Anti-HLA alloantibodies of the IgA isotype in re-transplant candidates part II: Correlation with graft survival.

We reported previously on the widespread occurrence of anti-HLA alloantibodies of the IgA isotype (anti-HLA IgA) in the sera of solid-organ re-transplantation (re-tx) candidates (Arnold et al., ). Specifically focussing on kidney re-tx patients, we now extended our earlier findings by examining the impact of the presence and donor specificity of anti-HLA IgA on graft survival. We observed frequent concurrence of anti-HLA IgA and anti-HLA IgG in 27% of our multicenter collective of 694 kidney re-tx patients. This subgroup displayed significantly reduced graft survival as evidenced by the median time to first dialysis after transplantation (TTD 77 months) compared to patients carrying either anti-HLA IgG or IgA (TTD 102 and 94 months, respectively). In addition, donor specificity of anti-HLA IgA had a significant negative impact on graft survival (TTD 74 months) in our study. Taken together, our data strongly indicate that presence of anti-HLA IgA, in particular in conjunction with anti-HLA-IgG, in sera of kidney re-tx patients is associated with negative transplantation outcome.

Functional Fc gamma receptor gene polymorphisms and donor-specific antibody-triggered microcirculation inflammation.

Fc-dependent effector mechanisms may contribute to antibody-mediated rejection (ABMR), and distinct gene polymorphisms modifying the function of Fc gamma receptors (FcγRs) may influence the capability of donor-specific antibodies (DSAs) to trigger inflammation. To evaluate the relevance of functional FcγR variants in late ABMR, 85 DSA-positive kidney allograft recipients, who were recruited upon antibody screening of 741 prevalent patients, were genotyped for polymorphisms in FcγRIIA (FCGR2A-H/R131 ; rs1801274), FcγRIIIA (FCGR3A-V/F158 ; rs396991), and FcγRIIIB (FCGR3B-neutrophil antigen 1 ([NA1]/NA2; rs35139848). Individuals with high-affinity FCGR3A-V158 alleles (V/V158 or V/F158 ) showed a higher rate (and extent) of peritubular capillaritis (ptc) in protocol biopsies than homozygous carriers of the lower-affinity allele (ptc score ≥1: 53.6% vs 25.9%; P = .018). Associations were independent of C1q-binding to DSA or capillary C4d. In parallel, there was a trend toward increased macrophage- and injury-repair response-associated transcript subsets. Kidney function over 24 months, however, was not different. In support of a functional role of FcγRIIIA polymorphism, NK92 cells expressing FCGR3A-V158 produced >2 times as much interferon gamma upon incubation with HLA antibody-coated cells as those expressing FCGR3A-F158 . FcγRIIA and FcγRIIIB polymorphisms were not associated with allograft morphology. Our data suggest that the presence of high-affinity FcγRIIIA variants may favor DSA-triggered microcirculation inflammation.

Effects of weak/non-complement-binding HLA antibodies on C1q-binding.

It is unknown under what conditions and to what extent weak/non-complement (C)-binding IgG subclasses (IgG2/IgG4) can block C1q-binding triggered by C-binding IgG subclasses (IgG1/IgG3). Therefore, we investigated in vitro C1q-binding induced by IgG subclass mixtures targeting the same HLA epitope. Various mixtures of HLA class II specific monoclonal antibodies of different IgG subclasses but identical V-region were incubated with HLA DRB1*07:01 beads and monitored for C1q-binding. The lowest concentration to achieve maximum C1q-binding was measured for IgG3, followed by IgG1, while IgG2 and IgG4 did not show appreciable C1q-binding. C1q-binding occurred only after a critical amount of IgG1/3 has bound and sharply increased thereafter. When both, C-binding and weak/non-C-binding IgG subclasses were mixed, C1q-binding was diminished proportionally to the fraction of IgG2/4. A 2- to 4-fold excess of IgG2/4 inhibited C1q-binding by 50%. Very high levels (10-fold excess) almost completely abrogated C1q-binding even in the presence of significant IgG1/3 levels that would usually lead to strong C1q-binding. In sensitized renal allograft recipients, IgG subclass constellations with ≥ 2-fold excess of IgG2/4 over IgG1/3 were present in 23/66 patients (34.8%) and overall revealed slightly decreased C1q signals. However, spiking of patient sera with IgG2 targeting a different epitope than the patient's IgG1/3 synergistically increased C1q-binding. In conclusion, if targeting the same epitope, an excess of IgG2/4 is repressing the extent of IgG1/3 triggered C1q-binding in vitro. Such IgG subclass constellations are present in about a third of sensitized patients and their net effect on C1q-binding is slightly inhibitory.

