Flow Cytometry

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Flow Cytometry Thomas A. Blair, Andrew L. Frelinger, III and Alan D. Michelson Center for Platelet Research Studies, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA, United States

INTRODUCTION 627 MEASUREMENT OF PLATELET ACTIVATION 628 Markers of Platelet Activation 628 Platelet Activation in Clinical Disorders 633 Reduced Circulating Activated Platelets and Platelet Hyporeactivity 634 DIAGNOSIS OF SPECIFIC DISORDERS 636 Platelet Surface Glycoprotein Deficiencies 636 Platelet-Type von Willebrand Disease 636 Scott Syndrome 636 Storage Pool Disease 636 Heparin-Induced Thrombocytopenia 636 MONITORING OF ANTIPLATELET AGENTS 636 MONITORING OF THROMBOPOIESIS 637 BLOOD BANK APPLICATIONS 638 Quality Control of Platelet Concentrates 638 Other Blood Bank Applications 638 PLATELET-ASSOCIATED IgG 638 PLATELET COUNT 638 Platelet Count in Humans 638 Platelet Count in Mice 638 OTHER RESEARCH APPLICATIONS 638 Platelet Survival, Tracking, and Function In Vivo 638 Monocyte and Neutrophil Procoagulant Activity With and Without Surface-Adherent Platelets 639 F-Actin 639 Calcium Flux 639 Fluorescence Resonance Energy Transfer 640 Platelet Recruitment to Growing Thrombi 640 Bacteria-Platelet Interactions 641 ADVANCES IN FLOW CYTOMETRY 641 Mass Cytometry 641 Imaging Flow Cytometry 642 REFERENCES 643

INTRODUCTION Flow cytometry is widely used for the functional and phenotypic characterization of platelets. The technology is capable of detecting multiple specific characteristics on cells in suspension (Table 35.1), with limited sample volume and in a time range of seconds. Before flow cytometric analysis, single cells in suspension are fluorescently labeled, typically with a fluorescently conjugated monoclonal antibody. In the flow cytometer, the suspended cells pass through a flow cell and, at a rate of up to 20,000 cells per second, through the focused beam of one or more lasers. After the laser light activates the fluorophore at the Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00035-7 Copyright © 2019 Elsevier Inc. All rights reserved.

excitation wavelength, detectors process the emitted fluorescence and light scattering properties of each cell. The intensity of the emitted light is proportional to the antigen density or the characteristics of the cell being measured. Flow cytometry is amenable to evaluation of platelet function in a variety of matrices, including physiologic buffers, as with washed platelets, plasma, as with platelet-rich plasma, and in whole blood. However, studies that utilize flow cytometric assays of washed platelets or platelet-rich plasma are, like other assays of platelet function, potentially susceptible to artifactual in vitro platelet activation as a result of the obligatory separation procedures. Various antiplatelet agents (e.g., apyrase [adenosine diphosphate (ADP) scavenger], indomethacin [cyclooxygenase inhibitor] or prostaglandin E1 [PGE1, which indirectly stimulates adenyl cyclase and reduces Ca2+ signaling]) can be used to circumvent platelet preactivation during these separation procedures. However, the use of these compounds is not always desirable and makes the experimental conditions less physiological. The detection of activated platelets in whole blood by flow cytometry was therefore a major advance.1 A typical schema of sample preparation for whole blood flow cytometric analysis of platelets is shown in Fig. 35.1. The anticoagulant is usually buffered sodium citrate, although other anticoagulants can be used.2 The purpose of the initial dilution is to minimize the formation of platelet aggregates, as the use of high concentrations of platelets allows for greater auto-activation to occur. While platelets can be identified on the basis of their forward- and side-light scatter properties alone, this can be confounded by the presence of other small particles with similar light scatter properties. Therefore, a minimum of two monoclonal antibodies is recommended for platelet testing, each conjugated with a different fluorophore, so that one antibody can be used as a “platelet identifier” in combination with light scatter properties to identify platelets accurately. A wide variety of fluorophores are available for antibody conjugation (e.g., phycoerythrin [PE], fluorescein isothiocyanate [FITC], peridinin chlorophyll protein [PerCP], PE-cyanine (Cy) 5, PerCP-Cy5.5). The “test” monoclonal antibody (recognizing the antigen to be measured) is added at a saturating concentration. The “platelet identifier” monoclonal antibody (e.g., glycoprotein [GP] Ib-, GPIX-, integrin αIIb-, or integrin β3-specific) is added at a near-saturating concentration. Physiological agonists can be used in the assay, including thrombin, thrombin receptor-activating peptide (TRAP), ADP, collagen, the complement fraction C5b-9, and thromboxane A2 analogs. Nonphysiologic agonists include phorbol myristate acetate, convulxin, fucoidan, rhodocytin, and the calcium ionophore A23187. Samples may be analyzed immediately after staining or may be stabilized by fixation, typically with a final concentration of 1% paraformaldehyde. The antibodies can be added after fixation, provided fixation does not interfere with antibody binding.2,3 An additional permeabilization step or the use of cell-permeable fluorescent proteins is required to evaluate intracellular platelet markers such as granular components, cytoskeletal elements, or specific phosphoproteins. Samples are then analyzed in a flow cytometer. After identification

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TABLE 35.1 Applications of Flow Cytometry to the Study of Platelets Application

Examples

Measurement of platelet activation (circulating activated platelets, platelet hyperreactivity, or platelet hyporeactivity)

Activation-dependent increases in surface glycoprotein expression Leukocyte-platelet aggregates Platelet-derived extracellular vesicles VASP phosphorylation and other signal transduction molecules Platelet-platelet aggregates Fibrinogen binding Bernard-Soulier syndrome Glanzmann thrombasthenia Platelet-type von Willebrand disease Scott syndrome Storage pool disease Heparin-induced thrombocytopenia COX-1 antagonists: arachidonic acid-dependent platelet activation P2Y12 antagonists: ADPdependent platelet activation GPIIb-IIIa antagonists: fibrinogen or PAC1 binding Immature platelets Quality control of platelet concentrates Identification of leukocyte contamination in platelet concentrates Immunophenotyping of platelet HPA-1a Detection of maternal and fetal anti-HPA-1a antibodies Platelet cross-matching Immune thrombocytopenias Alloimmunization

Diagnosis of specific disorders

Monitoring of antiplatelet agents

Monitoring of thrombopoiesis Blood bank applications

Platelet-associated IgG Platelet counting Other research applications

Platelet survival, tracking, and function in vivo F-actin Calcium flux Fluorescence resonance energy transfer Platelet recruitment to sites of thrombosis Viability (calcein) Bacteria-platelet interactions Mass cytometry Imaging flow cytometry

of platelets both by their characteristic light scatter (forwardscatter [FSC], which is proportional to cell dimension/size and side-scatter [SSC], which is proportional to the structural complexity/granularity of the cell) and by binding of the platelet identifier monoclonal antibody (indicated by [for example] PE positivity), binding of the (for example) FITC-conjugated test monoclonal antibody is determined by analyzing 5000–10,000 individual platelets. Refs. 4, 5 contain specific methodological protocols of whole blood flow cytometric assays of platelet function, together with a discussion of methodological issues. There are many advantages to whole blood flow cytometric analysis of platelet function. Platelets are directly analyzed in their physiological milieu of whole blood (including red cells and white cells, both of which affect platelet activation6,7). The minimal manipulation of the samples prevents artifactual

in vitro activation and potential loss of platelet subpopulations.1,8–10 Both the activation state and reactivity of circulating platelets can be determined. Flow cytometry permits the detection of a spectrum of activation-dependent modifications, such as changes in the platelet surface membrane (e.g., changes in surface receptor copy number, conformation, and phosphatidylserine expression), changes in the platelet cytoskeleton, and intracellular changes (e.g., calcium mobilization or phosphorylation of intracellular proteins). Furthermore, as new monoclonal antibodies directed against novel functional epitopes are developed, they can easily be incorporated into the assay. A subpopulation of as few as 1% partially activated platelets can be detected by whole blood flow cytometry.10,11 Only minuscule volumes (
5 μL) of blood are required,1,8 making whole blood flow cytometry particularly advantageous for neonatal studies and for studies of other patients for whom available sample volume is limited.12 The platelets of patients with profound thrombocytopenia can also be accurately analyzed.13 Finally, flow cytometric evaluation of platelets is easily adaptable to animal studies if the antibody reagents are available.14–16 However, non-antibody reagents that are not species-specific (e.g., autologous ligands [FITCfibrinogen], calcium indicators, F-actin probes [phalloidin], nucleotide dyes [thiazole orange], viability dyes [calcein], and annexin V) are particularly useful in animal studies. There are some disadvantages to flow cytometric analysis of platelet function. First, flow cytometers are expensive instruments to purchase and maintain, although the introduction in recent years of low cost, turnkey systems with userfriendly software has allowed the technology to become widely available. Second, for a clinical assay, sample preparation can be quite complicated, although the development of kits (e.g., BioCytex, Marseilles, France) has simplified some of the assays. Third, to avoid ex vivo platelet activation, blood samples should be processed within approximately 30 min of drawing for many assays.1 For the evaluation of some platelet receptors, this time issue can be circumvented by immediate fixation.2 Finally, the number of antigens that can be simultaneously analyzed is inherently limited by emission spectra overlap; however, this is mainly a limitation in the context of platelet research as opposed to clinical diagnosis of platelet disorders.17

MEASUREMENT OF PLATELET ACTIVATION In the absence of an added exogenous platelet agonist, whole blood flow cytometry can determine the activation state of circulating platelets, as judged by the binding of an activationdependent monoclonal antibody. In addition to this assessment of platelet function in vivo, inclusion of an exogenous agonist in the assay enables analysis of the reactivity of circulating platelets in vitro. In the latter application, whole blood flow cytometry is a physiological assay of platelet function in that an agonist results in a specific functional response by the platelets: a change in the surface expression of a physiological receptor (or other antigen or bound ligand), as determined by a change in the binding of a monoclonal antibody. In addition, as discussed below, whole blood flow cytometric enumeration of monocyte-platelet aggregates (MPAs) and procoagulant platelet-derived extracellular vesicles are also sensitive markers of in vivo platelet activation.

