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Background: Early administration of convalescent plasma obtained from blood donors who have recovered from coronavirus disease 2019 (Covid-19) may prevent disease progression in acutely ill, high-risk patients with Covid-19.Methods: In this randomized, multicenter, single-blind trial, we assigned patients who were being treated in an emergency department for Covid-19 symptoms to receive either one unit of convalescent plasma with a high titer of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or placebo. All the patients were either 50 years of age or older or had one or more risk factors for disease progression. In addition, all the patients presented to the emergency department within 7 days after symptom onset and were in stable condition for outpatient management. The primary outcome was disease progression within 15 days after randomization, which was a composite of hospital admission for any reason, seeking emergency or urgent care, or death without hospitalization. Secondary outcomes included the worst severity of illness on an 8-category ordinal scale, hospital-free days within 30 days after randomization, and death from any cause.Results: A total of 511 patients were enrolled in the trial (257 in the convalescent-plasma group and 254 in the placebo group). The median age of the patients was 54 years; the median symptom duration was 4 days. In the donor plasma samples, the median titer of SARS-CoV-2 neutralizing antibodies was 1:641. Disease progression occurred in 77 patients (30.0%) in the convalescent-plasma group and in 81 patients (31.9%) in the placebo group (risk difference, 1.9 percentage points; 95% credible interval, -6.0 to 9.8; posterior probability of superiority of convalescent plasma, 0.68). Five patients in the plasma group and 1 patient in the placebo group died. Outcomes regarding worst illness severity and hospital-free days were similar in the two groups.Conclusions: The administration of Covid-19 convalescent plasma to high-risk outpatients within 1 week after the onset of symptoms of Covid-19 did not prevent disease progression. (SIREN-C3PO ClinicalTrials.gov number, NCT04355767.). [ABSTRACT FROM AUTHOR]

Background: Despite prophylactic platelet transfusions, hypoproliferative thrombocytopenia is associated with bleeding; historical risk factors include hematocrit (HCT) ≤ 25%, activated partial thromboplastin time ≥ 30 s, international normalized ratio ≥ 1.2, and platelets ≤ 5000/μL., Methods: We performed a post hoc analysis of bleeding outcomes and risk factors in participants with hematologic malignancy and hypoproliferative thrombocytopenia enrolled in the American Trial to Evaluate Tranexamic Acid Therapy in Thrombocytopenia (A-TREAT) and randomized to receive either tranexamic acid (TXA) or placebo., Results: World Health Organization (WHO) grade 2+ bleeding occurred in 46% of 330 participants, with no difference between the TXA (44%) and placebo (47%) groups (p = 0.66). Overall, the most common sites of bleeding were oronasal (18%), skin (17%), gastrointestinal (11%), and genitourinary (11%). Among participants of childbearing potential, 28% experienced vaginal bleeding. Platelets ≤5000/μL and HCT < 21% (after adjusting for severe thrombocytopenia) were independently associated with increased bleeding risk (HR 3.78, 95% CI 2.16-6.61; HR 2.67, 95% CI 1.35-5.27, respectively). Allogeneic stem cell transplant was associated with nonsignificant increased risk of bleeding versus chemotherapy alone (HR 1.34, 95% CI 0.94-1.91)., Discussion: The overall rate of WHO grade 2+ bleeding was similar to previous reports, albeit with lower rates of gastrointestinal bleeding. Vaginal bleeding was common in participants of childbearing potential. Platelets ≤5000/μL remained a risk factor for bleeding. Regardless of platelet count, bleeding risk increased with HCT < 21%, suggesting a red blood cell transfusion threshold above 21% should be considered to mitigate bleeding. More investigation is needed on strategies to reduce bleeding in this population., (© 2024 The Author(s). Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background: Tranexamic acid (TXA) is an antifibrinolytic agent that reduces bleeding in a multitude of clinical settings from postpartum hemorrhage to trauma. TXA may have clinical effects unrelated to bleeding; plasminogen, the target of TXA, alters immune responses, and TXA appears to decrease the risk of infection in patients undergoing cardiac surgery, as well as joint arthroplasty., Objectives: To address whether TXA alters rates of infection and inflammatory outcomes in patients with hematologic malignancies., Methods: We performed a post hoc analysis of outcomes of patients randomized to receive either TXA or placebo in the double-blinded, multicenter American Trial to Evaluate Tranexamic Acid Therapy in Thrombocytopenia (Clinicaltrials.gov identifier: NCT02578901)., Results: TXA did not change the overall rate of infections, but the rate of severe infections (Common Toxicology Criteria for Adverse Events grade 3+) was lower in patients who received TXA compared with the placebo group. Patients who experienced grade 3+ infections had higher rates of World Health Organization grade 2+ bleeding and red blood cell transfusion requirements than patients who did not experience a grade 3+ infection, irrespective of treatment group. TXA did not impact other inflammatory outcomes such as mucositis, rash, or graft vs host disease., Conclusion: Patients with hematologic malignancies who received TXA had less severe infections than those who received placebo with no difference in overall rate of infection or other inflammatory outcomes. Further investigation is needed on the impact of TXA on infections in this population., (© 2024 The Author(s).)