[Recent development of hypertension and acute renal failure in a 59-year-old woman]. / Neu aufgetretene Hypertonie und akutes Nierenversagen bei einer 59-jährigen Patientin.

We report on a 59-year-old woman who presented with nausea, fatigue, arterial hypertension, and acute renal failure. Clinical examination, laboratory findings of blood and urine and abdominal sonography were inconclusive. Renal biopsy revealed infiltration by a diffuse large B­cell lymphoma. In fluorodeoxyglucose positron emission tomography/computed tomography tracer enrichment was demonstrated in both kidneys, skeletal system and retroperitoneal lymph nodes. Renal function improved already after the first cycle of R­CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). After chemotherapy, a complete remission, normalization of blood pressure and renal function were achieved.

Identification of the novel HLA-C*07:315 allele.

The HLA-C*07:315 allele is mostly identical to HLA-C*07:09 but has a non-synonymous substitution of C to G in exon 2.

16(th) IHIW: anti-HLA alloantibodies of the of IgA isotype in re-transplant candidates.

In this multicentre study, sera from 803 retransplant candidates, including 775 kidney transplant recipients, were analysed with regard to the presence and specificity of anti-HLA alloantibodies of the IgA isotype using a modified microsphere-based platform. Of the kidney recipients, nearly one-third (n = 237, 31%) had IgA alloantibodies. Mostly, these antibodies were found in sera that also harboured IgG alloantibodies that could be found in a total of 572 (74%) of patients. Interestingly, IgA anti-HLA antibodies were preferentially targeting HLA class I antigens in contrast to those of the IgG isotype, which targeted mostly both HLA class I and II antigens. Donor specificity of the IgA alloantibodies could be established for over half of the 237 patients with IgA alloantibodies (n = 124, 52%). A further 58 patients had specificities against HLA-C or HLA-DP, for which no information regarding donor typing was available. In summary, these data showed in a large cohort of retransplant candidates that IgA alloantibodies occur in about one-third of patients, about half of these antibodies being donor specific.

Human cytomegalovirus infection leads to elevated levels of transplant arteriosclerosis in a humanized mouse aortic xenograft model.

Recent findings emphasized an important role of human cytomegalovirus (HCMV) infection in the development of transplant arteriosclerosis. Therefore, the aim of this study was to develop a human peripheral blood lymphocyte (hu-PBL)/Rag-2(-/-) γc(-/-) mouse-xenograft-model to investigate both immunological as well as viral effector mechanisms in the progression of transplant arteriosclerosis. For this, sidebranches from the internal mammary artery were recovered during coronary artery bypass graft surgery, tissue-typed and infected with HCMV. Then, size-matched sidebranches were implanted into the infrarenal aorta of Rag-2(-/-) γc(-/-) mice. The animals were reconstituted with human peripheral blood mononuclear cells (PBMCs) 7 days after transplantation. HCMV-infection was confirmed by Taqman-PCR and immunofluorescence analyses. Arterial grafts were analyzed by histology on day 40 after transplantation. PBMC-reconstituted Rag-2(-/-) γc(-/-) animals showed splenic chimerism levels ranging from 1-16% human cells. After reconstitution, Rag-2(-/-) γc(-/-) mice developed human leukocyte infiltrates in their grafts and vascular lesions that were significantly elevated after infection. Cellular infiltration revealed significantly increased ICAM-1 and PDGF-R-ß expression after HCMV-infection of the graft. Arterial grafts from unreconstituted Rag-2(-/-) γc(-/-) recipients showed no vascular lesions. These data demonstrate a causative relationship between HCMV-infection as an isolated risk factor and the development of transplant-arteriosclerosis in a humanized mouse arterial-transplant-model possibly by elevated ICAM-1 and PDGF-R-ß expression.