Markers of Platelet Activation Activation-dependent Monoclonal Antibodies Laboratory markers of platelet activation include activationdependent conformational changes in integrin αIIbβ3 (the GPIIb-IIIa complex, CD41/CD61), exposure of granule

Flow Cytometry

629

35

Fig. 35.1 A typical schema of sample preparation for analysis of platelets by whole blood flow cytometry. Abbreviations: ADP, adenosine diphosphate; TRAP, thrombin receptor activating peptide.

membrane proteins, platelet surface binding of secreted platelet proteins, and development of a procoagulant surface (Table 35.2). The two most widely studied types of activationdependent monoclonal antibodies are those directed against conformational changes in αIIbβ3 and those directed against granule membrane proteins. The integrin αIIbβ3 complex, a receptor for fibrinogen, von Willebrand factor, vitronectin, and fibronectin, is essential for platelet aggregation (see Chapter 12). Whereas most monoclonal antibodies directed against αIIbβ3 bind to resting platelets, monoclonal antibody PAC1 is directed against the fibrinogen binding site exposed by a conformational change in αIIbβ3 of activated platelets (Table 35.2).18 Thus, PAC1 only binds to activated platelets, not to resting platelets. Despite the fact that fibrinogen is a competitive inhibitor of PAC1 binding, there is no difference in the levels of PAC1 binding to ADPstimulated washed platelets compared to platelets in PRP (where fibrinogen is approximately 3 mg/mL).1 This has been attributed to a greater apparent affinity of PAC1 (Kd ¼ 5 nM) than fibrinogen (Kd ¼ 250 nM) for the fibrinogen receptor.1 A similar reagent, JON/A-R-phycoerythrin (JON/APE) which discriminates between resting and activated αIIbβ3 on murine platelets has recently been described.45 Interestingly, JON/A, a rat monoclonal antibody directed against mouse αIIbβ3, when conjugated to R-phycoerythrin (PE), binds with high affinity to αIIbβ3 on activated but not resting mouse platelets whereas

the same antibody conjugated to FITC bound equally well to resting and activated murine platelets.45 Other αIIbβ3-specific activation-dependent monoclonal antibodies are directed against either ligand-induced conformational changes in αIIbβ3 (ligand-induced binding sites, LIBS)19 or receptorinduced conformational changes in the bound ligand (fibrinogen) (receptor-induced binding sites, RIBS)22 (Table 35.2). Rather than αIIbβ3-specific monoclonal antibodies, FITCconjugated fibrinogen can also be used in flow cytometric assays to detect the activated form of platelet surface αIIbβ3,46,47 but the concentration of unlabeled plasma fibrinogen and unlabeled fibrinogen released from platelet α granules must also be considered in these assays. The most widely studied type of activation-dependent monoclonal antibodies directed against granule membrane proteins are P-selectin (CD62P)-specific. P-selectin (see Chapter 16) is a component of the α granule membrane of resting platelets that is only expressed on the platelet surface membrane after α granule secretion. Therefore, a P-selectin-specific monoclonal antibody only binds to degranulated platelets, not to resting platelets. The activation-dependent increase in platelet surface P-selectin is not reversible over time in vitro.48,49 However, in vivo circulating degranulated platelets rapidly lose their surface P-selectin, but continue to circulate and function.15,50 Platelet surface P-selectin is therefore not an ideal marker for the detection of circulating degranulated

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TABLE 35.2 Activation-Dependent Monoclonal Antibodies, i.e., Antibodies That Bind to Activated But Not Resting Platelets Activation-Dependent Platelet Surface Change Conformational changes in integrins Activation-induced conformational changes in integrin αIIbβ3 resulting in exposure of the fibrinogen binding site Exposure of ligand-induced binding sites (LIBS) on integrin αIIbβ3

Prototypic Antibodies

References

PAC1

18

PMI-1, LIBS1, LIBS6 2G5, 9F9, F26 IAC-1

19–21

Exposure of receptor-induced binding sites (RIBS) on bound fibrinogen Activation-induced conformation changes in integrin α2β1 resulting in exposure of the collagen binding site Exposure of granule membrane proteins P-selectin (α-granules) S12, AC1.2, 1E3 GMP-33 (α-granules) RUU-SP 1.77 CD63 (dense granules and lysosomes) CLB-gran/ 12 LAMP-1 (lysosomes) H5G11 LAMP-2 (lysosomes) H4B4 CD40 ligand TRAP1 Lectin-like oxidized LDL receptor-1 JTX68 (LOX-1) Platelet surface binding of secreted platelet proteins Thrombospondin P8, TSP-1 Multimerin JS-1 Development of a procoagulant surfacea Factor V/Va binding V237 Factor X/Xa binding 5224 Factor VIII binding 1B3

22–24 25

26–28 29, 30 31 32 33 34 35

36, 37 38, 39 40 41 42

a

Development of a procoagulant platelet surface can also be detected by the binding of annexin V or lactadherin to phosphatidylserine.43,44

platelets, unless (a) the blood sample is drawn immediately distal to the site of platelet activation, (b) the blood sample is drawn within 5 min of the activating stimulus, or (c) there is continuous activation of platelets. CD63 (Table 35.2) is a membrane protein of dense granules and lysosomes and, like P-selectin, it is only expressed upon the surface of activated platelets.51 CD63 is a less sensitive marker of activation than P-selectin, because it is present at a lower copy number and a greater level of platelet activation is required for its exposure. However, unlike P-selectin, this glycoprotein is not prone to proteolysis and may therefore provide a more stable marker of platelet activation. The length of time that other activation-dependent surface markers remain expressed on the platelet surface in vivo has not yet been definitively determined.

the percent of neutrophils with adherent infused platelets; and (3) the in vivo half-life of detectable circulating MPAs (approximately 30 min) is longer than both the in vivo half-life of NPAs (approximately 5 min) and the previously reported15 rapid loss of surface P-selectin from nonaggregated infused platelets. All these findings suggested that measurement of circulating MPAs may be a more sensitive indicator of in vivo platelet activation than either circulating NPAs or circulating P-selectinpositive nonaggregated platelets. We therefore performed 2 clinical studies in patients with acute coronary syndromes.52 First, after percutaneous coronary intervention (PCI), there was an increased number of circulating MPAs (and, to a lesser extent, NPAs), but not P-selectin-positive platelets, in peripheral blood. Second, of patients presenting to an emergency department with chest pain, patients with acute myocardial infarction had more circulating MPAs than patients without acute myocardial infarction and normal controls. However, circulating P-selectin-positive platelets were not increased in chest pain patients with or without acute myocardial infarction.52 In summary, we have demonstrated by five independent means (in vivo tracking of activated platelets in baboons (Fig. 35.3),52 human PCI,52 human acute myocardial infarction,52 stable coronary artery disease53 (discussed in section “acute coronary syndromes”), and human chronic venous insufficiency54 (discussed in section “peripheral vascular disease”) that circulating MPAs are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin. Monocyte subsets and MPAs play an important role in driving the development of atherosclerosis and thrombosis.52,55–63 Monocyte subsets are heterogeneous and comprise three biologically distinct subpopulations that are classified as CD14 ++CD16  (classical monocytes, Mon1), CD14 ++CD16 + (intermediate monocytes, Mon2), and CD14 + CD16 ++ (nonclassical monocytes, Mon3).64 Any one of these monocyte subpopulations can adhere to platelets to form MPAs. In recent years, the individual contribution of monocyte subsets and subset-specific MPAs to thrombosis has been explored. Flow cytometric studies have reported that in unstable angina patients, increased CD14 ++CD16 + (Mon2) counts and CD14 ++CD16 + (Mon2) subset specific MPAs are associated with higher-risk of death or myocardial infarction.63 While in ST elevation myocardial infarction (STEMI) patients, high levels of CD14 ++CD16 + (Mon2) subset specific MPAs predict the degree of left ventricular systolic dysfunction post STEMI and are associated with a worse outcome in acute heart failure secondary to ischemic heart disease.61,65 A recent study by Brown et al.66 also demonstrated that CD14 ++CD16 (Mon2) subset specific MPAs were elevated in patients with diffuse coronary artery disease. Future studies are required to determine whether CD14 ++CD16 + (Mon2) monocytes may be a therapeutic target to combat thrombosis.

