Diagnosis of Inherited Platelet Disorders on a Blood Smear

Inherited platelet disorders (IPDs) are rare diseases featured by low platelet count and defective platelet function. Patients have variable bleeding diathesis and sometimes additional features that can be congenital or acquired. Identification of an IPD is desirable to avoid misdiagnosis of immune thrombocytopenia and the use of improper treatments. Diagnostic tools include platelet function studies and genetic testing. The latter can be challenging as the correlation of its outcomes with phenotype is not easy. The immune-morphological evaluation of blood smears (by light- and immunofluorescence microscopy) represents a reliable method to phenotype subjects with suspected IPD. It is relatively cheap, not excessively time-consuming and applicable to shipped samples. In some forms, it can provide a diagnosis by itself, as for MYH9-RD, or in addition to other first-line tests as aggregometry or flow cytometry. In regard to genetic testing, it can guide specific sequencing. Since only minimal amounts of blood are needed for the preparation of blood smears, it can be used to characterize thrombocytopenia in pediatric patients and even newborns further. In principle, it is based on visualizing alterations in the distribution of proteins, which result from specific genetic mutations by using monoclonal antibodies. It can be applied to identify deficiencies in membrane proteins, disturbed distribution of cytoskeletal proteins, and alpha as well as delta granules. On the other hand, mutations associated with impaired signal transduction are difficult to identify by immunofluorescence of blood smears. This review summarizes technical aspects and the main diagnostic patterns achievable by this method

The thrombopoietin-receptor agonist eltrombopag for the treatment of inherited thrombocytopenias

