Evaluation of seven PCR-based assays for the analysis of microchimerism

doi:10.1016/S0009-9120(98)00059-9

Clinical Biochemistry

Volume 31, Issue 8, November 1998, Pages 641-645

https://doi.org/10.1016/S0009-9120(98)00059-9 Get rights and content

Abstract

Objective: The presence of small numbers of cells of donor origin in the circulation of recipients of organ transplants (microchimerism) may correlate with immunologic tolerance. As part of our ongoing studies on microchimerism, we evaluated the utility of seven PCR-based assays for the detection of the less abundant DNA in paired mixtures (100 ng total DNA).

Design and methods: DNA samples were screened to identify pairs informative for one or more PCR assays. DNA mixtures from the informative pairs were then analyzed using at least one assay. The assays were based on the X-Y homologous region; a Y chromosome microsatellite locus; three autosomal microsatellite loci; the D1S80 minisatellite locus; and sequence specific oligonucleotide probe (SSOP) analysis of the HLA DRB1 locus.

Results: About 0.1% of male DNA against a background of female DNA was detectable using primers for the X-Y homologous region, but the sensitivity was increased to 0.0001% using nested primers for the Y chromosome microsatellite marker. Analysis of the minor DNA component was difficult with the three autosomal microsatellite assays because of the presence of shadow bands. Similar problems with the D1S80 assay were resolved using more stringent PCR conditions, and the sensitivity was 0.1%. Using the DRB1 locus, we were able to detect 1% DNA in the mixed samples.

Conclusions: These studies show that: (a) nested PCR for the Y chromosome is the most sensitive assay for the detection of microchimerism; (b) D1S80 is a useful marker for microchimerism; (c) additional optimization of analytical conditions is required if autosomal microsatellite markers and the SSOP assay are to be used for microchimerism analysis.

Section snippets

Introduction the ultimate goal in organ transplantation is to promote the development of immunologic tolerance, so that the need for long-term immunosuppression is reduced or eliminated. although several potent immunosuppressive agents have been developed, their use has led to a number of side effects and complications (1). the identification of strategies for the induction of tolerance in clinical transplantation is, therefore, highly desirable. a variety of procedures, including blood or bone marrow infusions, have been used to induce tolerance to allografts (2). however, the success of these procedures has been limited, and the mechanisms for their mode of action is a subject of much debate (3). it has been proposed that the exchange of migratory leukocytes between the transplanted organ and the recipient (microchimerism) leads to the development of long-term tolerance, and that this may allow for the withdrawal or reduction of immunosuppression (4)

A variety of PCR-based techniques have been used for the detection of donor-type cells or DNA in the peripheral circulation of organ transplant recipients 5, 6, 7, 8, 9, 10, 11. However, the reliable detection and quantitation of minute amounts of donor DNA against a large background of recipient DNA has been difficult. Additionally, there is a need for a battery of well-characterized assays for the detection of donor DNA, because one assay may not be sufficient or informative for a given

Sample preparation

DNA was isolated from peripheral blood samples from laboratory personnel (male and female) using an inorganic procedure (18). The DNA samples were quantitated by agarose gel electrophoresis and ethidium bromide staining, using known concentrations of HinDIII-digested lambda DNA as a standard. The DNA samples were screened using one or more PCR assays to identify informative pairs. DNA mixtures from the informative pairs were then prepared such that the percentage of one DNA ranged from 0 to 10

Analysis of Y chromosome DNA

Using the X-Y homologous primer pair, approximately 1% of male DNA against a background of female DNA was readily detectable by agarose gel electrophoresis and ethidium bromide staining of the PCR product. The detection sensitivity was increased to approximately 0.1% when [³²Pα-dCTP] was included in the PCR reaction mixture (data not shown). With the nested PCR assay for the Y chromosome microsatellite marker, and by including radioactive dCTP in the reaction mixture, we were able to readily

Discussion

The hypothesis that the presence of small numbers of donor cells in organ transplant recipients may be associated with immunologic tolerance has led investigators to actively induce microchimerism in transplant recipients by, for example, injection of bone marrow cells of donor origin (2). However, several investigators have failed to detect microchimerism in short-term or long-term recipients of organ transplants 19, 20. This may in part be due to technical difficulties in detecting minute

Acknowledgements

This work was supported in part by the Showalter Trust, University Surgical Associates, and Fujisawa, Inc. (USA).

