The use of flow cytometry in the diagnosis of heparin-induced thrombocytopenia (HIT)

YTMRV-50581; No of Pages 8 Transfusion Medicine Reviews xxx (xxxx) xxx

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The use of flow cytometry in the diagnosis of heparin-induced thrombocytopenia (HIT) Elvira Maličev Blood Transfusion Center of Slovenia, Ljubljana, Slovenia

a r t i c l e

i n f o

Available online xxxx Keywords: heparin-induced thrombocytopenia functional assays flow cytometry platelet activation laboratory diagnosis HIT

a b s t r a c t Heparin-induced thrombocytopenia (HIT) affects some of the patients exposed to heparin. It is mediated by antibodies that recognize neoepitopes on platelet factor 4 (PF4)/heparin complexes. A HIT diagnosis requires both clinical and laboratory evaluation and remains a challenge. Since many patients develop antibodies in response to heparin, but only a few of them generate anti-PF4/heparin antibodies capable of activating platelets which consequently cause clinical complications, the performance of serologic assays is not enough to diagnose HIT. Functional assays can identify pathogenic antibodies capable of platelet activation, but they are more demanding and their limited availability contributes to the problem of diagnosing HIT. Restricted laboratories usually collect sera of multiple patients to perform functional assays only once or twice a week; hence, a HIT diagnosis can take several days. The use of flow cytometry appears to be a promising alternative in the confirmation of pathogenic antiPF4/heparin antibodies. Flow cytometric assays detect either activation markers on a healthy donor’s platelet surfaces or platelet derived microparticles formed after platelet incubation with a patient’s serum. Flow cytometers are readily available in many clinical laboratories, so this technology introduces the possibility of an earlier HIT diagnosis. The objective of this review was to collect findings on flow cytometric HIT confirmations to the present date, and to review the currently available flow cytometric assays used in the diagnosis of HIT. © 2019 Elsevier Inc. All rights reserved.

Contents Pathogenesis of HIT . . . . . . . . . . . . . . . . Diagnosis. . . . . . . . . . . . . . . . . . . . . Principles of functional assays for the diagnosis of HIT Donor platelets for functional assays . . . . . . . . Flow cytometric assays for the diagnosis of HIT . . . Detection of platelets . . . . . . . . . . . . . . . Detection of activated platelets . . . . . . . . . . . Generation of platelet microparticles . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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Heparin remains a commonly used anticoagulant in prophylaxis and treatment. Heparin-induced thrombocytopenia (HIT) is an adverse side effect which usually develops within 5 to 10 days of heparin exposure [1,2]. Although thrombocytopenia and thrombosis occur only in 0.2%

E-mail address: [email protected].

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to 3% of patients exposed to the drug, it can induce serious consequences in these patients if not diagnosed. On the other hand, the overdiagnosis of HIT leads to the unnecessary exposure to alternative nonheparin anticoagulants [1-4]. HIT starts with the generation of specific antibodies, followed by platelet activation by these antibodies, the generation of procoagulant platelet microparticles, platelet aggregation, and the activation of

