Investigation of Bleeding Disorders: When and How Should We Test Platelet Functions?

Abstract

Introduction

Inherited platelet disorders (IPDs) are rare conditions with diverse underlying pathophysiology which should be suspected in patients presenting with mucocutaneous bleeding or hemorrhages upon hemostatic challenges, in the presence or not of thrombocytopenia. Identifying IPDs is critical for providing appropriate care, preventing misdiagnosis, and avoiding unnecessary interventions, such as splenectomy. Syndromic IPDs, which may be associated with severe complications like kidney failure, infection, and malignancies, underscore the importance of accurate diagnosis and tailored management.

Results

Diagnosing IPDs remains challenging, requiring a comprehensive approach that integrates clinical assessment, evaluation of the bleeding history using standardized tools, like the ISTH-BAT, and first-line laboratory tests, such as light transmission aggregometry and flow cytometry. Second-line and specialized tests, including transmission electron microscopy, genetic analysis, and biochemical studies, may provide further insight in complex cases. Technological advancements, including multicolor flow cytometry and microfluidic tools, may in perspective improve IPD diagnostics by providing high-throughput and precise laboratory assays. In particular, mass cytometry and multi-omics may contribute to unraveling IPD pathophysiology, identifying novel markers, and refining disease classification. The application of artificial intelligence shows potential for improving diagnostic accuracy through the automated analysis of platelet morphology and function, from flow cytometry and digital microscopy assays, and for improving the understanding of pathogenic mechanisms of IPD through the examination of big data.

Conclusion

This review summarizes current IPD platelet function testing strategies, emphasizing the need for a structured, tiered approach and examining emerging technologies and AI applications that could revolutionize diagnostic workflows, leading to personalized care and to an expanded understanding of IPDs.

Keywords

flow cytometry - inherited platelet function disorders - light transmission aggregometry

Introduction

Inherited platelet disorders (IPDs) encompass a range of rare conditions, from mild to life-threatening, with diverse underlying pathophysiology. They can be divided into inherited thrombocytopenias (ITs), characterized by low platelet counts, and inherited platelet function disorders (IPFDs), characterized by dysfunctional platelets due to defects in receptors, granules, or signal transduction mechanisms.[1] However, the distinction between disorders of platelet number and function is often unclear because a significant number of IPFD often show also thrombocytopenia. IPDs typically present with mucocutaneous bleeding and exhibit significant phenotypic and genotypic heterogeneity. Some IPDs are syndromic, involving other organs or systems, and may be associated with adverse prognosis due to hematological malignancies, kidney failure, or pulmonary fibrosis.

Around 60 distinct IPD forms, linked to defects in at least 75 genes, have been described. The exact prevalence of IPDs, and in particular of IPFDs, remains uncertain due to underreporting and the lack of population-based studies. Estimates suggested a prevalence ranging from 2 per million to over 1 per 100.[2] In a worldwide survey carried out among expert centers a few years ago, approximately 14,000 patients underwent platelet function testing for a suspected IPFD yearly, with over 5,600 new diagnoses made, indicating that these disorders may be more common than previously thought.[3] Indeed, more recent studies, based on exome and genome sequencing of unselected large subject populations, suggest prevalences higher than previously expected: a study of over 125,000 exomes from the gnomAD consortium found that 0.329% of individuals carry a clinically relevant platelet-related loss-of-function gene variant[4] and a recent study of over 140,000 UK biobank subjects showed that up to 2.5% of individuals carry variants associated with platelet disorders.[5]

The great heterogeneity in IPD clinical phenotype and pathogenesis poses a significant diagnostic challenge. This article aims to suggest when platelet studies should be performed in patients with bleeding for a suspected IPD and to review the general approach to IPD patients.

When to Suspect an IPD

Patients with Bleeding

IPD-affected individuals often present with symptoms and signs of mucocutaneous bleeding, such as bruising, epistaxis, and heavy menstrual bleeding. Rare manifestations are muscle hematomas, central nervous system bleeds, hematuria, and ovulation bleeding. They frequently also manifest abnormal bleeding upon hemostatic challenges, including childbirth, surgery, or dental procedures.[6] Trauma- or surgery-related bleeding typically occurs soon after a hemostatic challenge, although in some peculiar cases, such as the Quebec platelet disorder (QPD), the onset may be delayed by 1 to 2 days, reflecting increased fibrinolysis.[7]

Since subjective reporting and physician evaluation of bleeding symptoms can be very variable, standardized bleeding assessment tools (BATs) have been developed and successfully validated for patients with a suspected bleeding disorder.[8]

The utility of the ISTH bleeding assessment tool (ISTH-BAT), initially validated for suspected von Willebrand disease (VWD), has been recently confirmed in patients with IPD. In particular, the best cut-off discriminating IPFD from HC was >4 for females and >3 for males (with high negative predictive values of 90.8 and 90.2%, respectively). If a patient with a mucocutaneous bleeding diathesis has an ISTH-BAT BS >6 and preliminary laboratory screening has excluded VWD and blood clotting factor defects, the patient has ≥99% probability of being affected by an IPFD (positive predictive value of 98%, negative predictive value of 85%).[9] Moreover, it was shown to be highly predictive of subsequent postsurgical[6] and spontaneous bleeding.[10]

A recent study showed that the degree of impairment of platelet function assays is associated with the severity of bleeding in patients with IPFD.[11]