Multiple randomized, controlled clinical trials have yielded discordant results regarding the efficacy of convalescent plasma in outpatients, with some showing an approximately 2-fold reduction in risk and others showing no effect. We quantified binding and neutralizing antibody levels in 492 of the 511 participants from the Clinical Trial of COVID-19 Convalescent Plasma in Outpatients (C3PO) of a single unit of COVID-19 convalescent plasma (CCP) versus saline infusion. In a subset of 70 participants, peripheral blood mononuclear cells were obtained to define the evolution of B and T cell responses through day 30. Binding and neutralizing antibody responses were approximately 2-fold higher 1 hour after infusion in recipients of CCP compared with saline plus multivitamin, but levels achieved by the native immune system by day 15 were almost 10-fold higher than those seen immediately after CCP administration. Infusion of CCP did not block generation of the host antibody response or skew B or T cell phenotype or maturation. Activated CD4+ and CD8+ T cells were associated with more severe disease outcome. These data show that CCP leads to a measurable boost in anti-SARS-CoV-2 antibodies but that the boost is modest and may not be sufficient to alter disease course.

The American Trial Using Tranexamic Acid (TXA) in Thrombocytopenia (A-TREAT, NCT02578901) demonstrated no superiority of TXA over placebo in preventing World Health Organization (WHO) grade 2 or higher bleeding in patients with severe thrombocytopenia requiring supportive platelet transfusion following myeloablative therapy for hematologic disorders. In this ancillary study, we sought to determine whether this clinical outcome could be explained on the basis of correlative assays of fibrinolysis. Plasma was collected from A-TREAT participants (n = 115) before the initiation of study drug (baseline) and when TXA was at steady-state trough concentration (follow-up). Global fibrinolysis was measured by 3 assays: euglobulin clot lysis time (ECLT), plasmin generation (PG), and tissue-type plasminogen activator (tPA)-challenged clot lysis time (tPA-CLT). TXA was quantified in follow-up samples by tandem mass spectrometry. Baseline samples did not demonstrate fibrinolytic activation by ECLT or tPA-CLT. Furthermore, neither ECLT nor levels of plasminogen activator inhibitor-1, tPA, plasminogen, alpha2-antiplasmin, or plasmin-antiplasmin complexes were associated with a greater risk of WHO grade 2+ bleeding. TXA trough concentrations were highly variable (range, 0.7-10 μg/mL) and did not correlate with bleeding severity, despite the fact that plasma TXA levels correlated strongly with pharmacodynamic assessments by PG (Spearman r, -0.78) and tPA-CLT (r, 0.74). We conclude that (1) no evidence of fibrinolytic activation was observed in these patients with thrombocytopenia, (2) trough TXA concentrations varied significantly between patients receiving the same dosing schedule, and (3) tPA-CLT and PG correlated well with TXA drug levels., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Evidence of the effectiveness of prophylactic use of tranexamic acid (TXA) in thrombocytopenia is lacking. To determine whether TXA safely reduces bleeding incidence in patients undergoing treatment for hematologic malignancies, a randomized, double-blind clinical trial was conducted from June 2016 through June 2020. Of 3120 screened adults, 356 patients were eligible and enrolled, and 337 patients (mean age, 53.9; 141 [41.8%] women), randomized to 1300 mg TXA orally or 1000 mg TXA through IV (n = 168) vs placebo (n = 169) thrice daily for maximum 30 days. Three hundred thirty patients were activated when their platelet counts fell below 30 000 per µL; 279 (83%) had complete outcome ascertainment. World Health Organization (WHO) grade ≥2 bleeding was observed in the 30 days following activation in 50.3% (73/145) and 54.2% (78/144) of patients in the TXA and placebo groups, with an adjusted odds ratio of 0.83 (95% confidence interval [CI], 0.50-1.34; P = .44). There was no statistically significant difference in the mean number of platelet transfusions (mean difference, 0.1; 95% CI, -1.9 to 2.0), mean days alive without grade ≥2 bleeding (mean difference, 0.8; 95% CI, -0.4 to 2.0), thrombotic events (6/163 [3.7%] TXA, 9/163 [5.5%] placebo), or deaths due to serious bleeding. Most common adverse events were: diarrhea (116/164 [70.7%] TXA and 114/163 [69.9%] placebo); febrile neutropenia (111/164 [67.7%] TXA, 105/163 [64.4%] placebo); fatigue (106/164 [64.6%] TXA, 109/163 [66.9%] placebo); and nausea (104/164 [63.4%] TXA, 97/163 [59.5%] placebo). Among patients with hematologic malignancy undergoing chemotherapy or hematopoietic stem cell transplantation, prophylactic treatment with TXA compared with placebo did not significantly reduce the risk of WHO grade ≥2 bleeding., (© 2022 by The American Society of Hematology.)

