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Inherited thrombocytopenias (IT) are genetic diseases characterized by low platelet count, sometimes associated with congenital defects or a predisposition to develop additional conditions. Next-generation sequencing has substantially improved our knowledge of IT, with more than 40 genes identified so far, but obtaining a molecular diagnosis remains a challenge especially for patients with non-syndromic forms, having no clinical or functional phenotypes that raise suspicion about specific genes. We performed exome sequencing (ES) in a cohort of 116 IT patients (89 families), still undiagnosed after a previously validated phenotype-driven diagnostic algorithm including a targeted analysis of suspected genes. ES achieved a diagnostic yield of 36%, with a gain of 16% over the diagnostic algorithm. This can be explained by genetic heterogeneity and unspecific genotype-phenotype relationships that make the simultaneous analysis of all the genes, enabled by ES, the most reasonable strategy. Furthermore, ES disentangled situations that had been puzzling because of atypical inheritance, sex-related effects or false negative laboratory results. Finally, ES-based copy number variant analysis disclosed an unexpectedly high prevalence of RUNX1 deletions, predisposing to hematologic malignancies. Our findings demonstrate that ES, including copy number variant analysis, can substantially contribute to the diagnosis of IT and can solve diagnostic problems that would otherwise remain a challenge.

Prolonged cytopenias are a non-specific sign with a wide differential diagnosis. Among inherited disorders, cytopenias predisposing to leukemia require a timely and accurate diagnosis to ensure appropriate medical management, including adequate monitoring and stem cell transplantation prior to the development of leukemia. We aimed to define the types and prevalences of the genetic causes leading to persistent cytopenias in children. The study comprises children with persistent cytopenias, myelodysplastic syndrome, aplastic anemia, or suspected inherited bone marrow failure syndromes, who were referred for genetic evaluation from all pediatric hematology centers in Israel during 2016-2019. For variant detection, we used Sanger sequencing of commonly mutated genes and a custom-made targeted next-generation sequencing panel covering 226 genes known to be mutated in inherited cytopenias; the minority subsequently underwent whole exome sequencing. In total, 189 children with persistent cytopenias underwent a genetic evaluation. Pathogenic and likely pathogenic variants were identified in 59 patients (31.2%), including 47 with leukemia predisposing syndromes. Most of the latter (32, 68.1%) had inherited bone marrow failure syndromes, nine (19.1%) had inherited thrombocytopenia predisposing to leukemia, and three each (6.4%) had predisposition to myelodysplastic syndrome or congenital neutropenia. Twelve patients had cytopenias with no known leukemia predisposition, including nine children with inherited thrombocytopenia and three with congenital neutropenia. In summary, almost one third of 189 children referred with persistent cytopenias had an underlying inherited disorder; 79.7% of whom had a germline predisposition to leukemia. Precise diagnosis of children with cytopenias should direct follow-up and management programs and may positively impact disease outcome.

Several therapeutic agents can cause thrombocytopenia by either immune-mediated or non-immune-mediated mechanisms. Non-immune-mediated thrombocytopenia is due to direct toxicity of drug molecules to platelets or megakaryocytes. Immune-mediated thrombocytopenia, on the other hand, involves the formation of antibodies that react to platelet-specific glycoprotein complexes, as in classic drug-induced immune thrombocytopenia (DITP), or to platelet factor 4, as in heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT). Clinical signs include a rapid drop in platelet count, bleeding or thrombosis. Since the patient's condition can deteriorate rapidly, prompt diagnosis and management are critical. However, the necessary diagnostic tests are only available in specialized laboratories. Therefore, the most demanding step in treatment is to identify the agent responsible for thrombocytopenia, which often proves difficult because many patients are taking multiple medications and have comorbidities that can themselves also cause thrombocytopenia. While DITP is commonly associated with an increased risk of bleeding, HIT and VITT have a high mortality rate due to the high incidence of thromboembolic complications. A structured approach to drug-associated thrombocytopenia/thrombosis can lead to successful treatment and a lower mortality rate. In addition to describing the treatment of DITP, HIT, VITT, and vaccine-associated immune thrombocytopenia, this review also provides the pathophysiological and clinical information necessary for correct patient management.