A complete physical examination should include careful examination of the skin and musculoskeletal, cardiac, and neurologic systems for possible syndromic manifestations.[1] [12]

Information on food history or peculiar nutrients, including the use of herbal remedies, and drug use, including antibiotics, nonsteroidal anti-inflammatory drugs, and others, should be collected.[13]

A detailed family history may help establish whether there is a familial bleeding tendency and may contribute to understand if the disorder is inherited or acquired. However, the absence of bleeding in family members does not exclude inherited disorders because recessive forms, de novo mutations, or mosaicisms must be considered.[14]

Patients with Thrombocytopenia

Newly diagnosed thrombocytopenia is more commonly due to acquired conditions, such as immune thrombocytopenia (ITP), thrombocytopenia associated with autoimmune diseases, increased platelet consumption, splenomegaly, bone marrow suppression (due to infections or drugs) or failure, rather than to inherited conditions. Key clinical factors in favor of inherited thrombocytopenia include the age of onset and the chronicity of the condition. While platelet counts below 100,000/μL are to be considered pathologic, counts between 100,000/μL and 150,000/μL do not necessarily indicate a hematologic disorder. However, a platelet count consistently below 150,000/μL, especially in a patient with a family history of thrombocytopenia or unresponsive to standard treatments for ITP, suggests an inherited form. Additionally, patients with low platelets and disproportionate bleeding, particularly if young and otherwise healthy, should be suspected of an IPD.[15] In women with uncomplicated pregnancies and a platelet count below 100,000/μL, a cause other than pregnancy complications should be considered[16] including possible inherited thrombocytopenia when previous blood counts are not available.

Why Should an IPD Be Diagnosed

Establishing the conclusive diagnosis of IPD is essential to provide to patients personalized treatment and prognostic prediction and to undertake genetic counseling and screening of family members. This is particularly important for disorders associated with a high bleeding risk and for those associated with potentially ominous syndromic manifestations, such as myelofibrosis, lung fibrosis, renal insufficiency, and malignancy.

Although in the majority of IPDs treatment is symptomatic and it does not differ depending on the underlying molecular defect, a precise IPD diagnosis may help personalize treatment at least in some cases.

For instance, thrombopoietin receptor agonists can increase the platelet count and reduce the bleeding diathesis in congenital amegakaryocytic thrombocytopenias caused by thrombopoietin mutations and in MYH9-related disease (MYH9-RD) but also in some IPFD associated with thrombocytopenia, like ITGA2B/ITGB3-RT and WAS.[17] Hematopoietic stem cell transplantation is an option in patients with amegakaryocytic thrombocytopenia, amegakaryocytic thrombocytopenia with radioulnar synostosis (RUSAT-1 and RUSAT-2), WAS, and in some IPFD with very severe platelet function defects, like GT, leukocyte adhesion deficiency III (LAD-III), biallelic Bernard–Soulier syndrome (bBSS), and GPS.[14]

IPD associated with a reduced platelet number should be recognized to avoid misdiagnosis of ITP. Indeed, in most reported case series of inherited thrombocytopenias, at least 10% of the patients were for a misdiagnosed refractory ITP splenectomized[17] [18] and many more underwent unnecessary treatment with steroids. Other patients (e.g., those with ANKRD26-related thrombocytopenia) have been misdiagnosed as having myelodysplastic syndrome and received chemotherapy.[19]

What Should Be the Diagnostic Approach to IPDs

First-Line Tests

Current guidelines suggest a tiered approach to IPD diagnosis.[20] When clear abnormalities emerge from the initial careful clinical evaluation and/or the bleeding score is ≥6,[9] preliminary laboratory investigations should be performed, including full blood count, prothrombin time, activated partial thromboplastin time, and screening tests for von Willebrand factor (VWF) (VWF antigen, ristocetin cofactor activity, and factor VIII), to exclude VWD or a coagulopathy. If these turn out normal, a streamlined panel of laboratory tests should be undertaken ([Fig. 1]). Not all the tests have been validated for the diagnosis of IPD and guidelines are available only for some of them ([Table 1]).

Table 1 Availability of guidelines and validation of platelet laboratory tests

Test	Guidelines/standardization available	Validated for diagnosis of IPD	When to apply
ISTH-BAT	Yes (PMID: 20626619)	Yes (PMID: 31750621)	Initial clinical evaluation
Blood smear examination	No	No	First step
Light transmission aggregometry	Yes (PMID: 23574625)	Yes (PMID: 19143930)	First step and second step
Granules content and release	Expert opinion published (PMID: 35770723)	No	First step
Flow cytometry	Consensus recommendations published (PMID: 34580997)	Yes (PMID: 29337406)	First step
Mixing tests	No	No	Second step
Thromboxane B2	No	No	Second step
TEM	Yes (PMID: 29863946)	Yes (PMID: 29508516, 29863946)	Second step
Immunofluorescence on blood smears	Yes (PMID: 32079152)	Yes (PMID: 36732160)	Second step
Clot retraction	No	No	Second step
Genetic analysis	ACMG guidelines (PMID: 25741868)	TIER-1 gene list (PMID: 31179617)	First step or third step
PFA-100	No	No	Supporting test
Bleeding time	No	No	Supporting test
Biochemical studies	No	No	Supporting test/Research purposes

Abbreviations: ISTH-BAT, International Society for Thrombosis and Haemostasis Bleeding Assessment Tool; TEM, transmission electron microscopy.