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leukocytes and endothelial cells. The production of platelet derived microparticles increases thrombin generation which leads to an increased risk of thrombosis [1,5-7]. Although it is difficult to diagnose HIT through clinical information alone, diagnosis should begin by calculating the pretest probability of HIT prior to receiving any laboratory test results [1,7-9]. Calculations based on clinical events like the degree of thrombocytopenia, the timing of the onset of thrombocytopenia, thrombosis or other clinical sequelae, and the likelihood of other causes of thrombocytopenia. In cases where an intermediate or high probability of HIT is suspected, a laboratory diagnostic test has to be performed [10-11]. Currently, we do not have an ideal laboratory test for HIT. The generation of anti-PF4/heparin antibodies starts after heparin binds to endogenous platelet protein, platelet factor 4 [PF4], which is released from activated platelets [12,13]. Highly sensitive commercial immunoassays detect anti-PF4/heparin antibodies in a patient’s serum. Negative results eliminate HIT and no further laboratory test is needed. Positive results obtained by immunoassays require more attention because the immunoassays cannot discriminate between the presence of pathogenic and non-pathogenic anti-PF4/heparin antibodies, and many patients who test positive for anti-PF4/heparin antibodies do not develop thrombocytopenia or thrombosis [1-3]. Therefore, in the case of a positive immunoassay result, one of the functional laboratory assays should confirm the diagnosis of HIT. Functional assays detect the activation or aggregation of healthy donor platelets in the presence of patient serum. Functional assays are mostly in house tests and are not as widespread as immunological assays. The main reasons for their rarity are their technical complexity, time-consumption, the use of radioactivity, and the need for healthy platelet donors as well as special laboratory equipment. Functional assays are performed in specialized laboratories that collect sera from different clinics and analyze samples only once or twice a week. Such an approach prolongs the time until diagnosis. Novel laboratory assays that produce results more quickly and which are less technically complex would be very welcome. Flow cytometry is a technology that has become indispensable in hematological and immunological diagnostics. Therefore, flow cytometers are widespread in different clinical laboratories. The idea of using flow cytometry in HIT diagnosis is not new, but in recent years it has become a really promising way to confirm HIT. Pathogenesis of HIT Of all patients exposed to heparin, only a subset of them generates anti-PF4/heparin antibodies. And only a small group of patients with detected anti-PF4/heparin antibodies generate such anti-PF4/heparin antibodies that are capable of activating platelets [2,14]. This, as yet, not fully understood phenomenon, makes diagnosis of HIT more difficult and requires a comparison of the clinical picture with the results of different laboratory tests. Platelet factor 4 (PF4) is a positively charged protein tetramer stored in platelet α-granules [12]. After platelet activation, PF4 is released from platelets (Fig. 1). When PF4 and heparin concentrations are in a stoichiometric ratio, PF4 binds to negatively charged heparin and forms large PF4/heparin complexes with exposed neoepitopes [14,15]. The formation of these immunogenic complexes induces the generation of specific anti-PF4/heparin antibodies, which can be platelet-activating antibodies, or platelet non-activating anti-PF4/heparin antibodies [7,13]. Anti-PF4/heparin IgG antibodies bound to PF4/heparin complexes activate platelets by cross-linking platelet FcγRIIA receptors leading to the release of more PF4 and the formation of more immune complexes [7,16,17]. Anti-heparin/PF4 antibodies also bind to the surface of monocytes and endothelial cells and activate them. The consequence of platelet activation is the formation of platelet aggregates and thrombocytopenia [7,18]. Further activation of the clotting cascade results in thrombin generation and increases the risk of thrombosis [7,19].

