doi:10.3181/0802-RM-35
© 2008 Society for Experimental Biology and Medicine
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Josep-Maria Pujal1 and
David Gallardo
Translational Research Laboratory, Institut Català d’Oncologia, Hospital Duran i Reynals, L’Hospitalet de Llobregat, Barcelona, Spain
1 Translational Research Laboratory, Institut Català d’Oncologia, Hospital Duran i Reynals, Avda Gran Via s/n, Km 2.7, 08907 L’Hospitalet de Llobregat, Barcelona, Spain. E-mail: jmpujal{at}idibell.org
Abstract |
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TOP Abstract Introduction Methods Results Conclusions References |
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Peripheral blood microchimerism after pregnancy or solid organ transplantation has been widely studied, but a consensus on its detection has not yet been adopted. The objective of this study was to establish a panel of reproducible molecular polymerase chain reaction (PCR)–based methods for detection and quantification of foreign cells in an individual. We analyzed length polymorphisms generated by short tandem repeat (STR) and variable number tandem repeat (VNTR) markers. Human leukocyte antigen (HLA)-A and -B polymorphisms were detected by reference strand conformation analysis (RSCA). Class II polymorphisms on HLA-DRB1 locus were analyzed both by classical PCR–sequence-specific primers (SSP) and by quantitative PCR (Q-PCR). Also, sex-determining region-y gene (SRY) gene allowed specific male donor discrimination and quantification by Q-PCR in female recipients. Binomial statistical distribution analysis was used for each molecular technique to determine the number of PCR replicates of each sample. This analysis allowed the detection of the lowest detectable microchimerism level, when present. We could detect microchimerism in more than 96% and more than 86% of cases at levels as low as 1:105 and 1:106 donor per recipient cells (DPRC), respectively, using Q-PCR for SRY or for nonshared HLA-DRB1 alleles. These techniques allowed as low as 1 genome-equivalent cell detection. Lower levels (nanochimerism) could be detected but not quantified because of technique limitations. However, classical PCR methods allowed detection down to 1:104 DPRC for HLA-DRB1 PCR-SSP. The clinical application of these techniques in solid organ transplanted recipients showed microchimerism levels ranging from 1:104 to 1:106 DPRC after kidney or heart transplantation, and 1 log higher (1:103 to 1:106 DPRC) after liver transplantation. Inconclusion, the standardization of molecular microchimerismdetection techniques will allow for comparable interpretationof results in microchimerism detection for diagnostic or researchstudies.
Keywords: microchimerism, real-time PCR, detection, quantification
Introduction |
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TOP Abstract Introduction Methods Results Conclusions References |
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Hematopoietic chimerism can be defined as the presence of atleast two different DNA sources in a same individual. It canbe identified following natural or artificial cell transferduring of after pregnancy in the mother but also in the fetus,after blood transfusion, after stem cell transplantation, oreven after solid organ transplantation.
Molecular chimerism monitoring after stem cell transplantationis a useful tool. It is routinely performed in laboratoriesto detect disease relapse after hematopoietic stem cell transplantation.In these cases, it can also be referred to as macrochimerismquantification because donor cell amounts range from 1% to 100%of the total peripheral blood cells of the recipient. From thisviewpoint, microchimerism can be defined as less than 1% offoreign cells present in an individual. Presence of microchimerismcan be a result of cell transfer from the fetus or the motherbefore and during delivery, after solid organ transplants, oreven after blood transfusions.
Microchimerism detection is used for precocious detection of SRY DNA in the peripheral blood of the mother during pregnancy, which allows for the sex of the fetus to be determined (1–3) and, moreover, demonstrates that microchimerism detection can be used as a noninvasive tool for prenatal diagnosis (4, 5).