[Fundus leucaemicus as first manifestation of chronic myeloid leukemia. Diagnosis and monitoring with OCT under treatment with imatinib and interferon-alpha]. / Fundus leucaemicus als Erstmanifestation einer chronischen myeloischen Leukämie. Diagnosestellung und Verlaufskontrolle mit OCT unter Therapie mit Imatinib und Interferon-alpha.

Fundus leucaemicus with reduction of visual acuity can be one of the first signs of chronic myeloid leukemia (CML). The ocular manifestations are unspecific, but characteristic for severe systemic diseases. Optical coherence tomography (OCT) is helpful for documentation and quantification of the pathological retinal changes. However, peripheral blood counts and differential haemograms are seminal for the diagnosis of CML and can be important for survival. First line therapy for CML is the application of the tyrosine kinase inhibitor imatinib (Glivec). Ophthalmological adverse effects of this therapy, such as periorbital edema, are possible. Therefore regular ophthalmic monitoring should be performed.

Prevalence and specificity of immunoglobulin G and immunoglobulin A non-complement-binding anti-HLA alloantibodies in retransplant candidates.

The role of complement-binding donor-directed anti-human leukocyte antigen (HLA) antibodies in graft rejection is well established, whereas the prevalence and relevance of non-complement-binding (NCB) anti-HLA antibodies are less well defined. The aim of our study was to establish a sensitive and reliable test system for the detection and the specification of these NCB anti-HLA antibodies. Sera from 60 patients awaiting retransplantation were analysed for the presence of anti-HLA class I alloantibodies with complement-dependent cytotoxicity (CDC) tests. Immunoglobulin (Ig)G(all) anti-HLA class I and class II alloantibodies were differentiated on generic level by plate-based solid phase enzyme-linked immunosorbent assay. Subsequently, a modified bead-based (Luminex) assay was applied, allowing the investigation of IgG(2/4) NCB isotypes as well as IgA(1/2). The anti-HLA specificities of the NCB alloantibodies were determined and compared with known mismatches from previous transplants. Seventeen of the 60 sera (28%) were positive in the CDC increasing to 26 of 60 (43%) in the class I and 33 of 60 (55%) in the class II plate-based assay. Using the modified bead-based system 24 of 60 sera (40%) contained NCB IgG(2/4), which were mostly donor specific. In addition, a high prevalence of NCB IgA antibodies was detected (26 of 60 sera), which occurred independently of IgG(2/4) NCB, and half of which were donor specific. NCB anti-HLA alloantibodies, including the IgA isotype, can reliably be detected using the modified bead-based test system. These NCB alloantibodies had a high prevalence in retransplant candidates and were mostly donor specific.

Distinct tumour necrosis factor alpha, interferon gamma, interleukin 10, and cytotoxic T cell antigen 4 gene polymorphisms in disease occurrence and end stage renal disease in Wegener's granulomatosis.

BACKGROUND: Cytokines and T cell regulatory proteins play an important role in the pathogenesis of Wegener's granulomatosis (WG). OBJECTIVE: To investigate cytokine and cytotoxic T cell antigen-4 (CTLA4) gene polymorphisms and HLA class II alleles in generalised WG. METHODS: The distribution of cytokine and cytotoxic T cell antigen 4 (CTLA4) gene polymorphisms and HLA class II alleles was analysed in 32 patients with generalised WG and 91 healthy controls. Genotyping was carried out for HLA-DRB1 and HLA-DQB1 and for polymorphism of the genes encoding TNF alpha (-238, -308, -376), TGF beta (codon 10 and 25), IFN gamma (+874), IL6 (-174), IL10 (-592, -819, -1082), CTLA4 (-318, +49), and the (AT)(n) repeats of the CTLA4 gene. In addition, stratification analysis was carried out according to the presence (n = 15) or absence (n = 17) of end stage renal disease. RESULTS: An increase in the IFN gamma +874 T/T (odds ratio (OR) = 3.14) and TNF alpha -238 G/A (OR = 5.01) genotypes was found in WG patients. When ESRD positive and negative patients were compared, the IFN gamma +874 A/A and the CTLA4 -318 C/C genotypes were found more often in the ESRD subgroup (OR = 10.6 and OR = 2.25). WG patients without ESRD had a higher frequency of the IL10 GCC/ACC promotor genotype (OR = 0.13) and long CTLA4 (AT)(n) repeats (OR = 0.4). No effect was seen for HLA-DR and -DQ markers. CONCLUSIONS: Disease susceptibility and clinical course in WG may be associated with distinct polymorphisms of cytokine and CTLA4 genes.

