Mismatches Research Articles

The effect of HLA mismatch on highly sensitized renal allograft recipients

We examined the effects of increasing human leukocyte antigen (HLA) mismatches (MM) on long-term graft outcomes in patients transplanted with a panel reactive antibody (PRA) >80% over a 10-yr period. A total of 142 recipients were divided into three groups based on the number of HLA MM with their allograft (0-2, 3-4 and 5-6 MM; Groups I, II and III). All patients received the same immunosuppression protocol. The higher MM groups had a higher incidence of rejection (4.4% vs. 11.4% vs. 31.3%, p < 0.01). A multivariate analysis showed that rejection was the only significant variable affecting graft loss (OR = 7.45, p = 0.01). There was a trend toward more CMV infection and worse graft function with higher MM. Kaplan-Meier five-yr graft survival estimates were 100% vs. 81% vs. 74% for Groups I, II and III, respectively (p = 0.14). In patients with PRA levels >80%, a higher HLA MM is associated with higher incidence of acute rejection. Acute rejection was the only significant variable affecting graft loss. We found a trend toward more CMV infections and worse graft outcomes with higher MM. Closer HLA matching and immunologic monitoring needs to be considered to improve graft outcomes among sensitized recipients.

Donor-specific differences in long-term outcomes of myeloablative transplantation in adults with Philadelphia-negative acute lymphoblastic leukemia

We analyzed long-term outcomes of myeloablative stem cell transplantation (SCT) in 292 adults with Philadelphia (Ph)-negative acute lymphoblastic leukemia (ALL). Donors were related (RD; n=132), unrelated (URD; n=68; 30 well-matched (WM), 19 partially matched (PM), 19 mismatched (MM)) and autologous (AUTO; n=92). After a median follow-up of 85 months, the risk of relapse was higher for AUTO-SCT than for RD-SCT (P<0.001). MM-URD-SCT yielded higher risk of non-relapse mortality than RD-SCT (P=0.010). As a result, disease-free survival (DFS) at 5 years was inferior using AUTO (46.1%; P=0.010) or MM-URD (26.3%; P=0.036), whereas DFS from other donor sources was approximately equivalent (53.5% for RD, 63.3% for WM-URD and 57.0% for PM-URD). Other factors associated with poorer DFS included SCT beyond first complete remission (CR), older age and adverse cytogenetics. In a pairwise comparison of outcomes between RD-SCT and AUTO-SCT for patients in first CR, the inferiority of AUTO-SCT was observed, particularly in high-risk patients. Conversely, in standard-risk patients, AUTO-SCT yielded comparable outcomes to RD-SCT. SCT using RD, WM-URD or PM-URD may be considered the best donor sources for adult high-risk Ph-negative ALL.

The impact of HLA matching on long-term transplant outcome after allogeneic hematopoietic stem cell transplantation for CLL: a retrospective study from the EBMT registry

We analyzed 368 chronic lymphocytic leukemia patients who underwent allogeneic hematopoietic stem cell transplantation reported to the EBMT registry between 1995 and 2007. There were 198 human leukocyte antigen (HLA)-identical siblings; among unrelated transplants, 31 were well matched in high resolution ('well matched' unrelated donor, WMUD), and 139 were mismatched (MM), including 30 matched in low resolution; 266 patients (72%) received reduced-intensity conditioning and 102 (28%) received standard. According to the EBMT risk score, 11% were in scores 1-3, 23% in score 4, 40% in score 5, 22% in score 6 and 4% in score 7. There was no difference in overall survival (OS) at 5 years between HLA-identical siblings (55% (48-64)) and WMUD (59% (41-84)), P=0.82. In contrast, OS was significantly worse for MM (37% (29-48) P=0.005) due to a significant excess of transplant-related mortality. Also OS worsened significantly when EBMT risk score increased. HLA matching had no significant impact on relapse (siblings: 24% (21-27); WMUD: 35% (26-44), P=0.11 and MM: 21% (18-24), P=0.81); alemtuzumab T-cell depletion and stem cell source (peripheral blood) were associated with an increased risk. Our findings support the use of WMUD as equivalent alternative to HLA-matched sibling donors for allogeneic HSCT in CLL, and justify the application of EBMT risk score in this disease.