It has been hypothesised that fetal or mother-engrafted stem cells could be involved either in autoimmunity or in tolerance to fetal or maternal derived tissues, respectively (6–12) or even in organ repair (13). Solid organ transplants may also benefit from microchimerism detection, as demonstrated by some authors who have shown that the presence of donor circulating cells after transplantation is directly correlated to a significantly lower incidence of acute rejection (14–17) or even leads to tolerance (18). Many studies based on microchimerism detection after solid organ transplantation have been published. However, they have led to contradictory results because many different detection techniques and different control time points have been used to detect donor cells (19–21). So far, consensus has not been established, but various strategies have been suggested by numerous authors (22–34). Therefore, to have a potentialclinical impact based on precocious diagnostics, it is fundamentalto arrive at a concordance about molecular microchimerism detectiontechniques to assure comparability of results.
Compliance of at least four essential factors must be appliedfor the detection of this minority of donor circulating cellswithin the blood of the recipient. The first factor is to previouslydetermine and characterize DNA molecular disparities betweenthe donor and the recipient to recognize donor discrimination.The second factor is the specific detection of this donor polymorphismwithout false positives. The third factor is to validate thesensitivity of the improved technique, allowing the specificdetection of as small amounts of donor cells as possible. Andthe last essential factor is to validate the reproducibilityof the technique.
Here we enumerate various molecular polymerase chain reaction(PCR)-based techniques and their validation for specificity,sensitivity, reproducibility, and feasibility for microchimerismdetection. Also, to validate our results in a clinical setting,we have determined and quantified microchimerism presence insolid organ transplanted recipients.
Methods |
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TOP Abstract Introduction Methods Results Conclusions References |
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Artificial Mixed Microchimerism Synthesis.
To allow determination of efficiency, sensitivity, and specificity,we used different sources of control DNA receiving either adonor or a recipient label. Mixed chimeras were prepared bymixing 10-fold serial dilutions of donor-labeled DNA that rangedfrom 500 ng to 0.5 pg in a constant amount of recipient-labeledDNA of 200 to 500 ng.
Patients.
To assure the validity of all the techniques used, 20 healthy control subjects were enrolled to create a battery of artificially controlled microchimeras. Also, to validate results in a clinical setting, 119 transplanted recipients (single kidney [n = 84], heart [n = 27], or liver [n = 8]) were included for longitudinal microchimerism detection at 0, 2 (M2), 6 (M6), 12 (M12), and 18 (M18) months, forming the prospective cohort. The time 0 analysis corresponded to blood extraction before transplantation to examine the presence of pre-transplant microchimerism (from any source) and to allow comparison with M2 to M18 analysis. Also, 33 transplanted recipients (single kidney [n = 15], double kidney [n = 10], or liver [n = 8]) were included for only onesingle transversal microchimerism determination, forming theretrospective cohort. All recipients received cadaver donororgans and were transplanted at the Hospital Universitari deBellvitge (Barcelona, Spain) between March 2001 and January2006.
Blood Samples and DNA Extraction for Microchimerism Study.
Peripheral blood samples were collected in K3EDTA and analyzedin three different fractions: whole peripheral blood (PB), mononuclearcells (MNC), and granulocytic cells (GC). The separation ofthese last two fractions allowed an increase in donor-cell detectionsensitivity. All MNC and GC fractions were isolated by Ficoll–Hypaque(lymphocyte isolation solution; Beckman Coulter, Germany) gradientdensity separation. Genomic DNA was extracted from each fractionusing the QIAamp Blood Kit (Qiagen, Hilden, Germany), followingthe manufacturer’s recommendations.
Theoretical Number of PCR Replicates.
Not all PCR replicates were found to be positive for positivecontrol DNA at extreme dilutions, thus revealing the initialrate of PCR success for this extreme dilution. We found thatspecific detection of this DNA was statistically measurableby the probability of a positive PCR event in all PCR performedfor the same positive control sample. To determine the numberof PCR replicates that would be needed to find at least onesingle positive PCR event using a positive control sample, thebinomial distribution probability test was applied.
This statistical test was used to determine the theoreticalnumber of PCR replicates to be achieved (with an acceptablestatistical probability of detection) for a specific initialrate of PCR success on a potential positive sample.
Short Tandem Repeat (STR) Analysis.