Fig. 1 Diagnoses made using a step-by-step approach. ARC, arthrogryposis, renal dysfunction, and cholestasis; bBSS, biallelic Bernard–Soulier syndrome; CHS, Chediak–Higashi syndrome; GPS, Gray platelet syndrome; GT, Glanzmann thrombasthenia; mBSS, monoallelic Bernard–Soulier syndrome; HPS, Hermansky–Pudlak syndrome; LAD-III, leukocyte adhesion defect III; MYH9-RD, MYH9-related disease; PT-VWD, platelet-type von Willebrand disease; PSD, primary secretion defect; SPD, storage pool disease; WAS, Wiskott–Aldrich syndrome; XLT, X-linked thrombocytopenia.

A mildly reduced platelet count should not discourage further IPD testing. Moreover, given that rare cases of combined IPFD and blood clotting disorders have been reported[2] [21] [22] the investigation of platelet function may still be advisable in patients with mild VWD or clotting factor disorders with disproportionate or unusual bleeding manifestations.[12]

First-line laboratory investigations should include the examination of a peripheral blood smear, light transmission aggregometry (LTA), the assessment of platelet granular content and release, and the flow cytometric evaluation of the main platelet surface glycoproteins.[20]

Platelet Count and Peripheral Blood Smear

The first step in the investigation of platelet disorders involves the measurement of platelet count and size using automated counters or phase-contrast microscopy. A low count should always be confirmed by phase contrast microscopy to check for possible clumps, pseudo-thrombocytopenia,[23] [24] [25] [26] or satellitism.[27]

Platelet size should be measured by the mean platelet volume (MPV) provided by automated counters or by the mean platelet diameter (MPD) obtained by image analysis of peripheral blood smears.[28] Recently, the assessment of platelet size using forward scatter (FSC) on flow cytometry (FC) was proven to discriminate some subtypes of IT.[29] Microthrombocytopenia characterizes Wiskott–Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), CYCS-related thrombocytopenia (RT), FYB-RT, PTPRJ-RT, and ARCP1B-RT, while macrothrombocytopenia characterizes several IPD, typically BSS, both bi- and monoallelic, MYH9-RD, and the Paris-Trousseau syndrome ([Fig. 2]). A MPV greater than 12.4 fL has 83% sensitivity and 89% specificity for distinguishing inherited thrombocytopenia from ITP.[18] An increased immature platelet fraction (IPF), which suggests increased platelet turnover, is common in ITP but is also frequently found in inherited macrothrombocytopenias. IPF can be measured by FC or using automated hematology analyzers,[30] but large platelets or the presence of small platelet aggregates may artifactually increase the IPF.[31] Although the mechanisms underlying the increase in IPF values in inherited macrothrombocytopenias have not been fully elucidated, this parameter can be used to distinguish these forms from thrombocytopenia caused by bone marrow failure.[30]

Fig. 2 Diagnostic approach for suspected cases of inherited platelet disorders. ACTB-RT, Baraitser–Winter syndrome 1 with macrothrombocytopenia; ARC, arthrogryposis, renal dysfunction, and cholestasis; bBSS, biallelic Bernard–Soulier syndrome; CAMT, congenital amegakaryocytic thrombocytopenia; CDC42-RT, Takenouchi–Kosaki syndrome with thrombocytopenia; CHS, Chediak–Higashi syndrome; FLNA-RT, thrombocytopenia with filaminopathy; FPDMM, familial platelet disorder with propensity to myeloid malignancies; GNE-RT, myopathy associated with thrombocytopenia; GPS, Gray platelet syndrome; GT, Glanzmann thrombasthenia; KDSR-RT, thrombocytopenia and erythrokeratoderma; mBSS, monoallelic Bernard–Soulier syndrome; HPS, Hermansky–Pudlak syndrome; LAD-III, leukocyte adhesion defect III; MYH9-RD, MYH9-related disease; PT-VWD, platelet-type von Willebrand disease; PSD, primary secretion defect; QPD, Quebec platelet defect; RD, related disorder; RT, related thrombocytopenia; RUSAT, amegakaryocytic thrombocytopenia with radioulnar synostosis; SPD, storage pool disease; STSL, sitosterolemia with macrothrombocytopenia; TAR, thrombocytopenia with absent radii; TCPT/JBS, Paris–Trousseau thrombocytopenia/Jacobsen syndrome; THPO-RT, congenital amegakaryocytic thrombocytopenia caused by variants in THPO; VCF, velocardiofacial syndrome; WAS, Wiskott–Aldrich syndrome; XLT, X-linked thrombocytopenia. In very rare cases without thrombocytopenia; ^#Some cases present thrombocytopenia.

The assessment by light microscopy of stained peripheral blood smears may allow to identify specific morphologic abnormalities, like large pale platelets suggesting gray platelet syndrome (GPS) and very large, granulated platelets orienting toward BSS. Dohle-like bodies in leukocytes point to MYH9-RD, while giant white blood cell granules suggest Chediak–Higashi syndrome (CHS). Abnormal red blood cell morphology may indicate GATA-1 mutations (anisocytes and poikilocytes) or Stormorken syndrome (asplenia evidenced by Howell–Jolly bodies) or ETV6-RT (red cell macrocytosis), and platelet clumps may suggest platelet-type VWD. Other reported abnormalities which may be suggestive of some IPD are neutropenia in some forms of HPS or DIAPH-RT, eosinophilia in ARPC1B-RT, and stomatocytosis in sitosterolemia.

