Acquired Platelet Function Disorders Research Articles

Mepacrine Flow Cytometry Assay for the Diagnosis of Platelet δ-granule Defects: Literature Review on Methods-Towards a Shared Detailed Protocol.

Accurate assessment of platelet secretion is essential for the diagnosis of inherited or acquired platelet function disorders and more specifically in identifying δ-storage pool disease. Mepacrine, a fluorescent dye, specifically accumulates in platelet δ-granules. The mepacrine flow cytometry (mepacrine FCM) assay has been used for more than half a century in the clinical laboratory as a diagnostic tool for platelet δ-granule disorders. The assay requires a small volume of blood, can be performed in thrombocytopenic patients, provides rapid assessment of δ-granule content and secretion, and, thus, enables differentiation between storage and release defects. There is however a broad heterogeneity in methods, reagents, and equipment used. Lack of standardization and limited data on analytical and clinical performances have led the 2022 ISTH SSC (International Society on Thrombosis and Haemostasis Scientific and Standardization Committee) Subcommittee on Platelet Physiology expert consensus to rate this assay as simple but of uncertain value. Yet, the data used by experts to formulate the recommendations were not discussed and even not mentioned. Guidance for laboratory studies of platelet secretion assay would be very helpful for clinical laboratories and health authorities especially considering the implications of the new In Vitro Diagnostic Regulation in Europe. The purpose of the present work was to review the reported methodologies for the mepacrine FCM assay and to offer an example of detailed protocol. This would help standardization and pave the way for more rigorous comparative studies.

Autoimmune disorders of platelet function: systematic review of cases of acquired Glanzmann thrombasthenia and acquired delta storage pool disease.

Acquired platelet function disorders (PFD) are rare bleeding diseases that should be suspected in all patients with unexplained mucocutaneous bleedings of recent onset, with no previous history of haemorrhages, and with normal coagulation test and platelet count. Drug-induced platelet function bleeding disorders are the most frequent PFDs and can easily be identified on the basis of recent administration of platelet-inhibiting drugs. Apart from these, the most challenging acquired PFDs are those caused by autoimmune mechanisms. In fact, demonstration of autoantibodies inhibiting platelet function may be difficult in most non-specialised centres. Among autoimmune PFDs (aPFDs), acquired Glanzmann thrombasthenia (aGT), which is caused by autoantibodies that bind to platelet αIIbβ3 integrin, inhibiting its function, is the most frequent. aGT can be associated with underlying haematological malignancies or autoimmune diseases but can also be idiopathic. More rarely, other immune-mediated PFDs can occur, such as acquired delta storage pool disease (aδSPD). Treatment of aPFDs must rely on the control of acute and chronic bleedings, treatment of the underlying disease in secondary forms, and immunosuppressive treatment for autoantibody reduction or eradication. aPFDs may completely resolve upon treatment of any underlying disease that may be present. In primary aPFDs, and in the majority of secondary forms, treatment relies on immunosuppressive therapies.Here we present a systematic review of previously described immune-mediated aGT and aδSPD cases. Clinical and laboratory characteristics, treatments for the control of bleedings and for the eradication of autoantibodies, and responses to treatments are also discussed. Although no guidelines are available for the management of these very rare conditions, presentation of all cases reported so far can help clinicians in the diagnosis and treatment of these life-threatening diseases.

Acquired Glanzmann thrombasthenia: a rare disorder

Qualitative platelet function defects may be congenital, e.g., Glanzmann thrombasthenia, or acquired. Acquired platelet function disorders are more common than reported. Glanzmann thrombasthenia, an inherited platelet function disorder, is caused by mutations in the ITGA2B and ITGB3 genes encoding the αIIbβ3 integrin. An acquired form of GT is also seen, which is caused by antibodies to platelet integrin αIIbβ3. This affects fibrinogen binding and blocks platelet aggregation. These patients have low platelet count with a moderate-to-severe bleeding tendency, while some patients may have normal platelet counts. It is commonly found in association with autoimmune disorders, hematological malignancies, and infections. Glanzmann thrombasthenia-like state can also be seen in immune thrombocytopenic purpura (ITP) due to presence of antibodies to αIIbβ3. These patients present with severe bleeding even with mild thrombocytopenia. We describe a patient of ITP with borderline low platelet count and severe bleeding, who posed a diagnostic challenge. However, an accurate diagnosis and suitable management helped to avoid catastrophic bleeding.