Inherited thrombocytopenias (ITs) are a group of rare disorders characterized by reduced platelet count and variable bleeding phenotype. IT patients often require platelet transfusions to raise platelet counts before surgery or invasive procedures. Moreover, some subjects have clinically significant spontaneous bleeding, which may benefit from a durable improvement of their thrombocytopenia. The hypothesis that thrombopoietin-mimetic drugs, which are currently approved for several forms of acquired thrombocytopenia, can increase platelet count in ITs is appealing, but evidence is scarce. In fact, only one prospective study enrolling patients affected with MYH9-related disease, MYH9-RD, has been conducted so far. In this trial, a short course of the oral thrombopoietin-receptor agonist eltrombopag was given to 12 patients, and 11 of them showed a significant increase in platelet count. From 2010, to all MYH9‐RD patients followed at our Institute who needed elective surgery and had a platelet count too low for a safe procedure, a short‐term eltrombopag cycle has been offered, and 11 consecutive surgical procedures (including major surgeries) were carried out after eltrombopag administration so far. Eltrombopag successfully and safely replaced platelet transfusions prior surgery in 10 out of 11 cases. We then conducted a novel prospective, phase II clinical trial to investigate the efficacy of eltrombopag in a wider group of ITs. We enrolled 24 patients affected with MYH9-RD, ANKRD26-related thrombocytopenia, (ANKRD26-RT), X-linked thrombocytopenia/ Wiskott-Aldrich syndrome, monoallelic Bernard-Soulier syndrome, or ITGB3-related thrombocytopenia. The average pre-treatment platelet count was 40.4 ×109/L. Patients received a 3- to 6-week course of eltrombopag in a dose-escalated manner. Of 23 patients evaluable for response, 11 (47.8%) achieved a major response (platelet count >100 ×109/L), ten (43.5%) a minor response (platelet count at least twice as much as the baseline value), and two patients (8.7%) did not respond. The average increase of platelet count compared to baseline was 64.5 ×109/L. Four patients with clinically significant spontaneous bleeding received a long-term eltrombopag administration (16 additional weeks): all of them obtained remission of mucosal hemorrhages, which persisted throughout the treatment period. The treatment was globally well tolerated: five patients reported mild adverse events and one a moderate adverse event. In our study, a variable extent of platelet response was observed, either among different forms of IT and among different patients suffering from the same disease. By exploiting a silk-based miniaturized 3D bone marrow tissue model, which recapitulates ex vivo platelet biogenesis of subjects with different ITs, we demonstrated that the ex vivo model can predict the in vivo clinical platelet response to eltrombopag in individual patients affected with MYH9-RD and ANKRD26-RT. In fact, the number of platelets recovered in the ex vivo model under standardized conditions - including exposure to eltrombopag - was significantly correlated with the increase in platelet count that we had observed in vivo in the same subjects after eltrombopag treatment. In conclusion, eltrombopag was safe and effective in increasing platelet count and reducing bleeding symptoms in different forms of IT. With regard to the less rare IT, i.e. MYH9-RD, short‐term eltrombopag should be considered as the first‐line treatment to prepare patients with severe thrombocytopenia for elective surgery. Despite these encouraging results, caution is recommended when using eltrombopag, particularly in patients with ITs predisposing to hematological malignancies. In a near future, tools that mimic the bone marrow ex vivo can be used to predict the individual response to this and others drugs stimulating platelet production, thus informing therapeutic choices in a personalized view.Inherited thrombocytopenias (ITs) are a group of rare disorders characterized by reduced platelet count and variable bleeding phenotype. IT patients often require platelet transfusions to raise platelet counts before surgery or invasive procedures. Moreover, some subjects have clinically significant spontaneous bleeding, which may benefit from a durable improvement of their thrombocytopenia. The hypothesis that thrombopoietin-mimetic drugs, which are currently approved for several forms of acquired thrombocytopenia, can increase platelet count in ITs is appealing, but evidence is scarce. In fact, only one prospective study enrolling patients affected with MYH9-related disease, MYH9-RD, has been conducted so far. In this trial, a short course of the oral thrombopoietin-receptor agonist eltrombopag was given to 12 patients, and 11 of them showed a significant increase in platelet count. From 2010, to all MYH9‐RD patients followed at our Institute who needed elective surgery and had a platelet count too low for a safe procedure, a short‐term eltrombopag cycle has been offered, and 11 consecutive surgical procedures (including major surgeries) were carried out after eltrombopag administration so far. Eltrombopag successfully and safely replaced platelet transfusions prior surgery in 10 out of 11 cases. We then conducted a novel prospective, phase II clinical trial to investigate the efficacy of eltrombopag in a wider group of ITs. We enrolled 24 patients affected with MYH9-RD, ANKRD26-related thrombocytopenia, (ANKRD26-RT), X-linked thrombocytopenia/ Wiskott-Aldrich syndrome, monoallelic Bernard-Soulier syndrome, or ITGB3-related thrombocytopenia. The average pre-treatment platelet count was 40.4 ×109/L. Patients received a 3- to 6-week course of eltrombopag in a dose-escalated manner. Of 23 patients evaluable for response, 11 (47.8%) achieved a major response (platelet count >100 ×109/L), ten (43.5%) a minor response (platelet count at least twice as much as the baseline value), and two patients (8.7%) did not respond. The average increase of platelet count compared to baseline was 64.5 ×109/L. Four patients with clinically significant spontaneous bleeding received a long-term eltrombopag administration (16 additional weeks): all of them obtained remission of mucosal hemorrhages, which persisted throughout the treatment period. The treatment was globally well tolerated: five patients reported mild adverse events and one a moderate adverse event. In our study, a variable extent of platelet response was observed, either among different forms of IT and among different patients suffering from the same disease. By exploiting a silk-based miniaturized 3D bone marrow tissue model, which recapitulates ex vivo platelet biogenesis of subjects with different ITs, we demonstrated that the ex vivo model can predict the in vivo clinical platelet response to eltrombopag in individual patients affected with MYH9-RD and ANKRD26-RT. In fact, the number of platelets recovered in the ex vivo model under standardized conditions - including exposure to eltrombopag - was significantly correlated with the increase in platelet count that we had observed in vivo in the same subjects after eltrombopag treatment. In conclusion, eltrombopag was safe and effective in increasing platelet count and reducing bleeding symptoms in different forms of IT. With regard to the less rare IT, i.e. MYH9-RD, short‐term eltrombopag should be considered as the first‐line treatment to prepare patients with severe thrombocytopenia for elective surgery. Despite these encouraging results, caution is recommended when using eltrombopag, particularly in patients with ITs predisposing to hematological malignancies. In a near future, tools that mimic the bone marrow ex vivo can be used to predict the individual response to this and others drugs stimulating platelet production, thus informing therapeutic choices in a personalized view

Platelet Counting and Measurement of Platelet Dimensions

Since the introduction of automated blood cell analyzers, platelet counting and platelet dimension measurements are greatly improved in accuracy and disposability. No doubts in considering their relevance evident in routine clinical practice, particularly in diagnosis and management of many bleeding and thrombotic imbalances. Besides the impedance-based one, the first performed, and to date the most diffused, several others automated counting methods have been developed making platelet recognition increasingly more accurate. Platelet counting is now obtained not only by size but also by density and surface glycoproteins’ expression. Although these technological improvements, spurious abnormalities of platelet count such as pseudothrombocytopenia and pseudothrombocytosis can still occur, posing serious diagnostic difficulties. Therefore, in few selected clinical settings, the manual microscopy techniques are still considered of great utility to correctly identify and enumerate platelets. Moreover, the widespread automated cell blood counters make today available additional platelet parameters like the mean platelet volume and the amount of young platelets, calculated as reticulated platelets or immature platelet fraction. Although methodological issues remain to be solved, the availability of these further platelets’ parameters can also drive effectively the diagnosis of common or rare platelet disorders such as immune thrombocytopenia and inherited thrombocytopenias. The relevance in diagnosis and prognosis of these parameters is still under investigations in a large number of platelet diseases and in disorders not primarily affecting platelets