Abstract: High-quality evidence guiding optimal transfusion and other supportive therapies to reduce bleeding is needed to improve outcomes for patients with either severe bleeding or hemostatic disorders that are associated with poor outcomes. Alongside challenges in performing high-quality clinical trials in patient populations who are at risk of bleeding or who are actively bleeding, the interpretation of research evaluating hemostatic agents has been limited by inconsistency in the choice of primary trial outcomes. This lack of standardization of primary endpoints or outcomes decreases the ability of clinicians to assess the validity of endpoints and compare research results across studies, impairs meta-analytic efforts, and, ultimately, delays the translation of research results into clinical practice. To address this challenge, an international panel of experts was convened by the National Heart Lung and Blood Institute and the US Department of Defense on September 23 and 24, 2019, to develop expert opinion, consensus-based recommendations for primary clinical trial outcomes for pivotal trials in pediatric and adult patients with six categories in various clinical settings. This publication documents the conference proceedings from the workshop funded by the National Heart Lung and Blood Institute and the US Department of Defense that consolidated expert opinion regarding clinically meaningful outcomes across a wide range of disciplines to provide guidance for outcomes of future trials of hemostatic products and agents for patients with active bleeding., Competing Interests: A complete list of conflicts of interest and working group members is provided in Supplementary Appendix., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Approximately 2,500 pediatric hematopoietic cell transplants (HCTs), most of which are allogeneic, are performed annually in the United States for life-threatening malignant and nonmalignant conditions. Although HCT is undertaken with curative intent, post-HCT complications limit successful outcomes, with pulmonary dysfunction representing the leading cause of nonrelapse mortality. To better understand, predict, prevent, and/or treat pulmonary complications after HCT, a multidisciplinary group of 33 experts met in a 2-day National Institutes of Health Workshop to identify knowledge gaps and research strategies most likely to improve outcomes. This summary of Workshop deliberations outlines the consensus focus areas for future research.

Immune reconstitution after hematopoietic stem cell transplantation (HCT) beyond 1 year is not completely understood. Many transplant recipients who are free of graft-versus-host disease (GVHD) and not receiving any immunosuppression more than 1 year after transplantation seem to be able to mount appropriate immune responses to common pathogens and respond adequately to immunizations. However, 2 large registry studies over the last 2 decades seem to indicate that infection is a significant cause of late mortality in some patients, even in the absence of concomitant GVHD. Research on this topic is particularly challenging for several reasons. First, there are not enough long-term follow-up clinics able to measure even basic immune parameters late after HCT. Second, the correlation between laboratory measurements of immune function and infections is not well known. Third, accurate documentation of infectious episodes is notoriously difficult. Finally, it is unclear what measures can be implemented to improve the immune response in a clinically relevant way. A combination of long-term multicenter prospective studies that collect detailed infectious data and store samples as well as a national or multinational registry of clinically significant infections (eg, vaccine-preventable severe infections, opportunistic infections) could begin to address our knowledge gaps. Obtaining samples for laboratory evaluation of the immune system should be both calendar and eventdriven. Attention to detail and standardization of practices regarding prophylaxis, diagnosis, and definitions of infections would be of paramount importance to obtain clean reliable data. Laboratory studies should specifically address the neogenesis, maturation, and exhaustion of the adaptive immune system and, in particular, how these are influenced by persistent alloreactivity, inflammation, and viral infection. Ideally, some of these long-term prospective studies would collect information on long-term changes in the gut microbiome and their influence on immunity. Regarding enhancement of immune function, prospective measurement of the response to vaccines late after HCT in a variety of clinical settings should be undertaken to better understand the benefits as well as the limitations of immunizations. The role of intravenous immunoglobulin is still not well defined, and studies to address it should be encouraged., (Published by Elsevier Inc.)