Several factors appear to have an influence on the risk for HIT. The risk is lower with low molecular heparin than with unfractionated heparin, since low molecular heparin is less likely to induce the conformational changes in PF4 that induce the formation of anti-PF4/heparin antibodies [20,21]. Polymorphism and the number of FcγRIIA on the platelet surface, and plasma proteins can also modulate the risk for HIT [22,23]. In a recent publication it was shown that low fibronectin levels are associated with a higher risk of PF4/heparin immunization and might also be a risk factor in the clinical breakthrough of HIT if platelet-activating anti-PF4/heparin antibodies are present [22]. Immunization triggered by heparin is common, more often seen in surgical patients than medical patients [3,24]. Among surgical patients, those having major surgical procedures, have a greater risk for developing anti-PF4/heparin antibodies than patients undergoing minor surgical procedures [25]. From 50–80% of cardiac surgery patients develop an immune response [24]. The causes that contribute to the immunogenicity of PF4/heparin complexes are not well known, however some studies suggest a connection between the immune reaction with prior bacterial infections. It was shown that PF4 binds to lipid A on the surface of Gram-negative bacteria, thereby exposing the same neoantigen as with heparin [26]. A greater number and variety of studies will need to be undertaken to explain the high frequency of the immune response in patients exposed to heparin. For example, recently it has been demonstrated that B cells rather than other leukocytes bind ultra-large complexes of PF4/heparin. The mechanism of binding involves the activation of complement by formation of PF4/heparin complexes followed by the binding of PF4/heparin/complement complexes to B cells via CD21 [27]. Diagnosis HIT presents with two major sequelae: thrombocytopenia and thrombosis. Diagnosis starts with the clinical assessment of the pretest probability of HIT [1,2,7,18]. For the purposes of clinical evaluation, scoring systems such as the 4Ts or HIT Expert Probability (HEP) score are used [10,11]. The most commonly used 4Ts score calculates the degree of thrombocytopenia, the timing of thrombocytopenia with respect to heparin exposure, the occurrence of thrombotic complications, and the presence of alternative explanations for thrombocytopenia [11]. Calculated 4T scores are grouped as follows: 0-3, low probability; 4-5, intermediate probability; 6-8, high probability of HIT [9,11]. Patients with an intermediate or high score should undergo laboratory diagnostics, since a diagnosis cannot be confirmed without laboratory testing, which includes simple immunoassays and more complicated functional assays [2,7,18]. The generation of anti-PF4/heparin antibodies occurs in 8–80% of patients exposed to heparin [4,24,28]. The presence of anti-PF4/heparin antibodies in a serum can be rapidly detected in different laboratory assays. Laboratory assays to detect anti-PF4/heparin antibodies are immunoassays, which generally have a very high degree of sensitivity, however they lack specificity. Many types of immunoassays are in use, like the enzyme-linked immunosorbent assay (ELISA) [15,29], the chemoluminiscence assay [30], the particle gel immunoassay [31], and the lateral flow immunoassay [32]. The most commonly used immunoassay is still the ELISA, which tests for PF4/heparin or PF4/polyvinyl sulfonate reactive antibodies. ELISA has an excellent negative predictive value of 98 to 99%, but a poor positive predictive value, and relying only on the result of the ELISA can contribute to misdiagnosis and unnecessary treatment [29,33,34]. As ELISA and some other immunoassays have a high negative predictive value, no further testing is needed in a case of negative result, except in cases where a high clinical probability of HIT has been determined. On the other hand, the performance of immunoassays is not enough to diagnose HIT in the case of a positive result. In ELISA, a clinically more relevant approach than reporting results as positive or negative might be to report the optical density (OD) level [35]. A higher antibody titre, as measured by an

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Fig. 1. Platelet activation in HIT: On platelet activation PF4 is released from platelet alfa granules. Tetramers of PF4 bind with heparin, forming large PF4/heparin complexes. Exposed neoepitopes induce production of anti-PF4/heparin antibodies. Anti-PF4/heparin antibodies of the type IgG can activate platelets through Fcγ receptors on platelet surfaces. Detection of CD62P with Annexin V or phosphatidylserine with anti-CD62P antibodies after platelet activation, or detection of platelet-derived microparticles after platelet activation, are the most common flow cytometric assays for the confirmation of HIT.

ELISA OD, predicts the greater likelihood of the presence of anti-PF4/ heparin platelet activating antibodies. However, as ELISA OD levels may differ among laboratories even when using the commercial assays of the same company, we have to be careful when using OD predictive values in clinical interpretation [35]. This limitation could be overcome by the normalization of the OD level of the respective laboratory. OD correlation curves may also differ between divers patient groups, e.g. between patients having undergone major surgery and patients in intensive care units [35]. Last but not least, a correlation between the OD level and the result of a functional assay is not equivalent to a correlation between the OD level and clinical HIT [35]. However, experts agree that functional or platelet-activating assays can have a much higher specificity than assays that only detect the presence of antiPF4/heparin antibodies, and that functional assays still have to be performed as HIT confirmatory tests. Principles of functional assays for the diagnosis of HIT When platelet activating antibodies in serum recognize PF4/heparin complexes they bind to FcRIIa receptors on the donor platelets and induce platelet activation in vitro. Changes in donor platelets after activation can be detected in different ways. All functional tests require a source of fresh platelets from healthy donors which are incubated with patient serum or plasma in the presence of various doses of heparin. If antibodies are present in the