HLA‐matched kidney transplantation in the era of modern immunosuppressive therapy

AbstractBACKGROUNDThe effect of HLA match on renal graft survival has become controversial as has the policy of mandatory sharing of kidneys.METHODWe performed a retrospective analysis of HLA matched (M) and mismatched (MM) kidney transplants in our center. Tacrolimus, mycophenolic acid, and steroids were used as maintenance therapy and basiliximab induction was added for high‐risk patients.RESULTA total of 229 kidney transplants were included with median follow‐up of 5.1 years. The 5‐year death‐censored graft survival by Kaplan‐Meier method was significantly higher in the M group than in the MM group for deceased‐donor kidney transplants (log‐rank, p = .018). This graft survival advantage was detected in patients with a peak panel reactive antibody (PRA) greater than 20% (p = .023), but not in those with a PRA level of less than 20% (p = .32). The graft survival was not statistically different for live donor kidney transplants (p = .077). A mismatched kidney was an independent risk for graft loss (hazard ratio: 2.27, 95% confidence interval: 1.009–5.09, p = .047) and acute rejection was a significant cause of graft loss in mismatched deceased‐donor transplants (p = .035).CONCLUSIONAcute rejection remains a significant cause of graft loss in HLA‐6‐antigen mismatched deceased‐donor kidney transplants. Our data support mandatory sharing of HLA‐matched kidneys in sensitized patients with a PRA level greater than 20%.

420: The Impact of Repeat HLA Mismatch on Pulmonary Retransplantation

Pulmonary retransplantation (retxp) remains contraversial due to poor outcomes historically (<50% survival at 1 y). T cell depletion prior to retxp followed by steroid minimization regimen was performed at our institution with retxp survival 91% at 1y and 76% at 2y. We analyzed whether repeat HLA mismatches (MM) had a detrimental effect on outcome.

Re-Exposure of Cord Blood (CB) to Non-Inherited Maternal HLA Antigens (NIMA) Improves Transplant Outcome in Patients (pts) with Hematologic Malignancies and May Reduce Relapse.

Abstract 661CB hematopoietic stem cell transplantation (CBT) can be successful even if donor and recipient are not fully matched for human leukocyte antigens (HLA). This may result, at least in part, from tolerance-inducing events during pregnancy, but this concept has not been tested to date. Hence we analyzed the impact of fetal exposure to NIMA of the HLA-A, -B antigens or -DRB1 alleles on the outcome of 1121 pts with hematologic malignancies. All pts received single CB units provided by the NYBC, for treatment of ALL (N=451), AML (N=376), CML (N=116), MDS (N=79), other (N=99); 22% were transplanted in advanced stage. Median age was 9.7 years (range: 0.1-67); 29% of recipients were >16 years. Most pts (96%) received myeloablative cytoreduction. Sixty-two pts received fully matched grafts while 1059 received units mismatched (MM) for one or two HLA antigens. Of these, 79 (7%) had a MM antigen which was identical to a donor NIMA (Example: Pt: A1, A3; CBU: A1, A2; mother-CBU: A1, A3; A3 is NIMA). NIMA match was found in 25 recipients with one HLA MM and 54 of those with two MM. The NIMA match was identified after the transplant and was not used in unit selection. In multivariate analyses, NIMA matched transplants (NMTs), showed faster neutrophil recovery (RR=1.3, p=0.043), even for grafts with cell dose <3×107 (RR=1.6, p=0.053). There was no difference in the incidence of acute (grade II-IV) or chronic GvHD. 3-year relapse risk (cumulative incidence 22%) was reduced compared to 1 or 2 HLA MM no NIMA matched transplants, especially in pts with myelogenous malignancies given units with 1 HLA MM (RR=0.2, p=0.074). Further, 3-year transplant-related mortality was reduced (RR=0.7, p=0.034), particularly in pts ≥5 years old (RR=0.5, p=0.006), as was the 3-year overall mortality (RR= 0.7, p=0.029 and RR=0.6, p=0.015, respectively). As a result, in the NMTs, treatment failure (relapse or death) was significantly lower, particularly in pts ≥5 years (RR=0.7, p=0.019) and DFS was significantly improved (figure) and was similar to that of the 0 HLA MM group. These findings are the first indication that donor exposure to NIMA can improve post-transplant survival in unrelated CBT and might reduce relapse. We propose to include the NIMA of CB units in search algorithms. Thus, for pts lacking fully HLA matched grafts, HLA MM but NIMA matched CB units could be selected preferentially, since no adverse effects were seen. This strategy of selecting HLA MM grafts with optimal outcome effectively “expands” the current CB Inventory several-fold. [Display omitted] Patient GroupNRR(95% Cl)p value0 MM360.5(0.3–0.8)0.0051 MM / NIMA Match180.4(0.2–0.9)0.0262 MM / NIMA Match400.8(0.5–1.2)0.3091 MM / No NIMA Match229reference group2 MM / No NIMA Match4871.1(0.9–1.3)0.365 Disclosures:No relevant conflicts of interest to declare.