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Organ-specific patterns of donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism in lung, kidney, and liver transplant recipients

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Cited by (19)

Microchimerism in Peripheral Blood and Urine in Renal Transplant Recipients: Preliminary Results
2008, Transplantation Proceedings
The role of microchimerism in peripheral blood and urine of renal transplant recipients remains a matter of debate, depending on the sensitivity of the methods used for detection. We studied 17 female renal transplant recipients who had received renal allografts from male donors. Polymerase chain reaction (PCR) was applied to blood and urine for the microsatellite markers D1S80, DYZ1, TH01, and kαi SE33. Detection of DYZ1 that is present only on the Y chromosome was considered proof for microchimerism. No microchimerism was detected in peripheral blood, whereas it could be detected in the urine of 8/17 (48%) patients. There were no differences between patients with and without microchimerism regarding patient age, dialysis vintage, immunosuppression, time post-transplantation, and allograft function as measured using serum creatinine, creatinine clearance, and proteinuria. Two patients in each group showed chronic allograft dysfunction. These findings raise questions regarding the role of microchimerism in renal transplantation.
Microchimerism Evaluation in Recipients of Living-Related or Unrelated Deceased Allograft Renal Transplants
2006, Transplantation Proceedings
Citation Excerpt :
We may hypothesize that kidney-resident stem cells did not survive owing to inadequate temperature storage or time-related apoptosis caused by the long ischemia time (from 23–40 hours). Another explanation may be related to the possibility that microchimerism was underestimated due to microchimerism waves, that is, detected in the nadir of the curve.10 Further larger studies evaluating two or more consecutive samples may be required to provide an answer to these hypotheses.
The presence of microchimerism in the peripheral blood of solid organ graft recipients has been associated with long-term solid organ acceptance, immunologic tolerance, and less aggressive immunosuppressive therapy. Molecular biology assays are among the most sensitive methods to detect microchimerism, primarily to evaluate Y chromosome sequences in females as indirect evidence of circulating male nucleated donor cells. We screened for the presence of the SRY sequence region in peripheral blood of 13 female recipients of male kidney grafts: 5 living-related and 8 deceased grafts.² Only patients who received grafts from related living donors exhibited microchimerism. Five of 13 patients studied exhibited better graft outcomes, including the 4 who were positive for the SRY sequences.
Short tandem repeat (STRs) and sex specific Amelogenin analysis of blood samples from neurosurgical female transfused patients
2005, Journal of Clinical Forensic Medicine
Blood transfusion and its effects on DNA profiles of an individual is an important issue in the forensic context. In the present study, the effects of unfiltered whole male blood transfusion in seven female subjects were investigated by PCR-based assays on serial post-transfusion blood samples. PCR analysis of specific short tandem repeat (STR) primer pair to CSF1PO, TPOX, TH01, F13A01, FESFPS, vWA and sex specific Amelogenin loci for banding pattern of alleles generated from the pre- and post-transfusion of seven female recipients and their corresponding donors were studied. Pre- and post-transfusion DNA profiles of all the seven female recipients were found to be consistent with no evidence of the male and female donor genetic material. It is concluded that currently used PCR-based STRs and sex specific Amelogenin loci DNA profiling techniques in forensic science are reliable and informative for paternity and identity purposes in situations of transfusion of 1 or 2 U of unfiltered whole male and female blood (>72 h old) to female recipient up to 24 h post-transfusion.
Quantitative polymerase chain reaction-based assay with fluorogenic Y-chromosome specific probes to measure bone marrow chimerism in mice
2002, Journal of Immunological Methods
A quantitative duplex polymerase chain reaction (PCR) method using the LightCycler™ detection system was developed to generate a reproducible and convenient method to quantitatively measure chimeric states in murine transplantational models using male and female BALB/c mice. Materials and methods: DNA mixtures isolated from male and female murine BALB/c bone marrow cells were analyzed with Y-chromosome and control autosomal GAPDH specific primers by either monoplex or duplex real-time quantitative PCR using the LightCycler™ detection system to determine chimeric states. Results: High specific Y-chromosome and control autosomal GAPDH primers gave a detection sensitivity of approximately 0.01% using LightCycler™ PCR. Analysis of standard curve distribution of diluted DNA samples accurately matched the proportional dilutions. Y6 primers run on male DNA samples against a female background showed amplification slope initiations reflecting the amounts of male DNA contained in these mixtures. Mixed samples run with GAPDH primers showed amplification corresponding to 100% DNA amounts of limiting dilution runs which were used as controls. Calculated engraftment generated by monoplex PCR lacked reproducibility. Quantitation using duplex PCR using specific detection probes for both Y-chromosome Y6 and autosomal GAPDH primer amplicons allowed precise and reproducible calculations of engraftment levels. Conclusion: Duplex PCR using specific detection probes for Y-chromosome Y6 and autosomal GAPDH primer amplicons allows for rapid, precise and specific engraftment determination after transplantation of male BALB/c marrow cells into female BALB/c recipients.