Leukocyte-Platelet Aggregates

Procoagulant Platelets and Platelet-Derived Extracellular Vesicles

P-selectin mediates the initial adhesion of activated platelets to monocytes and neutrophils by binding to its counter-receptor, P-selectin glycoprotein ligand 1 (PSGL-1), which is constitutively expressed on the surface of leukocytes (see Chapter 16). MPAs and neutrophil-platelet aggregates (NPAs) are readily identified by whole blood flow cytometry (Fig. 35.2).5 Tracking of autologous infused biotinylated platelets in baboons by three color whole blood flow cytometry enabled us52 to directly demonstrate in vivo (Fig. 35.3) that: (1) platelets degranulated by thrombin very rapidly (within 1 min) form circulating aggregates with monocytes and neutrophils; (2) the percent of monocytes with adherent infused platelets is greater than

As determined by flow cytometry, in vitro activation of platelets by some agonists (e.g., C5b-9, collagen/thrombin, and the calcium ionophore A23187) in the presence of extracellular calcium ions results in platelets and platelet-derived extracellular vesicles (defined by low forward angle light scatter and binding of a platelet-specific monoclonal antibody) that are procoagulant (determined by binding of monoclonal antibodies to activated factors V, VIII, or X, or by detection of phosphatidylserine exposure by annexin V or lactadherin).40,42–44,51,67 These findings suggest that procoagulant platelets and platelet-derived extracellular vesicles may have an important role in the assembly of the “tenase”

35 Fig. 35.2 Whole blood flow cytometric analysis of monocyte-platelet aggregates and neutrophil-platelet aggregates in a normal donor after activation with thrombin receptoractivating peptide (TRAP) 20 μM. Monocytes (blue) and neutrophils (green) were identified by their characteristic light scatter properties and the binding of FITCconjugated CD14-specific monoclonal antibody TUK4 (left panel). Platelet-positive monocytes (i.e., monocyte-platelet aggregates) were identified by the binding of the PE-conjugated αIIb (CD41)-specific monoclonal antibody 5B12 in the monocyte region (upper right panel). Platelet-positive neutrophils (i.e., neutrophil-platelet aggregates) were identified by the binding of the PE-conjugated αIIb (CD41)-specific monoclonal antibody 5B12 in the neutrophil region (lower right panel). SSC-H, side scatter height.

Platelets activated preinfusion 80

Monocyte-platelet aggregates Neutrophil-platelet aggregates Platelet surface P-selectin

70

Percent

60 50 40 30 20 10

0 0

10

20

30

40

50

60 120

Time (min) Platelets not activated preinfusion 80

Monocyte-platelet aggregates Neutrophil-platelet aggregates Platelet surface P-selectin

70

Percent

60 50 40 30 20 10

0 0

10

20

30 Time (min)

40

50

60 120

Fig. 35.3 Tracking of autologous infused biotinylated platelets in baboons by three color whole blood flow cytometry. Baboons were infused with autologous, biotinylated platelets that were (upper panel) or were not (lower panel) thrombin-activated preinfusion. Surface P-selectin on the infused platelets and participation of the infused platelets in circulating monocyte-platelet and neutrophil-platelet aggregates was determined by 3-color whole blood flow cytometric analysis of peripheral blood samples drawn at the indicated time points. The “0” time point refers to blood samples taken immediately preinfusion. Platelet surface P-selectin is expressed as mean fluorescence intensity (MFI), as a percentage of the fluorescence of a preinfusion maximally activated (10 U/mL) thrombin control sample. Monocyte-platelet and neutrophil-platelet aggregates are expressed as the percent of all monocytes and neutrophils with adherent infused platelets. Data are mean  SEM. (Reproduced with permission from Ref. 52.)

PART III Clinical Tests of Platelet Function

and “prothrombinase” components of the coagulation system in vivo. There has been increasing interest in platelet-derived extracellular vesicles because of their potential use as biomarkers and their apparent roles in health and disease. For example, murine studies have demonstrated that extracellular vesicles are involved in venous thrombogenesis, with extracellular vesicles derived from leukocytes correlating negatively with thrombus weight and extracellular vesicles from platelets correlating positively with thrombus weight.68 Furthermore, platelet-derived extracellular vesicle levels in blood are increased in diseases such as rheumatoid arthritis, ITP, sickle cell disease, uremia, cancer, multiple sclerosis, antiphospholipid syndrome, systemic lupus erythematosus, and in patients with HIT.69–72 Consequently, flow cytometric methods for the direct detection, enumeration and characterization of platelet-derived extracellular vesicles in whole blood have been developed.73–78 In the past, a lack of standardized methods hampered the comparison of results between different studies. However, the accuracy by which platelet-derived extracellular vesicles can be quantified and characterized has been significantly improved in recent years by (i) identification of preanalytical processes that influence extracellular vesicle release in samples and consequently drive variability across studies and (ii) the development of standardized protocols for whole blood sample processing and analysis of extracellular vesicles by flow cytometry.72,79–81 Megakaryocytes are also known to produce extracellular vesicles; however, there are surface markers that clearly distinguish megakaryocyte-derived extracellular vesicles from extracellular vesicles derived from activated platelets.72,82–84 Flow cytometric studies have shown that megakaryocyte-derived extracellular vesicles are negative for CD62P and LAMP-1, but positive for surface GPVI and CLEC-2, whereas extracellular vesicles released from activated platelets express surface CD62P, LAMP-1, and CLEC2, but do not express GPVI.72,82–84 Similarly, extracellular vesicles derived from other cell types can be differentiated from plateletderived extracellular vesicles by surface expression of unique antigens. For example, leukocyte-derived extracellular vesicles are CD45-positive, endothelial-derived extracellular vesicles are CD144/CD106/CD62E-poisitve, and erythrocyte-derived extracellular vesicles are CD235 +.85,86 Platelet-derived extracellular vesicles are discussed in more detail in Chapter 22.

Phosphorylation of Intracellular Proteins In addition to extracellular markers of platelet activation, flow cytometry can be used to detect and quantify phosphorylation of specific intracellular platelet proteins using phosphorylation-specific monoclonal antibodies.87 For example, flow cytometric evaluation of the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) provides a measure of the activity of P2Y12, a platelet ADP receptor. Under basal conditions VASP is nonphosphorylated. (PGE1 activates the cyclic adenosine monophosphate cascade leading to VASP phosphorylation, whereas the cascade is inhibited by ADP through the P2Y12 receptor. When platelets are treated with ADP and/or PGE1, the degree of VASP phosphorylation correlates with P2Y12 activity, reported as the platelet reactivity index (PRI).87,88 The VASP assay (BioCytex, Marseilles, France) can be used to monitor the efficacy of antiplatelet drugs that target this receptor (as discussed in section “monitoring of antiplatelet agents” and in Chapter 36).89,90 Clopidogrel is a thienopyridine that undergoes in vivo biotransformation by hepatic cytochrome P450 (CYP) enzymes to yield active metabolites that irreversibly inhibit the platelet P2Y12 ADP receptor (Chapter 51). Heterozygotes and

homozygotes for loss-of-function CYP2C19 alleles have lower levels of the active clopidogrel metabolite,91,92 diminished platelet inhibition,93,94 and higher rates of adverse cardiovascular events when compared to noncarriers following treatment with a standard 75 mg maintenance dose of clopidogrel.95 The ELEVATE-TIMI 56 study,96 a multicenter, randomized, double-blind clinical trial, was conducted to establish whether higher maintenance doses of up to 300 mg daily of clopidogrel could improve platelet reactivity in patients with the CYP2C19 genotype (CYP2C19*2). VASP PRI demonstrated that (i) increasing the maintenance dose of clopidogrel to 225 mg daily in CYP2C19*2 heterozygotes achieved levels of platelet reactivity similar to that seen with the standard 75 mg dose in noncarriers (Fig. 35.4) and (ii) that administering doses as high as 300 mg daily to CYP2C19*2 homozygotes did not result in comparable degrees of platelet inhibition when compared to noncarrier platelet function.96 Co-administration of proton pump inhibitors (PPI) with clopidogrel reduces the risk of gastrointestinal bleeding in patients who have been dosed with clopidogrel.97 However, PPIs are known inhibitors of CYP2C19. PPIs may therefore prevent hepatic conversion of clopidogrel to its active metabolite and consequently block or reduce inhibition of the platelet P2Y12 ADP receptor in vivo.97 Not all PPIs inhibit CYP2C19 to the same extent.98–100 We therefore conducted a randomized, open-label, 2-period, crossover study of healthy subjects (n ¼ 160, age 18 to 55 years, homozygous for the CYP2C19 genotype) to determine the effects of four different PPIs (dexlansoprazole, lansoprazole, omeprazole, and esomeprazole) on the steady-state pharmacokinetics and pharmacodynamics of clopidogrel (75 mg).101 This study demonstrated that VASP PRI values were not different when clopidogrel was co-administered with or without dexlansoprazole or lansoprazole, whereas VASP PRI values were greater than the prespecified no-effect limit when clopidogrel was coadministered with omeprazole or esomeprazole (Fig. 35.5).101 VASP platelet reactivity index 20

Least squares differences (95% Cl) (%)

632

P <0.001

15

P = 0.12

10 5

P = 0.07

0

P <0.001 –5 –10 –15

75

150

225

300

Clopidogrel dose (mg) Fig. 35.4 Difference in platelet reactivity between CYP2C19*2 heterozygotes treated with increasing doses of clopidogrel versus noncarriers treated with 75 mg clopidogrel daily. Data are reported as least squares differences and 95% confidence intervals for platelet reactivity between CYP2C19*2 heterozygotes at clopidogrel doses of 75 mg (n ¼ 76), 150 mg (n ¼ 73), 225 mg (n ¼ 75), and 300 mg (n ¼ 73) and noncarriers at 75 mg of clopidogrel (n ¼ 237). Differences in least squares means were tested using asymptotic methods (normal z test). VASP indicates vasodilator-stimulated phosphoprotein phosphorylation assay. (Reproduced with permission from Ref. 96.)

Flow Cytometry

633

100

VASP PRI (%)

60 40 20

LS-Mean difference VASP PRI (%)

15.56

80

35

15.71

15 11.04

11.44

10 8.17

5

6.52

7.18 4.10

2.05

0

0.03

0 lo p lo a lo n p + e O PZ C lo p C a lo lo p ne + EP Z C lo p a l C lo o n e p + LP C Z lo p C a lo lo p ne + D PZ

OPZ 80 mg

EPZ 40 mg

LPZ 30 mg

–0.86

DPZ 60 mg

C

C

4.95

(A)

(B)

Fig. 35.5 Pharmacodynamics of clopidogrel in the presence and absence of various proton pump inhibitors. (A) Vasodilator-stimulated phosphoprotein (VASP) platelet reactivity index (PRI) (box and whisker limits). (B) Least squares mean differences with and without proton pump inhibitors and corresponding 90% confidence intervals. Dashed line represents upper no-effect boundary. Clop, clopidogrel; DPZ, dexlansoprazole; EPZ, esomeprazole; LPZ, lansoprazole; OPZ, omeprazole. (Reproduced with permission from Ref. 101.)

