidated, is better for identifying the included animal species. This method should be useful for quality control of crude drugs and Kampo drugs.

Bloodstain examination and DNA typing from hand-washed bloodstains on clothes.

We investigated whether bloodstain examination and DNA typing can be performed on washed bloodstains on clothes. Blood was dropped onto T-shirts made from 100% cotton or 100% polyester. After drying, the T-shirts were hand-washed with handwashing soap, dishwashing detergent, laundry detergent, soap, or just water until the bloodstains could not be seen. After drying the T-shirts, DNA and RNA were extracted simultaneously from the bloodstained areas using commercial kits. RNA was reverse-transcribed to DNA, and then the detection of the mRNAs for HBB, ACTB, and 18S rRNA was examined. DNA was quantified via real-time PCR, and then STR typing was performed with a commercial kit. The luminol and leucomalachite green tests were used as preliminary bloodstain tests, and an immuno-chromatography kit was used to identify human bloodstains. DNA could be extracted from all washed bloodstains, but more DNA was extracted from cotton T-shirts than from polyester T-shirts. STR typing was successful for all bloodstains without issues such as PCR inhibition. In the human bloodstain identification test using mRNA, almost all bloodstains produced a Ct value for HBB and all bloodstains produced a Ct value for 18S rRNA, whereas few bloodstains produced a Ct value for ACTB. All bloodstains reacted positively to luminol, but some were negative for leucomalachite green. Most of the bloodstains did not react positively in the human bloodstain identification test using the immuno-chromatography kit. The results suggest that human bloodstain identification and DNA typing can still be performed after clothes with bloodstains are washed.

The origin identification method for crude drugs derived from arthropods and annelids using molecular biological techniques.

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.

Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing.

We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209-16,400) and B- (positions 30-284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.

Molecular cytogenetic analysis of a hydatidiform mole with coexistent fetus: a case report.

BACKGROUND: A hydatidiform mole with a coexisting fetus is a rare condition that commonly occurs as either a partial mole with fetus or a twin pregnancy comprising a complete mole and normal fetus. In the former case, the fetus is triploid, and in the latter case, the fetus is diploid with different alleles from those of the mole. Because there is a difference in the persistent trophoblastic disease incidence between the two, an accurate diagnosis is required. CASE PRESENTATION: We present a case of a 34-year-old Japanese woman who was pregnant with a hydatidiform mole and two coexisting fetuses. At 17 weeks of gestation, hemorrhage-induced progressive anemia in the mother prompted the decision to terminate the pregnancy, after which no complications occurred. Molecular cytogenetic analysis revealed that one of the fetuses was a normal diploid fetus with the same allele in the fetus and placenta. The hydatidiform mole was revealed to be a mosaic of two diploids, and the other coexisting fetus was a normal diploid that shared one of the mole alleles. CONCLUSIONS: This was presumed to be a rare case of twin pregnancy by triploid embryo formation, followed by loss of an allele due to postzygotic diploidization, development of a diploid fetus, and development of another fetus from a separate embryo. Because of the existence of cases such as this one with a diploid fetus, but without a normal pregnancy coexistent with a complete hydatidiform mole, diagnosis by genetic analysis is required for prognosis.

Establishment of widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals using molecular techniques.

We established widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals. Twenty-one samples including 17 kinds of crude drug derived from animals were examined. DNA was extracted from most of the crude drugs by adjustment of the QIAamp® DNA Mini Kit. DNA extraction was performed successfully using phenol to remove impurities after applying a proteinase treatment. DNA extraction was performed successfully by decalcification treatment using ethylenediaminetetraacetic acid (EDTA), before applying the proteinase treatment for crude drugs having high calcium content, such as those from oyster shell and cuttlefish bone. DNA could not be extracted from sea-ear shell using the EDTA decalcification treatment, but was extracted successfully using a TBONE EX KIT. The mitochondrial 16S ribosomal RNA (rRNA) gene region was amplified, and Basic Local Alignment Search Tool (BLAST) analysis was performed after sequencing. Polymerase chain reaction (PCR) products of approximately 600 bp in length were obtained from all samples except donkey glue, one of the two seahorses, and longgu. Drug origins were determined in all samples by sequence analysis based on the BLAST results, and match rates were >97 %. Moreover, 16 samples had a match rate >99 %. Our DNA extraction methods were widely applicable to evaluation of many crude drugs derived from animals, and proved very useful for identifying the origins of such drugs.