Chemical gradient‐mediated melting curve analysis for genotyping of SNPs

This report describes a microfluidic solid-phase chemical gradient-mediated melting curve analysis method for SNP analysis. The method is based on allele-specific denaturation to discriminate mismatched (MM) from perfectly matched (PM) DNA duplexes upon exposure to linear chemical gradient. PM and MM DNA duplexes conjugated on beads are captured in a microfluidic gradient generator device designed with dams, keeping the beads trapped perpendicular to a gradient generating channel. Two denaturants, formamide and urea, were tested for their ability to destabilize the DNA duplex by competing with Watson-Crick pairing. Upon exposure to the chemical gradient, rapid denaturing profile was monitored in real time using fluorescence microscopy. The results show that the two duplexes exhibit different kinetics of denaturation profiles, enabling discrimination of MM from PM DNA duplexes to score SNP.

A distribution-free convolution model for background correction of oligonucleotide microarray data

IntroductionAffymetrix GeneChip® high-density oligonucleotide arrays are widely used in biological and medical research because of production reproducibility, which facilitates the comparison of results between experiment runs. In order to obtain high-level classification and cluster analysis that can be trusted, it is important to perform various pre-processing steps on the probe-level data to control for variability in sample processing and array hybridization. Many proposed preprocessing methods are parametric, in that they assume that the background noise generated by microarray data is a random sample from a statistical distribution, typically a normal distribution. The quality of the final results depends on the validity of such assumptions.ResultsWe propose a Distribution Free Convolution Model (DFCM) to circumvent observed deficiencies in meeting and validating distribution assumptions of parametric methods. Knowledge of array structure and the biological function of the probes indicate that the intensities of mismatched (MM) probes that correspond to the smallest perfect match (PM) intensities can be used to estimate the background noise. Specifically, we obtain the smallest q2 percent of the MM intensities that are associated with the lowest q1 percent PM intensities, and use these intensities to estimate background.ConclusionUsing the Affymetrix Latin Square spike-in experiments, we show that the background noise generated by microarray experiments typically is not well modeled by a single overall normal distribution. We further show that the signal is not exponentially distributed, as is also commonly assumed. Therefore, DFCM has better sensitivity and specificity, as measured by ROC curves and area under the curve (AUC) than MAS 5.0, RMA, RMA with no background correction (RMA-noBG), GCRMA, PLIER, and dChip (MBEI) for preprocessing of Affymetrix microarray data. These results hold for two spike-in data sets and one real data set that were analyzed. Comparisons with other methods on two spike-in data sets and one real data set show that our nonparametric methods are a superior alternative for background correction of Affymetrix data.

Kidney Transplantation: Charles Nicolle Hospital Experience

The aim of our retrospective study was to analyze the short- and long-term follow-up of 298 renal transplantations performed between June 1986 and May 2005. All were first transplantations except 4 cases, with 54 from cadaveric and 244 from living donors. The recipients included 196 males and 102 females of overall mean age of 31.21 ± 8.9 years (range, 16–61 years). A combination of prednisolone and azathioprine was presented for 212 patients or mycophenolate mofetil for 86 patients. Polyclonal or monoclonal antibodies were used as induction therapy in 183 cases. Cyclosporine was administered to 188 cases and tacrolimus only to 16. HLA matching was 0 mismatches (MM) in 65 cases; 1 or 2 MM in 113; 3 MM in 99; and ≥4 MM in 21. Acute tubular necrosis occurred in 45 cases. One hundred eighteen patients experienced at least 1 acute rejection episode: 102 cases (41.8%) among living and 16 (29.6%) among cadaveric kidneys donor ( P = .0007). The actuarial patient and graft survival rates at 1, 5, 10, 15, and 20 years were 95.9%, 87.4%, 77.5%, 65.6%, and 60.8%, and 94.9%, 84.5%, 75.4%, 65.4%, and 53%, respectively. Sixty-three patients died and 72 patients returned to dialysis. Our results were comparable to experienced centers. However, the member of kidney transplantations does not match the increased number of patients on renal replacement therapy. It is advisable to promote obtaining organs from brain-dead donors.