A panel of six Cy5-labeled STR markers (TPOX, HUMH01, CSFP01,LPL, FVW, and F13A) was used for microchimerism detection. Aprevious informative marker screening step was required foreach donor-recipient pair using multiplexed PCR for TPOX-HUMTH01-CSFP01and also for LPL-FVW-F13A. All PCR primers and annealing conditionsare described in Table 1. PCR was carried out in a PTC-100 thermocycler(MJ Research, Inc., Reno, NV). PCR products were run in a 6%denaturing polyacrylamide gel using AlfExpress II (AmershamPharmacia, Uppsala, Sweden) automated DNA sequencer and analyzedwith AlfWin Fragment Analyzer software (Amersham Pharmacia).
Variable Number of Tandem Repeat (VNTR) Analysis.
A panel of six Cy5-labeled VNTR markers (Apo-B, D1S80, D4S20,D16S83, D17S30, and D19S20 as described in Table 1) was alsoused for microchimerism detection with a previous informativescreening step, but for this analysis, multiplexed PCR reactioncould not be achieved because of loss of efficiency. PCR productswere run in a 12% nondenaturing polyacrylamide gel using thesame automated DNA sequencer described above.
Human Leukocyte Antigen (HLA) Class I and Class II Polymorphism Analysis.
HLA-A and -B typing and donor detection were performed by reference strand conformation analysis (RSCA) (35). Class II typing for HLA-DRB1 and microchimerism-specific detection were both performed using Olerup primers (Table 1) by sequence-specific primer (SSP)-PCR (36). All HLA disparities were graded from 0 to 6 (out of 6),showing full match to full mismatch, respectively. Like VNTRanalysis, all Cy5-labeled PCR products resulting from HLA studywere analyzed in AlfExpress II automated sequencer.
For real-time HLA-DRB1 SSP-quantitative PCR (Q-PCR), the sameprimers were adopted (without Cy5-added modification). All Q-PCRwere performed in a LightCycler v2.0 (Roche, Germany) real-timethermocycler, and results were analyzed with the LC Softwarev4.0 (Roche, Germany), allowing the use of nonlinear inflexion-reproduciblestandard curve points at extreme dilution points.
Sex Mismatch Analysis.
SRY male specific primers (37) (Table 1) were used for microchimerismdetermination in a transplanted female receiving an organ froma male. To assure the absence of previous microchimerism interference,an initial pre-transplant analysis step of women was required.Gender-specific PCR amplification and analysis were carriedout by real-time PCR as described above.
Results |
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TOP Abstract Introduction Methods Results Conclusions References |
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Adopted Number of PCR Repetitions for Each Sample.
Binomial statistical distribution analysis applied to each moleculartechnique helped to determine the number of PCR replicates ofeach sample for the detection of lowest detectable microchimerismlevel, when present, within the maximum number of recipients(Fig. 1).
To explain the utility of this test, we here describe an example encountered when amplifying the SRY gene by Q-PCR. In artificially controlled chimera testing, the minimum amount of “donor” DNA detected was one genome-equivalent cell (GEC) (= 6.6 pg) in a PCR reaction containing 105 GEC (= 660 pg) of recipient-labeled DNA. Thus, the low-down detection limit was 1:105 donor perrecipient cells (DPRC). In this test, we observed that 50% ofthe PCR reactions gave positive results; consequently, the initialrate of PCR success was 1/2. We then wondered how many PCR replicatesof the same sample were needed to assure that at least one controlPCR gave a positive amplification when microchimerism was present.This test allowed a choice of the number of PCR replicates,which is a function of the final probability of truthfulnessof the test. In other words, when choosing five as the numberof replicates, it is expected that at least one will turn outto be a positive PCR reaction of the five replicate reactionsin more than 96% of cases (dotted lines in Fig. 1).
After all, this example shows that the detection of SRY by Q-PCR allowed the detection down to 1:105 DPRC (0.001% of microchimerism) in more than 96% (p(Positive) = 0.96) of recipients, althoughit required up to five replicates of the same sample.
By means of the same SRY test, we were able to detect 1:106 DPRC when achieving a mixed chimera containing 0.6 pg and 660 pg of donor and recipient DNA, respectively. With an initial rate of PCR success of 1/3 and five as the chosen number of replicates, the veracity of the test descended to 86%. Consequently, 1:106 DPRC could be detected in 86% of recipients with fivePCR replicates using SRY specific and quantitative detection.