Niche availability provided by stromal cells is critical to thymus function. Thymi with diminished function contain fewer stromal cells, whereas thymi with robust function contain proliferating stromal cell populations. Here, we show that the thymus, brain, and testes-associated gene (Tbata; also known as SPATIAL) regulates thymic epithelial cell (TEC) proliferation and thymus size. Tbata is expressed in thymic stromal cells and interacts with the enzyme Uba3, thereby inhibiting the Nedd8 pathway and cell proliferation. Thymi from aged Tbata-deficient mice are larger and contain more dividing TECs than wild-type littermate controls. In addition, thymic reconstitution after bone marrow transplantation occurred more rapidly in Rag2(-/-)Tbata(-/-) mice than in Rag2(-/-)Tbata(+/+) littermate controls. These findings suggest that Tbata modulates thymus function by regulating stromal cell proliferation via the Nedd8 pathway.

Interleukin-7 receptor (IL-7R) levels are tightly controlled during ontogeny: high on double-negative (DN) cells, absent on double-positive (DP) cells, and high once again on thymocytes undergoing positive selection. To determine if loss of IL-7-mediated survival signals in DP cells is necessary for normal antigen-specific selection, we created T-lineage-specific IL-7R alpha chain (IL-7Ralpha) transgenic (Tg) mice in which IL-7R is expressed throughout ontogeny. There was no effect of the IL-7Ralpha Tg on negative selection. Surprisingly, however, although the thymi of IL-7Ralpha Tg mice were comparable at birth, there was a decrease in thymocyte number as the mice aged. This was found to be due to competition between DN and IL-7R-expressing DP cells for endogenous IL-7, which resulted in decreased levels of Bcl-2 in DN cells, increased DN apoptosis, and decreased DN cell number. Therefore, the down-regulation of IL-7R on DP cells is an "altruistic" act required for maintaining an adequate supply of local IL-7 for DN cells.

To study interleukin-7 (IL-7) in early thymocyte development, we generated mice transgenic (Tg) for the IL-7 gene under control of the lck proximal promoter. Founder line TgA, with the lowest level of IL-7 overexpression, showed enhanced alphabeta T-cell development. In contrast, in the highest overexpressing founder line, TgB, alphabeta T-cell development was disturbed with a block at the earliest intrathymic precursor stage. This was due to decreased progenitor proliferation as assessed by Ki-67 staining and in vivo bromodeoxyuridine (BrdU) incorporation. Bcl-2 was up-regulated in T-cell-committed progenitors in all Tg lines, and accounted for greater numbers of double positive (DP), CD4 single positive (SP), and CD8SP thymocytes in TgA mice where, in contrast to TgB mice, thymocyte progenitor proliferation was normal. Mixed marrow chimeras using TgB(+) and congenic mice as donors, and experiments using anti-IL-7 monoclonal antibody (MAb) in vivo, confirmed the role of IL-7 protein in the observed TgB phenotype. In conclusion, at low Tg overexpression, IL-7 enhanced alphabeta T-cell development by increasing thymocyte progenitor survival, while at high overexpression IL-7 reduces their proliferation, inducing a dramatic block in DP production. These results show for the first time in vivo a dose effect of IL-7 on alphabeta T-cell development and have implications for IL-7 in the clinical setting.

Background: The sensitive cross-match (XM) techniques that have been introduced for clinical transplantation can detect anti-donor immune reactivity despite a negative standard National Institute of Health (NIH) cross-match. One of them uses anti-kappa human light chain globulins (AHG). But there is some discussion about the clinical consequences of a positive AHG-XM in the historical sera that became negative in the sera collected just before the transplantation (pretransplant sera). This study was intended to assess the risk of kidney graft failure associated with a positive historic but negative pretransplant AHG-XM in allosensitized patients having a negative historic NIH-XM., Methods: This retrospective study includes 90 consecutive renal transplants in immunized patients performed at one center between 1985 and 1991. All of the patients had negative historical and pretransplant standard NIH lymphocytotoxic cross-matches and received the same immunosuppressive regimen. The AHG-XMs were done retrospectively using peak historic and sera collected on the day of the transplantation., Results: The AHG cross-match (AHG-XM) was positive in 17 patients, although the standard NIH cross-match was negative. Fourteen of them had a positive historical but negative pretransplant AHG-XM. The actuarial graft survival in this group of 14 patients was 100% at 1 year and 78% at 9 years compared with 90 and 67%, respectively, in patients with negative historical AHG-XM. In addition, the number of rejection episodes per patient as well as renal function at 1, 2, and 5 years were similar in the two groups. IgG anti-donor HLA class I accounted for the XM positivity in 12 of the 14 patients; most rapidly lost all antibody reactivity by NIH technique in an average time of 8 months before the transplantation. In conclusion, this study suggests that transplant patients having a negative historic NIH-XM but a positive historic AHG-XM may not be at high risk of graft failure especially if there is a well-documented sera history showing a marked decrease in PRA level before transplantation and a negative pretransplant AHG-XM.

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.

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.

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.

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 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.

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.

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.