Platelets are anucleate cells that can be studied by benchtop flow cytometers and today there are several types of flow cytometric assays for platelets. Platelet structure studies in clinical flow cytometry mostly target rare congenital platelet glycoprotein deficiencies (e.g., Glanzmann-thrombasthenia, and Bernard-Soulier syndrome) that can be considered as the prototypes for platelet flow analysis, since the identification of complete or partial deficiency of these molecules provides the proper diagnosis. Another area of platelet flow cytometry is the detection of activated platelets. Mostly it means the gold standard platelet P-selectin assay or the investigation of PAC-1 binding-an assay where the activation dependent epitope of the GPIIb/IIIa receptor is investigated-but the measurement of platelet-leukocyte aggregates and microparticle analysis have gained increasing role. A unique platelet subtype is the subgroup designated as coated platelets that are formed by the simultaneous activation with collagen and thrombin. In clinical practice further functional assays are also informative tests: the monitoring of clopidogrel resistance by measuring the intracellular phosphorylated VASP and identifying heparin induced thrombocytopenia (HIT) by the use of patient plasma. The flow cytometric HIT test utilize the detection of phosphatidylserine expression on normal platelets or the formation of microparticles in the presence of the patient's plasma and therapeutic concentration of heparin, thus it seems reasonable to suggest it for the study of the functionality of the HIT antibodies. Nevertheless, there are also limitations to these assays as several of them require fresh samples, thus sample transport to specialized laboratories is not always feasible.

Monoclonal B-cell lymphocytosis (MBL) is a type of chronic lymphocytic leukemia (CLL) and offers a unique window into the initial stages of leukemogenesis. Even though the vast majority of MBL patients are clinically stable, a subgroup develops CLL, which highlights the significance of understanding the molecular processes that modulate this development. Recent developments in molecular genetics have shown recurrent NOTCH1, SF3B1, TP53, and ATM mutations that determine clonal dynamics and the development of disease. In a like manner, the epigenetic dysregulation, where DNA methylation patterns and chromatin remodeling are aberrant, plays a role in lineage plasticity and altered outlines of gene expression during the transition phase between MBL and CLL. The significant role of non-coding RNAs (ncRNAs) in this development is intensively highlighted by growing evidence. MicroRNAs (miR-15a/16-1, miR-34a, and miR-155) and long non-coding RNAs (DLEU2, MALAT1, NEAT1) control apoptosis, DNA damage response and immune signaling, and transcriptional networks involved in leukemogenesis, respectively. The circular RNA is involved as a sponge in competing endogenous RNA interactions and enhances oncogenic signaling. Combining genetic, epigenetics, and ncRNA signatures provides an attractive chance to optimize risk stratification of MBL, forecast progression to CLL and establish minimally invasive biomarkers in liquid biopsy format. Though the application of ncRNA-targeted therapeutics is mostly experimental, initial translational research indicates that there are lines along which molecularly guided therapy can be pursued. This review provides a synthesis of the existing knowledge of the molecular evolution of MBL to CLL with a special focus on the interactions between DNA modifications, epigenetic control, and ncRNA biology and outlines how these findings can be used to provide more accurate diagnostics, prognostication, and subsequent targeted treatment.

Unfractionated heparin (UFH) has long been an anticoagulant of choice. Despite the increasing use of low molecular weight heparin derivatives and more recently direct oral anticoagulants, UFH is still prescribed to prevent or treat thrombosis in specific populations at high risk of thrombosis or bleeding, including patients with severe renal impairment, critically ill patients, or those undergoing cardiac catheterization/surgery. Due to its narrow therapeutic index, with a risk of hemorrhage in case of overdosing and a risk of thrombosis in case of underdosing, monitoring its anticoagulant activity is considered standard of care even though scientific data remains limited. Despite being used for decades, some unanswered questions remain, particularly regarding the biological monitoring of its efficacy. Which assay can be used? In practice, the most widely used tests are plasma-based, such as the aPTT, a clotting assay, or the anti-Xa activity, a chromogenic substrate-based assay, whereas whole blood tests such as the ACT are limited to specific situation such as extracorporeal circulation. Which therapeutic ranges should be used? An aPTT prolongation of 1.5-2.5 × control, or an anti-Xa activity of 0.30-0.70 IU/mL, corresponding to 0.2-0.4 U/mL by protamine titration, ranges are commonly used, despite limited evidence-based data. Furthermore, these assays are not standardized due to some degree of heterogeneity in the sensitivity of reagents to UFH. Moreover, some pre-analytical questions remain, particularly regarding the collection tube to be used for example, citrate or CTAD anticoagulant solution, full- or partial-draw (half-filled) tubes, and the maximum time between collection and analysis.

Bleeding disorder of unknown cause (BDUC) constitutes the largest group of patients presenting with a mild-to-moderate bleeding tendency in tertiary care settings. Their clinical bleeding phenotype is characterized by mucocutaneous bleeding, as well as bleeding following surgical challenges or childbirth, and is associated with impaired health-related quality of life. Iron deficiency, with or without anemia, is common, particularly among women, who comprise up to 80% of BDUC cohorts and frequently report heavy menstrual bleeding. Diagnosing BDUC requires a rigorous exclusion of established hemostatic and non-hemostatic causes of bleeding. Common inherited bleeding disorders, including coagulation factor deficiencies (CFD), von Willebrand disease (VWD), and platelet function disorders (PFD), must be systematically excluded. CFD evaluation should extend beyond routine assays (prothrombin time, activated thromboplastin time, thrombin time), as clinically relevant mild reductions in factors VIII, IX, and XI may occur despite normal screening tests; and factor XIII deficiency is not detected by global assays. VWD assessment mandates measurement of VWF antigen and activity, with additional studies to define subtype when indicated. For PFD, light transmission aggregometry remains the reference gold standard. Substantial diagnostic overlap exists among these entities and BDUC, and repeated testing is often required. Investigations for rare causes such as hyperfibrinolysis or excess natural anticoagulants are typically limited to patients with distinctive phenotypes or strong family histories. Although the pathogenesis of BDUC remains incompletely understood, continued investigation into platelet biology, global hemostasis, and vascular contributions holds promise for uncovering therapeutic targets, ultimately improving management for this prevalent yet understudied condition.

Heritable platelet function disorders (HPFD) are a diverse group of bleeding disorders characterised by a primary qualitative defect in platelet function rather than platelet number. HPFD may be broadly categorised according to the severity of bleeding, with Glanzmann thrombasthenia and Bernard Soulier syndrome classically considered severe HPFD, with well-described clinical and laboratory phenotypes. The remaining non-severe HPFD are less completely characterised and provide additional challenges in diagnosis despite advances in standardising diagnostic approaches for HPFD. Distinguishing patients with non-severe HPFD from patients who do not have a bleeding disorder may be aided by employing bleeding assessment tools early in patient assessment, although there may be limitations according to patient factors. Distinguishing patients with non-severe HPFD from those with acquired platelet function disorders and from those with other heritable disorders of haemostasis (including bleeding disorder of unknown cause) requires more sophisticated clinical and laboratory tools, including platelet light transmission aggregometry, secretion and flow cytometry assays, which can categorise many HPFD to the level of the defective platelet pathway and some to the level of an individual protein. The increasing availability of high-throughput genetic sequencing has made genetic diagnosis possible for some patients with non-severe HPFD; however, this is limited by difficulties with standardisation of laboratory phenotyping and the challenges of distinguishing pathogenic from benign genetic variation. This review will focus on updates in diagnostic approaches for patients with non-severe HPFD since the last IJLH education article on this topic in 2014.