Given its importance in orienting IPD diagnosis, the blood smear should be inserted among the initial screening tests and should always be performed.

Platelet Aggregation

LTA is still the most widely used test of platelet function and a crucial assay in the diagnostic workup of most IPFD.[3] Preanalytical variables may have a strong impact on platelet function testing; thus, particular attention must be paid during blood sampling and sample preparation. Blood samples should be drawn with minimal or no venostasis using a needle of at least 21 gauge into the buffered anticoagulant sodium citrate (109 or 129 mM, although currently the most used is 109 mM). Moreover, results could be inaccurate when the platelet count in the PRP is lower than 150 × 10⁹/L.[32] Efforts are ongoing for standardization[33] and accreditation of LTA.[34]

Agonists should include epinephrine, ADP, collagen, arachidonic acid (AA), and ristocetin. An extended panel of agonists for LTA should include α-thrombin with gel-filtered platelets (for GPS diagnosis),[35] thrombin receptor-activating peptides (TRAP-6 and protease-activated receptor-4-activating peptide), the GPVI-specific agonists convulxin (also acting via α₂β₁) and collagen-related peptide (CRP) (acting on GPVI) and the synthetic thromboxane analog U46619 (acting on the TP receptor). The use of these agonists is suggested for second-step tests by the ISTH guideline[20]; however, they may all be included among agonists tested at the first step.

Glanzmann thrombasthenia patients show absent aggregation to all agonists except high-dose ristocetin (which may be reversible), while patients with BSS do not respond to ristocetin but show normal or sometimes reduced aggregation to the other agonists, in part due to the macrothrombocytopenia that makes difficult to obtain PRP with an adequate platelet concentration. GPVI deficiency gives absent or severely reduced LTA with collagen and GPVI agonists, such, as CRP or convulxin, while α₂β₁ deficiency gives no response to collagen but an almost normal response to CRP[36] [37]; a P2Y₁₂ defect shows a reduced and reversible response to ADP and impaired aggregation in response to collagen and AA; GPS shows impaired aggregation in response to TRAP6; patients with storage pool disease or platelet secretion defects display a decreased secondary aggregation in response to epinephrine and ADP and decreased aggregation in response to collagen and in some cases to ristocetin[38]; increased response to a low dose of ristocetin is suggestive of platelet-type VWD, although it may also characterize type 2B VWD. Platelets from patients with abnormalities of thromboxane (Tx)A₂ synthesis do not aggregate in response to AA but respond to the synthetic thromboxane analog U46619, while defects of TPα, the TxA₂ receptor, will show the absence of aggregation to AA and to U46619.[20] [39] [40]

For as concerns epinephrine, a significant fraction of healthy individuals show abnormal aggregation possibly due to variations in α2 adrenoreceptor number[41] or to the presence of SNV in PEAR1 receptor[42]; therefore, the diagnostic utility of an impaired response to epinephrine remains uncertain, although it has been reported in the QPD.[43]

It is anyway a good practice to repeat LTA that showed abnormal findings to exclude transient interfering factors, using the same instrument and reagents, 10 to 15 days later,[44] also in case LTA clearly suggested a diagnosis.

Alternative Methods to Study Platelet Activation

Attempts to automate certain steps of the LTA procedure have recently been made. This is exemplified by the Sysmex CS series that enables to automate the selection of agonists and their concentrations without the need for highly experienced personnel.[45] This method requires a smaller volume of platelet-rich plasma than traditional methods (140 vs. 200–500 μL), but it has higher costs for reagents and consumables.[46] [47]

An alternative test is impedance aggregometry which can measure aggregation in whole blood (WBA) with different devices. However, the sensitivity of WBA for IPFD is low and its diagnostic utility is limited to GT and a few other severe IPFDs.[46]

In the landscape of high-throughput screening tests, the Optimul 96-well platelet aggregation assay has emerged as a valuable tool. This method measures platelet aggregation in a 96-well plate using simultaneously a range of agonists, including ADP, collagen, and ristocetin, with small sample volumes, making it a promising assay for the diagnosis of platelet defects,[48] although its diffusion is limited by the fact that the Optimul plates containing lyophilized agonists are not commercially available and must be prepared in-house.

A flow-cytometric platelet aggregation assay, measured by the increase of forward scatter/side scatter over time,[49] has been employed in patients with a suspected IPD, although it is still very rarely applied even in expert laboratories.[50]

FC is also a validated and widely used method for studying platelet activation. The exposure of specific activation antigens on the platelet surface, such as the GPIIb–IIIa activation epitope (PAC1), P-selectin (CD62P), and LAMP-3 (CD63), is measured after stimulation of whole blood with a set of agonists[51] [52]; also ristocetin-induced VWF binding to platelets can be used for the diagnosis of PT-VWD.[53] [54] For FC, no minimal platelet count cutoff in whole blood is required, provided that 5,000 to 10,000 platelet events can be collected, although reduced platelet activation responses might be observed at very low platelet counts (i.e., < 10 × 10⁹/L).[55]