STR and VNTR Polymorphisms.
We managed to reach donor-specific detection levels of 0.1%to 0.05 %. This sensitivity varied depending on the marker used.Higher informativity rates were reached for HumtH01, D1S80,D17S30, Fvw, D4S95, CSFP01, and Apo-B markers, with an informativityrate of 89.6%, 76.4%, 76.0%, 72.6%, 72.4%, 68.9%, and 60.4%,respectively (marker informativity may vary, depending on theexamined population). Also, acceptable informativity rates of52.0% and 51.7% were reached for F13A and TPOX, respectively.
Semiquantification of donor alleles was achieved in some casesby adding the area of the resulting donor and recipient fluorescencepeaks and calculating the percentage of donor peaks.
Because VNTR contained longer repeated sequences, differencesin length were more important than those obtained with STR markers,therefore leading to the phenomenon of preferential amplificationof smaller alleles, which could either interfere or enhancedonor-specific detection. Shorter alleles were preferentiallyamplified compared with longer alleles because they have highernumber of repetitions, thus leading to great differences inthe resulting amplicon amount within the same number of PCRcycles and the same polymerase concentration limiting factor.If at least one donor allele contained fewer repetitions thanboth recipient alleles, PCR reaction enhanced donor-specificamplification in mixed chimeras. Inversely, if one or both recipientalleles contained fewer repetitions, the amplification of theultra minority of donor alleles was interfered by smaller allelepreferential amplification artefact. Donor-estimated quantificationcould be achieved in the enhanced cases by adding peaks of allareas and calculating the percentage of the donor peaks.
HLA-A and -B Nonshared Alleles.
RSCA technique was performed for HLA-A and -B loci typing. As previously described (38, 39), we found that this techniquewas also useful for HLA matching between individuals, withoutthe need of using a molecular typing ladder, but required runningthe two pre-transplant samples (donor and recipient) along withthe replicates of the tested sample of the (posttransplant)recipient.
The sensitivity of this technique (1%) was not sufficient formicrochimerism detection; however, it was useful for macrochimerismdetection in HLA class I mismatched bone marrow transplants.
Amplification of HLA-DRB1 Mismatched Samples with Classic PCR Methods.
The study of HLA-DRB1 polymorphisms required a previous typing step for donor and recipient in order to find which nonshared DRB1 polymorphism could differentiate donor from recipient. Olerup primers (36) allowed the differentiation of with allelesor groups of alleles (01, 03-11-13–14, 04, 07, 09, 10,08–12, 15–16). All SSP-PCR were realized along withthe addition of human growth hormone (HGH) gene primer set atlimiting concentration, allowing the visualization of the positivecontrol reaction (HGH primers used were 5′-gcc ttc cca acc attccc tta -3′ and 5′-tca cgg att tct gtt gtg ttt c -3′ [429 bpfragment] as direct and reverse primers, respectively).
Direct SSP-PCR and polyacrylamide electrophoresis allowed specificdetection down to 40 pg of donor DNA in a mixed chimera with100 ng of recipient DNA (Fig. 2A and 2B).
Nested PCR allowed the detection of 4 pg of donor DNA. Triplicatesof tested samples and controls eliminated false positives andallowed nested PCR validation, but easy contaminations and additionof multiple controls made the simple nonnested SSP-PCR preferable.
An assay of seminested PCR with three primers (5′ common DR,SSP and common reverse primer) was realized, but sensitivitydid not reach higher microchimerism detection (only 0.5%). Theamplification of common DR fragment served as PCR internal positivecontrol fragment and allowed DRB1 background amplification (similarlyas nested), thus SSP primers could found more donor templateamount to amplify (Fig. 2C).
Quantification of SRY in Gender Mismatched Transplants.