TNF-α plays a role in hepatocyte apoptosis in Niemann-Pick type C liver disease

Niemann-Pick type C (NPC) is a fatal autosomal recessive lysosomal storage disease clinically characterized by neurodegeneration and liver disease. Heterogeneous mutations in the NPC1 and NPC2 genes cause impaired egress of free cholesterol from lysosomes, leading to accumulation of cholesterol and glycosphingolipids. Key features of NPC liver disease include hepatic apoptosis, inflammation, and fibrosis. It is unclear what signaling events regulate these disease processes in NPC. We hypothesize that tumor necrosis factor alpha (TNF-alpha), which is involved in both proinflammatory and apoptotic signaling cascades, is a key mediator of inflammation, apoptosis, and fibrosis in NPC liver disease. In this study, we evaluated the role of TNF-alpha signaling in NPC liver disease by utilizing NPC1-specific antisense oligonucleotides to knock down NPC1 expression in control and TNF-alpha knockout mice. In the absence of TNF-alpha, NPC1 knockdown produced liver disease with significantly less inflammation, apoptosis, and fibrosis.

11: HLA Mismatches Influence Lung Transplant Recipient Survival, Bronchiolitis Obliterans, and Rejection – Implications for Donor Lung Allocation

Lung transplantation currently does not consider human leukocyte antigen (HLA) compatibility as a factor in matching donors and recipients. We previously reported that fewer individual Class I (HLA-A, B) and Class II (HLA-DR) antigen mismatches (MM) resulted in minor improvements in lung transplantation. In the current study, we evaluated whether the effects of total HLA MM, Class I MM and Class II MM were more important determinants of 1) recipient survival, 2) freedom from acute rejection (AR), and 3) the development of bronchiolitis obliterans (BO).

Allocation of Deceased Donor Kidneys in São Paulo, Brazil: Effect of Human Leukocyte Antigen Compatibility on Graft Survival

In Brazil, organ transplantation has been regulated by a federal law since 1997. This law was created to guarantee equal access to treatment on a national scale. Deceased donor organ procurement and sharing are centralized and controlled by the Health Department of each state of the nation, following a regional allocation policy. In São Paulo, time on the waiting list was the main criterion adopted to allocate deceased donor kidneys up to January 1, 2002. After that, HLA mismatches (MM) were the main criterion. The aim of this study was to investigate the impact of HLA compatibility on graft survival among 3312 consecutive kidney recipients. The 2-year kidney graft survival rates were compared among recipients transplanted based on the waiting time policy and based on HLA MM. Better results were observed in the HLA MM group (78.1% vs 64.9%; P < .0001). Regarding kidney allocation based on HLA MM, recipients transplanted with 0 HLA-A, -B, or -DR MM showed significantly better 5-year survival rates than those with 1–2 or 3–4 or 5–6 HLA-A, -B, or -DR MM (70.36% vs 64.71% vs 58.07% vs 55.64%; P < .050). We concluded that HLA compatibility is a feasible criterion to allocate deceased donor kidneys in Brazil.

The Clinical Significance of Matching for Alleles at the Low Expression HLA Loci DP, DQ and DRB3/4/5 in Unrelated Hematopoietic Stem Cell Transplantation