Cytofluorimetric Platelet Analysis

Blood platelets are highly specialized cells that drive hemostatic events and tissue repair mechanisms at the site of vascular injury. Their peculiar morphology and certain functional characteristics can be analyzed by flow cytometry (FCM). Specifically, platelet activation, a hallmark of prothrombotic states and inflammatory conditions, is associated with changes in expression of both surface and intracellular antigens that are recognized by specific monoclonal antibodies. Assessment of platelet activation status as ex vivo or in vitro reactivity to specific agonists has become relevant in particular conditions (namely, cardiovascular diseases, hematological malignancies, monitoring of pharmacological antiaggregation). In addition, aberrant surface marker expression that characterizes inherited and acquired platelet function disorders is also detected by FCM. This review discusses the main applications of FCM in platelet analyses, which are relevant for both research and clinical settings.

Angeborene Thrombozytenfunktionsstörungen

Inherited thrombocytopathies are much less frequent in comparison to acquired platelet function disorders. However, congenital disorders can lead to severe bleeding tendency and are often not diagnosed. They are induced by different platelet defects based on disorders of platelet adhesion, receptors, secretion and signal transduction. In some cases they are associated with thrombocytopenia, giant platelets and various comorbidities. This article gives an overview regarding diverse defects, their diagnosis and treatment options.

Stellenwert der Thrombozytenfunktionsdiagnostik

Numerous laboratory tests are in use to detect congenital or acquired platelet function disorders. Platelet aggregometry, using ADP, collagen, arachidonic acid or ristocetin as inductor is the standard test system for diagnosis. It is also used to detect platelet non-response to antiplatelet therapy. Studies have demonstrated that laboratory assessment of platelet non response to aspirin or clopidogrel is associated with adverse outcomes, and they indicate the importance of adjusting antiplatelet therapy in patients with a low degree of platelet inhibition. Nevertheless, a standardized method for identifying these patients is still missing.

Therapeutic options for perioperatively acquired platelet dysfunctions

Increased intra-operative and postoperative blood loss might be caused by acquired platelet function disorders. In particular because conventional coagulation analyses and platelet count fail to detect impaired platelet function, implementation of bedside-tests for platelet function in the peri-operative period is desirable according to the results of retrospective studies. Following adequate adjustment of basic conditions of haemostasis (e.g. temperature, pH, Ca2+-concentration, haematocrit) a pharmacological approach with desmopressin (1-desamino-8-d-arginine vasopressin; DDAVP) or tranexamic acid potentially represents a low cost alternative to platelet transfusions with minor side effects.

Erworbene Plättchenfunktionsstörungen

Given the high consumption of pharmacological agents in western societies, it is not surprising at all that drugs represent the most common cause of acquired platelet dysfunction. While acetylsalicylic acid, clopigogrel and integrin alphaIIbbeta3 (GPIIb-IIIa) receptor antagonists are well-known as prototypes of antiplatelet drugs, other widely used agents including non-steroidal anti-inflammatory drugs, antibiotics, serotonin reuptake inhibitors, and volume expanders can also impair platelet function and cause or aggravate haemorrhages. Besides pharmacological agents, certain clinical conditions are often associated with qualitative platelet disorders and bleeding diathesis. Consequently, in contrast to inherited platelet disorders, acquired platelet function defects are much more frequent in clinical practice and deserve special attention. Their pathogenesis is widespread and heterogeneous with various, sometimes overlapping abnormalities. Moreover, acquired platelet dysfunctions can occur at any age and range in severity from mild to life-threatening haemorrhages. Due to their heterogeneity, acquired platelet function disorders will be classified and discussed according to the underlying clinical setting or disease.