The new techniques of genetic analysis have made it possible to identify many new forms of inherited thrombocytopenias (IT) and study large series of patients. In recent years, this has changed the view of IT, highlighting the fact that, in contrast to previous belief, most patients have a modest bleeding diathesis. On the other hand, it has become evident that some of the mutations responsible for platelet deficiency predispose the patient to serious, potentially lifethreatening diseases. Today's vision of IT is, therefore, very different from that of the past and the therapeutic approach must take these changes into account while also making use of the new therapies that have become available in the meantime. This review, the first devoted entirely to IT therapy, discusses how to prevent bleeding in those patients who are exposed to this risk, how to treat it if it occurs, and how to manage the serious illnesses to which patients with IT may be predisposed.

Chemotherapy-induced thrombocytopenia (CIT) is a common complication of the treatment of non-hematologic malignancies. Many patient-related variables (e.g., age, tumor type, number of prior chemotherapy cycles, amount of bone marrow tumor involvement) determine the extent of CIT. CIT is related to the type and dose of chemotherapy, with regimens containing gemcitabine, platinum, or temozolomide producing it most commonly. Bleeding and the need for platelet transfusions in CIT are rather uncommon except in patients with platelet counts below 25x109/L in whom bleeding rates increase significantly and platelet transfusions are the only treatment. Nonetheless, platelet counts below 70x109/L present a challenge. In patients with such counts, it is important to exclude other causes of thrombocytopenia (medications, infection, thrombotic microangiopathy, post-transfusion purpura, coagulopathy and immune thrombocytopenia). If these are not present, the common approach is to reduce chemotherapy dose intensity or switch to other agents. Unfortunately decreasing relative dose intensity is associated with reduced tumor response and remission rates. Thrombopoietic growth factors (recombinant human thrombopoietin, pegylated human megakaryocyte growth and development factor, romiplostim, eltrombopag, avatrombopag and hetrombopag) improve pretreatment and nadir platelet counts, reduce the need for platelet transfusions, and enable chemotherapy dose intensity to be maintained. National Comprehensive Cancer Network guidelines permit their use but their widespread adoption awaits adequate phase III randomized, placebo-controlled studies demonstrating maintenance of relative dose intensity, reduction of platelet transfusions and bleeding, and possibly improved survival. Their potential appropriate use also depends on consensus by the oncology community as to what constitutes an appropriate pretreatment platelet count as well as identification of patient-related and treatment variables that might predict bleeding.

Over the last 100 years the role of platelets in hemostatic events and their production by megakaryocytes have gradually been defined. Progressively, thrombocytopenia was recognized as a cause of bleeding, first through an acquired immune disorder; then, since 1948, when Bernard-Soulier syndrome was first described, inherited thrombocytopenia became a fascinating example of Mendelian disease. The platelet count is often severely decreased and platelet size variable; associated platelet function defects frequently aggravate bleeding. Macrothrombocytopenia with variable proportions of enlarged platelets is common. The number of circulating platelets will depend on platelet production, consumption and lifespan. The bulk of macrothrombocytopenias arise from defects in megakaryopoiesis with causal variants in transcription factor genes giving rise to altered stem cell differentiation and changes in early megakaryocyte development and maturation. Genes encoding surface receptors, cytoskeletal and signaling proteins also feature prominently and Sanger sequencing associated with careful phenotyping has allowed their early classification. It quickly became apparent that many inherited thrombocytopenias are syndromic while others are linked to an increased risk of hematologic malignancies. In the last decade, the application of next-generation sequencing, including whole exome sequencing, and the use of gene platforms for rapid testing have greatly accelerated the discovery of causal genes and extended the list of variants in more common disorders. Genes linked to an increased platelet turnover and apoptosis have also been identified. The current challenges are now to use next-generation sequencing in first-step screening and to define bleeding risk and treatment better., (Copyright© 2020 Ferrata Storti Foundation.)

