Platelet Phenotyping and Function Testing in Thrombocytopenia

Abstract

Patients who suffer from inherited or acquired thrombocytopenia can be also affected by platelet function defects, which potentially increase the risk of severe and life-threatening bleeding complications. A plethora of tests and assays for platelet phenotyping and function analysis are available, which are, in part, feasible in clinical practice due to adequate point-of-care qualities. However, most of them are time-consuming, require experienced and skilled personnel for platelet handling and processing, and are therefore well-established only in specialized laboratories. This review summarizes major indications, methods/assays for platelet phenotyping, and in vitro function testing in blood samples with reduced platelet count in relation to their clinical practicability. In addition, the diagnostic significance, difficulties, and challenges of selected tests to evaluate the hemostatic capacity and specific defects of platelets with reduced number are addressed.

Keywords: bleeding; flow cytometry; platelet count; platelet disorders; platelet function tests; thrombocytopenia.

Figure 1

Current view of important thrombocytopenia-causing genes associated with platelet function defects. Gene names (blue italic font), activating responses (black arrow), inhibitory effects (red blunted arrow), and gene mutation consequences (red line) are indicated, and further details are listed in Table S1 [18,19,20,21,22]. MYH9-associated platelet dysfunction according to a case report [23]. VWF: Von Willebrand factor; ADP: adenosine diphosphate; PAR: proteinase-activated receptor; PLA₂: phospholipase A₂; PKA: protein kinase A; STIM-1: stromal interaction molecule-1; TMEM16F: transmembrane protein 16F; TxA₂: thromboxane A₂; TP: TxA₂ receptor; AA: arachidonic acid; PGI₂: prostacyclin; DTS: dense tubular system; GP: glycoprotein; PS: phosphatidylserine.

Figure 2

Flow cytometric analysis of the human platelet phenotype ex vivo and functional capacity in vitro in thrombocytopenia. Flow cytometry allows for a comprehensive analysis of common platelet phenotype and function defects in samples of whole blood and platelet-rich plasma, even with a very low platelet count. Commonly observed platelet granule defects (e.g., storage pool deficiency and primary secretion defects) in inherited and acquired thrombocytopenias can be detected by the surface expression of granule membrane markers stained by fluorochrome-conjugated antibodies (*) or by the antibody staining of the α-granule cargo protein TSP-1 and δ-granule cargo molecules ADP and ATP with mepacrine. Quantitative defects in platelet surface receptors, as prominently observed in acquired thrombocytopenias, are detected by staining with fluorochrome-conjugated antibodies (*), too. The use of different platelet agonists enables the detection of activated integrin αIIbβ3 (antibody PAC-1 (*) or the binding of fluorochrome-conjugated fibrinogen, indicative of platelet aggregation), VWF-binding (anti-VWF antibody staining (*), indicative of VWF-mediated platelet agglutination and biallelic BSS, platelet type VWD, and acquired VWD), and the exocytosis of granules by staining of granule membrane proteins with fluorochrome-conjugated antibodies (*). *Antigens of platelet receptors and granule membranes detected by fluorochrome-conjugated antibodies. Fluorochrome-conjugated annexin-V or lactadherin is used to quantify anionic phospholipid exposure ex vivo or in response to agonists, which determines the recruitment of coagulation factors, leading to platelet-based thrombin generation. Distinct signaling defects are detected by phospho-specific antibodies after permeabilization of the platelet surface membrane. Phospho-specific antibodies against phosphorylated VASP at S239 detect a cytosolic increase in levels of inhibitory cAMP, leading to the activation of PKA. A defective release of Ca²⁺ ions from intracellular Ca²⁺ stores (e.g., dense tubular system (DTS)) can be detected by Ca²⁺-sensitive fluorescent Fluo and Fura dyes. VWF: Von Willebrand factor; cAMP: cyclic adenosine monophosphate; ADP: adenosine diphosphate; CRP-XL: cross-linked collagen-related peptide; PAR: proteinase-activated receptor; PKA: protein kinase A; TxA₂: thromboxane A₂; TP: TxA₂ receptor; AA: arachidonic acid; PLA₂: phospholipase A₂; PKA: protein kinase A; STIM-1: stromal interaction molecule-1; PGI₂: prostacyclin; DTS: dense tubular system; GP: glycoprotein; PS: phosphatidylserine; TRAP-6: thrombin receptor activating peptide-6; TLT-1: trem-like transcript-1; TMEM16F: transmembrane protein 16F; TSP-1: thrombospondin-1; LAMP-1: lysosome-associated membrane glycoprotein-1; PMA: phorbol 12-myristate 13-acetate; VASP: vasodilator-stimulated phosphoprotein; PKC: protein kinase C. PKA and PKC are some examples of protein kinases, but there are many more protein kinases involved.

Figure 3

Platelet phenotype and function analysis of a patient with biallelic Bernard–Soulier syndrome (BSS) by flow cytometry and calibrated automated thrombography. (a) Representative flow cytometry histogram of platelets labelled with anti-GPIbα antibody SZ2-fluorescein isothiocyanate (FITC) from a donor with GPIbα-deficiency (biallelic Bernard-Soulier syndrome (BSS)) in comparison to platelets from a day control and to platelets labelled with immunoglobulin G1 (IgG1)-FITC as negative control and flow cytometric detection of antigen binding sites (ABSs) of fluorochrome-labelled antibodies against major receptors on platelets in citrated whole blood compared to healthy controls (n = 10, means ± SD). (b) Flow cytometric analysis of ristocetin-induced VWF-binding (labeling with anti-VWF-FITC antibody) to platelets from BSS patient compared to controls (n = 10, means ± SD). (c) Thrombogram (calibrated automated thrombography) of thrombin-induced thrombin generation in platelet-rich plasma from a BSS patient compared to a day control with adjusted platelet mass. MFI: mean fluorescence intensity.

Figure 4

Platelet function analysis of a patient with the May–Hegglin anomaly (pathogenic MYH9 variant E1841K) by flow cytometry and calibrated automated thrombography. (a) Flow cytometric analysis of mepacrine uptake in resting patient platelets and thrombin- or convulxin (GPVI-agonist)-induced mepacrine release compared to a day control. (b) Flow cytometric analysis δ-granule/lysosome exocytosis expressed as CD63 surface expression of patient and day control platelets in response to increasing concentrations of thrombin and convulxin, respectively. (c) Thrombogram (calibrated automated thrombography) and quantitation of the endogenous thrombin potential (ETP) of tissue factor (TF) or thrombin-stimulated patient and control platelets in platelet-rich plasma; three replicates; n = 3; * p < 0.05 versus corresponding controls. AU: arbitrary units.