serum, they activate platelets at pharmacologic heparin concentrations (0.1-0.3 IU mL -1). In a parallel reaction, high heparin concentrations (10-200 IU mL -1) should disrupt PF4/heparin complexes and inhibit anti-PF4/heparin antibody-induced platelet activation [36]. Therefore, to confirm a tested sample is positive, platelet activation must occur at a low, but not at a high heparin concentration. Functional assays are more complex than screening immunoassays and are performed by only a minority of specialized laboratories. They have higher diagnostic specificity than immunoassays, but they are technically demanding. Platelet activation can be detected after labelling platelets with radioactive serotonin and measuring this radioactivity in supernatant after platelet degranulation [37]. The serotonin release assay (SRA) is highly specific but time consuming and uses radioactivity. The SRA result is not available the same day that the test is ordered. The connection between the likelihood of a positive SRA and the concentration of anti-PF4/heparin antibodies has been established and it was found that patients with a low optical density value (OD, 0.4-1.0) have only a 5% chance of a positive SRA assay, whereas increasing OD values increases the probability of positive SRA results [37]. Visually determining platelet aggregates after platelet activation or a heparin-induced platelet aggregation assay (HIPA) [38] is another way to confirm the presence of platelet activating antibodies in serum. The HIPA result is expressed as the presence or absence of a visual donor platelet aggregation. The assay is highly specific but also reported to be less sensitive than the SRA.

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An ideal functional assay for confirming HIT is still not available, however, new methods are emerging and some of them involve the use of flow cytometry. The advantages of flow cytometric assays in the diagnosis of HIT over other functional assays is their nonradioactivity, objective analysis, rapid turnaround time, and the fact that flow cytometers are also used other diagnostic purposes, which means that no additional laboratory equipment is needed. The development of reliable flow-cytometric assays will require further investigation and analysis, but the need not to send serum samples to specialized laboratories will shorten the time to diagnosis. Donor platelets for functional assays As platelets from different healthy donors can respond differently to anti-PF4/heparin antibodies, the variability of donor platelets must be considered. It is recommended that a check be done of the reactivity of donor platelets and to choose donor platelets with higher sensitivity levels to perform the platelet activating assay [40]. An alternative option is to include random platelets from more than one source, which could affect the sensitivity of an assay. In each assay, a negative and positive control needs to be included, and it may be better to use relatively “weak” HIT serum as a positive control than a “strong” reactive one [40]. Sensitivity can also be affected by the method of donor platelet preparation. Donor platelets for flow cytometric tests can be used as platelet-rich plasma (PRP) after the low speed centrifugation of fresh whole blood. The other possibility is to use washed donor platelets. Platelet washing is a timeconsuming procedure which is technically more demanding and requires experience to avoid platelet activation. However, in studies comparing platelet responses to anti-PF4/heparin antibodies, it has been reported that platelet activation assays like SRA and HIPA that use washed donor platelets are more sensitive in the detection of HIT antibodies than PRPbased assays [22,40,41]. Certainly is the reason the presence of plasma proteins that interferes with activation of platelets by PF4/heparin antibody complexes in vitro. They found that reduced fibronectin levels in washed platelet assays increased the sensitivity of the SRA. Fibronectin inhibits PF4 and PF4/heparin binding to platelets, anti-PF4/heparin antibody binding to PF4/heparin complexes and anti-PF4/heparin antibody-induced platelet activation as a result of PF4/heparin complex disruption [22]. Functional flow cytometric assays for HIT usually use PRP of one or more platelet donors. So far no study comparing PRP and washed platelets in flow cytometric assays by detection of surface platelet activation markers has been described in the literature. Therefore, one of the things that still need to be done regarding flow cytometric assays for HIT is to compare anti-PF4/heparin antibody activation of washed donor platelets and platelets within PRP. In general, a lot of attention has to be paid to pre-analytic procedures for platelet activating assays, including blood drawing and donor platelet preparation. Donor platelets should be processed immediately after blood collection to avoid spontaneous activation. Since platelets are very sensitive, activation can also occur when platelets are shaken or exposed to a low temperature. Flow cytometric assays for the diagnosis of HIT Flow cytometry is a powerful technology for the rapid identification, quantification and detection of the functional abilities of cells. Flow cytometers have become indispensable pieces of equipment in clinical laboratories today, and they are routinely used in the diagnosis of various health disorders and in quality control analyses of cell products. As in the case of leukocytes and erythrocytes, flow cytometry is also a useful tool for platelet studies. Different endpoints in flow cytometric methods provide a quantitative assessment of the properties of platelets, such as an increase in the expression of surface receptors, the detection of exposed molecules after degranulations, the release of microparticles or the release of molecules from granules. A determination of the ratio between resting and activated platelets is usually