Analysis of mainland Japanese and Okinawan Japanese populations using the precision ID Ancestry Panel.

We typed 165 AIMs in 49 mainland Japanese and 47 Okinawa Japanese using the Precision ID Ancestry Panel (Thermo Fisher Scientific). None of the 165 SNPs showed significant deviation from Hardy-Weinberg equilibrium in the mainland Japanese. One SNP (rs3943253) showed significant deviation from Hardy-Weinberg equilibrium in Okinawa Japanese. Fisher's exact tests showed that the genotype frequencies of 14 loci were significantly different (p<0.05) between the two populations before correction for multiple testing. After Bonferroni correction, only rs671 remained statistically significant (p<0.0003). This SNP is located in the ALDH2 gene. The mutant A allele is associated with increased side effects after alcohol intake. The frequency of the GG genotype (wild type) was higher in the Okinawa Japanese (78.7%) than in mainland Japanese (34.7%; Bonferroni corrected P<0.001). For 31 (63.3%) of the mainland Japanese and 42 (89.4%) of Okinawa Japanese, the highest population likelihood was obtained with the Japanese reference population. However, only in a few individuals, the likelihoods were significantly different from those calculated using reference data from neighboring populations. The likelihoods for mainland Japanese and Okinawa Japanese were not significantly different from each other for any of the investigated individuals. STRUCTURE and PCA analyses showed that mainland Japanese, Okinawa Japanese, and East Asians could not be differentiated with the Precision ID Ancestry Panel.

A human genotyping trial to estimate the post-feeding time from mosquito blood meals.

Mosquitoes occur almost worldwide, and females of some species feed on blood from humans and other animals to support ovum maturation. In warm and hot seasons, such as the summer in Japan, fed mosquitoes are often observed at crime scenes. The current study attempted to estimate the time that elapsed since feeding from the degree of human DNA digestion in mosquito blood meals and also to identify the individual human sources of the DNA using genotyping in two species of mosquito: Culex pipiens pallens and Aedes albopictus. After stereomicroscopic observation, the extracted DNA samples were quantified using a human DNA quantification and quality control kit and were genotyped for 15 short tandem repeats using a commercial multiplexing kit. It took about 3 days for the complete digestion of a blood meal, and genotyping was possible until 2 days post-feeding. The relative peak heights of the 15 STRs and DNA concentrations were useful for estimating the post-feeding time to approximately half a day between 0 and 2 days. Furthermore, the quantitative ratios derived from STR peak heights and the quality control kit (Q129/Q41, Q305/Q41, and Q305/Q129) were reasonably effective for estimating the approximate post-feeding time after 2-3 days. We suggest that this study may be very useful for estimating the time since a mosquito fed from blood meal DNA, although further refinements are necessary to estimate the times more accurately.

Identification of canine saliva using mRNA-based assay.

A dog saliva analysis in addition to a bite-mark analysis may be important for evidence when a crime involves a dog bite. In this study, the utility of detecting canine saliva-specific mRNAs to identify canine saliva was evaluated. Canine saliva swabs (n = 20), urine swabs (n = 20), body surface swabs (n = 20), whole blood samples (n = 10), human saliva (n = 20), human skin surface swabs (n = 20), and human whole blood (n = 20) were tested. The saliva-specific genes encoding statherin (STATH), carbonic anhydrase VI (CA-VI), and dog allergens (Canf1 and Canf2) were analyzed as candidate genes. Moreover, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as confirmation of canine mRNA extraction. STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs were detected in 19/20, 1/20, 11/20, 4/20, and 20/20 saliva samples, respectively. The STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs did not exhibit cross-reactivity with samples of human origin. This mRNA-based assay was also able to detect canine saliva in mock forensic samples. The results of this study indicated that the detection of STATH mRNA is useful for the identification of canine saliva, and GAPDH is a suitable marker for canine mRNA extraction.

Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA.

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples.

Sudden death of a child from myocardial infarction due to arteritis of the left coronary trunk.

An eight-year-old Japanese boy developed abdominal pain, followed by convulsion and loss of consciousness. He was taken to an emergency room but could not be resuscitated. At autopsy, the left main coronary trunk (LMT) demonstrated an increase in caliber with severe luminal narrowing, and the left anterior descending branch (LAD) subsequent to the LMT showed severe stenosis. Microscopically, the intima of the LMT demonstrated severe fibrosis and infiltration of lymphocytes and histiocytes suggesting vasculitis, and the small lumen was occupied by a fresh thrombus. The LAD showed significant intimal thickening with strong lymphocytic inflammation at the edge of the thickening. The left ventricle showed widespread myocardial infarction in the recovery stage. There were no findings of atherosclerosis, vasculitis or fibrocellular changes in the ascending aorta or intravisceral arteries other than the LMT and the LAD under investigation. The increase in the caliber of the LMT and the limitation of arteritis to the LMT and the subsequent branch suggested Kawasaki disease (KD), but it was atypical that the patient had no clinical history consistent with KD. The present case showed no findings suggesting classical polyarteritis nodosa (cPAN) at the acute or scar stage in the other vessels being investigated, and cPAN in childhood is rare compared to KD. A nonspecific inflammatory reaction (single organ vasculitis, SOV) was also considered as a possible cause, but it is difficult to determine whether the cause of the coronary stenosis in the present case was SOV because the sampling of arteries was insufficient. If forensic pathologists make unusual findings suggesting vasculitis at autopsy, the collection of a sufficient number of vessels of various sizes is warranted.

A novel method for sex determination by detecting the number of X chromosomes.

A novel method for sex determination, based on the detection of the number of X chromosomes, was established. Current methods, based on the detection of the Y chromosome, can directly identify an unknown sample as male, but female gender is determined indirectly, by not detecting the Y chromosome. Thus, a direct determination of female gender is important because the quality (e.g., fragmentation and amelogenin-Y null allele) of the Y chromosome DNA may lead to a false result. Thus, we developed a novel sex determination method by analyzing the number of X chromosomes using a copy number variation (CNV) detection technique (the comparative Ct method). In this study, we designed a primer set using the amelogenin-X gene without the CNV region as the target to determine the X chromosome copy number, to exclude the influence of the CNV region from the comparative Ct value. The number of X chromosomes was determined statistically using the CopyCaller software with real-time PCR. All DNA samples from participants (20 males, 20 females) were evaluated correctly using this method with 1-ng template DNA. A minimum of 0.2-ng template DNA was found to be necessary for accurate sex determination with this method. When using ultraviolet-irradiated template DNA, as mock forensic samples, the sex of the samples could not be determined by short tandem repeat (STR) analysis but was correctly determined using our method. Thus, we successfully developed a method of sex determination based on the number of X chromosomes. Our novel method will be useful in forensic practice for sex determination.

Evaluation of forensic examination of extremely aged seminal stains.

The results of forensic tests, such as semen identification and short tandem repeat (STR) analysis of extremely aged seminal stains from unsolved sex crimes can provide important evidence. In this study we evaluated whether current forensic methods could be applied to seminal stains that were stored at room temperature for 33-56years (n=2, 33years old; n=1, 41years old; n=1, 44years old; n=1, 56years old). The prostatic acid phosphatase (SM-test reagent), microscopic (Baecchi stain method) and semenogelin (RSID™ Semen Laboratory Kit) tests were performed as discriminative tests for semen. In addition, the mRNA levels of the semen-specific proteins semenogelin 1 (SEMG1) and protamine 2 (PRM2) were investigated. STRs were analyzed using the AmpFlSTR® Identifiler™ PCR Amplification Kit. All samples were positive in the prostatic acid phosphatase and semenogelin tests, and sperm heads were identified in all samples. The staining degree of the aged sperm heads was similar to that of fresh sperm. Although SEMG1 mRNA was not detected in any sample, PRM2 mRNA was detected in three samples. In the STR analysis, all loci were detected in the 33-years-old sample and five loci were detected in the 56-years-old sample. We confirmed that current forensic examinations - including STR analysis - could be applied to extremely aged seminal stains. These results could be useful for forensic practice.