Rekombinanter Faktor VIIa bei Patienten mit Plättchenfunktionsstörungen oder Thrombozytopenien

Apart from on-label indications, recombinant factor VIIa (rFVIIa) is increasingly administered for treatment of life threatening bleeding events when appropriate standard therapy fails. Case reports and short treatment series document the efficacy and safety of rFVIIa to achieve haemostasis in patients with platelet function disorders and thrombocytopenias of various origin. An established on-label indication for the use of rFVIIa is given in patients with Glanzmann thrombasthenia with refractoriness to transfusions of platelet concentrates. Bolus applications of rFVIIa at dosages between 80 and 120 microg/kg body weight every 1.5 to 3 h are also administered successfully in patients with Bernard-Soulier syndrome, platelet storage pool defects, and other acquired platelet function disorders. In patients with Glanzmann thrombasthenia, at least three bolus injections are required to achieve effective haemostasis. In approximately half of the patients with thrombocytopenias, a single bolus of rFVIIa has been shown to be sufficient in managing otherwise untreatable bleeding complications. In these patients, haemostasis was achieved even at platelet counts <20,000/microl, although the efficacy of rFVIIa increases at higher platelet concentrations.

Platelet function analyzer (PFA)‐100® closure time in the evaluation of platelet disorders and platelet function – a rebuttal

Platelet function analyzer (PFA)‐100® closure time in the evaluation of platelet disorders and platelet function – a rebuttal

Determination and Treatment of Disorders of Primary Haemostasis

SummaryPerioperative bleeding complications due to disorders of primary haemostasis are often underestimated. Routine determination of primary haemostasis is still problematic. The in vivo bleeding time (BT) shows low sensitivity and high variability. In this contribution the results and experiences with the IVBT having been obtained in various studies and during 10 years of routine use are reported. Patients and Methods: Blood donors before and after ASA ingestion, patients with thrombocytopenia as well as congenital and acquired platelet function disorders. Monitoring of desmopressin efficacy. IVBT with Thrombostat 4000 (tests with CaCl2 = TST-CaCl2 and ADP = TST-ADP) and PFA-100 (test cartridges with epinephrine = PFA-EPI and ADP = PFA-ADP). Results and Conclusions: IVBT becomes abnormal with platelet counts &lt;100,000/μl. With platelet counts &lt;50,000/μl the results are mostly outside the methodical range. IVBT proved clearly superior to BT in von Willebrand syndrome (vWS). All 16 patients with vWS were detected by PFA-EPI, whereas with BT 7 of 10 patients with moderate and 1 of 6 patients with mild forms of vWS were spotted. The majority of acquired and congenital platelet function disorders with relevant bleeding tendency were detectable by IVBT. Sometimes diagnostic problems arose in case of storage pool defect. Four to 12 h after ingestion of a single dose of 100 mg ASA the TST-CaCl2 became abnormal in all cases, the PFA-EPI only in 80%. However, the ASA sensitivity of TST-CaCl2 proved even too high when looking for perioperative bleeding complications in an urological study. Therefore, the lower ASS sensitivity of the PFA-100 seems to be rather advantageous for the estimation of a real bleeding risk. The good efficacy of desmopressin in the majority of cases with mild thrombocytopenia, congenital and acquired platelet function disorders and even ASS-induced platelet dysfunction could be proven by means of the IVBT. Thus IVBT may help to increase the reliability of the therapy. However, the IVBT with the PFA-100 is not yet fully developed. Nevertheless, routine use can be recommended when special methodical guidelines are followed.

Analysis of glycoprotein Ia, Ib, IIb and IV RNA in platelets: quantitative determination using fluorescence-based polymerase chain reaction.

The aim of this study was to analyze the RNA level of glycoprotein (GP) receptors in platelets. We have therefore established a quantitative fluorescence-based polymerase chain reaction (PCR) to analyze GP Ia, Ib, IIb, and IV RNA. Isolation of platelet RNA was performed by guanidium isothiocyanate/phenol chloroform extraction. An internal standard consisting of cRNA copies from plasmid pAW109 was included before reverse transcription in each RNA sample and PCR amplification was performed using fluorescence-labeled primers. Subsequently, PCR fragments were separated by gel electrophoresis and quantitation of the GP-specific fragments was done by measuring the fluorescence intensities in comparison to the internal standard. Relative amount of GP RNA/platelet were calculated taking into account the number of platelets used for isolation of platelet RNA and the platelet size as determined by flow-cytometric analysis. Using this method we analyzed the GPIa, Ib, IIb and IV RNA content of platelets in healthy blood donors. In parallel experiments the number of GP cell surface receptors was measured by flow-cytometric analysis and correlated with the GP-specific RNA content. This method may be useful to study the GP-specific RNA content in platelets as well as in other tissues, such as megakaryocytes, especially in patients with congenital or acquired platelet function disorders.