Single or multiple mismatches at HLA-A, B, C, and DRB1 have a detrimental effect on outcome of bone marrow transplantation using unrelated donors. Most of these loci encode for products that are expressed with high density on the surface of many cell types. In contrast, HLA-DRB3, DRB4, DRB5, DQ, DP encode for class II products that are expressed at low levels (LEL); some investigations suggest that mismatches in some of these loci have a minimal impact on outcome. We hypothesized that the influence of the LEL is weak and cumulative and only demonstrable in combination with mismatches (MM) in other loci. National Marrow Donor Program data from 3853 US transplants performed from 1988–2003 were analyzed to investigate this hypothesis. Patients had AML, ALL, CML or MDS and received myeloablative conditioning regimens. Most patients received calcineurin-based GvHD prophylaxis with T-replete grafts (79%). Nearly all received marrow grafts (94%). Median follow-up was 6 years. HLA loci were typed retrospectively by high resolution methods. MM was defined as the difference in the antigen recognition site of the HLA molecule of the patient and donor and computed in GvH and HvG vector, the highest vector mismatch was assigned as overall mismatch for a locus. Because multiple comparisons were made, only p-values <0.01 were considered significant. All analyses were adjusted for patient and transplant characteristics. Transplants were stratified according to match grade in the high expression loci (HEL) as 8/8, 7/8 and <7/8; the MM in LEL were computed as 0, 1, 2 and 3 or more MM. A greater degree of mismatch in the HEL was associated with a greater degree of MM in the LEL (p<0.0001); 3 or more MM in the LEL were found in 6, 11 and 21 percent of the patients receiving transplants matched in 8/8, 7/8 and <7/8 HEL groups, respectively. In the 7/8 group, multiple mismatches in the LEL loci were significantly more common in the patients presenting a mismatch in HLA-DRB1 than in the HLA class I loci (p <0.0001); 3 or more MM in the LEL loci were found in 33 percent of the patients presenting one mismatch in HLA-DRB1 while only 8 percent of the patients with one mismatch in either HLA-A, B or C loci presented 3 or more MM in the LEL loci.RESULTS: In the 8/8 HEL group, mismatches in LEL did not significantly associate with mortality. In the 7/8 HEL group, patients with 3 or more MM in LEL in the GvH vector had a significantly higher risk for mortality and treatment related mortality than the subgroups with 0MM (RR=1.44; 95% Confidence Interval (CI):1.07–1.94; p=0.015; and RR=1.63; CI:1.17–2.28; p=0.004) and 1MM (RR=1.46; CI:1.12–1.90; p=0.005; and RR=1.50; CI:1.12–2.01; p=0.006) in LEL, and a similar but not significant trend was observed when compared to the 2MM subgroup. No single LEL appeared to have a more pronounced effect on clinical outcome, when mismatched, versus the other LEL.CONCLUSION: The presence of 3 or more mismatches at LEL may adversely affect clinical outcome after 7/8 matched transplantation. Mismatches at HLA-DRB1 were associated with the occurrence of additional LEL mismatches. Prospective evaluation of matching for HLA-DRB3, 4, 5, DQ and DP loci may be warranted to reduce post-transplant risks in donor recipient pairs matched for 7/8 HEL.

14: Human Leukocyte Antigen (HLA) Matching and Blood Group Identity/Compatibility and Heart Transplant Survival across Twenty Years: What Matters Early, What Matters Late?

Nonself HLA is stimulus for rejection. The fewer donor-recipient HLA mismatches (MM) the better the outcome. Heart transplant patients (HTxpts) are not HLA matched prospectively. Studies on HLA and 2-10y HTx outcome are contradictory. We studied long term survival of HTxpts related to total HLA-A+B+DRMM; specific locus A, B or DRMM; and identical(IDENT)vs compatible(COMP) blood group (BLD G).

HLA Locus-Specific Outcomes in Double Umbilical Cord Blood Reduced Intensity Transplantation (DCBT) in Adults.