Sex-determining region-y gene (SRY) determination was efficient only in females receiving a male organ and no previous typing was necessary for macrochimerism detection. Only male and female DNA controls were required to assure nonfalse results. We reached quantification down to 5 pg of donor (male) DNA in a mixed chimera with recipient (female) DNA, allowing the detection of one genome equivalent cell (GEC) within a total of 105 GEC (Fig. 3). Becausethis technique allowed quantification down to one single celllevel, we paid special attention to quantification and normalizationwith a control gene.
Any part of conserved DNA could allow normalization by detectionof one or both alleles contained in all the cells, but we choseprimers amplifying the beta-2-microglobulin (B2m) gene. As allthe cells contained the B2m gene, both donor and recipient DNAwere amplified, and thus quantified. Standard curves were createdamong DNA dilutions of donor, recipient, and a mixed chimeraof the donor-recipient couple.
Using a male control, we analyzed the correlation between SRY and B2m with the same set of positive samples and we found 2.04 (±0.6) [SRY] = [B2m]. Thus, the correction factor ( =2.04 ± 0.6) for SRY detection could be applied to allthe samples.
We analyzed inter-PCR variations and the overall standard deviationof all PCR internal controls used reached 0.82% (mean crossingpoint (Cp) = 20.80 ± 0.17). Also, to reduce intra-PCRvariation, the resulting mean Cp of each replicated sample wasused for calculation of the detected SRY amount (the same procedurewas used with the control gene B2m), consequently, the proportionof SRY within the total of cells (B2m amount) could be calculatedfollowing the formula:
(The B2m amount corresponding to male cells fraction was usuallyinsignificant compared with the B2m amount of the recipientcell fraction.)
Previous microchimerism detection in pre-transplant samples was necessary to keep in mind the possible posttransplant false positive (fetal microchimerism, blood transfusion, previous transplantation). For example, we were able to detect fetal microchimerism in a female (carrying a boy) at levels lower than 1:106 DPRC, but posttransplant (kidney transplant) study gave one SRY-log more (addition of SRY amounts to first detection) because of donor microchimerism, at a quantifiable resulting amount higher than 1:105 DPRC (data not shown).
Although lower levels of microchimerism could be detected, nostatistically reproducibility could be achieved, and the numberof replicates was too high to validate it in a clinical setting.Thus, this nondetected microchimerism could be named nanochimerismand was usually undetected by actual molecular techniques describedhere.
Quantification of HLA-DRB1 Mismatched Alleles.
Real-time quantification of donor HLA-DRB1 non-shared alleleswas possible with a previous typing of each donor-recipientcouple, using the standard method described here before. Thequantification method used was very similar to that used forSRY quantification, only nonshared alleles were specificallyamplified (SSP-PCR). Normalization was also realized with acontrol gene (B2m) for each DRB1 allele. The sensitivity ofthe detection varied depending on the amplified allele and the”recipient” allele background. These results are resumed inTable 2.
Validation of Techniques in a Clinical Setting: Microchimerism Detection in Solid Organ Transplanted Recipients.
We first determined the number of DPRC in kidney, heart, andliver transplanted recipients 2 months after transplantationin the prospective group using the SRY or HLA-DRB1 techniquesdescribed above and summarized in Table 3. All microchimerismdeterminations in those recipients demonstrate that almost thehalf of recipients showed donor circulating cells in their peripheralblood within the first 60 days after transplantation (incidencesof microchimerism-positive recipients are shown in Table 4).Heart and kidney transplanted recipients displayed similar microchimerismincidences (51.9% and 55.3%, respectively), whereas almost allliver transplanted recipients (87.5%) showed microchimerism.
We also analyzed a retrospective cohort of double kidney, singlekidney, and liver transplanted recipients in a transversal analysis(also shown in Table 4) at no specific time point. This demonstratedthat larger organs such as the liver showed higher microchimerismlevels than smaller organs such as the kidney.
Therefore, we could detect and quantify 5.4:105 DPRC (range 5.0:104 to 1.2:106), 1.6:104 DPRC (range 4.9:104 to 1.2:106), 1.3:105 DPRC (range 2.6:105 to 2.4:106), and 3.1:104 DPRC (range 1.5:103 to 1.6:106) in single kidney, double kidney, heart,or liver recipients, respectively.