detected through activation-dependent changes on platelet surfaces or by the formation of platelet microparticles. Before a sample is analyzed with a flow cytometer, platelets or platelet components in suspension are fluorescently labelled, usually with fluorescently conjugated monoclonal antibodies. Detection of platelets The platelet population can be distinguished from other cells in blood by their light-scattering properties, which are linked to cell size and granularity. Due to their small size and variation in shape, platelets are identified using logarithmic settings for light scatter properties rather than the linear settings more useful for other blood cells. Their more precise identification by flow cytometry can be done by the detection of specific platelet glycoproteins (GP), like GP IIb/IIIa (CD41), GPIX (CD42a), GPIb (CD42b) or GPIIIa (CD61) [39]. Using platelet specific antibodies allows us to avoid including cells other than platelets in analyses. Anti-CD41 and anti-CD61 monoclonal antibodies against membrane glycoproteins, which are present on resting and activated platelets, are the most frequently used for platelet identification. To avoid spontaneous platelet activation and nonspecific staining with fluorochrome-conjugated antibodies, we have to process platelet samples soon after preparation. Detection of activated platelets The rapid changes which happen upon platelet activation in cell surface protein expression can be detected by using different fluorochrome-conjugated molecules. Although only size and granularity characteristics on flow cytometric graphs can be used to identify the platelet population, double-labelling of platelets is usually used for HIT confirmation with flow cytometric tests: first, antibodies that bind to all platelets, and second, antibodies or other molecules specific to activated platelets. This combination enables a more precise determination of the resting as well as the activated state of the platelets in the sample. Flow cytometric functional assays for HIT described in the literature are listed in Table 1 and Table 2. According to literature, the most common platelet activation markers in studies of HIT confirmation are Annexin V and CD62P. Protein Annexin V is a positively charged protein and could be used as a marker which binds with high affinity to anionic phospholipids like phosphatidylserine in the presence of calcium. Under physiological conditions, negatively charged phosphatidylserine is mainly present in the inner layer of the plasma membrane [42]. This asymmetrical distribution of phospholipids in the plasma membrane is dynamically maintained by several enzymes. During cell activation, the increased concentration of cytosolic calcium affects enzymes, resulting in an exposure of phospholipids on platelet surfaces [43]. Then fluorescentlabelled Annexin V binds to exposed phospholipids and distinguishes activated platelets from other platelets. In 1997, Tomer reported on a flow cytometric test for HIT which detected activated platelets after binding of Annexin V [44]. The author used anti-CD41a antibodies for platelet identification and Ca-ionophore to prepare a positive control for the assay. The study describes the flow cytometric method in comparison to the established SRA. After parallel analysis of 18 positive patients and 10 negative controls the test had a high correlation with the SRA, showing 100% specificity and 95% sensitivity. It was also reported that ELISA and SRA (performed with PRP) were both negative in the patient with clinical confirmed HIT, while the result of flow cytometric assay (also performed with PRP) was positive [45]. In 2001, Poley et al prospectively evaluated samples from patients in parallel, using HIPA test as a reference and the test showed a good agreement with the HIPA. They used Annexin V and pooled platelets from multiple donors [46]. Currently, two commercial functional flow cytometric assays for HIT are on the market and one of them, HITAlert™ Kit, which probably uses Annexin V, achieves 81% sensitivity and 100% specificity

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Table 1 Flow cytometric studies for the detection of activation markers on a platelet surface or detection of serotonin content upon activation with anti-PF4/heparin antibodies Flow cytometric platelet activation marker

No. of samples

Conclusion

Donor platelets used

References

Annexin V

18 HIT patients and 10 controls 18 HIT patients; 1 patient ELISA and SRA negative 248 HIT suspected patients and 97 controls

100% specificity, 95% sensitivity; SRA as reference method

PRP one donor PRP one donor

Tomer A. Br J Haematol. 1997. [44] Tomer et al. Am J Hematol 1999. [45]