Platelet Granule Content and Release

The assessment of platelet granules should be performed among first-line tests because LTA may give normal results in patients with a secretion defect.[20] [56]

The most commonly used method for α-granule release is the measurement of platelet surface P-selectin (CD62P) by FC after in vitro stimulation with different agonists, such as ADP, thrombin receptor-activating peptide (TRAP)-6, convulxin, AA, and the thromboxane analog U46619.[3] [56] This marker, however, may have some diagnostic limitations because in some patients with GPS, platelet surface P-selectin was found to be normally expressed.[57]

Several other proteins (fibrinogen, VWF, multimerin-1, clotting factor V, PAI-1, α2-antiplasmin, uPA, TGF-β, PDGF, VEGF, and some chemokines, including PF4) may be measured in the supernatant of a PRP sample at the end of LTA recording or in platelet lysates, as markers of α-granule release and content, by several methods, including enzyme-linked immunosorbent assay (ELISA) or HPLC. Some of these, which are present at undetectable or very low levels in plasma (e.g., PF4, β-TG, multimerin-1), may be preferable as selective markers of α-granule release.[58] [59]

The most widely used method to measure δ-granules release is lumiaggregometry, which allows to assess ATP release simultaneously to LTA, thus shortening the execution time.[3] [56]

ATP secretion may, however, also be measured independently from LTA in the supernatant of stimulated platelets by luminometry or high-performance liquid chromatography.

In case of defective ATP secretion, the distinction between δ-granule deficiency (δ-SPD) and impaired release (secretion defects) requires the measurement of the total platelet adenine nucleotide content by HPLC-based or luciferin/luciferase-based assays of platelet lysates.[56]

The gold standard is the measurement of ¹⁴C-5-HT release from prelabeled platelets, but it is used rarely in clinical laboratories due to the requirement of radioisotopes.[56] [60] 5-HT release can also be measured by FC by specific intracytoplasmic staining with antiserotonin antibody or in the supernatant of a PRP sample at the end of LTA recording by ELISA, ortho-phthalaldehyde assay, HPLC, and mass spectrometry.[56]

Another frequently employed test measures the uptake and release of mepacrine, a fluorescent dye rapidly taken up by platelet δ-granules, by FC,[61] although there is insufficient information to recommend its use[56]; moreover, being a probe binding to ADP and ATP, it is not strictly specific for δ-granules.

Finally, platelet granules can be counted by transmission electron microscopy (TEM) using whole mount (WM) preparations or platelet thin sections (TS). WM-TM is the gold standard for the quantification of δ granules, while TS-TEM is preferred for counting platelet α-granules and/or for the identification of platelet structural alterations.[62] [63] [64]

Defective α-granular content and release are described in GPS, QPD, arthrogryposis renal dysfunction and cholestasis syndrome, Stormorken, GATA1-RT, GFI1B-RT, and FPD/AML (frequently associated with defective δ-granule content and release). Defective δ-granular content and release are described in δ-SPD and, in HPS, CHS, WAS, thrombocytopenia with absent radii (TAR), SLFN14-RT, FLI1-RT.

Combined α- and δ-granule content and release deficiency are described in IKZF5-RT, while defective release is described in WAS as a primary secretion defect.

Assessment of Platelet Surface Glycoproteins by Flow Cytometry

FC can be used to detect platelet glycoprotein defects and alterations in the expression of platelet procoagulant activity. The measurement of glycoprotein receptor expression is crucial for the diagnosis of platelet adhesive protein defects, such as Glanzmann thrombasthenia and BSS, and should be performed using antibodies toward GPIIb (CD41), GPIIIa (CD61), GPIbα (CD42b), and GPIX (CD42a).[20] [51] Given that large platelets may show increased expression of GPs due to increased volume, in IPDs with macrothrombocytopenia (for instance, in monoallelic BSS [mBSS] or ITGA2B/ITGB3-RT) it is recommended to calculate GPs expression as a ratio versus another GP, for instance versus CD41.

FC can be used also when there is significant thrombocytopenia and with limited sample volume, and this may be of special help in the investigation of pediatric or neonatal subjects.[50]

High-affinity fluorescent probes for surface phosphatidylserine (like annexin V, lactadherin) may allow to detect impaired (Scott syndrome) or enhanced (Stormorken syndrome) procoagulant activity syndromes.[65] [66] [67]

An expanded FC panel should include antibodies against GPIa/IIa (CD29 and CD49b), GPIV (CD36), and GPVI to detect any abnormalities in these platelet surface glycoproteins, orienting toward genetic defects of the collagen receptors or toward FPDMM in the case of defective expression of GPIa/IIa.[68]

As previously said for LTA, these antibodies are advised for second-step tests by the ISTH[20]; however, a simplified approach would be to include them in the first step. There are no indications on the necessity to repeat the test to confirm the defect.

Second-Line Tests

In case enhanced ristocetin-induced platelet aggregation was detected during first-step laboratory investigations, suggesting either VWD type 2B (2B-VWD) or platelet-type VWD (PT-VWD), mixing tests either by LTA or by FC with patient and control platelets and plasma may help to discriminate a plasmatic (2B-VWD) from a platelet (PT-VWD) defect.[54]

The measurement of TxB₂ in serum should be performed when an aspirin-like platelet defect is suspected (cPLA2, COX-1, or Tx-synthase defects) but is also a clue for the intake of aspirin or nonsteroidal anti-inflammatory drugs.[69] TxB₂ can be measured using enzyme-linked immunosorbent assays (ELISA) or radioimmunoassay in either serum or in the supernatant of AA-stimulated platelets, both of which offer high sensitivity and specificity.