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4(+) T cell activation but is unable to prevent immune responses mediated by CD8(+) T cells. However, even in the absence of CD8(+) T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40(-/-) (H2(b)) recipients were transplanted with MHC-mismatched BALB/c (H2(d)) aortas. Transplant arteriosclerosis was evident in both CD40(-/-) and CD40(+/-) mice (intimal proliferation was 59 +/- 5% for CD40(-/-) mice vs 58 +/- 4% for CD40(+/-) mice) in the presence or absence of CD8(+) T cells (intimal proliferation was 46 +/- 7% for CD40(-/-) anti-CD8-treated mice vs 50 +/- 10% for CD40(+/-) anti-CD8-treated mice), confirming that CD8(+) T cells are not essential effector cells for the development of this disease. In CD40(-/-) recipients depleted of CD8(+) T cells, the number of eosinophils infiltrating the graft was markedly increased (109 +/- 24 eosinophils/grid for CD40(-/-) anti-CD8-treated mice vs 28 +/- 7 for CD40(+/-) anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40(-/-) recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 +/- 5 eosinophils/grid for CD40(-/-) anti-CD8(+), anti-IL-4-treated mice vs 109 +/- 24 for CD40(-/-) anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 +/- 5% for CD40(-/-) anti-CD8(+)-, anti-IL-4-treated mice vs 46 +/- 7% for CD40(-/-) anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8(+) T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Successful removal of a giant recurrent chondrosarcoma of the thoracic wall in a patient with hereditary multiple exostoses.

Chondrosarcoma represents the most common malignant tumour of the chest wall, with a tendency for local recurrence after resection. Here we report the successful complete resection of a giant, local recurrent chondrosarcoma of the chest wall (max. diameter 25 cm), in a patient with hereditary multiple exostoses, who had had a wide resection 8 years before. Clinical features and surgical management are described.

Differential role for competitive reverse transcriptase-polymerase chain reaction and intracellular cytokine staining as diagnostic tools for the assessment of intragraft cytokine profiles in rejecting and nonrejecting heart allografts.

The early and reliable diagnosis of allograft rejection is a difficult task and the assessment of cytokine expression in the grafts can be a helpful parameter. We have compared competitive reverse transcriptase-polymerase chain reaction (RT-PCR) with intracellular cytokine staining by flow cytometry as tools to measure cytokine expression in rejecting and nonrejecting murine cardiac allografts. Both techniques gave comparable results for cytokine expression in rejecting allografts and syngeneic controls. Grafts from mice pretreated with anti-CD4 antibody and donor-specific blood transfusion showed a marked reduction in cytokine expression, as assessed by competitive RT-PCR, even though a cellular infiltrate was present in the graft. In contrast, the cytokine production measured by intracellular cytokine staining of the isolated graft-infiltrating cells was high and exceeded even that of the rejecting allografts. We conclude that intracellular cytokine staining of graft-infiltrating leukocytes by flow cytometry does not necessarily reflect accurately the cytokine milieu in the graft. This technique might therefore have a limited clinical application in contrast to competitive RT-PCR for the differentiation between graft acceptance and graft rejection.

Intragraft interleukin-4 mRNA expression after short-term CD154 blockade may trigger delayed development of transplant arteriosclerosis in the absence of CD8+ T cells.