These data suggest that the potential of PPIs to attenuate the efficacy of clopidogrel could be minimized by the use of dexlansoprazole or lansoprazole rather than esomeprazole or omeprazole.101

Platelet-Platelet Aggregates Platelet-platelet aggregates can be measured by flow cytometry on the basis of light scattering properties. However, if the platelets are aggregated, the amount of antigen per platelet cannot be determined by flow cytometry.15,102 This is because flow cytometry measures the amount of fluorescence per individual particle, irrespective of whether the particle is a single platelet or an aggregate of an unknown number of platelets. However, an approximate estimate of aggregate size can be made by analyzing the increased platelet-specific fluorescence.

Shed Blood Because of the minuscule volumes of blood required, whole blood flow cytometry can be used to analyze the shed blood that emerges from a standardized bleeding time wound.8,23,103,104 The time-dependent increase in the platelet surface expression of P-selectin in this shed blood reflects in vivo activation of platelets.8,23,103,104 Immediate fixation (prior to antibody incubation) is obligatory, in order to observe these time-dependent changes. The assay can be used to demonstrate deficient platelet reactivity in response to an in vivo wound, for example during cardiopulmonary bypass.103 In addition, by tracking of infused platelets with biotin or PKH2 (see section “platelet survival, tracking, and function in vivo”), shed blood can be used to detect the functional participation of the infused platelets in in vivo platelet aggregates.15

Platelet Activation in Clinical Disorders Acute Coronary Syndromes Platelets play an important role in the pathogenesis of coronary artery disease, including unstable angina and acute myocardial infarction (see Chapter 26). Whole blood flow cytometric studies have demonstrated circulating activated platelets, as determined by activation-dependent monoclonal antibodies, in patients with stable angina, unstable angina, and acute

myocardial infarction.53,105–108 In addition, as determined by activation-dependent monoclonal antibodies, PCI results in platelet activation in coronary sinus blood.109,110 Flow cytometric analysis of platelet activation-dependent markers may be useful for the determination of optimal antiplatelet therapy in clinical settings, e.g., in acute coronary syndromes111–113 and after coronary stenting.114,115 Flow cytometric analysis of platelet activation markers before PCI can predict an increased risk of acute and subacute ischemic events after PCI.116–119 As a specific example, acute coronary syndrome patients with elevated VASP PRI are at increased risk for adverse thrombotic events.90,120–123 Flow cytometrically detected exposure of LIBS is strongly associated with the development and progression of heart transplant vasculopathy.124 The Impact of Transfusion of Red Blood Cell on Platelet Activation and Aggregation Studied with Flow Cytometry Use and Light Transmission Aggregometry (TRANSFUSION-2) cross-sectional observational study employed flow cytometry to demonstrate that VASP PRI was significantly elevated following red blood cell (RBC) transfusion of anemic patients with acute coronary syndromes and other cardiac diseases.125 The majority of patients participating in the study were receiving antiplatelet medications.125 These data show that following RBC transfusion, there is an increase in platelet reactivity that is driven in part by upregulation of the P2Y12 receptor/ADP pathway. These findings may account for the excess of ischemic events observed in patients with acute coronary syndrome who are treated with PCI and P2Y12 inhibitors.125 The PlA2 polymorphism of GPIIIa has been reported to be associated with ischemic coronary syndromes.126 Flow cytometry has been used to demonstrate that (a) PlA2-positive platelets display a lower threshold for activation and (b) platelets heterozygous for PlA alleles show increased sensitivity to antiplatelet drugs.126 Circulating leukocyte-platelet aggregates are increased in stable coronary artery disease,53,126,127 unstable angina,105,128 acute myocardial infarction,52,128–131 and cardiopulmonary bypass.132 Circulating leukocyte-platelet aggregates also increase after PCI,52 with a greater magnitude in patients experiencing late clinical events.133 As discussed in section “leukocyte-platelet aggregates,” circulating MPAs (but not NPAs) are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin in the clinical settings of stable coronary artery disease,53 PCI,52 and acute myocardial infarction.52 Furthermore, circulating MPAs are an early marker of acute myocardial infarction.131,134

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In addition, increased surface expression of the platelet collagen receptor GPVI is observed in acute coronary syndromes.135–137 Finally, platelet-derived extracellular vesicles are increased in acute coronary syndromes138 and cardiopulmonary bypass.23,139

Cerebrovascular Ischemia Platelets play an important role in the pathogenesis of ischemic cerebrovascular disease (Chapter 57). Increased circulating Pselectin-positive, CD63-positive, activated αIIbβ3-positive platelets, platelet-derived extracellular vesicles, and MPAs have been reported in acute cerebrovascular ischemia.134,140–148 This platelet activation can be evident 3 months after the acute event, suggesting the possibility of an underlying prothrombotic state.142,144–146,149,150 Furthermore, increased expression of surface P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation.151 In addition, following transient ischemic attack or stroke, elevated platelet surface expression of GPVI has also been reported.152 Platelet-derived extracellular vesicles are increased after transient ischemic attacks.23,153 Increased platelet-derived extracellular vesicles and procoagulant activity occur in symptomatic patients with prosthetic heart valves and provided a potential pathophysiological explanation of cerebrovascular events in this patient group.154 Enhanced systemic platelet degranulation is associated with progression of intima-media thickness of the common carotid artery in patients with,155 or without,156 type 2 diabetes (as determined by platelet surface CD63 and CD40 ligand, and by platelet surface P-selectin, respectively).

Peripheral Vascular Disease Circulating activated platelets and platelet hyperreactivity (as determined by P-selectin expression, platelet aggregates, and platelet-derived extracellular vesicle formation) are increased in patients with peripheral arterial disease compared with healthy volunteers.157,158 Furthermore, as the severity of peripheral artery disease increases, a corresponding increase in platelet activity is observed.159 In accordance with this, circulating MPAs and NPAs are significantly greater in the early postoperative period in patients with peripheral vascular disease who go on to develop later graft occlusion.160 With regard to peripheral venous disease, Peyton et al.54 demonstrated an increased presence of MPAs in the lower extremity veins of patients with chronic venous stasis ulceration, as compared to control individuals without venous disease. Interestingly, these changes were present not only in blood drawn from the lower extremity veins of affected individuals, but also in blood drawn from arm veins, suggesting that the changes are systemic rather than localized to the lower extremities.54 Powell et al.161 further characterized these findings as being related to the presence of chronic venous disease rather than the presence of venous ulceration, since increased numbers of MPAs were noted in patients with all classes of venous disease, not just in patients with deep venous valvular insufficiency. Furthermore, increased levels of MPAs were noted even in patients with only superficial venous stasis disease manifested by the presence of varicose veins. Even more intriguing is the fact that the number of MPAs remains elevated 6 weeks after total correction of the venous insufficiency by stripping of the abnormal veins, leaving normal venous physiology as documented by postoperative duplex scanning.162 This finding suggests an underlying predisposition to the development of chronic venous disease

in these patients, perhaps mediated by monocyte-platelet interactions.

Immune Thrombocytopenia Recently, our group conducted a cross-sectional study of 57 pediatrics patients with immune thrombocytopenia (ITP) and demonstrated that platelet function tests, independent of platelet count, are associated with bleeding severity (assessed by standardized bleeding score) in ITP.163 More specifically, after adjustment for platelet count, higher levels of TRAPstimulated percent P-selectin- and activated integrin αIIbβ3positive platelets (as determined by flow cytometry) were found to be significantly associated with a lower bleeding score, whereas higher levels of immature platelet fraction (IPF, determined using a Sysmex XE-2100 hematology analyzer, see section “Monitoring of Thrombopoiesis”), TRAP-stimulated platelet surface CD42b, unstimulated platelet surface P-selectin, and platelet FSC (as determined by flow cytometry) were associated with a higher bleeding score.163 A follow-up crosssectional study of 15 children with ITP at two visits separated by 10 months confirmed that platelet function in ITP, independent of platelet count, was consistent over time and was associated with both concurrent and subsequent bleeding severity (Fig. 35.6).164 Flow cytometry may therefore be a useful tool to evaluate markers of future bleeding risk in ITP.

Other Clinical Disorders Associated With Platelet Hyperreactivity and/or Circulating Activated Platelets There are numerous other conditions in which whole blood flow cytometric measurement of platelet hyperreactivity, circulating activated platelets, and/or circulating leukocyte-platelet aggregates may prove to have a clinical role, including diabetes mellitus (as discussed in Chapter 27),155,165–168 metabolic syndrome,169 atrial fibrillation,170–172 cystic fibrosis (Fig. 35.7),173 preeclampsia,174,175 placental insufficiency,176 migraine,177 nephrotic syndrome,178 hemodialysis,179 sickle cell disease (Chapter 31),180–183 systemic inflammatory response syndrome,184 septic multiple organ dysfunction syndrome,185,186 antiphospholipid syndrome,187 systemic lupus erythematosus,187 rheumatoid arthritis,187,188 inflammatory bowel disease,189 myeloproliferative disorders,190,191 and Alzheimer disease.192 High levels of circulating MPAs can predict rejection episodes after orthotopic liver transplantation.193 Uremic patients with thrombotic events have higher numbers of circulating platelet-derived extracellular vesicles than those without thrombotic events.194

Reduced Circulating Activated Platelets and Platelet Hyporeactivity In addition to the detection of increased circulating activated platelets and platelet hyperreactivity discussed in the previous section “Platelet Activation in Clinical Disorders,” whole blood flow cytometry may be useful in the clinical assessment of reduced circulating activated platelets and platelet hyporeactivity—although there are few published studies in this area.

