High resolution HLA class I and II typing and CTLp frequency in unrelated donor transplantation: a single-institution retrospective study of 69 BMTs.

UNLABELLED: The results of unrelated donor transplantation (URD-BMT) are difficult to analyze since the continuous advances in HLA typing technology allow the detection of new mismatches unknown at the time of transplantation. We sought to confirm that matched recipient-donor pairs are in fact often mismatched when advanced HLA typing techniques are used. We retrospectively studied the impact of the results of high resolution HLA typing for HLA class I (-A, -B, -C) and HLA class II (-DR, -DQ, -DP) loci, and cytotoxic T lymphocyte precursor (CTLp) frequency, on the outcome of 69 URD-BMT procedures. At the time of transplant, six (6/69) and two (2/69) donor-recipient pairs were mismatched for HLA class I (-A and -B by serology) and HLA class II, respectively, while one pair was mismatched for both HLA class I and II. Using high resolution DNA typing, HLA class I mismatches were found in 31 (45%) pairs and HLA class II mismatches in nine (13%) pairs. Twenty-three of the 69 pairs were HLA-C mismatched. Low CTLp frequencies were found among the 19 HLA class I matched pairs tested, and also in 5/14 mismatched pairs (of whom three had severe aGVHD). The overall survival of the cohort was 28 +/- 6%. Among the 33 patients who were fully matched with their donors, the survival rate was 66% in the 18 patients with a standard hematological risk and 9% in the 15 high risk patients. Only two of the 33 patients developed severe aGVHD, and only one had graft rejection. Among the 36 mismatched pairs, the survival rate was 31% in the 13 patients with a standard hematological risk and 8% in the 23 high risk patients. Sixteen of these 36 patients died from severe aGVHD and four had graft failure or rejection. Three of the 10 patients with only an HLA-C mismatch died from severe aGVHD, and two had graft rejection. IN CONCLUSION: (1) donor-recipient matching based on high resolution HLA class I and II DNA typing is associated with significantly better outcome after URD-BMT; (2) the results of URD-BMT with classical GVHD prevention are comparable to those of geno-identical BMT when donor and recipient are fully matched for HLA-A, -B, -C, -DRB1 and -DQB1 on the basis of high resolution typing; (3) CTLp frequencies do not correlate constantly with HLA class I matching, and our results fail to show that CTLp assay can distinguish between permissible and non-permissible class I mismatches; (4) clinical trials involving donor-recipient pairs with known HLA class I mismatches are needed to improve aGVHD prevention without increasing graft failure rate.

A novel HLA-B*39 allele (HLA-B*3916) due to a rare mutation causing cryptic splice site activation.

A novel HLA-B*39 variant, found in an African patient with sickle cell anemia undergoing bone marrow transplantation is described. Initially suspected by inconsistent serological typing (B-blank, Bw6), then recognized by PCR-SSP, and finally characterized by nucleotide sequencing, this novel allele is designated HLA-B*3916. It differs from HLA-B*3910 by a point mutation (G to C) at position 17 of exon 3 causing glutamine to histidine change at codon 96 of alpha(2) domain, a conserved position among HLA class I alleles. cDNA sequence analysis further revealed the presence of both normally and abnormally spliced mRNA species in established cell lines. The abnormal species correspond to partial truncation of exon 3 presumably due to the nucleotide change in exon 3, which constitutes a new consensus acceptor splice site within this exon. We postulate that the observed blank is essentially the consequence of qualitative change in a critical region of this novel antigen as abnormal mRNA species are relatively less abundant than normal species. Because the residue 96 of the HLA class I heavy chain is directly involved in interaction with alpha(2)m, another interesting possibility is that an aminoacid change in this position would perturb such interaction and consequently could affect the serological specificity of B*3916, or its expression or both.

Clonality analysis of hematopoiesis and thrombopoietin levels in patients with essential thrombocythemia.

Essential thrombocythemia (ET) is a myeloproliferative disorder, characterized by sustained thrombocytosis. Diagnosis requires the elimination of all known causes of thrombocytosis. ET is believed to be a clonal disorder, and we investigated the frequency of a clonal hematopoiesis in this disease with the aim of using this as a positive diagnostic criterion. However, a non-random inactivation pattern can be encountered in normal females which mimics clonal hematopoiesis. In addition, the percentage of normal females with skewed lyonization seems higher using techniques based on the difference in DNA methylation, compared to G6PD enzyme polymorphism. Recently, new techniques based on transcript analysis have been developed. We report here the results of clonality studies of hematopoiesis in 53 ET patients using two different techniques based on DNA and RNA polymorphisms, and T-lymphocytes as a control tissue of lyonization. The majority of ET patients showed monoclonal hematopoiesis in the presence of polyclonality of T-lymphocytes. Because all ET patients did not show the same clonal pattern of hematopoiesis, we searched for inappropriate secretion of thrombopoietin (TPO) in patients with polyclonal disease. This assay was performed in 48 patients, of whom 9 showed polyclonal hematopoiesis and 27 monoclonal hematopoiesis. We found no difference in TPO levels between ET patients and normal controls, nor between patients with polyclonal hematopoiesis and those with monoclonal hematopoiesis. Our results confirm the high frequency of monoclonal hematopoiesis in ET, the usefulness of RNA markers, and the possibility of using T-lymphocytes as a control tissue for X-chromosome inactivation patterns. On the other hand, TPO levels are not decreased even in ET patients with high platelet counts, suggesting an increased production or decreased clearance of TPO in this disease.