TEM allows a high-resolution visualization of the platelet ultrastructure, which makes it particularly useful for assessing abnormalities in the number and morphology of platelet granules,[56] [63] such as in GPS (the absence or severe reduction of α-granules), Hermansky–Pudlak syndrome (HPS; the absence or severe reduction of δ-granules), or α/δ-storage pool deficiencies. A simplified TEM preparation (i.e., WM TEM) is particularly appropriate for δ-granule counting.[70] Moreover, it can be useful to detect structural abnormalities, such as the presence of giant granules in the Paris–Trousseau syndrome[71] or the presence of ubiquitin/proteasome-rich particulate cytoplasmic structures (PaCSs) in the platelets and megakaryocytes of ANKRD26-RT.[72]

Recently, standardized procedures, reference ranges, and image interpretation criteria for TEM have been published, validating this technique for the diagnosis of IPD.[63] Nevertheless, TEM remains mainly a research tool due to the complexity of sample preparation and the limited availability of electron microscopy in centers.

Recently, a simple approach based on the staining of peripheral blood smears using fluorescent-labeled monoclonal antibodies directed toward specific platelet proteins (platelet antigens, granular proteins, cytoskeletal proteins) and examination by fluorescence microscopy has been claimed to allow diagnosis in 25 to 30% of patients with a suspected IPD.[73] This test has been employed for the diagnosis of 9 IPD: MYH9-RD, BSS, mBSS, GT, TUBB1-RT, GFI1B-RT, FLNA-RT, δ-SPD, and WAS/XLT. Moreover, it allowed to identify platelet abnormalities in other 9 IPD[74] and is currently undergoing independent external validation by an international working group. This test may represent a valuable method when gold standard tests are not available, it can be performed remotely on stored and shipped samples, and it requires only very small blood amounts.

Clot retraction evaluates the ability of platelets to contract a fibrin clot, a process mediated by the interaction between α_IIbβ₃ and fibrinogen. Clot retraction is defective in GT, and it has been reported to be impaired also in the Stormorken and Scott syndromes, WAS, ETV6-RT.[69]

Genetic Analysis

High-throughput sequencing (HTS) has transformed the diagnostic approach to IPD, offering deep insight into the molecular mechanisms involved. Genetic testing now enables the analysis of a broad range of genes linked to platelet formation and function, significantly improving diagnostic accuracy. HTS methods, such as multigene panels, whole-exome sequencing (WES), and whole-genome sequencing (WGS), provide fast, cost-efficient, and accurate diagnoses, especially in cases with complex or unclear phenotypes. For example, HTS identifies variants in genes like RUNX1, ANKRD26, and ETV6, which do not display a platelet phenotype sufficiently characteristic to allow diagnosis.[20]

The diagnostic yield of genetic testing in IPD, however, ranges from 50% for inherited thrombocytopenias (IT) to only 25% for IPFDs, with success increasing in the presence of careful anamnesis and of supporting platelet laboratory results.[22] [75]

The advantages and challenges of NGS are outside the scope of this review and will not be discussed elsewhere.

Tests Not Included in the Diagnostic Workup

The skin bleeding time (BT) and PFA-100 are still used for suspected mild moderate bleeding disorders (MMBDs).[3] [8] However, these methods are not recommended for guiding further investigations due to their poor reproducibility, sensitivity, and specificity and for the invasiveness of BT.[20] [76] A recent retrospective analysis of patient records and laboratory results from 1,473 consecutive subjects with suspected mucocutaneous MMBD confirmed that these two tests should not be recommended indiscriminately for the initial screening of patients with clinical suspicion of a MMBD because of insufficient accuracy, but also suggested that a greater alteration of the BT may orient toward an IPFD rather than VWD.[77] BT should thus be considered as an optional assay of potential utility when performed by skilled operators under carefully standardized conditions.

Biochemical studies are crucial for investigating the molecular mechanisms of platelet dysfunction in some specific IPD; however, they cannot be included in the diagnostic workup because they are available only in very few specialized centers for research purposes. Among these tests, receptor ligand-binding assays evaluate the interaction between platelet receptors and their ligands to assess for possible receptor defects, such as for P2Y₁₂ and TP-receptor deficiencies,[78] although these assays are not used in a diagnostic setting but only in research laboratories due to the use of radioactivity. Platelet spreading and adhesion assays, under static and flow conditions, measure the ability of platelets to adhere to specific extracellular matrix proteins, like fibrinogen, collagen, or VWF, providing insights into integrin function. Defective spreading characterizes GT, WAS, LADIII, CalDAG-GEFI-related disorder, and autosomal dominant GT, while increased spreading can be observed in FLNA-related thrombocytopenia. Impaired deposition of platelets on a collagen-coated surface under flow conditions has been reported in patients with GT, HPS, GPS, BSS, GPIV, and GPVI defects.[79] The measurement of second messengers, like calcium ions (Ca²⁺), cAMP, and cGMP, reveals defects in signaling pathways involving G-protein-coupled receptors such as P2Y₁₂, Gαs abnormalities, or phospholipase Cβ2 deficiency.[80] Western blotting is useful to detect defects in platelet signal transduction or the abnormal presence of MYH10 in platelets, a biomarker for FPD/AML and FLI1-related disorders.[81] LTA using agonists such as platelet-activating factor (PAF), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 might enable to characterize rarer forms of IPFDs. For example, impaired aggregation with PMA, a direct stimulator of protein kinase C, and with platelet-activating factor suggest a defect in a protein kinase C isozyme.[69]

These tests can also be performed after the identification of a gene variant of uncertain significance (VUS) to assess its pathogenicity or to get insight in the molecular mechanisms of the disorder. The expression of genetic variants in cellular models moreover is the best option to assess for VUS pathogenicity[1]; however, this requires complex and costly assays, exclusively performed in research settings.

Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are advanced viscoelastic testing methods that offer a global view of the clotting process and potentially allow the monitoring of platelet function.[82] These techniques evaluate the viscoelastic properties of the blood clot in real-time, providing insights into clot formation, stabilization, and dissolution. Clot strength, highlighted by the trace amplitude, reflects the function and count of platelets, and their interaction with fibrinogen and clotting factors. The TEG's Platelet Mapping assay has been reported to detect platelet dysfunction induced by aspirin and clopidogrel; thus, it might in principle detect platelet dysfunction in at least some IPD.[47] [83] However, to the best of our knowledge, no reports on this are available yet.

Advances in IPD Diagnostic Techniques

Some innovative technologies hold promise for IPD diagnosis, however further standardization of devices and protocols is required to fully integrate them into routine clinical practice.

One of these are microfluidic devices that simulate physiological flow conditions more accurately than previously used methods. These devices enable the study of platelet adhesion and aggregation under controlled shear stress on standardized surfaces closely mimicking blood flow in vessels. One of the first was the IMPACT Cone and Plate(let) Analyzer, a system that evaluates platelet aggregation by rotating a cone over a polystyrene plate, simulating the shear stress conditions that platelets experience in the circulation. This platform was reported to be of help in detecting conditions like GT and BSS.[84]

Digital microfluidics and other laboratory-on-a-chip technologies enable precise, small-volume testing. The Total Thrombus Formation Assay System (T-TAS) uses two microchips, the platelet (PL) chip and the atheroma (AR) chip, to analyze thrombus formation. The PL chip is designed to assess primary hemostasis, and was reported to be useful in detecting VWD and platelet storage pool disease.[85] AR-modified chips with a different chamber depth (T-TAS HD) have been developed for the evaluation of the hemostatic function in patients with thrombocytopenia and seem to discriminate between thrombocytopenic patients with or without a bleeding tendency.[86] [87] Further studies are required to clarify whether this method may provide advantages over currently used tests.

A multimicrospot microfluidic test using nine different surfaces composed of key physiological platelet-adhesive proteins has been designed and applied to the study of patients with GT, HPS, MYH9-RD, and GPS.[88] This test is thus potentially useful for the diagnosis of patients with suspected bleeding disorders; however, the complexity of sample preparation and results interpretation, high costs, and the fact that it is not commercially available represent limitations to its application in diagnosis.

FC has also evolved with the development of multicolor FC and imaging flow cytometry (IFC), offering more detailed and high-throughput analysis of platelet receptor expression and function. Multicolor FC allows the simultaneous measurement of various intracellular signaling events by labeling platelets with different fluorescent dyes, enabling the study of multiple protein phosphorylation pathways critical for platelet function. This technique allowed to identify a specific defective pattern of phosphorylation of signal transduction proteins in patients with δ-storage pool deficiency.[89] IFC combines FC with microscopy, allowing to capture high-resolution images of individual platelets thus distinguishing different platelet subpopulations[90] and monocyte-platelet aggregates.[91] However, to the best of our knowledge, it has not been applied to studies on IPD patients so far.

Another significant innovation is mass cytometry (MC) which enables the simultaneous analysis of numerous platelet surface markers and functional proteins by using heavy metal-tagged antibodies, which are detected through mass spectrometry. This technology has allowed the discovery of specific platelet subpopulations and has revealed previously unrecognized alterations of some surface glycoproteins of GT platelets, such as an increased expression of CD9 and CD63 or the reduced expression of CD31 and GPVI.[92]

Advances in IPD Diagnostics Using Artificial Intelligence

Artificial intelligence (AI) refers to the creation of artificial systems capable of performing tasks that mimic human intelligence. This is accomplished by equipping computers with the ability to learn from data, a process commonly known as machine learning (ML). AI is increasingly being applied in life science, and may revolutionize the way complex biological processes, such as platelet function and aggregation, are analyzed.[93] Given the complex diagnostic challenges that IPD present, ML and convolutional neural networks (CNNs) may offer an innovative solution to these challenges.