Survival of transfused donor white blood cells in HIV-infected recipients
2001, Blood
Citation Excerpt :
Regardless of the assay used, the detection of rare populations of cells in blood remains challenging. Assays range in sensitivity from 0.1% male DNA in a female DNA background using single primer pairs for the Y chromosome to 0.0001% with nested primers.35 Different primers may lead to different amplification efficiencies, and inadvertent mispriming of associated material, such as pseudogenes in the recipient, has led to false-positive results because of misinterpretation of the bands as of donor origin.36
The appearance and expansion of donor white blood cells in a recipient after transfusion has many potential biologic ramifications. Although patients with HIV infection are ostensibly at high risk for microchimerism, transfusion-associated graft-versus-host disease (TA-GVHD) is rare. The purpose of this study was to search for sustained microchimerism in such patients. Blood samples were collected from 93 HIV-infected women (a subset from the Viral Activation Transfusion Study, an NHLBI multicenter randomized trial comparing leukoreduced versus unmodified red blood cell [RBC] transfusions) before and after transfusions from male donors. Donor lymphocytes were detected in posttransfusion specimens using a quantitative Y-chromosome–specific polymerase chain reaction (PCR) assay, and donor-specific human leukocyte antigen (HLA) alleles were identified with allele-specific PCR primers and probes. Five of 47 subjects randomized to receive nonleukoreduced RBCs had detectable male lymphocytes 1 to 2 weeks after transfusion, but no subject had detectable male cells more than 4 weeks after a transfusion. In 4 subjects studied, donor-specific HLA haplotypes were detected in posttransfusion specimens, consistent with one or more donors' cells. None of 46 subjects randomized to receive leukoreduced RBCs had detectable male lymphocytes in the month after transfusion. Development of sustained microchimerism after transfusion in HIV-infected patients is rare; HIV-infected patients do not appear to be at risk for TA-GVHD.
Fetal microchimerism in primary biliary cirrhosis
2000, Journal of Hepatology
Background/Aims: Recent studies have suggested a role of fetal microchimerism in the pathogenesis of scleroderma. The present study investigated the potential role of fetal microchimerism in primary biliary cirrhosis (PBC), a closely related disease.
Methods: A quantitative nested polymerase chain reaction was used to detect Y-chromosome sequences in the peripheral blood or the liver of PBC women and controls having male children and no transfusion or miscarriage history.
Results: Male microchimerism was found in the peripheral blood from 45% (9 of 20) of PBC women and 25% (5 of 20) of healthy controls matched for the number of male children and age of the youngest son (p=0.28), and in the liver-biopsy specimens from 33% (5 of 15) of PBC women and 32% (8 of 25) of controls. The level of chimerism did not differ between patients and controls either in blood or in liver. Microchimerism was not related to the severity of the disease but was more frequent in PBC patients with anticentromere antibodies (p=0.049).
Conclusions: Fetal microchimerism does not seem to play a major role in most cases of PBC. However, the association with anticentromere antibodies suggests a possible role in the subgroup of patients with CREST syndrome or scleroderma.

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AnalyticalEvaluation of seven PCR-based assays for the analysis of microchimerism

Abstract

Section snippets

Sample preparation

Analysis of Y chromosome DNA

Discussion

Acknowledgements

Lancet

Lancet

Blood

Lancet

Lancet

Posttransplant diabetes mallitus—a review

Transplantation

Cell migration and chimerism after whole organ transplantationthe basis of graft acceptance

Hepatology

The role of cell migration and microchimerism in the induction of tolerance after solid organ transplantation

Postgrad Med J

Identification of donor-derived cells in organ transplants by in vitro amplification of Y-chromosome gene using polymerase chain reaction

Cell Transplant

Peripheral blood chimerism in renal allograft recipients transfused with donor bone marrow

Transplantation

Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant patient

Transplantation

Organ-specific patterns of donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism in lung, kidney, and liver transplant recipients

Transplantation

Analytical
Evaluation of seven PCR-based assays for the analysis of microchimerism