X-chromosome inactivation in healthy females: incidence of excessive lyonization with age and comparison of assays involving DNA methylation and transcript polymorphisms.

Skewed lyonization in healthy females represents the major disadvantage of X-chromosome-based clonality assays. Because most techniques are based on the difference in DNA methylation between active and inactive X-chromosomes, incomplete DNA digestion may occur, giving an unreliable clonality result. Here, we compare two different techniques carried out in healthy females belonging to three age groups and in a group of patients with essential thrombocythemia. The first technique involved the human androgen receptor gene, the second the transcript analysis of the iduronate-2-sulfatase, P55, and glucose-6-phospate dehydrogenase genes. Results between both techniques were concordant in most cases except in neonates, and the same pattern was observed in all fractions in healthy females. We conclude that: (a) clonality assays involving DNA and RNA polymorphisms are usually concordant except in neonates; (b) appropriate control tissue embryologically related to the sample must be chosen to eliminate excessive lyonization; (c) acquired skewing increases with age, whereas nonrandom lyonization is a rare phenomenon.

Clonality markers in polycythaemia and primary thrombocythaemia.

Our current understanding of the pathogenesis of the myeloproliferative disorders is based on the assumption that they represent a clonal disorder resulting from transformation of a haematopoietic stem cell. Clonality assays exploit the fact that female cells have only one active X-chromosome. Methods for determining X chromosome inactivation have been devised at the protein (G6PD isoenzymes); DNA (HUMARA; phosphoglycerate kinase (PGK); hypoxanthine-phosphoribosyl transferase (HPRT) and RNA (G6PD; P55; IDS) levels. In this type of RNA assay the product of the active X chromosome is quantified by studying polymorphisms present in mRNA. The presence of skewed lyonization in normal females is a potential limitation to the method, although the use of T cells as a control makes it possible to distinguish clonal haematopoiesis from skewed lyonization. However, the phenomenon of acquired skewing in normal elderly women means that X-inactivation patterns in elderly patients must be interpreted with caution. Clonality studies have been conducted in polycythaemia vera (PV) and essential thrombocythaemia (ET) patients. They usually demonstrate a clonal X-inactivation pattern in granulocytes and/or platelets but a polyclonal pattern in T cells. However, in both ET and PV a significant minority of patients exhibit polyclonal haematopoiesis with polyclonal patterns in granulocytes/platelets. Female patients with polyclonal haematopoiesis differ from those with clonal haematopoiesis in terms of age and platelet count and possibly in their requirements for treatment. This new technology for the investigation of the MPD seems promising for understanding certain clinical aspects of these diseases and may be introduced for evaluation of new modalities of treatment.

Clonality analysis of hematopoiesis in essential thrombocythemia: advantages of studying T lymphocytes and platelets.

Essential thrombocythemia (ET) is a myeloproliferative disorder characterized by a sustained elevation of the platelet count in the absence of other causes of thrombocytosis. ET is difficult to diagnose, and the demonstration of clonal hematopoiesis may be of value. However, clonality analysis of hematopoietic cells based on the study of the X-chromosome inactivation pattern is complicated by the observation that some normal females present skewed lyonization. Moreover, DNA methylation of X-linked genes in hematopoietic cells may differ from that in other tissues. Appropriate controls for skewed lyonization are therefore critical for the study of clonality. We developed two techniques based on X-chromosome inactivation and polymerase chain reaction (PCR) analysis of polymorphisms, to study clonality in ET patients. Reverse transcriptase-PCR analysis of IDS, P55, and G6PD mRNAs was used to examine the different hematopoietic cell lineages including platelets in patients heterozygous for these polymorphisms and analysis of the HUMARA gene methylation pattern permitted us to study clonality in all nucleated cell fractions of the other patients. Using both types of assay and T lymphocytes as a control tissue for lyonization, clonal hematopoiesis was demonstrated in 28 patients. In 14 patients, the granulocytes were polyclonal; among these patients, platelets were monoclonal in 3 cases, polyclonal in 7 cases, and in the remaining 4 cases this fraction could not be studied because the patients were homozygotes for all RNA markers. No conclusion about clonality could be drawn in 6 cases. Polyclonal hematopoiesis was found in all the cases of reactive thrombocytosis. These findings confirm the high frequency of monoclonal hematopoiesis in ET, the utility of studying platelets, and the possibility of using T lymphocytes as a control tissues for X-chromosome inactivation patterns.

Fusidic acid induced acute immunologic thrombocytopenia.