Very Low Birth Weight Preterm Neonates Compared to adults, the platelets of very low birth weight preterm neonates are markedly hyporeactive to thrombin, ADP/ epinephrine and thromboxane A2, as determined by flow cytometric detection of (a) the exposure of the fibrinogen binding site on αIIbβ3, (b) fibrinogen binding, (c) the increase in platelet surface P-selectin, and (d) the decrease in platelet surface GPIb.12,195 Furthermore, procoagulant platelet-derived

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35

Fig. 35.6 Association of platelet tests with subsequent bleeding severity, independent of platelet count, in ITP patients. Odds ratios and confidence intervals per 1 SD unit (defined below) increase in the indicated parameter. One SD unit for each test is as follows: Immature platelet fractions, 1%; FSC, no agonist, 166.7 forward light scatter units; Act. integrin αIIbβ3-positive platelets, 20 μM ADP, 3.25%, 1.5 μM TRAP, 17.44%; 20 μM TRAP, 2.96%; P-selectin-positive platelets, 0.5 μM ADP, 17.43%, 20 μM ADP, 10.19%, 1.5 μM TRAP, 22.6%; 20 μM TRAP, 4.19%; Platelet surface P-selectin MFI, no agonist, 4.98; Platelet surface GPIbα MFI, 1.5 μM TRAP, 83.2 MFI, 20 μM TRAP, 54.94 MFI. MFI is reported in arbitrary geometric mean fluorescence units. (Reproduced with permission from Ref. 164.)

Fig. 35.7 Cystic fibrosis (CF) patients have increased circulating monocyte- and neutrophil-platelet aggregates and increased platelet responsiveness to ADP and TRAP compared with healthy controls. Whole blood from CF patients and healthy controls was incubated with or without agonist and analyzed for (A) monocyte-platelet aggregates and (B) neutrophil-platelet aggregates. Data are mean  SEM; n ¼ 18. *P <0.05 versus healthy controls. + P <0.01 versus healthy controls. (Reproduced with permission from Ref. 173.)

extracellular vesicles and calcium ionophore A23187-induced platelet surface binding of factor V/Va are decreased in preterm neonates compared with adults.73,195 When monitored for 2 weeks after birth, extremely low birth weight neonate platelets remained hyporesponsive in comparison to adult platelets; however, neonatal platelet function significantly improved from day 0–1 through 10–14, as demonstrated by platelet surface P-selectin, GPIb and factor V/Va binding (Fig. 60.1 in Chapter 60).195 This platelet hyporeactivity of preterm neonates, as judged by both activation-dependent platelet surface changes and the generation of procoagulant platelet-derived extracellular vesicles, may contribute to the age-dependent propensity of very low birth weight neonates to intraventricular hemorrhage.196

Hematologic Malignancies Prediction of hemorrhage in thrombocytopenic patients has been problematic because of the lack of laboratory markers or validated clinical assessment tools.13,197 The clinical decision to prophylactically transfuse platelets is therefore usually based solely on the platelet count (see Chapter 64). However, low levels of expression of platelet surface P-selectin, as determined by whole blood flow cytometry, have been reported to be a prognostic marker for hemorrhage in acute myeloid leukemia.197 Flow cytometric studies have demonstrated platelet hyporeactivity in patients with essential thrombocythemia198 and active myeloma.199 However, more in-depth studies are

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required to determine suitable biomarkers that may be used to predict bleeding episodes in these patients.

DIAGNOSIS OF SPECIFIC DISORDERS Platelet Surface Glycoprotein Deficiencies Bernard-Soulier Syndrome Bernard-Soulier syndrome is an inherited deficiency of the GPIb-IX-V complex (Chapter 48). Flow cytometric analysis with GPIb-, GPIX-, and GPV-specific monoclonal antibodies provides a rapid and simple means for the diagnosis of the homozygous and heterozygous states of Bernard-Soulier syndrome.200 Whole blood flow cytometry allows analysis of platelets without attempting the technically difficult procedure of physically separating the giant Bernard-Soulier syndrome platelets from similarly sized red and white blood cells. Because light scatter (especially forward light scatter) correlates with platelet size, light scatter gates may need to be adjusted in the flow cytometric analysis of giant platelet syndromes such as Bernard-Soulier syndrome. This adjustment may result in overlap of the light scatter of giant platelets with red and white blood cells. It is therefore essential to include in the assay a platelet-specific monoclonal antibody as a platelet identifier. For Bernard-Soulier syndrome platelets, this identifier antibody obviously cannot be GPIb-, GPIX-, or GPV-specific.

Glanzmann Thrombasthenia Glanzmann thrombasthenia is an inherited deficiency of integrin αIIbβ3 (Chapter 48). Flow cytometric analysis with αIIb- and β3-specific monoclonal antibodies provides a rapid and simple means for the diagnosis of the homozygous and heterozygous states of Glanzmann thrombasthenia.200,201 In addition, a panel of activation-dependent monoclonal antibodies can be used to evaluate patients with defects in platelet aggregation,20 secretion,202 or procoagulant activity.203

Platelet-Type von Willebrand Disease Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder which is due to a mutation in the gene encoding for platelet GPIbα resulting in enhanced affinity for von Willebrand factor.204 PT-VWD is often mistakenly diagnosed as type 2B VWD because of the similarities between these two conditions. Giannini et al.204 used a flow cytometric assay to demonstrate the increased affinity of von Willebrand factor for GPIbα and to differentiate PT-VWD and type 2B VWD through mixing tests.

Scott Syndrome Hemostasis requires the exposure of cell membrane phosphatidylserine to provide a catalytic surface where clotting factors can interact with cofactors to drive thrombin generation and clot formation. Scott syndrome (Chapter 48), a very rare inherited bleeding disorder, is caused by a scramblase defect that impairs the externalization of phosphatidylserine on the platelet membrane following activation.205 Scott syndrome patients suffer severe bleeding due to altered thrombin generation and impaired clot formation. The clinical diagnosis of Scott syndrome is relatively difficult, because nearly all standard coagulation tests are normal. However, increased residual prothrombin in patient serum is a good indicator of the disease.

Whole blood flow cytometry assays have been developed that use fluorochrome-conjugated annexin V with calcium ionophores to detect externalized phosphatidylserine on the membrane of activated platelets.206 This technique provides a simpler and more rapid means to diagnose Scott syndrome.

Storage Pool Disease Inherited dense granule storage pool deficiency, a relatively common cause of a mild hemorrhagic diathesis, cannot be reliably diagnosed by standard platelet aggregometry.207 The conventional method to establish the diagnosis of storage pool disease is to label the platelets with the fluorescent dye mepacrine and then measure platelet fluorescence by microscopy.208 This assay is based on the selective binding of mepacrine to adenine nucleotides in dense granules. The assay is not ideal for the clinical laboratory because it is subjective, tedious, and examines only a small number of platelets. In contrast, dense granule storage pool deficiency can be accurately diagnosed by a simple, rapid, one-step flow cytometric assay in a clinical laboratory.209,210 The method shows good correlation with fluorescent light microscopic methods, but improves the detection of the mepacrineloaded platelets by quantitatively measuring fluorescence on a large number (5000) of platelets.209,210 Acquired dense granule storage pool deficiency, which occurs in myeloproliferative disorders and end-stage renal failure, can also be diagnosed by flow cytometry.209,211 An alternative approach to the flow cytometric diagnosis of storage pool disease is to measure intraplatelet serotonin (5-hydroxytryptamine) with a serotonin-specific monoclonal antibody.212,213 Storage pool disease is discussed in detail in Chapter 48.

Heparin-Induced Thrombocytopenia Unlike normal sera and sera from patients with quinine- or quinidine-induced thrombocytopenia, sera from patients with HIT generate procoagulant platelet-derived extracellular vesicles from normal platelets.214 This observation has been used to develop a rapid, specific, and sensitive flow cytometric assay for the diagnosis of HIT.215,216 Alternative flow cytometric approaches to the diagnosis of HIT have been described.217 HIT is discussed in detail in Chapter 41.

MONITORING OF ANTIPLATELET AGENTS The in vivo effect of the thienopyridines (clopidogrel, ticlopidine, and prasugrel) (Chapter 51) on platelet function can be monitored by the VASP assay (Figs. 35.4 and 35.5).88,89,218 We monitored the VASP PRI in PCI patients administered a loading dose of clopidogrel or prasugrel.219 At all time points (2, 6, and 24 h postadministration), the average PRI of prasugrel-treated patients was significantly lower than the average PRI for clopidogrel-treated patients (Fig. 35.8).219 In acute coronary syndrome patients administered a pre-PCI loading dose of prasugrel or clopidogrel followed by maintenance dosing of the same drug, the mean VASP PRI was significantly lower for prasugrel versus clopidogrel treatment at both 1–2 h postloading dose and after 30 days of maintenance dosing (Fig. 35.9).220 Thus, utilizing the VASP flow cytometric assay, we have shown in the setting of PCI that prasugrel results in a greater inhibition of ADP-mediated platelet function than clopidogrel. Ticagrelor is described as a direct-acting, reversibly binding inhibitor of the platelet P2Y12 receptor221 approved for the prevention of thrombotic events in patients with acute coronary syndromes (Chapter 51). However, residual platelet inhibition persists after discontinuation of ticagrelor when plasma levels

Flow Cytometry

100

VASP PRI (%)