Pooled PRP multiple donors PRP four donors

Poley et al. Eur J Haematol 2001. [46]

Annexin V

Annexin V

18 HIT patients were both FCA and SRA positive; ELISA and SRA negative patient was FCA positive 214 negative, 17 indeterminate, 17 HIPA positive patients. From 17 HIPA indeterminate: 14 FCA negative and 3 indeterminate

PF4/heparin-coated beads, Annexin V

13 HIT suspected patients Combined analysis: PF4/heparin-coated beads for the detection of anti-PF4/heparin and 6 controls antibodies + Annexin V for activated platelets

Annexin V

37 HIT suspected patients 81% sensitivity and 100% specificity; reference method SRA

PRP one donor

Annexin V

346 HIT suspected patients 4 HIT suspected patients and 6 controls

88.2% sensitivity, 99.1% specificity; reference method clinical HIT diagnosis

PRP

FCA can replace SRA

PRP 15 donors

Gobbi et al. Cytometry B Clin Cytom 2004. [50] Solano et al. Blood Coagul Fibrinolysis 2013. [47] Garritsen et al. Int J Lab Hematol 2014. [48] Gobbi et al. Br J Haematol 2003. [49]

Whole blood one donor PRP probably one donor

Jy et al. Thromb Haemost.1999 [51] Vitale et al. Cytometry. 2001 [52]

serotonin flow cytometric detection CD62P CD62P Annexin V CD62P

PF4-dependent CD62P expression CD62P

12 HITT patients, 30 HIT 11 of 12 HITT patients and 5 of 30 HIT patients were positive patients and 65 controls 85% CD62P- positive and 40% Annexin V-positive HIT patients 13 HIT patients, 15 non-HIT patients and 10 controls 41 HIT suspected patients 83% specificity, reference method HIPA

PRP 4 separate donors Pooled PRP 91 HIT suspected patients 11 of 16 PCA positive and SRA negative patients were HIT positive (HIT positive: intermediate 4Ts score and a PF4 ELISA OD ≥ 2.0, or a high 4Ts score and a PF4 ELISA of 2 or 3 donors OD ≥ 1.0) 290 HIT suspected 90% sensitivity, 94% specificity; reference clinical HIT diagnosis PRP one patients donor

Maličev et al. Int J Lab Hematol 2016. [56] Padmanabhan et al. Chest 2016. [58] Amiral J. Emosis. 2018. [59]

HIT: Heparin-Induced Thrombocytopenia; HITT: Heparin-Induced Thrombocytopenia and Thrombosis; SRA: Serotonin Release Assay; HIPA: Heparin-Induced Platelet Aggregation Assay; FCA: Flow Cytometric Assay; PRP: Platelet-Rich Plasma.

in the diagnosis of HIT using an SRA as a reference after analysing 37 HIT suspected patients [47]. In the next study, clinical data were used as a reference for a direct comparison with flow cytometric results and HITAlert™ Kit showed 88.2% sensitivity and 99.1% specificity [48]. A non-radioactive method for the detection of the serotonin content in activated and resting platelets by flow cytometry was also presented.

Platelets were identified by CD41a surface staining and their serotonin content was measured by specific anti-serotonin intracellular staining, while their activation was independently shown by Annexin V binding [49]. Later, in 2004, Gobbi et al described a method of HIT confirmation based on the antibody detection by PF4/heparin-coated beads and at the same time the determination of platelet activation by Annexin V [50].

Table 2 Flow cytometric testing for HIT by detection of platelet-derived microparticles upon activation with anti-PF4/heparin antibodies Flow cytometric platelet activation marker

No. of samples

Conclusion

Donor platelets used References

anti-CD42b positive microparticles

202 HIT suspected patients

96% agreement between FCA and SRA

Washed platelets

/

53 HIT suspected patients, 15 HITT patients and 19 controls 53 HIT suspected patients

92% agreement between FCA and HIPA

/

88.9% sensitivity and 100% specificity; reference clinical HIT diagnosis.