Over 75% of severely thrombocytopenic neonates receive platelet transfusions, though little evidence supports this practice, and only 10% develop major bleeding. In a recent randomized trial, giving platelet transfusions at a threshold platelet count of 50x10 9 /L compared to a threshold of 25x10 9 /L was associated with an increased risk of major bleeding or mortality. This finding highlights the need for improved and individualized guidelines on neonatal platelet transfusion, which require accurate prediction of bleeding risk. Therefore, the objective of this study was to develop a dynamic prediction model for major bleeding in thrombocytopenic preterm neonates. This model allows for calculation of bleeding risk at any time-point during the first week after the onset of severe thrombocytopenia. In this multicenter cohort study, we included neonates with a gestational age <34 weeks, admitted to a neonatal intensive care unit, who developed severe thrombocytopenia (platelet count <50x10 9 /L). The study endpoint was major bleeding. We obtained predictions of bleeding risk using a proportional baselines landmark supermodel. Of 640 included neonates, 71 (11%) had a major bleed. We included the variables gestational age, postnatal age, intrauterine growth retardation, necrotizing enterocolitis, sepsis, platelet count and mechanical ventilation in the model. The median cross-validated c-index was 0.74 (interquartile range, 0.69-0.82). This is a promising dynamic prediction model for bleeding in this population that should be explored further in clinical studies as a potential instrument for supporting clinical decisions. The study was registered at www.clinicaltrials.gov (NCT03110887)., (Copyright© 2019 Ferrata Storti Foundation.)

Severe thrombocytopenia (≤50×10 9 platelets/L) due to hematological malignancy and intensive chemotherapy is associated with an increased risk of clinically significant bleeding. Since the bleeding risk is not linked to the platelet count only, other hemostatic factors must be involved. We studied platelet function in 77 patients with acute leukemia, multiple myeloma or malignant lymphoma, who experienced chemotherapy-induced thrombocytopenia. Platelets from all patients - independent of disease or treatment type - were to a variable extent compromised in Ca 2+ flux, integrin a β activation and P-selectin expression when stimulated with a panel IIb of 3 agonists. The patients' platelets were also impaired in spreading on fibrinogen. Whereas the Ca 2+ store content was unaffected, the patients' platelets showed ongoing phosphatidylserine exposure, which was not due to apoptotic caspase activity. Interestingly, mitochondrial function was markedly reduced in platelets from a representative subset of patients, as evidenced by a low mitochondrial membrane potential ( P <0.001) and low oxygen consumption ( P <0.05), while the mitochondrial content was normal. Moreover, the mitochondrial impairments coincided with elevated levels of reactive oxygen species (Spearman's rho=-0.459, P =0.012). Markedly, the impairment of platelet function only appeared after two days of chemotherapy, suggesting origination in the megakaryocytes. In patients with bone marrow recovery, platelet function improved. In conclusion, our findings disclose defective receptor signaling related to impaired mitochondrial bioenergetics, independent of apoptosis, in platelets from cancer patients treated with chemotherapy, explaining the low hemostatic potential of these patients., (Copyright© 2018 Ferrata Storti Foundation.)

Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B -related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B -related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b -null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b -null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content., (Copyright© Ferrata Storti Foundation.)

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia., (Copyright© Ferrata Storti Foundation.)

Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy and the course of pregnancy did not differ from that of healthy subjects in terms of miscarriages, fetal bleeding and pre-term births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but 2 of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8% to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of abnormal blood loss was increased with respect to the general population. However, no mother died or had to undergo hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery below 50 × 10(9)/L., (Copyright© Ferrata Storti Foundation.)

The cut off for hemoglobin or hematocrit that indicates the need for an isotopic red cell mass study was investigated in 179 patients with a presumptive diagnosis of polycythemia vera or essential thrombocythemia. Hematocrit showed better diagnostic accuracy than hemoglobin. Hemoglobin over 18.5 g/dL in males or over 16.5 g/dL in females showed a high specificity indicating that red cell mass study could be avoided in such cases, but it showed low sensitivity leading to 46% false negatives. The best value of hematocrit to indicate a red cell mass study was 0.50 L/L in males (specificity 75%, sensitivity 87.5%) and 0.48 L/L in females (specificity 73%, sensitivity 94%). Lowering the hematocrit threshold to 0.48 L/L in males increased sensitivity up to 95%. A red cell mass study should be performed in patients with suspected diagnosis of essential thrombocythemia or polycythemia vera and with hematocrit between 0.48 L/L and 0.52 L/L.