BACKGROUND: It has recently been shown that, although anti-CD154 induces CD4+ T-cell tolerance, it is unable to prevent allograft rejection mediated by CD8+ T cells. We have also shown that anti-CD154 monotherapy does not protect the graft from the development of transplant arteriosclerosis even in the absence of CD8+ T cells. This study was designed to investigate and characterize possible mechanisms responsible for the development of transplant arteriosclerosis after CD154 blockade in the absence of CD8+ T cells. METHODS: C57BL/6 (H2b) recipients received a fully MHC-mismatched BALB/c donor aorta (H2d). Animals were either treated with anti-CD154 monoclonal antibody (mAb) in the presence or absence of CD8 T cells. Histology, morphometric measurements, immunohistochemistry, and the production of alloantibodies (IgM, IgG1, IgG2a) were analyzed on days 14, 30, and 50 after transplantation. Cytokine production within the graft was investigated by competitive reverse transcription-polymerase chain reaction on day 14. RESULTS: Combined treatment with anti-CD154 and a depleting CD8 mAb resulted in a delay in the development of transplant arteriosclerosis (intimal proliferation: 33+/-10% vs. 67+/-11% untreated control, day 30) but ultimately did not prevent its progression (intimal proliferation: 55+/-10% vs. 78+/-9% untreated control, day 50). Although there was a significant decrease in the number of CD4+, CD11b+, and CD40+ graft-infiltrating cells and a reduction in the formation of donor-specific IgG1 alloantibodies in recipients treated with anti-CD154 and anti-CD8 mAbs, mRNA for interleukin (IL)-4 was increased, suggesting a shift in the intragraft cytokine profile towards a Th2-like pattern. CONCLUSIONS: Our data provide evidence that short-term CD154 blockade is insufficient to prevent transplant arteriosclerosis, even in combination with CD8+ T-cell depletion. Moreover, the increased expression of the Th2 cytokine interleukin-4 within the graft may be responsible for the development of transplant arteriosclerosis in the long term.

CD8+ T cells contribute to the development of transplant arteriosclerosis despite CD154 blockade.

BACKGROUND: The CD40-CD154 receptor-ligand pair plays a critical role in allograft rejection by mediating the activation of endothelial cells, antigen-presenting cells, and T cells. Blockade of this interaction prevents acute allograft rejection and leads to prolonged allograft survival in numerous experimental models, but in most cases indefinite graft survival is not achieved due to evolving transplant arteriosclerosis. In this study, we have used a model of transplant arteriosclerosis to investigate whether CD4+ and CD8+ T cells are differentially affected by CD154 blockade. METHODS: BALB/c (H2d) aortic grafts were transplanted into C57BL/6 (H2b) recipients treated with anti-CD154 monoclonal antibody in the presence or absence of CD8+ T-cell depletion. Histology and morphometric measurements were performed on day 30 after transplantation. RESULTS: Only combined treatment with anti-CD154 and anti-CD8 monoclonal antibodies resulted in a significant reduction of intimal proliferation (33 +/-10% vs. 67+/-14%; untreated control). Administration of either antibody alone did not produce this effect. Thymectomy did not alter the degree of intimal proliferation observed in any of the treatment groups. CONCLUSIONS: Our data provide direct evidence that CD8+ T cells are not targeted effectively by CD154 blockade and that the transplant arteriosclerosis seen after CD154 blockade is not due to recent thymic emigrant T cells.

CD40-CD40 ligand-independent activation of CD8+ T cells can trigger allograft rejection.

In experimental transplantation, blockade of CD40-CD40 ligand (CD40L) interactions has proved effective at permitting long-term graft survival and has recently been approved for clinical evaluation. We show that CD4+ T cell-mediated rejection is prevented by anti-CD40L mAb therapy but that CD8+ T cells remain fully functional. Furthermore, blocking CD40L interactions has no effect on CD8+ T cell activation, proliferation, differentiation, homing to the target allograft, or cytokine production. We conclude that CD40L is not an important costimulatory molecule for CD8+ T cell activation and that following transplantation donor APC can activate recipient CD8+ T cells directly without first being primed by CD4+ T cells.

Islet allograft rejection can be mediated by CD4+, alloantigen experienced, direct pathway T cells of TH1 and TH2 cytokine phenotype.