Personalized reference intervals for platelet count reduce the number of subjects with unexplained thrombocytopenia

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Spontaneous splenic rupture due to extramedullary haematopoiesis in a patient with inherited thrombocytopenia

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A novel homozygous <i>GFI1B </i>variant in 2 sisters with thrombocytopenia and severe bleeding tendency

Genetic variants in growth factor-independent 1B (GFI1B), encoding transcription factor GFI1B, are causative of platelet-type bleeding disorder-17. Presently, 53 cases of GFI1B associated inherited thrombocytopenia (IT) have been published, however only three were homozygous. The bleeding- and platelet phenotypes of these patients depend on location and inheritance pattern of the GFI1B variant. We report a novel homozygous GFI1B (Thr174Ile) variant located in the first Zinc finger domain of GFI1B in two sisters of Palestinian ancestry born to consanguineous parents. They experienced severe bleeding tendency at moderately reduced platelet counts. Flow cytometry and immunofluorescent microscopy confirmed the diagnostic features of GFI1B associated IT: a reduced content of alpha granules and aberrant expression of the stem cell marker CD34 on platelets. Transcription factor GFI1B is differentially expressed during hemato- and lymphopoiesis. In addition, to platelet function investigations, we present results of lymphoid subgroup analyses and deformability of red cells measured by ektacytometry

Validation of immunofluorescence analysis of blood smears in patients with inherited platelet disorders

Background: Inherited platelet disorders (IPDs) are rare diseases characterized by reduced blood platelet counts and/or impaired platelet function. Recognizing IPDs is advisable but often challenging. The diagnostic tools include clinical evaluation, platelet function tests, and molecular analyses. Demonstration of a pathogenic genetic variant confirms IPDs. We established a method to assess the platelet phenotype on blood smears using immunofluorescence microscopy as a diagnostic tool for IPDs. Objectives: The aim of the present study was to validate immunofluorescence microscopy as a screening tool for IPDs in comparison with genetic screening. Methods: We performed a blinded comparison between the diagnosis made using immunofluorescence microscopy on blood smears and genetic findings in a cohort of 43 families affected with 20 different genetically confirmed IPDs. In total, 76% of the cases had inherited thrombocytopenia. Results: Immunofluorescence correctly predicted the underlying IPD in the vast majority of patients with 1 of 9 IPDs for which the typical morphologic pattern is known. Thirty of the 43 enrolled families (70%) were affected by 1 of these 9 IPDs. For the other 11 forms of IPD, we describe alterations of platelet structure in 9 disorders and normal findings in 2 disorders. Conclusion: Immunofluorescence microscopy on blood smears is an effective screening tool for 9 forms of IPD, which include the most frequent forms of inherited thrombocytopenia. Using this approach, typical changes in the phenotype may also be identified for other rare IPDs

The Copenhagen founder variant GP1BA c.58T&gt;G is the most frequent cause of inherited thrombocytopenia in Denmark

BACKGROUND: The classic Bernard‐Soulier syndrome (BSS) is a rare inherited thrombocytopenia (IT) associated with severe thrombocytopenia, giant platelets, and bleeding tendency caused by homozygous or compound heterozygous variants in GP1BA, GP1BB, or GP9. Monoallelic BSS (mBSS) associated with mild asymptomatic macrothrombocytopenia caused by heterozygous variants in GP1BA or GP1BB may be a frequent cause of mild IT. OBJECTIVE: We aimed to examine the frequency of mBSS in a consecutive cohort of patients with IT and to characterize the geno‐ and phenotype of mBSS probands and their family members. Additionally, we set out to examine if thrombopoietin (TPO) levels differ in mBSS patients. PATIENTS/METHODS: We screened 106 patients suspected of IT using whole exome‐ or whole genome sequencing and performed co‐segregation analyses of mBSS families. All probands and family members were phenotypically characterized. Founder mutation analysis was carried out by certifying that the probands were unrelated and the region around the variant was shared by all patients. TPO was measured by solid phase sandwich ELISA. RESULTS: We diagnosed 14 patients (13%) with mBSS associated with heterozygous variants in GP1BA and GP1BB. Six unrelated probands carried a heterozygous variant in GP1BA (c.58T>G, p.Cys20Gly) and shared a 2.0 Mb region on chromosome 17, confirming that it is a founder variant. No discrepancy of TPO levels between mBSS patients and wild‐type family members (P > .05) were identified. CONCLUSION: We conclude that the most frequent form of IT in Denmark is mBSS caused by the Copenhagen founder variant