Background: Bernard-Soulier syndrome is a very rare form of inherited thrombocytopenia that derives from mutations in GPIbα, GPIbβ, or GPIX and is typically inherited as a recessive disease. However, some years ago it was shown that the monoallelic c.515C>T transition in the GPIBA gene (Bolzano mutation) was responsible for macrothrombocytopenia in a few Italian patients., Design and Methods: Over the past 10 years, we have searched for the Bolzano mutation in all subjects referred to our institutions because of an autosomal, dominant form of thrombocytopenia of unknown origin., Results: We identified 42 new Italian families (103 cases) with a thrombocytopenia induced by monoallelic Bolzano mutation. Analyses of the geographic origin of affected pedigrees and haplotypes indicated that this mutation originated in southern Italy. Although the clinical expression was variable, patients with this mutation typically had a mild form of Bernard-Soulier syndrome with mild thrombocytopenia and bleeding tendency. The most indicative laboratory findings were enlarged platelets and reduced GPIb/IX/V platelet expression; in vitro platelet aggregation was normal in nearly all of the cases., Conclusions: Our study indicates that monoallelic Bolzano mutation is the most frequent cause of inherited thrombocytopenia in Italy, affecting 20% of patients recruited at our institutions during the last 10 years. Because many people from southern Italy have emigrated during the last century, this mutation may have spread to other countries.

Background: The current gold-standard for diagnosing heparin-induced thrombocytopenia is the detection of platelet-activating antibodies by means of functional assays which, since they are time consuming and not widely available, are not suited to guiding acute treatment decisions. The objective of our study was to assess the ability of more rapid immunoassays to predict the presence of functionally relevant anti-platelet factor 4/heparin-antibodies., Design and Methods: We analyzed 1,291 of 1,383 (93.4%) patients consecutively evaluated for suspected heparin-induced thrombocytopenia at our institution. Clinical pre-test probability was defined by the 4T-score. Anti-platelet factor 4/heparin-antibodies were measured with three immunoassays (ID-H/PF4-PaGIA, Asserachrom-HPIA, and GTI-PF4) and their functional relevance was assessed by a two-point heparin-induced platelet aggregation test. Performance of the immunoassays was evaluated by receiver operating characteristic analysis., Results: Among 1,291 patients, 96 (7.4%) had a positive heparin-induced platelet aggregation-test: 7 of 859 (0.8%) with a low, 50 of 358 (14.0%) with an intermediate, and 39 of 74 (52.7%) with a high 4T-score. Receiver operating characteristics analysis indicated that best immunoassay thresholds for predicting a positive platelet aggregation test were: Titer of 4 or more (ID-H/PF4-PaGIA), optical density more than 0.943 (Asserachrom-HPIA) and more than 1.367 (GTI-PF4). A 100% negative predictive value was observed at the following thresholds: Titer of 1 or under (ID-H/PF4-PaGIA), optical density less than 0.300 (Asserachrom-HPIA) and less than 0.870 (GTI-PF4). A 100% positive predictive value was reached only by ID-H/PF4-PaGIA, at titers of 32 or over. Positive and negative likelihood ratios were calculated for results between the thresholds with 100% negative or positive predictive value., Conclusions: We show that: i) negative and weak positive results of immunoassays detecting anti-platelet factor 4/heparin-antibodies exclude heparin-induced thrombocytopenia; ii) anti-platelet factor 4/heparin-antibody titers of 32 or over (ID-H/PF4-PaGIA) have a 100% positive predictive value for functionally relevant antibodies; iii) combining the clinical pre-test probability with the likelihood ratio of intermediate immunoassay results allows assessment of post-test probability for heparin-induced thrombocytopenia in individual patients.

Primary myelofibrosis shows histological and pathogenetic overlap with essential thrombocythemia and polycythemia vera. Several diagnostic classifications have been proposed for primary myelofibrosis, although little is known about their clinical utility. In a comparison of three recent classifications, overall concordance was 79%. Inclusion of raised serum lactate dehydrogenase categorized 9% of patients as primary myelofibrosis when other criteria were not met. Although mean serum lactate dehydrogenase levels were higher in patients with primary myelofibrosis, levels were also increased in the majority of patients with essential thrombocythemia or polycythemia vera, and significant overlap was observed. A positive correlation with higher leukocyte and platelet count, and disease duration in primary myelofibrosis, suggests that serum lactate dehydrogenase is a biomarker for disease bulk and/or cellular proliferation. In conclusion, raised lactate dehydrogenase lacks specificity for primary myelofibrosis, consistent with the concept of a phenotypic continuum between essential thrombocythemia, polycythemia vera and primary myelofibrosis.