80

Clopidogrel 600 mg 75

60

68.4 ∗∗∗ 21.5

40 20

64.3

∗∗∗ P<0.0001 ∗∗∗

Prasugrel 60 mg

7.4

∗∗∗ 10.3

0 0

4

8

12

16

20

h

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Fig. 35.8 Platelet function in patients undergoing percutaneous coronary intervention treated with prasugrel or clopidogrel, as measured by the VASP platelet reactivity index (PRI). Patients scheduled for PCI were administered a loading dose of prasugrel 60 mg or clopidogrel 600 mg
1 h before cardiac catheterization. Citrated blood samples were drawn at baseline and 2, 6, and 24 h after treatment, and analyzed flow cytometrically using the VASP kit (BioCytex). Blue circles and lines indicate prasugrel measurements; green triangles and lines, clopidogrel measurements. Data are mean  SD. ***P < 0.0001 by 2 sample t-test for prasugrel versus clopidogrel at the corresponding time point. (Reproduced with permission from Ref. 219.)

with very high affinities for integrin αIIbβ3 (disintegrins230 or cyclic RGD peptides231); or (d) fibrinogen binding after platelet activation (detected by a polyclonal antifibrinogen antibody).232 Another type of direct method measures the binding of an antibody directed against the integrin αIIbβ3 antagonist.233,234 Indirect methods measure either: (a) the integrin αIIbβ3 antagonist-induced binding of an anti-LIBS antibody;235 or (b) platelet aggregation, as determined by light scatter.226 Through the use of arachidonic acid as the agonist, flow cytometry can also be used to monitor aspirin therapy.236,237 The monitoring of antiplatelet agents by platelet function tests is discussed in more detail in Chapter 36.

MONITORING OF THROMBOPOIESIS

Fig. 35.9 Distribution of individual VASP PRI values in prasugreland clopidogrel-treated patients. Patients with acute coronary syndrome received a loading dose of prasugrel 60 mg or clopidogrel 300 mg prior to PCI, followed by a daily maintenance dose (prasugrel 10 mg or clopidogrel 75 mg) for 30 days post-PCI. Citrated blood samples were drawn at baseline (prestudy drug, pre-PCI), 1–2 h postloading dose, and 30 days post-PCI (30 days of maintenance dosing), and analyzed for VASP PRI. P-values are from ANCOVA with factors for study site, baseline value, and treatment. Line represents mean for each group. (Reproduced with permission from Ref. 220.)

are undetectable.222–224 We recently evaluated the ex vivo effect of ticagrelor on platelet function and the potential mechanisms driving the incomplete reversibility of platelet inhibition following prolonged exposure to ticagrelor.225 In the study, fluorescently labeled antibodies targeting phosphorylated-VASP, activated integrin αIIbβ3 and P-selectin were used in conjunction with flow cytometry to assess irreversibility of the inhibitory effect on platelet function.225 The results demonstrated that platelets undergo rapid, reversible inhibition after short exposure to ticagrelor and a slower, less reversible inhibition of integrin αIIbβ3 activation and P-selectin expression, but not VASP dephosphorylation, with longer exposure to ticagrelor.225 This suggests that irreversible changes occur independent of VASP signaling.225 Flow cytometric methods can also be used to monitor integrin αIIbβ3 antagonists (abciximab, eptifibatide, and tirofiban) (Chapter 52) by measuring receptor occupancy by these drugs. These methods can be categorized as either direct or indirect. One type of direct method is a competitive binding assay with either: (a) biotinylated226 or FITC-conjugated227 αIIbβ3 antagonists; (b) blocking monoclonal antibodies;228,229 (c) peptides

Whole blood flow cytometric methods have been developed for the identification of young platelets (i.e., those containing mRNA) by their staining with thiazole orange.238–240 Because of the analogy to reticulocytes, these thiazole orange-positive platelets have been termed “reticulated platelets”241 and have been used to monitor thrombopoiesis.239,242 Thrombocytopenic patients whose bone marrow contains normal or increased numbers of megakaryocytes have significantly elevated proportions of circulating reticulated platelets.238 In contrast, the proportion of reticulated platelets in thrombocytopenic patients with impaired platelet production (reduced bone marrow megakaryocytes) does not differ from normal controls and the absolute number of reticulated platelets is significantly lowered.238 Measurement of reticulated platelets has been used as an aid in assessing bone marrow recovery after bone marrow transplantation.243 In addition, measurement of reticulated platelets may be useful in evaluating both treatment response and thrombotic risk in patients with thrombocytosis.244 However, there are persistent methodological issues with the flow cytometric measurement of reticulated platelets. Because thiazole orange also binds to ADP and ATP (contained in dense granules), important controls in the flow cytometric measurement of reticulated platelets are the demonstration that thiazole orange staining is (a) abolished by pretreatment of the sample with RNAase and (b) not abolished by pretreatment of the sample with thrombin. In fact, the higher thiazole orange signal of young platelets has been reported to be derived to a significant extent from their large volume and granule content,245–247 leading to the suggestion that platelet degranulation with TRAP should be an initial part of the assay for reticulated platelets.245 However, other investigators report that, under modified assay conditions, degranulation does not significantly change thiazole orange fluorescence and that RNAase demonstrates specificity of the thiazole orange staining.240 Automated methods to reliably quantify reticulated platelets, expressed as the IPF, have been developed, e.g., the XE-2100 and

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XN-1000 blood cell counters (Sysmex, Kobe, Japan).248–250 The IPF is identified by flow cytometry techniques and the use of a nucleic acid-specific dye in the reticulocyte/optical platelet channel. The clinical utility of this parameter was established in the laboratory diagnosis of thrombocytopenia due to increased platelet destruction, e.g., ITP.248,250 The measurement of IPF is discussed in greater detail in Chapter 32. As discussed earlier in section “immune thrombocytopenia,”, we recently demonstrated that, after adjustment for platelet count, higher levels of IPF (determined using a Sysmex XE-2100 hematology analyzer) were associated with a higher bleeding score in patients with ITP.163 However, further study is required to clarify the relationship between IPF and bleeding risk in ITP patients.250,251

stored platelet concentrates. The application of flow cytometry to study these surface changes is discussed in greater detail in section “Platelet Survival, Tracking, And Function In Vivo.”

Other Blood Bank Applications Flow cytometry can also be used to: identify leukocyte contamination and changes in other platelet activation markers in platelet concentrates261,262 (Chapter 64); immunophenotype platelet HPA-1a263 and other polymorphisms (Chapter 64); detect maternal and fetal anti-HPA-1a antibodies264 (Chapter 45); and crossmatch platelets, which may be useful for alloimmunized patients for whom HLA-compatible platelets are not readily available265,266 (Chapter 64).

BLOOD BANK APPLICATIONS

PLATELET-ASSOCIATED IgG

Quality Control of Platelet Concentrates

Measurement of platelet-associated IgG by flow cytometry may be useful in ITP267–269 and alloimmunization.270 A quantitative direct platelet immunofluorescence test for plateletassociated IgG has been proposed as a screening test for ITPs.271–273

Measurement of the platelet surface expression of P-selectin by flow cytometry is one of the most commonly applied measures of platelet activation in platelet concentrates stored in blood banks, and efforts have been made to standardize these measurements.252 However, we15 have demonstrated in a nonhuman primate model that infused, degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function in vivo. Thus, platelet surface P-selectin molecules, rather than degranulated platelets, are rapidly cleared. Our results15 were subsequently independently confirmed by Berger et al.,50 who found that the platelets of both wild-type and P-selectin knockout mice had identical life spans. When platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100 kDa P-selectin fragment in the plasma.50 Storage of platelets at 4°C caused a significant reduction in their life span in vivo, but again no significant differences were observed between the two genotypes. Thus, the results of Berger et al.50 confirm that P-selectin does not mediate platelet clearance. Furthermore, in a thrombocytopenic rabbit kidney injury model, Krishnamurti et al.253 reported that thrombin-activated human platelets: (a) lose platelet surface P-selectin in the (reticuloendothelial system-inhibited) rabbit circulation; (b) survive in the circulation just as long as fresh human platelets; and, most importantly; and (c) are just as effective as fresh human platelets at decreasing blood loss. In summary, these studies15,50,253 strongly suggest that the measurement of platelet surface P-selectin in platelet concentrates stored in the blood bank should not be used as a predictor of platelet survival or function in vivo. However, platelet surface P-selectin could still be a useful measure of quality control during processing, storage, and manipulation (filtration, washing).252 This is because, in contrast to the situation in vivo,15,50,253 the activation-dependent increase in platelet surface P-selectin is not reversible over time under standard blood banking conditions.254 Quality control of platelet concentrates is discussed in detail in Chapter 64. Recent flow cytometric studies, have suggested that platelet surface glycosylation and desialylation are more suitable markers than P-selectin for predicting platelet survival or function in vivo.255–259 Glycosylation of platelets has been found to prolong the circulation of functional short-term cooled platelets post-transfusion,255,256 and platelet desialylation drives a mechanism of platelet clearance in vivo.257–260 However, more work is required to standardize the measurement of these surface alterations on platelets and apply them to the quality control of

PLATELET COUNT Platelet Count in Humans The International Council for Standardization in Haematology and the International Society of Laboratory Hematology has recommended a flow cytometric reference method for platelet counting that utilizes erythrocyte counts, determined by an automated counter, as an internal reference standard.274,275 Platelet counting in humans is discussed in detail in Chapter 32.