Also, the latest microchimerism-positive detection could be achieved in a retrospective double-kidney transplanted woman at day 2665 after transplantation (3.8:105 DPRC), but no microchimerismdetermination could be achieved previous to transplantation(data not shown).
Conclusions |
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TOP Abstract Introduction Methods Results Conclusions References |
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Initially based on polymorphism detection used in forensic medicinefor cadaveric identification or paternity matching, hematopoieticchimerism detection of those polymorphisms became a useful toolin the transplantation and the autoimmunity fields.
In our study, and according to previously published data (38, 40), we observed that detection of donor-specific HLA-A and-B polymorphisms was suitable for macro-chimerism detectionin class I mismatched bone marrow transplants using RSCA technique.Also, this approach can be used with no previous HLA-typingstep because RSCA allowed direct matching and, thus, directdonor semi-quantification. In bone marrow class II HLA-DRB1mismatched transplants, classical PCR-SSP with a previous typingstep can be used for this purpose. Also, satellite length polymorphisms(extensive lists of STR or VNTR markers are now available) canbe used for macrochimerism detection, but prior informativeloci must be determined for each donor-recipient couple. Becausemacrochimerism is defined by the presence of 1% to 100% of foreigncells in a recipient, intrinsic technique variations up to 1%in RSCA, VNTR, or STR techniques did not result in statisticallysignificant variations. Moreover, detection of donor-cell engraftmentor relapse after bone marrow transplantation need to be time-monitoredand compared with previous results and clinical diagnosis.
We agree that microchimerism detection needed more accuratetechniques with lower intrinsic variations. In this way, STRor VNTR markers were suitable for micro-chimerism qualitativeor semiquantitative detection. Our results suggest that microchimerismquantification can be performed using HLA-DRB1 nonshared allelesusing classical SSP-PCR with polyacrylamide fluorescence detectionor quantitative SSP-PCR real-time quantification. We also arguethat it can be applied to mismatched HLA class I (A, B, or C)transplants. Also, male gender discrimination by quantitativereal-time PCR (SRY) allowed high sensitivity. Thus, we suggestthat HLA-DRB1 (classical and real-time PCR) and SRY (real-timePCR) techniques, showing strong reproducibility and sensitivity,could be used for microchimerism detection because both techniquescan cover the majority of transplant cases.
With concern to DPRC detected in recipients after solid organ transplantation, we can observe that smaller mass organs like kidney or heart displayed less DPRC than liver. This phenomenon can also be caused by additional properties of liver like continuous cell turnover (41–44). Moreover, liver can shelter a higheramount of cells during donor brain-death associated stress,which can be later returned to circulation. We have also observedin kidney and heart that posttransplant microchimerism is atransient phenomenon that trends to disappear within the firstyear.
In this study, we have performed a density-gradient separation mediated by Ficoll to separate mononuclear cells from granulocytes. It is possible that many cells migrating directly from the organ and different from lymphocytes, macrophages or granulocytes can be detected by PCR as donor cells. No flow cytometry was performed to identify which cells originate microchimerism. Moreover, microchimerism can also be a result of DNA contained in recipient cells or from apoptotic bodies as many authors have already published (45). Also, in unpublished data, we haveperformed a PCR directly from plasma to detect free-donor DNAin serum, but we did not obtain any positive result, leadingus to the conclusion that microchimerism was originated by circulatingcells or by DNA contained in recipient cells.
As we previously published (46), we believe that to includemolecular detection of microchimerism as a routine protocolin solid organ transplanted recipients can provide undeniableadditional data, involving clinical decisions, ultimately concerningthe fate of the transplanted organ.
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Acknowledgments |
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The authors thank J. M. Grinyó, N. Manito, J. Fabregat,and E. Ramos for their clinical assessment and P. Hernàndez,J. Klaustermeier, A. Villanueva, C. Maxwell, and G. Capellàfor their helpful contributions.
Footnotes |
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This study was partially financed by a Fundació MaratóTV3 2000 grant. The author (JMP) was the recipient of a grantFundació Crèdit Andorrà 2002–2005.
Received for publication February 1, 2008.
Accepted for publication April 17, 2008.
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