Background: Lack of adequate post-transfusion platelet count increments - platelet refractoriness - is a complication of chronic platelet support shown by 5-15% of chronic platelet recipients. INFORMATION SOURCES. To review the frequency, diagnosis, management and cost of platelet refractoriness, particularly as described in English literature published during 2000-2004 and searched with Pubmed., Results: Refractoriness is usually defined as the occurrence of 2-3 post-transfusion platelet count increments, corrected for the patient's size and number of administered platelets, at 10-60 minutes and at 18-24 hours post-transfusion below 4,500-5,000 and 2,500 platelets per microliter respectively. In most cases refractoriness is associated with clinical and pharmacological causes. In those cases in which refractoriness is due to immune factors, anti-HLA antibodies are most frequently implicated. Validated strategies to select effective platelets for alloimmunized refractory patients include the selection of HLA-matched platelet donors from HLA-typed donor registries and the use of manual or automated platelet cross-matching. Both strategies, which require significant organizational and financial resources, can provide successful platelet support in about 2/3 of transfusions., Interpretation and Conclusions: Unlike the less frequent cause of platelet refractoriness (anti-HLA alloimmunization) whose detrimental effect can be overcome by using HLA compatible platelets, the main causes of platelet refractoriness (patient's poor clinical condition and the use of drugs affecting platelet survival and function) remain largely unresolved.

Background and Objectives: The Italian Gruppo di Studio delle Piastrine recently developed a diagnostic algorithm to assist clinicians in the diagnosis of inherited thrombocytopenias. This algorithm is based on the simplest possible diagnostic investigations and can also be used in centers that are not highly specialized. The aim of the present study was to validate this diagnostic algorithm by applying it to a case series of genetic thrombocytopenias., Design and Methods: The diagnostic algorithm was applied retrospectively to 46 consecutive patients observed during the last five years at a single institution. Twenty-eight were affected by defined illnesses or their variants, while 18 had a disorder that did not fit the criteria for any known genetic thrombocytopenia. The study was based on the evaluation of clinical records and laboratory tests., Results: The diagnostic algorithm recognized: 4 homozygous and 4 heterozygous Bernard-Soulier syndromes, 11 MYH9-related diseases, one von WillebrandOs disease type 2B, one gray platelet syndrome and one X-linked thrombocytopenia with thalassemia. Moreover, it identified 4 patients with the clinical and laboratory features of heterozygous Bernard-Soulier syndrome not caused by mutations in the coding region of the GPIbalpha, GPIbbeta, GPIX or GPV genes, and two patients with the clinical phenotype of MYH9-related disease but without MYH9 mutations. Since the diagnostic flow chart did not allow prompt recognition of two subjects with MYH9-related disease, we introduced a small change to the previously proposed flow chart to obviate this defect., Interpretation and Conclusions: The diagnostic algorithm correctly diagnosed 26 of 28 patients with known disorders or phenotypic variants of known disorders. By a simple modification of the investigation sequence, its sensitivity reached 100%. The algorithm also identified 18 patients with new, as yet uncharacterized forms of genetic thrombocytopenia.

The Sebastian syndrome (SS) is a MYH9-related disorders, which are an extremely infrequent group of four autosomal dominant illnesses. SS consist of giant platelets, leukocyte inclusions and thrombocytopenia. To our knowledge, there are no case reports of this syndrome in South America. The propositus was a 35-year-old Argentine woman with a history of purpuric lesions in her lower limbs and thrombocytopenia. Idiopathic thrombocytopenia purpura (ITP) was previously diagnosed, but she did not respond to treatment with steroids. Family history failed to provide any evidence of hearing loss, easy bruising, nephritis, renal failure or cataracts. The patient and 11 members of her family were evaluated. The diagnosis of SS was established by demonstrating giant platelets, thrombocytopenia and leukocyte inclusions in peripheral smear in two relatives and by peripheral smear and electronic microscopy in the propositus. MYH9-related disorders should be suspected whenever a patient has a low platelet count or a bleeding diathesis of unknown origin. In these cases, the history, carefully peripheral smear exam, immunocytochemistry and electronic microscopy will be of great help. Differentiation ITP with SS is important to avoid unnecessary diagnostic studies and treatments.