Whole blood, one donor

40 ELISA positive patients: 17 Annexin V positive (12 of them showed thrombosis), and 14 positive with microparticle assay (10 of them showed thrombosis). The incidence of thromboembolic events in patients who tested strongly positive, weakly positive, and negative was 61 %, 40 %, and 29 %, respectively; reference clinical HIT diagnosis. HIT patients have increased levels of platelet-derived and leukocyte-derived microparticles. Leukocyte TF‐bearing microparticles are significantly associated with the thrombotic risk in HIT.

Selected highresponder PRP donors

Mullier et al. Thromb Res 2014. [65] Kerényi et al. Cytometry B Clin Cytom 2017.[66]

Selected and washed platelets from 2 donors Platelet free plasma from patients and control samples

Maeda et al. Thromb Haemost 2017. [67] Campello E et al. Cytometry B Clin Cytom 2018. [5]

Annexin V positive microparticles

microparticles identified by size-selection

405 HIT suspected patients

anti-CD42b positive microparticles

401 HIT suspected patients

45 HIT patients (12 Platelet- and leukocyte-derived with HITT), 45 microparticles positive for Annexin V, CD62P, CD62L, PF4 and non-HIT patients TF

Lee et al. Br J Haematol 1996. [63] Kounavi et al. Crit Care 2007. [64]

HIT, heparin-induced thrombocytopenia; HITT, heparin-induced thrombocytopenia and thrombosis; SRA, serotonin release assay; HIPA, heparin-induced platelet aggregation assay; FCA, flow cytometric assay; PRP, platelet-rich plasma; TF, tissue factor.

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The use of platelet activation marker CD62P in flow cytometric assays for HIT has also been reported on a number of times [45,51,52]. P-selectin or CD62P is a classical marker of platelet activation. Its detection on a platelet’s surface shows α-granule release which happens within seconds of platelet activation. P-selectin is a Ca2+ dependent receptor on the inner wall of α-granules. During platelet activation, it translocates to the plasma membrane and adding anti-CD62P antibodies to a platelet sample allows the quantitative determination of activated platelets [53-55]. Flow cytometric analysis of CD62P alone or in a combination with other markers has already been used for the assessment of platelet activation in vitro after their incubation with the serum of patients with HIT. In the study from 2001 it was showed that CD62P expression was positive in 85%, and Annexin V was positive in 40% of the HIT cases examined. The authors concluded that the expression of CD62P appears to be a better functional marker for the diagnosis of HIT than Annexin V [52]. Another study describes the flow cytometric assay based on the use of anti-CD61 and anti-CD62P antibodies, where 41 patients deemed positive with IgG-specific ELISA assays were selected for the flow cytometric assay and HIPA. The diagnostic specificity of the flow cytometric assay was calculated based on HIPA results and was 83%. The assay was performed in a 96-well microtiter plate and the platelet-activation threshold was set at 50% instead of 20%, which reduces the risk of a false-positive result and distinguishes the method from other flow cytometric assays [56]. Later, the correlation between quantitative ELISA results and the same flow cytometric assay outcomes were studied in patients suspected of having HIT and the results showed that, similarly to SRA, absolute OD values could predict the possibility of the presence of platelet-activating anti-PF4/heparin antibodies [57]. Padmanabhan et al used platelets pre-treated with PF4 as targets for antibody detection. They compared the PF4-dependent CD62P expression assay with SRA with identical target platelets, and the test had a higher diagnostic accuracy than the SRA [58]. In 2018, the second flow cytometric test, Emo-test HIT Confirm®, came onto the market with antiCD41 and anti-CD62P antibodies. Study results showed that this assay has at least the same specificity and sensitivity as the SRA [59]. For the commercial assays, fresh PRP from only one healthy donor is needed, without any prior selection of reactive platelets. A weakness of both commercial assays is that they do not include a positive control serum. Flow cytometry also allows for the determination of platelet activation by labelling with PAC-1 antibodies. Fibrinogen receptors undergo a conformational change during platelet activation and monoclonal antibody PAC-1 detects the active state of GP IIb/IIIa present on the platelet surface. PAC-1 is infrequently used for HIT diagnosis. The ability of fondaparinux and unfractionated heparin to induce platelet activation in the presence of HIT sera among different markers of platelet activation was researched. In a comparison of PAC1, anti-CD62P antibodies and Annexin V, Annexin V was the most sensitive for detecting heparin-dependent platelet antibodies [60]. After reviewing the literature about flow cytometric assays for HIT we can conclude that both commonly used platelet activation markers, CD62P and Annexin V, are suitable for the performance of the functional assay. There are still some limitations for clinical implementation, like unification of donor platelet preparation, which applies also to nonflow cytometric assays. Reported flow cytometric assays differ in the time required to perform the assay, from 30 min to 2 h. Above all, the correlation of flow cytometric results with clinical HIT diagnosis in a larger study still remains to be seen. Generation of platelet microparticles The evaluation of platelet-derived microparticles is an alternative method to determine platelet activation by flow cytometry. The platelet microparticles that bud off from platelet surfaces maintain similar surface marker profiles as the platelets. Using flow cytometry, platelet-derived microparticles can be identified by their light scatter characteristics by a size ranging from 0.1μm to 1.0μm and by the antigens such as CD41 or