Fusidic acid is used in hospitals as second-line therapy for multidrug-resistant staphylococcal infections. We report the first fully documented case of fusidic acid induced thrombocytopenia, in a 48-year-old patient. The thrombocytopenia was abrupt and severe but resolved spontaneously 7 d after drug withdrawal. The thrombocytopenia transiently relapsed 6 d later, when fusidic acid was reintroduced. Haemorrhagic signs were observed, but no severe bleeding occurred. Platelet transfusions failed to increase the platelet count. We detected an IgG platelet antibody in the patient's serum, that specifically recognized platelet glycoprotein IIb/IIIa only in the presence of fusidic acid. Fusidic acid induced thrombocytopenia should be considered as a possible cause for the thrombocytopenia frequently seen in the intensive care setting.

Posttransfusion purpura-like syndrome associated with CD36 (Naka) isoimmunization.

BACKGROUND: CD36 deficiency, which could lead to CD36 isoimmunization, has been reported in the Japanese population. CD36 isoantibody has been involved in platelet transfusion refractoriness. CASE REPORT: A 50-year-old woman originally from Corsica developed severe acute thrombocytopenia after massive transfusion. She was found to be CD36 deficient, and platelet immunoassays revealed a CD36 (Naka) platelet isoantibody. Although the involvement of another mechanism could not be entirely ruled out, the thrombocytopenia was attributed to posttransfusion purpura-like syndrome. The antibody was also involved in platelet transfusion refractoriness. CD36 deficiency was present in two members of the patient's family as well. Flow cytometry studies demonstrated the absence of CD36 expression on the surface of blood monocytes and cultured erythroblasts and megakaryocytes from one of the two CD36-deficient family members studied, but, in the absence of previous immunization, these CD36-deficient patients were not isoimmunized. In contrast, CD36 deficiency was not found in a population of 808 healthy blood donors in the Paris, France, area. CONCLUSION: CD36 isoantibody might be involved in some cases of posttransfusion purpura and platelet transfusion refractoriness. These findings also confirm the extremely low frequency of CD36 deficiency among whites.

Clonal analysis of haemopoietic cells in essential thrombocythaemia.

Essential thrombocythaemia (ET) is a myeloproliferative disease (MPD) predominantly involving the platelet lineage, with a diagnosis often difficult to establish in practice. The demonstration of a clonal haemopoiesis can contribute to diagnosis. The clonal origin of blood cells can be assessed in female patients using X-chromosome-linked polymorphisms, assuming a random inactivation of the X chromosome. The human androgen receptor gene HUMARA has been used for this purpose, taking advantage of a highly polymorphic trinucleotide repeat in the first exon. The close proximity of the polymorphic trinucleotide repeat to four methylation sites permits a clonal analysis based on the polymerase chain reaction. We have used this technique to study the clonality of haemopoietic cells in 32 patients with ET and 23 age-matched control heterozygotes for the HUMARA polymorphism. A monoclonal pattern of haemopoiesis was detected in unfractionated nucleated blood cells from 22 patients, suggesting that haemopoiesis is essentially monoclonal in a majority of cases in this disease. In some patients a monoclonality of granulocytes was documented, whereas the mononuclear fraction had a polyclonal pattern of X-inactivation. Finally, in two patients for whom a polyclonality had been found in unfractionated blood, analysis of G6PD transcripts in platelets revealed a clonal megakaryocytopoiesis. These findings confirm the heterogeneity of haematological abnormalities in ET patients and the potential utility of a multifaceted laboratory approach to investigate these patients.

HIV-associated thrombocytopenia: in vitro megakaryocyte colony formation in 10 patients.

In vitro bone-marrow megakaryocyte colony formation was studied in 10 patients with HIV-associated thrombocytopenia to investigate the mechanism of thrombocytopenia. Increased colony formation was observed in 3 patients and decreased growth in 7 patients. No relationship was noted between the growth potential of megakaryocyte progenitors and platelet count, number of CD4+ celts, platelet response to azidothymidine, and platelet count 7 days after culture. In all patients, megakaryocyte morphology was abnormal: blebbing of the membrane and abnormal chromatin with separated lobes of nuclei. Further studies are needed to determine if growth potential of megakaryocyte progenitors is useful in understanding the mechanism of thrombocytopenia in HIV-infected individuals.

In vitro megakaryocyte colony formation in patients with idiopathic thrombocytopenic purpura: differences between acute and chronic ITP.

In vitro megakaryocyte colony formation from the bone marrow of patients with acute idiopathic thrombocytopenic purpura (ITP) or chronic ITP was compared using a plasma clot system. The number of megakaryocyte colony-forming units (CFU-Meg) was significantly higher (p less than 0.05) in acute ITP compared to chronic ITP (54.3 +/- 68.4 vs. 12.9 +/- 15.3/10(5) nonadherent mononuclear cells, mean +/- SD), and significantly lower (p less than 0.05) in chronic ITP compared to controls (12.9 +/- 15.3 vs. 22.8 +/- 15.9). A significant correlation was observed between platelet recovery 7 and 30 days after culture, and the number of CFU-Meg (r = 0.49 and 0.45, respectively, p less than 0.05). An inverse correlation was observed between platelet count at the time of culture and the number of Megs per colony (r = -0.48, p less than 0.05). These results indicated a difference between acute and chronic ITP in the ability to promote in vitro Meg colony formation and may suppose a different immune mechanism for thrombocytopenia in these two disorders.