The use of two umbilical cord blood units provides effective stem cell replacement for patients receiving reduced intensity transplants. However, these unique transplants have two donors, both of which are usually HLA mismatched (MM) at 1 – 2 loci. There are few data exploring the effect of locus-specific allele-level mismatching on outcomes in recipients of DCBT. Therefore, we inquired whether compatibility determined by high-resolution typing at HLA-A, B, C, DR, DQ influences overall survival (OS), disease-free survival (DFS), engraftment, and acute graft versus host disease (aGVHD). Forty-three consecutive patients (19 – 64y) were recipients of a reduced intensity DCBT. Diseases treated were AML (14), ALL (1), NHL (13), HD (5), CLL (3), MDS (4), other (3). Conditioning regimen was fludarabine, melphalan, and rabbit antithymocyte globulin. Cord units were at least a 4/6 allele level HLA-A, B, DR match with the patient and each other with a minimum pre freeze combined cell dose of >3.7 × 107 TNC/kg. Twenty-one patients received cyclosporine and mycophenolate mofetil (CYA/MMF) and 22 patients received sirolimus and tacrolimus (SIR/TAC) for GVHD prophylaxis. We retrospectively studied patient/donor(s) HLA mismatching of both cords combined, and in the predominant cord, defined as the cord that contributes >60% peripheral blood leukocytes at 90 days. Patients receiving HLA-DR matched cord blood units had a lower risk of aGVHD grade II–IV (p=0.018) as shown in table 1. The results were consistent within each GVHD protocol. Moreover, patient and predominant cord matching follows the same trend, although not as robustly (p=0.064). This finding is particularly intriguing, given that high resolution matching for DR is known to be important for aGVHD in other transplant settings. We also found modest evidence that HLA-DQ matching was associated with less aGVHD, however this was not significant.Neutrophil engraftment was faster in patients who have better HLA-B matching to their combined cords (p=0.006). Median days to platelets >20K were also faster in patients who had no HLA-B MM (p=0.023). A similar effect was observed when compatibility between the patient and the predominant cord was assessed for neutrophils (p=0.035), but not platelets (p=0.124), see table 2.HLA-A or C MM had no observed effect on engraftment or aGVHD. HLA matching did not effect OS, DFS, or which cord blood unit eventually predominated. In conclusion,1.HLA-DR matching is associated with lower risk of aGVHD,2.HLA-B matching is associated with faster neutrophil engraftment,3.HLA matching does not affect OS, DFS, nor does it predict which cord will become predominant, although the power of the analysis is limited by the small sample size.Table 1HLA-DR Mismatch and aGVHDCombined CordsPredominant CordNumber of MM0 – 1≥20≥1Total aGVHD (% patients)14331932CYA/MMF21573057SIR/TAC021020Table 2HLA-B Mismatch and EngraftmentCombined CordsPredominant CordNumber of MM0–1≥10≥1ANC >500192217.521Platelets >20K40474144

Microfluidic Device for the Discrimination of Single-Nucleotide Polymorphisms in DNA Oligomers Using Electrochemically Actuated Alkaline Dehybridization

This work describes an integrated microfluidic (mu-fl) device that can be used to effect separations that discriminate single-nucleotide polymorphisms (SNP) based on kinetic differences in the lability of perfectly matched (PM) and mismatched (MM) DNA duplexes during alkaline dehybridization. For this purpose a 21-base single-stranded DNA (ssDNA) probe sequence was immobilized on agarose-coated magnetic beads, that in turn can be localized within the channels of a poly(dimethylsiloxane) microfluidic device using an embedded magnetic separator. The PM and MM ssDNA targets were hybridized with the probe to form a mixture of PM and MM DNA duplexes using standard protocols, and the hydroxide ions necessary for mediating the dehybridization were generated electrochemically in situ by performing the oxygen reduction reaction (ORR) using O2 that passively permeates the device at a Pt working electrode (Pt-WE) embedded within the microfluidic channel system. The alkaline DNA dehybridization process was followed using fluorescence microscopy. The results of this study show that the two duplexes exhibit different kinetics of dehybridization, rate profiles that can be manipulated as a function of both the amount of the hydroxide ions generated and the mass-transfer characteristics of their transport within the device. This system is shown to function as a durable platform for effecting hybridization/dehybridization cycles using a nonthermal, electrochemical actuation mechanism, one that may enable new designs for lab-on-a-chip devices used in DNA analysis.

Rapid discrimination of single-nucleotide mismatches using a microfluidic device with monolayered beads

A microfluidic device incorporating monolayered beads is developed for the discrimination of single-nucleotide mismatches, based on the differential dissociation kinetics between perfect match (PM) and mismatched (MM) duplexes. The monolayered beads are used as solid support for the immobilization of oligonucleotide probes containing a single-base variation. Target oligonucleotides hybridize to the probes, forming either PM duplexes or MM duplexes containing a single mismatch. Optimization studies show that PM and MM duplexes are easily discriminated based on their dissociation but not hybridization kinetics under an optimized buffer composition of 100 mM NaCl and 50% formamide. Detection of single-nucleotide polymorphism (SNP) using the device is demonstrated within 8 min using four probes containing all the possible single-base variants. The device can easily be modified to integrate multiplexed detection, making high-throughput SNP detection possible.

HLA-DR Matching Improves Survival After Heart Transplantation: Is it Time to Change Allocation Policies?