Platelet Count in Mice With advances in manipulation of the mouse genome, murine models have become increasingly important in our understanding of platelet disorders. However, standard methods for murine platelet counting require relatively large volumes of blood and therefore serial platelet counts cannot be followed over time in the same mouse. To circumvent this problem, we described a rapid, reproducible, and accurate flow cytometric method to determine the number, and activation state, of circulating platelets from a single mouse over extended periods of time.16 The method uses fluorescent staining of platelets in whole blood with a specific antibody and the addition of known numbers of fluorescent beads for standardization of the sample volume. Analysis of platelets obtained by tail bleeding indicated that this sampling procedure did not activate platelets, and only 5 μL of blood were required for platelet counting. Thus, the method can be used to follow the number and the activation state of circulating platelets from individual mice over extended periods of time and is applicable to a wide range of murine models of platelet disorders.16

OTHER RESEARCH APPLICATIONS Platelet Survival, Tracking, and Function In Vivo Multicolor whole blood flow cytometry can be used to track platelets in vivo and determine their survival and function (Fig. 35.3).15,52,255,276–283 In 3-color flow cytometry, the fluorescent labeling typically identifies: (1) platelets in whole blood (e.g., by a PE-conjugated GPIb-, GPIX-, αIIb-, or β3-specific monoclonal antibody), (2) the infused platelets (e.g., by prelabeling with PKH2, or biotin followed by

Flow Cytometry

the ex vivo addition of a streptavidin conjugate), and (3) an activation-dependent monoclonal antibody (e.g., FITCconjugated P-selectin-specific, or activated αIIb-, or β3-specific, monoclonal antibody). By these means, the in vivo function of tracked, infused platelets can be determined at multiple time points by multiple independent assays including, for example: participation in platelet aggregation in response to an in vivo wound, exposure of the fibrinogen binding site on αIIbβ3, adherence to Dacron in an arteriovenous shunt, and generation of procoagulant platelet-derived extracellular vesicles.15 Hughes et al.284 used 2-color whole blood flow cytometry with a FITCconjugated HLA-A2-specific monoclonal antibody to track and characterize transfused platelets by selecting donor/recipient pairs discrepant for HLA-A2. More recently, Li et al.257 tracked transfused 5-chloromethylfluorescein diacetate (CMFDA)labeled platelets treated with anti-GPIbα monoclonal antibodies in mice treated with asialofetuin (an Ashwell-Morell receptor inhibitor) at various time points using flow cytometry. By these means, Li et al.257 demonstrated that anti-GPIbα antibodies induce Fc-independent platelet activation, desialylation (assessed by fluorescein-conjugated Ricinus communis agglutinin I [RCA-I] lectin-binding, which targets exposed galactose residues following desialylation), and ultimately platelet clearance in the liver via hepatocyte Ashwell-Morell receptors. This was one of the first studies to demonstrate an alternate platelet clearance mechanism to the classic Fc-FcγR-dependent macrophage phagocytosis clearance mechanism in the spleen. Other flow cytometric studies, have shown that CD8 + T cells are able to induce platelet clearance in the liver via platelet desialylation258 in ITP patients and that platelet desialylation correlates with efficacy of first-line ITP therapies (dexamethasone (3–5 days) with/ without intravenous immunoglobulin (3–5 days), followed by daily low-dose prednisone).260 Hoffmeister et al.255 used flow cytometry to demonstrate a two-fold increase in binding of succinyl-wheat germ agglutin (S-WGA), a lectin specific for β-N-acetylglucosamine (βGlcNAc), to chilled murine platelets compared to room temperature murine platelets. The β2 integrins on hepatic resident macrophages were found to selectively recognize irreversibly clustered β-GlcNAc-terminating immature glycans on GPIb receptors on short-term (4 h)-cooled (0°C) platelets, which ultimately caused their rapid clearance from the circulation.255 By capping surface β-GlcNAc residues via enzymatic galactosylation, Hoffmeister et al.255 were able to decrease S-WGA binding to chilled platelets to levels that were comparable to the binding on room temperature platelets, and prevent the clearance of short-term-cooled platelets in vivo. Therefore, platelet glycosylation appears to be an effective means to restore the survival of circulating shortterm chilled murine platelets.255 Rumjantseva et al.256 extended the work of Hoffmeister et al.255 by studying the effects of long-term (48 h) refrigeration (4°C) on murine platelet survival in vivo. Long-term refrigerated platelets had higher galactose exposure as evidenced by increased binding of FITC-labeled RCA-I and Erythrina cristagalli agglutinin (ECA), compared to short-term cooled or room-temperature platelets.256 By transfusing CMFDA-labeled platelets into Ashwell-Morell receptor subunit knockout (Asgr1/ and Asgr2/) mice Rumjantseva et al.256 demonstrated a role for the Ashwell-Morell receptor in clearing long-term cooled platelets that expressed a high density of galactose residues. These findings are of clinical interest as platelets stored at room temperature are known to be more susceptible to bacterial contamination and consequently more likely to cause bacterial sepsis in patients.285,286 By using a combination of cold storage and antibacterial products to increase shelf-life, it may be possible to reduce bacterial contamination in platelet concentrates

639

and thereby reduce the frequent platelet shortages in blood banks. Inhibition of chilled platelet clearance in vivo by targeting both β2 integrins on macrophages and Ashwell-Morell receptors on hepatocytes may allow for cold storage of platelets.

Monocyte and Neutrophil Procoagulant Activity With and Without Surface-Adherent Platelets Monocytes and neutrophils form heterotypic aggregates with platelets via engagement of platelet surface P-selectin with leukocyte surface PSGL-1 (Chapter 16). The resultant intracellular signaling causes leukocyte surface expression of tissue factor287 and activation of leukocyte surface Mac-1 (integrin αMβ2, CD11b/CD18).288,289 The activation-dependent conformational change in monocyte surface Mac-1290,291 results in the binding of activated coagulation factor X (Xa) and/or fibrinogen to Mac-1.292–294 The platelet surface also binds coagulation factors via exposed negatively charged phospholipids such as phosphatidylserine (Chapter 21).44 Tissue factor from the vascular wall and platelets forms a complex with activated coagulation factor VII, facilitating the activation of factor X. Tissue factor is a key component of monocyte surface procoagulant activity.295 We have developed whole blood flow cytometry assays to measure bound tissue factor, coagulation factor Xa, fibrinogen, activated Mac-1 and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface-adherent platelets (Figs. 35.10 and 35.11).5,296 These methods are applicable to in vivo and in vitro studies of pharmacological agents targeted to either coagulation and/or cellular activation processes. The monocyte and neutrophil surface binding of tissue factor (Fig. 35.11), coagulation factor Xa, and fibrinogen (Fig. 35.10) is mainly dependent on platelet adherence to the monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac-1 is mainly independent of platelet adherence to the monocytes and neutrophils.296

F-Actin Platelet cytoskeletal rearrangement can be analyzed flow cytometrically by measuring F-actin content with NBD- or bodipyphallacidin or FITC-phalloidin.297,298 We analyzed the effect of APD791, an inverse agonist of the platelet serotonin receptor 5HT2A, on platelet cytoskeletal rearrangement. In canines treated with APD791, a significant decrease in serotoninstimulated F-actin polymerization (measured by the binding of fluorescently conjugated phalloidin) was observed in comparison to control animals (Fig. 35.12A).299 As expected, ADP-stimulated phalloidin binding was unaffected by APD791 treatment (Fig. 35.12C).

Calcium Flux In washed platelet preparations, platelet-rich plasma, or whole blood, flow cytometry can be used to measure platelet calcium flux, an important platelet second messenger.300,301 In parallel to F-actin measurements, we evaluated the effect of APD791, an inverse agonist of the platelet serotonin receptor 5HT2A, on platelet intracellular calcium levels. A significant decrease in serotonin-stimulated calcium flux (as measured by fluorescence of the calcium indicator dye Fluo-4) was observed in platelets from APD791-treated dogs, as compared to control animals (Fig. 35.12B).299 In contrast, as expected, ADPstimulated intracellular calcium flux was comparable in APD791-treated or control platelets (Fig. 35.12D).

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Fig. 35.10 Assay of monocyte and neutrophil procoagulant activity with and without surfaceadherent platelets. Upper panels: Monocytes and neutrophils were initially gated by a combination of light scattering and CD14 PECy5 fluorescence. Dim CD14, forward and 90° light scatter define neutrophils (blue, R1 + R3). Bright CD14 and 90° light scatter define monocytes (red, R2). Forward and 90° light scatter increase significantly upon formation of heterotypic aggregates. Lower panels: In this collagen-stimulated example, monocytes (red) and neutrophils (blue) were further gated into CD42anegative platelet-free (R6 and R8) and CD42a-positive platelet-bound (R5 and R7) subpopulations. Note monoclonal antibody F26 (directed against surfacebound fibrinogen) (X axis) increases with increased numbers of leukocyte-bound platelets (Y axis). (Reproduced with permission from Ref. 296.)

Fig. 35.11 Surface expression of tissue factor on platelet-bound monocytes and neutrophils and platelet-free monocytes and neutrophils. These whole blood flow cytometric assays required preincubations at 37°C to allow physiological ligand binding prior to adding the test antibody.232 Data are mean  SEM, n ¼ 10. *P <0.05 by paired t test for surface tissue factor with agonist compared with no agonist. + P <0.05 by paired t test for platelet-free monocytes compared with platelet-bound monocytes and for platelet-free neutrophils compared with platelet-bound neutrophils. There were insufficient platelet-free monocytes or neutrophils after collagen stimulation to generate these data points. (Reproduced with permission from Ref. 296.)