AI algorithms, when applied to digital morphology analyzers, i.e. advanced imaging systems used to capture and analyze the shapes and sizes of biological samples at a micro or nano scale, have demonstrated high accuracy in estimating platelet counts and in differentiating platelet subtypes, even in samples with interfering factors such as large platelets or RBC fragments.[94] Furthermore, CNNs trained on differential interference contrast (DIC) microscopy images of platelet spreading assays were able to automatically identify key morphological features, such as spread area and circularity,[95] suggesting that this technology might be applied to IPD diagnosis. Moreover, AI has been employed for the intelligent classification of platelet aggregates by agonist type. This approach, termed “the intelligent platelet aggregate classifier (iPAC),” employs high-throughput imaging FC coupled with a CNN to detect subtle morphological differences between platelet aggregates formed upon activation by distinct agonists. The differentiation of platelet aggregates based on their morphological features might offer a powerful diagnostic tool for platelet function disorders.[96] ML models have been applied to clinical datasets to predict the presence and severity of ITP using routine laboratory parameters, demographic features, and duration of disease demonstrating the potential of ML models to significantly enhance diagnostic accuracy for ITP and possibly its differentiation from IT.[97]

The integration of AI-driven models in routine diagnostics holds promise for improving the recognition of IPDs, reducing time to diagnosis, and allowing personalized treatment, thus prefigurating the development of fully automated diagnostic workflows enabling clinicians to make quicker and more appropriate decisions.

Finally, the ongoing application of multiomics to the investigation of platelet strongly relies on ML methods for the removal of confounding factors and will probably allow a leap forward in the understanding of the mechanisms of IPDs.[98] [99] [100]

A Representative Clinical Case

A 34-year-old woman came to our observation with a history of mucocutaneous hemorrhages, including severe episodes. She underwent surgery for strabismus at the age of 3, with postoperative bleeding, appendectomy at 14, without bleeding, and another eye surgery for strabismus at 16, also followed by significant bleeding. She gave birth in 2009 after prophylactic administration of tranexamic acid and desmopressin with no bleeding. She had constantly suffered from menometrorrhagia throughout her postpubertal life. Her ISTH-BAT BS was 8. There was no family history of bleeding. Physical examination showed some ecchymoses and no further alterations. Platelet count was normal (294 × 10⁹/L). Screening studies (prothrombin time, activated partial thromboplastin time, and VWF antigen and activity) excluded coagulation defects. BT was twice the normal (13 minutes; normal: 6.8 ± 2.3 minutes). PFA-100 closure time was 1.5 times longer than normal with the collagen/epinephrine (292 seconds; normal: 145 ± 51 seconds) and normal with the collagen/ADP cartridge (115 seconds; normal: 95 ± 31 seconds).[77]

Considering these results and her abnormal ISTH-BAT BS, we decided to proceed with IPFD laboratory testing.

Platelet size was assessed on blood smears[16] and resulted normal (98% normal, 2% large, 0% very large platelets; normal values: 95–100% normal, 0–4% large, 0–1% very large) and also morphology, excluding CHS, Griscelli syndrome. LTA revealed impaired aggregation in response to epinephrine, ADP, collagen, and AA excluding defects in which aggregation is severely impaired (e.g., GT or CALDAG-GEF1 disorder), and orienting toward a possible granule secretion or content defect. Platelet surface receptors α_IIbβ₃, GPIb/IX/V, α₂β₁, GPVI, and GPIV, as assessed by FC, were normal, thus excluding defects of surface glycoproteins. The content of α-granules, assessed by ELISA of β-TG on resting platelet lysates, was normal. The release of α-granules, assessed by ELISA of β-TG in the supernatant of a PRP sample at the end of LTA recording and by surface expression of P-selectin by FC after stimulation of platelets with 20-mM ADP, was only slightly reduced. These results excluded a defect of α-granule content, thus eliminating the hypothesis of a possible GPS, but suggested that δ-granule secretion might be defective with the consequent lack of amplification of platelet activation. The content and release of δ-granules, assessed by the measurement of ATP content and release by lumiaggregometry and by mepacrine uptake at FC, was severely reduced.

A diagnosis of δ-storage pool deficiency (SPD) was therefore formulated.

The patient was called back after 6 months to repeat platelet function testing which confirmed the defect. Due to the very peculiar light blue color of her eyes, we suspected HPS and suggested a specialistic ophthalmologic examination that revealed diffuse retinal hypopigmentation, absent foveal reflex and foveal hypoplasia pointing to a diagnosis of ocular albinism. Moreover, we quantified platelet δ-granules by TEM that showed defective content, with a mean of 0.4 δ-granules per platelet (normal values: 0.9–2 δ-granules/platelet). HPS was therefore diagnosed and, due to the possible prognostic implications of this diagnosis and the known genotype/phenotype correlation,[12] we decided to carry out targeted sequencing of 91 TIER-1 genes,[22] which showed the known pathogenic HPS1 c.1632C > A (p.Phe544Leu) variant, leading to the final diagnosis of HPS type 1.

The patient since then is regularly followed-up for possible extrahematological complications, such as pulmonary fibrosis and granulomatous colitis, but so far she has not shown any signs of involvement of these organs.

Conclusion

IPD represent a heterogeneous group of conditions with complex pathophysiology for which early diagnosis is critical to avoid mismanagement. Current diagnostic strategies rely on a tiered approach, utilizing a combination of clinical assessment, laboratory testing, and genetic analysis. Recent advances in AI and emerging technologies hold promise for refining IPD diagnostics, paving the way for more accurate, personalized patient management and enhanced understanding of platelet pathophysiology.

Conflict of Interest

PG: Speaker fees: Roche, Kedrion. Support for attending meetings and/or travel: STAGO.

LB and EF have no conflict of interest.

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Publication History

Received: 04 November 2024

Accepted: 09 February 2025

Article published online:
12 May 2025