Background: Although no epidemiological study has so far been performed, inherited thrombocytopenias are considered to be very rare based on the number of case reports in the literature. However, diagnosis of these disorders is often difficult and requires competences that are limited to specialized centers. We, therefore, suspect that inherited thrombocytopenias are underreported because their diagnosis is often missed., Objectives: The aim of this study was to develop a diagnostic algorithm that utilizes the simplest possible diagnostic tests and that can also be used in centers that are not highly specialized., Methods: The basic features of the diagnostic algorithm (definition and classification of hereditary thrombocytopenias, laboratory tests, sequence of investigations) were discussed by the members of the Italian Gruppo di Studio delle Piastrine during two meetings held in October 2000 and 2001. The sources of information were literature and personal experience. The final proposed diagnostic algorithm was produced by the authors of this paper and approved during a third meeting in May 2002., Perspectives: A definite diagnosis for patients with hereditary thrombocytopenias serves many purposes. It is essential to define the prognosis of the patients and to identify the best therapeutic approach. It also defines the risk of transmitting the disorder to progeny and, in many cases, allows prenatal diagnosis. Finally, several pieces of evidence indicate that not all the genetic thrombocytopenias have yet been identified. Exclusion of known disorders by accurate investigation is, therefore, the starting point for the discovery of these new illnesses.

Background and Objectives: Despite an extensive search for a definitive diagnostic assay for platelet autoantibodies, the laboratory diagnosis of immune thrombocytopenia (ITP) still remains a clinical challenge. Data in the literature have so far demonstrated that measurement of platelet-associated IgG (PAIgG) is sensitive, especially when flow cytometry is employed, but lacks adequate specificity. Measuring specific autoantibodies by antigen capture techniques increases specificity, but a large part of patients escape autoantibodies detection by such means too. The aim of the present study was to compare the diagnostic value of PAIgG with a modified antigen capture ELISA (MACE) in patients with primary and secondary immune thrombocytopenia and in patients with non-immune thrombocytopenia., Design and Methods: One hundred and four patients with a platelet count lower than 100x109/L were studied. Forty-two patients had primary ITP (P-ITP), 23 patients had ITP secondary to other immune diseases (S-ITP) and 39 patients had thrombocytopenia due to decreased platelet production (non-immune; NITP). PAIgG was measured by immunofluorescent flow cytometry, whereas specific platelet-associated autoantibodies (against GP IIb/IIIa, Ib/IX, Ia/Ia) were measured by a commercially available modified antigen capture assay (MACE, GTI, USA)., Results: The sensitivity of the PAIgG assay for ITP was 60%, the specificity was 77%, the positive predictive value was 81% and the negative predictive value was 54%. The sensitivity of MACE was 60%, specificity was 97%, the positive predictive value 97% and the negative predictive value 59%. We found a 73% concordance between PAIgG and MACE assays. Both PAIgG and MACE had significantly greater sensitivity in S-ITP than in P-ITP., Interpretation and Conclusions: Forty percent of patients with clinically diagnosed immune thrombocytopenia had no detectable platelet autoantibodies, possibly because of intrinsic methodological detection problems, different stages of disease, or absence of a true immune etiology.

Background and Objective: Flow cytometry (FC) to identify platelet-associated (PA) immunoglobulin (Ig) is a potentially useful diagnostic test for idiopathic thrombocytopenic purpura (ITP). However, the restricted application of PAIg measurement to thrombocytopenic populations primarily comprised of ITP patients will artificially enhance the test's diagnostic specificity. For this reason, we performed a prospective study in which the results of a sensitive technique for detecting PAIg, as is FC, were correlated to the cause of the thrombocytopenia., Design and Methods: A total of 118 patients with platelet counts <100 x 10(9)/L and 30 normal donors with a platelet count >200 x 10(9)/L were studied for PAIg employing a flow cytometer. Forty-two children and 20 adults were diagnosed as having immune thrombocytopenia and 27 children and 29 adults had nonimmune thrombocytopenia of different etiology., Results: Raised levels of PAIg were found in 56/62 patients with immune thrombocytopenia and in 34/56 patients with non-immune thrombocytopenia. Diagnostic values of PAIg for the detection of immune thrombocytopenia were: sensitivity 90.3% and specificity 39. 3%. An enzyme-linked immunoabsorbant assay (ELISA) for the detection of autoantibodies to platelet glycoprotein (GP) complexes was used in adults, 9 with immune-related thrombocytopenia and 16 with non-immune thrombocytopenia, in order to determine the true non-specific nature of the positive PAIg test. By ELISA, 8/9 patients with immune thrombocytopenia and 7/16 with non-immune thrombocytopenic disorders showed autoantibodies to platelet GP complexes., Interpretation and Conclusions: PAIg detection by FC constitutes a sensitive but non-specific assay thus making it unnecessary and inappropriate for establishing the diagnosis of ITP.