Fluorescence Resonance Energy Transfer

Platelet Recruitment to Growing Thrombi

Fluorescence resonance energy transfer (FRET) can be used to investigate the spatial separation or orientation of exoplasmic domains within receptor molecules,302 and to detect and characterize antiplatelet antibodies directed against HLA class 1 molecules or platelet-specific glycoproteins.303

A three-color flow cytometric method can be used for the simultaneous monitoring of two platelet populations that enables the study of the effects of stimuli (even very shortacting stimuli such as nitric oxide) on platelet recruitment to the growing thrombus.304

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Fig. 35.12 Inhibition of ex vivo serotonin-stimulated platelet function by APD791, an inverse agonist of the platelet 5HT2A receptor. Citrated blood samples were collected from dogs before or 1.5 h after administration of saline (control) or APD791. Mean values of serotonin-stimulated (A, B) or ADP-stimulated (C, D) F-actin polymerization (A, C; assessed by phalloidin binding) and increases in platelet cytosolic calcium levels (B, D; assessed by Fluo-4 fluorescence) for control and APD791-treated groups were measured flow cytometrically before and after treatment. Data were normalized to baseline and analyzed using repeated measures ANOVA with the Newman-Keuls post hoc test. **P < 0.01 versus control. (Reproduced with permission from Ref. 299.)

Bacteria-Platelet Interactions The binding of platelets to other cells, e.g., bacteria, and the functional consequences of this binding on both cell types, can be studied by multicolor flow cytometry.305,306

ADVANCES IN FLOW CYTOMETRY Mass Cytometry Mass cytometry is a next generation flow cytometry platform which utilizes elemental mass spectrometry to detect metalconjugated antibodies that are bound intracellularly or extracellularly to antigens of interest on single cells.307 The number of cellular parameters that can be simultaneously monitored by conventional fluorescence flow cytometry assays is inherently limited by fluorophore emission spectra overlap (Fig. 35.13A). In contrast, however, mass cytometry accurately discriminates metal isotopes of different atomic masses without channel overlap (Fig. 35.13A). This not only ameliorates the need for complex compensation matrices but enables simultaneous analysis of a much greater number of cellular features than fluorescence flow cytometry (Fig. 35.13A and B). Whereas standard fluorescence flow cytometry instruments are typically capable of simultaneously analyzing up to 13 different parameters, recent studies have employed mass cytometry to concurrently monitor 45 different cellular features (using metal-tagged antibodies, cell viability markers and DNA intercalators) to identify major immune populations in peripheral blood of humans.308 We recently developed a platelet-specific metal-tagged antibody panel capable of surveying 14 different platelet surface antigens simultaneously by mass cytometry.309 This enabled us to survey a significantly larger number of surface antigens compared to a typical flow cytometry platelet antibody

cocktail, which is capable of analyzing just 3 parameters (standardly containing antibodies against CD42b/CD41 or CD61 (“platelet identifier”) and CD62P and activated integrin αIIbβ3 (activation markers).309 We were also able to identify novel platelet subpopulations by mass cytometry in healthy donors and in patients with inherited platelet disorders (Glanzmann thrombasthenia and Hermansky-Pudlak syndrome).309 A typical schema of sample preparation for mass cytometric analysis of cells is shown in Fig. 35.13B. In brief, cells are incubated (or “stained”) with a panel of metal-tagged antibodies that target antigens of interest. A DNA intercalator is typically incorporated into the panel to allow determination of nucleated cells from nonnucleated cells. Cells are stained under resting or stimulating conditions and are fixed prior to analysis. The samples are washed to remove unbound antibody and salts and diluted to an appropriate cell concentration. Cells are then passed in a single-cell suspension into a nebulizer, which aerosolizes the cells into droplets for introduction into the mass cytometer. Upon entering the instrument, cells travel through an argon plasma at 7000°K which completely vaporizes and ionizes the cell and the attached antibodies into a cloud of single-atom ions. The size of the cloud is largely driven by gas expansion kinetics and is relatively independent of the cell size. The ion cloud is filtered by a quadrupole to remove common biological elements with a mass less than
75 Da, to leave only the heavy metal ions that were attached to the staining antibodies and antigens of interest on cells. The ions within the cloud are separated by their mass-to-charge ratio in a time-of-flight (TOF) mass spectrometer. Ion signals are integrated on a per-cell basis, resulting in single-cell measurements for analysis (Fig. 35.13B).307,310–312 There are some disadvantages to using mass cytometry to analyze cellular function. First, mass cytometers are

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Fig. 35.13 Comparison of fluorescence and mass spectra overlap and typical schema of sample preparation and analysis of single cells by mass cytometry. (A) Histograms demonstrating the emission spectra overlap that exists between different fluorophores within a fluorescence-labeled flow cytometry antibody panel compared to the metal mass overlap that exists for heavy metals within a mass cytometry metal-labeled antibody panel. (B) Sample preparation and analysis by mass cytometry schema. Cells are stained with a metal-labeled antibody cocktail targeting specific antigens of interest under resting or stimulating conditions. Cells are fixed prior to mass cytometry acquisition to preserve the cell state/antibody binding and washed to remove salts and unbound antibodies. Samples are then spiked with calibration beads, which act as internal controls for postacquisition normalization. The beads are used to minimize variance in the mass spectrometry detector, which, due to the accumulation of metal ions over time becomes less sensitive. This normalization consequently reduces variation over the time needed to collect data for each sample and also reduces sample-to-sample signal variation. Cells (and calibration beads) are passed in a single-cell suspension into a nebulizer at 500–1000 events/second. Upon entering the instrument, cells travel through an argon plasma that vaporizes and ionizes the cells, antibodies, and the attached heavy metal tags. Each cell is converted into an ion cloud and passes through a quadrupole filter, which removes all ion masses below approximately 75 Da. The remaining heavy-metal reporter ions in the ion cloud are separated by their mass-to-charge ratio as they accelerate towards a detector in a time-of-flight mass spectrometer. The time-resolved detector measures a mass spectrum that represents the identity and quantity of each isotopic probe on a single-cell basis.

expensive instruments to purchase and maintain, but a typical approach to overcome this issue is the development of a mass cytometry core facility which ultimately reduces costs. Second, there are no forward- or side-scatter light settings on a mass cytometer to determine cell size or granularity. However, novel methods to characterize cell size by mass cytometry using wheat germ agglutinin or osmium tetroxide as a stain have recently been reported.313 The instrument therefore typically relies on the use of a DNA intercalator and known unique surface markers to identify cells. For example, platelets in whole blood samples can be identified by their expression of surface CD41/CD61 and the fact that they are anucleate, and therefore are DNA-low compared to nucleated cells such as leukocytes which are DNA-high. Third, the current configuration of the mass cytometer instrument

means that it is a destructive technique with no possibility of recovery of cells for sorting.

Imaging Flow Cytometry Imaging flow cytometry (IFC) is an analytical platform that combines high-resolution fluorescence microscopy with high-throughput flow cytometry. IFC allows multiparametric fluorescent and morphological analysis of thousands of cellular events with single-cell resolution. Currently, IFCs are capable of acquiring up to 12 images simultaneously of each cell with event rates of up to
5000 objects per second. The imaging channels can capture label-dependent fluorescence markers (currently up to 10) as well as transmitted brightfield and laser

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Fig. 35.14 Imaging flow cytometry analysis of monocyte and neutrophil platelet aggregates. Monocytes are identified by monoclonal anti-CD14 (red), neutrophils are identified by SSC (pink), and platelets are identified by monoclonal anti-CD61 (green). Using imaging flow cytometry, monocyte and neutrophil platelet aggregates can be differentiated from coincidental events. (Unpublished data from the authors’ laboratory.)

side scatter (dark-field) information. IFC has primarily been used in the platelet research field to study (i) leukocyte-platelet and platelet-platelet aggregate formation (Fig. 35.14),314,315 and (ii) platelet extracellular vesicle generation.316 The primary advantage of IFC over conventional fluorescence flow cytometry is the ability of the platform to identify collected events by their real images. IFC can retrieve an image for each event (dot) on a two-dimensional dot-plot.317 The major disadvantage of IFC is that data analysis is often performed in a highly manual and subjective manner using limited image analysis techniques and complex software.317 REFERENCES 1. Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987;70:307–15. 2. Michelson AD, Barnard MR, Krueger LA, Frelinger 3rd AL, Furman MI. Evaluation of platelet function by flow cytometry. Methods 2000;21:259–70. 3. Michelson AD, Barnard MR, Benoit SE, Mitchell J, Knowles C, Ault KA. Characterization of platelet binding of blind panel mAb. In: Schlossman SF, Boumsell L, Gilks W, Harlan JW, Kishimoto T, Morimoto C, Ritz J, Shaw S, Silverstein RL, Springer TA, Tedder TF, Todd RF, editors. Leucocyte typing V. Oxford: Oxford University Press; 1995. p. 1207–10. 4. Krueger LA, Barnard MR, Frelinger 3rd AL, Furman MI, Michelson AD. Immunophenotypic analysis of platelets. Curr Protoc Cytom 2002; [Chapter 6:Unit 6.10]. 5. Gerrits AJ, Frelinger 3rd AL, Michelson AD. Whole blood analysis of leukocyte-platelet aggregates. Curr Protoc Cytom 2016;78: 6.15.1–6.15.10. 6. Santos MT, Valles J, Marcus AJ, Safier LB, Broekman MJ, Islam N, Ullman HL, Eiroa AM, Aznar J. Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. A new approach to platelet activation and recruitment. J Clin Invest 1991;87:571–80. 7. LaRosa CA, Rohrer MJ, Benoit SE, Rodino LJ, MR B, Michelson AD. Human neutrophil cathepsin G is a potent platelet activator. J Vasc Surg 1994;19:306–18 [discussion 318–319]. 8. Michelson AD, Ellis PA, Barnard MR, Matic GB, Viles AF, Kestin AS. Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound. Blood 1991;77:770–9. 9. Abrams C, Shattil SJ. Immunological detection of activated platelets in clinical disorders. Thromb Haemost 1991;65:467–73. 10. Michelson AD. Platelet activation by thrombin can be directly measured in whole blood through the use of the peptide GPRP and flow cytometry: methods and clinical studies. Blood Coagul Fibrinolysis 1994;5:121–31.

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