CD42a, CD42b, CD61. [61,62]. Moreover, they can expose characteristic activated platelet markers such as P-selectin, phosphatidylserine and PAC-1, and CD63 from dense granules. Using flow cytometry, platelet-derived microparticles can be detected in samples of whole blood, platelet-rich plasma (PRP) or platelet-poor plasma (PPP). The detection of platelet-derived microparticles is not a method in routine use. It is a demanding technical process, which includes careful blood sample drawing, stabilizing platelets to prevent further activation, ultracentrifugation and the determination of the size calibration and gating methods for flow cytometry. Due to variabilities in platelet-derived microparticle isolation as well as differences in flow cytometric settings, different results can be obtained from the one sample. Standardized protocols are still lacking, so we have to be careful in comparing the results of different studies. However, some studies have been done over the years. The flow cytometric platelet microparticle assay using anti-CD42b antibodies was evaluated against the SRA and the agreement between the two assays was 96% [63]. The flow cytometric platelet microparticle assay was also compared to the HIPA and a 92% agreement was calculated [64]. In another study, the results of the platelet microparticle assay and the results of the SRA were compared to the clinical outcome. Plateletderived microparticle concentrations were measured after their staining with anti-CD41 and Annexin V. Using the clinical outcome as a reference, the sensitivity and specificity of the flow cytometric platelet microparticle assay reached 88.9% and 100% while the sensitivity and specificity of the SRA were 88.9% and 95.5% [65]. In the next study, the results of platelet-derived microparticle analysis were comparable to results obtained after Annexin-V binding to platelets [66]. Maeda et al evaluated the association between the platelet microparticle assay with selected and washed donor platelets and clinical outcomes [67]. Campello et al confirmed the presence of CD62P positive microparticles and microparticles carrying PF4 derived from activated platelets in patients with HIT [5]. The flow cytometric analysis of platelet-derived microparticles requires skilled personnel and special laboratory equipment. Other standardized methods to measure microparticles and their association with platelet activation would probably be more appropriate rather than flow cytometry. Conclusion To date, flow cytometric assays have shown themselves to be a promising method in determining the activating capabilities of antiPF4/heparin antibodies in patient serum. HIT is usually confirmed after the analysis of healthy donor platelet surfaces, or less often, by detecting platelet-derived microparticles. New flow cytometric assays or their variants were mostly compared to other functional assays such as SRA or HIPA. In general, when taking the results of other functional assays as a reference, we have to take into account that the reported specificities of the reference assays can vary and are rarely 100%. A comparison of flow cytometric results to the clinical presentations of HIT patients is often lacking, especially in larger prospective studies. However, as flow cytometry is an established technology used in clinical laboratories, and taking into consideration the results achieved so far and the rapid turnaround time, flow cytometric functional assays for the confirmation of HIT are really promising. Conflict of interest statement None. Acknowledgements The author would like to thank to Eva Kocjan for providing Fig. 1 in this review.

Please cite this article as: E. Maličev, The use of flow cytometry in the diagnosis of heparin-induced thrombocytopenia (HIT), Transfusion Medicine Reviews, https://doi.org/10.1016/j.tmrv.2019.08.001

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