Background and Objective: Thrombocytopenia of peripheral origin is basically due to platelet destruction or splenic sequestration. Thrombopoietin (TPO) regulates platelet production stimulating megakaryocyte proliferation and maturation. The evaluation of TPO levels may be a useful tool in the diagnosis of thrombocytopenias of unknown origin. We tried to determine the value of TPO levels in some thrombocytopenias classically considered as peripheral., Design and Methods: Serum TPO levels and platelet counts were measured in 32 thrombocytopenic patients with liver cirrhosis (LC) and 23 with chronic hepatitis C (CHC) viral infection, in 54 patients with a clinical and serological diagnosis of autoimmune thrombocytopenic purpura (AITP), and in 88 patients infected with the human immunodeficiency virus (HIV)., Results: Patients with LC, AITP and HIV had lower platelet counts than patients with CHC. The degree of thrombocytopenia did not, however, correlate with the TPO levels. HIV infected patients (246+/-304 pg/mL) and AITP patients (155+/-76 pg/mL) had higher TPO levels than controls (121+/-58 pg/mL). TPO levels in patients with CHC (125+/-40 pg/mL) did not differ from those in control subjects, but were slightly decreased in patients with LC (104+/-56 pg/mL)., Interpretation and Conclusions: Reduced TPO production could be involved in the development of thrombocytopenia in LC patients, but not in patients with early stages of CHC viral infection. HIV and AITP patients had slightly raised levels of TPO. As TPO levels are normal or slightly increased in most peripheral thrombocytopenias, these data alone are not sufficient to distinguish the different types of peripheral thrombocytopenia. They may, however, be a useful tool for differentiating some central and peripheral thrombocytopenias.

A young man with bleeding symptoms, mild thrombocytopenia and abundant marrow megakaryocytes was classified as having idiopathic thrombocytopenic purpura. Neither prednisone therapy nor splenectomy modified the clinical picture. Subsequently, a severe defect of platelet aggregation and release reaction was demonstrated. Fifteen months after the onset of bleeding symptoms, fever and hepatomegaly appeared and the diagnosis of T cell non Hodgkin lymphoma was made on the basis of a histologic review of paraffin sections of the spleen. Chemotherapy induced remission of the lymphoma, disappearance of bleeding symptoms and normalization of the platelet count and function.

In vitro EDTA-induced platelet aggregation is a fairly rare event but can have serious clinical consequences producing pseudothrombocytopenia and pseudoleukocytosis. Sixteen specimens with previously recognized EDTA-induced platelet aggregation were collected in a new anticoagulant-antiaggregant mixture containing trisodium citrate 17 mmol/l, pyridoxal 5'-phosphate 11.3 mmol/l and Tris 24.76 mmol/l (CPT mixture) and analyzed at various times after venepuncture with four hematological instruments: Coulter Counter S-Plus STKR, Technicon H6000, Technicon H1 and Ortho ELT-8. In CPT-anticoagulated specimens the signals and instrumental flags of platelet clumping were absent, and the platelet number correlated very well with a microscopic count from a finger stick drawn into Unopette. The complete blood count was very similar in "normal" hematological specimens either collected in K3. EDTA or in CPT, although Technicon H1 and Ortho3 ELT-8 required a suitable calibration for MCV and hematocrit in the latter mixture. Mean platelet volume was stable for up to 24 h only in CPT-collected specimens, if it was measured on a Coulter Counter S-Plus STKR. In routine hematological practice CPT can be an alternative anticoagulant to K3. EDTA, most suitable for automated complete blood count and useful in avoiding EDTA-induced platelet clumping.