Identification of feces by detection of Bacteroides genes.

In forensic science, the identification of feces is very important in a variety of crime investigations. However, no sensitive and simple fecal identification method using molecular biological techniques has been reported. Here, we focused on the fecal bacteria, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron, and developed a novel fecal identification method by detection of the gene sequences specific to these bacteria in various body (feces, blood, saliva, semen, urine, vaginal fluids and skin surfaces) and forensic (anal adhesions) specimens. Bacterial gene detection was performed by real-time PCR using a minor groove binding probe to amplify the RNA polymerase ß-subunit gene of B. uniformis and B. vulgatus, and the α-1-6 mannanase gene of B. thetaiotaomicron. At least one of these bacteria was detected in the feces of 20 donors; the proportions of B. uniformis, B. vulgatus and B. thetaiotaomicron were 95, 85 and 60%, respectively. Bacteroides vulgatus was also detected in one of six vaginal fluid samples, but B. thetaiotaomicron and B. uniformis were not detected in body samples other than feces. Further, we applied this method to forensic specimens from 18 donors. Eighteen anal adhesions also contained at least one of three bacteria; B. uniformis, B. vulgatus and B. thetaiotaomicron were detected in 89, 78 and 56%, respectively, of the specimens. Thus, these bacteria were present at a high frequency in the fecal and forensic specimens, while either B. uniformis or B. vulgatus was detected in all samples. Therefore, B. uniformis and B. vulgatus represent more appropriate target species than B. thetaiotaomicron for the identification of fecal material. If B. vulgatus and/or B. uniformis are detected, it is likely that the sample contains feces. Taken together, our results suggest that the use of molecular biological techniques will aid the detection of feces in forensic practice, although it is possible that the samples contained both feces and vaginal fluid.

Fragment size analysis of free fetal DNA in maternal plasma using Y-STR loci and SRY gene amplification.

Free fetal DNA (ffDNA) in maternal plasma has now become a valuable source for noninvasive prenatal diagnosis. Being able to accurately identify the size of ffDNA in maternal plasma is essential for a noninvasive prenatal diagnosis. Furthermore, it is important to investigate the molecular characteristics related to apoptosis which gives rise to ffDNA. We investigated the fragment size of ffDNA in each sample more precisely, using both Y-STR and SRY primers, in 20 maternal plasma samples from the 17th to 39th weeks of gestation. PCR was conducted with Y-STR and SRY primers which can be used to amplify 100-524 bp fragments. In samples from 10 pregnant women carrying male fetuses, the maximum fragment size detected by Y-STR and SRY primers ranged from 219 to 313 bp. As a result, the mean average maximum fragment size of free fetal DNA detected by Y-STR and SRY primers was 286 +/- 28 bp. The Y-STR alleles detected in each maternal plasma DNA sample were all in agreement with the results of their cord blood samples. We concluded that the fragment size of ffDNA comprises 2 nucleosomal complexes or less, but not exceeding 3.

Gestational choriocarcinoma of Fallopian tube diagnosed with a combination of p57KIP2 immunostaining and short tandem repeat analysis: case report.

Tubal choriocarcinoma is an extremely rare condition and can be of gestational or non-gestational origin. The appropriate management of choriocarcinoma begins with the categorization of the tumor. However, it is difficult to discriminate the two types by routine histological examination. We report the first case of gestational choriocarcinoma of the Fallopian tube to be confirmed by a combination of p57(KIP2) immunostaining and DNA polymorphism analysis at 15 short tandem repeat loci, along with X and Y chromosome markers. The patient had no detectable metastasis or evidence of recurrence 15 months after treatment, which involved surgery without adjuvant chemotherapy. This case demonstrates the usefulness of a combination of p57(KIP2) immunostaining and DNA polymorphism analysis in determining the origin of extrauterine choriocarcinoma (i.e. gestational or non-gestational), which helps to determine the strategy for treatment of the disease.