HLA matching has improved outcome in kidney transplantation but is not considered in current allocation policies in heart transplantation. The aim of this single-center study was to assess the impact of HLA matching on long- term outcome after heart transplantation. The records of 240 consecutive heart transplant recipients (time period 1995 to 2002; mean age 51.8 +/- 11.7 years; mean follow-up 5.9 +/- 1.8 years) were analyzed retrospectively. According to the renal allocation policy, HLA mismatches (MM) on the major antigen loci HLA-A, HLA-B and HLA-DR were calculated, demonstrating 0 to 6 MM. Patients with primary graft failure were excluded from statistical analysis. Survival analysis revealed a statistically significant impact of HLA-DR MM on survival. Five-year survival was 90% in patients without HLA-DR MM (n = 10), 79% in patients with 1 HLA-DR MM (n = 113), and 68.1% in patients with 2 HLA-DR MM (n = 117) (1 MM vs 2 MM: p < 0.05). Freedom from cardiac allograft vasculopathy after 5 years was 89% in HLA-DR-identical recipients (n = 10), 61% in patients with 1 HLA-DR MM (n = 102), 54% in patients with 2 HLA-DR MM (n = 104). Conventional matching with 6 mismatches over the three major HLA antigen loci revealed a trend toward a higher relative risk for adverse outcome in patients with increased MM. HLA-DR matching had a significant impact on survival after heart transplantation (HTx) at our center. In the effort to achieve the best comparative use of scarce donor organs the inclusion of HLA-DR matching into allocation policies might improve long-term outcome after HTx.

GeneChip microarrays—signal intensities, RNA concentrations and probe sequences

GeneChip microarrays consist of hundreds of thousands of oligonucleotide probes. Thetransformation of their signal intensities into RNA transcript concentrations requires theknowledge of the response function of the measuring device. We analysed the ‘apparatus’function of perfect match (PM) and mismatched (MM) oligonucleotide probes of GeneChipmicroarrays after changes of the target concentration using the results of a spiked-inexperiment. In agreement with previous studies we found that a competitivetwo-species Langmuir-adsorption model describes the probe intensities well. Each PMand MM probe is characterized by two hybridization constants which specify thepropensity of the probe to bind specific and non-specific transcripts. The affinity fornon-specific hybridization is on average equal for PM and MM. The purine–pyrimidineasymmetry of base pair interaction strengths, however, causes a characteristicPM–MM intensity difference, the sign of which depends on the middle base of theprobe. The affinity for specific hybridization of the PM exceeds that of the MM onaverage by nearly one order of magnitude because the central mismatched baseonly weakly contributes to the stability of the probe/target duplexes. For thefirst time we differentiate between the free energy parameters related to the 64possible middle-triples of DNA/RNA oligomer duplexes with a central Watson–Crickpairing and a central mismatched pairing. Both the PM and MM probes respondto the concentration of specific transcripts, which can be estimated from thePM and MM probe intensities using the Langmuir-model. The analysis of thePM–MM intensity difference provides at least no loss of accuracy and precision of theestimated concentration compared with the PM-only estimates which in turnoutperform the MM-only estimates. The results show that the processing of thePM–MM intensity difference requires the consideration of a background term dueto non-specific hybridization, which is, however, reduced by nearly one orderof magnitude when compared with the respective background of the PM andMM probes. The calculation of the sequence-specific affinity constants using apositional-dependent nearest-neighbour model opens up the possibility to estimate targetconcentrations beyond the training set of several hundred of spiked-in probes.

High-resolution HLA typing by sequencing for HLA-A, -B, -C, -DR, -DQ in 122 unrelated cord blood/patient pair transplants hardly improves long-term clinical outcome

To determine the impact of high-resolution (HR) HLA typing with outcomes after UCBT, DNAs of 122 pairs were analysed for HLA class I and class II mismatches (MM) based on HR typing. For HLA-A, -B on low-resolution typing and -DRB1 on HR typing, the following MM situation resulted: no MM (13%), one MM (40%), two MM (36%), three MM (8%), four MM (3%). For A, B, C, DR and DQ based on HR typing the following MM occurred: No MM (4%), one MM (10%), two MM (15%), three MM (22%), four MM (25%), five MM (12%), six MM (6%), seven MM (3%), eight MM (2%). There was no significant association between number of MM (HR) for both HLA-A, -B and -DRB1 and HLA-A, -B, -C, -DRB1 and DQB1 and aGvHD grade II-IV. There was a trend that MM in class I HR were associated with neutrophil recovery; HLA-A locus typing analysed in HvG direction was associated with reduced cumulative incidence of engraftment (P=0.04), the same for C-KIR in HvG direction (P=0.01). No significant correlation was found between numbers of HLA-MM on the HR level with 2-year survival. The analysis shows that the degree of mismatching in UCBT is even higher than expected.