BACKGROUND: It is widely believed that Thl cells that secrete interferon-gamma are primarily involved in the rejection of allografts whereas Th2 cells [interleukin(IL) 4 and IL-10] are thought to be protective of this process. However, the exact role and specificity of these helper T lymphocytes in mediating allograft damage is presently unknown. METHODS: Th0, Th1, and Th2 cell lines specific for the class II MHC molecule H2IAb were adoptively transferred into T cell deficient, syngeneic, diabetic mice before transplantation of fully allogeneic C57BL/10 (H2b) or (CBKxBALB/c)F1 (H2k/d+Kb) islet grafts. T cells were 5-(and-6-)-carboxyfluorescein diacetate succinimidyl ester- (CFSE) labeled to allow detection, immunohistochemistry was performed, and IL-4 transcripts within the rejected islet grafts were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Adoptive transfer (IV) of Th0-, Th1-, and Th2 IAb-specific T cells resulted in rejection of H2b islet allografts. CFSE-labeling demonstrated that these T cells were able to home to the graft site. CD4+ T cells and CD11b+ macrophages were present within the graft after adoptive transfer of both Thl and Th2 cells. Interestingly, CD8+ T cells and B cells were absent from these rejecting grafts. Even when Th2 cells were introduced directly at the graft site, prompt rejection was still observed despite the presence of increased IL-4 mRNA expression within the islet allografts. CONCLUSIONS: Th2 and Th0 alloreactive CD4+ T helper cells can reject islet grafts with similar efficiency to Th1 cells. These results suggest that deviation of the immune response from a Th1 to Th2 phenotype will not be sufficient to allow successful engraftment of allogeneic organs or tissues.

Lead induced anaemia due to traditional Indian medicine: a case report.

Lead intoxication in adults without occupational exposure is a rare and unexpected event. The case of a western European is reported who had severe anaemia after ingestion of several ayurvedic drugs, obtained during a trip to India. Laboratory findings showed high blood lead concentrations, an increased urinary lead concentration, and an increased urinary excretion of delta-aminolaevulinic acid. Also, slightly increased urinary concentrations of arsenic and silver were found. Physicians should be aware that with growing international travel and rising self medication with drugs from uncontrolled sources the risk of drug induced poisoning could increase in the future.

Microchimerism after liver transplantation: prevalence and methodological aspects of detection.

BACKGROUND: Microchimerism after liver transplantation is a readily observed phenomenon. The immunological implications, however, remain unclear. Moreover, methodological approaches and their detection limits in the study of allogeneic microchimerism have not been studied in detail. METHODS: Therefore, the aim of this study was to evaluate the single-step and nested formats of the polymerase chain reaction/sequence-specific priming (PCR-SSP) approach under standardized conditions. For that purpose, a panel of recombinant plasmid clones was generated by PCR cloning. The panel contained the allelic sequences of the second exon of DRB1 covering all DR specificities on a low-resolution level. Using this panel, limiting dilution assays for various DR sequences in the presence and absence of competitor DNA were carried out to determine the minimal number of copies required for detection by single-step and nested PCR-SSP. Subsequently, 22 liver transplant recipients were analyzed in a retrospective study for the presence of allogeneic microchimerism by nested PCR-SSP. RESULTS: Although at least 10 copies of template DNA could be detected by nested PCR-SSP overall, single-step PCR-SSP was on average 10(2) to 10(3) times less sensitive. Upon the addition of human competitor DNA, the detection limits decreased on average by a factor of 10. In addition, sequence-specific differences in amplification efficiency could be appreciated. Using nested PCR-SSP, peripheral blood allogeneic microchimerism could be observed in 17 of 22 HLA-DR-mismatched liver recipients. Recombinants representing recipient DRB1 specificities were used to exclude false-positive results by lack of cross-reactivities of the donor-specific primers and to evaluate negative results due to sample-related reduced amplification efficiencies in microchimerism-negative recipients. In donor/recipient combinations that differed by at least one DR specificity, allogeneic microchimerism was seen in 87.5% of the cases. In five chimerism-negative cases, sample-related problems were detected in two cases. CONCLUSION: The optimization and standardization of the detection of genomic HLA sequences at low copy number may be greatly facilitated using a clonal reference system. Furthermore, a clonal reference system may be used to conduct cross-priming experiments to exclude false-positive results and may allow the determination of sample-specific detection limits for donor-derived HLA-DR specificities in chimerism-negative patients. Our evaluation of the PCR-SSP approach for the study of allogeneic microchimerism indicated that nested PCR-SSP provides the most sensitive format when HLA sequences are targeted. Yet, the detection sensitivity may vary between individual alleles and specificities. Allogeneic microchimerism in liver recipients can be observed in the majority of patients. However, the detection may be subject to the degree of mismatching, the HLA-DR alleles involved, and sample-related impaired PCR amplification efficiency.