The classic washed platelet assay for HIT is the serotonin release assay (SRA) (Sheridan et al., 1986; Warkentin et al., 1992). This assay was a modification of platelet-washing techniques in use at McMaster University that resuspended washed platelets in buffer containing physiological concentrations of calcium. The purpose was to avoid platelet activation artifacts associated with low calcium concentrations (Mustard et al., 1972; Kinlough-Rathbone et al., 1983) (see Chapter 1). The use of washed platelets is also central to certain other functional assays, such as the heparin-induced platelet activation (HIPA) assay (Greinacher et al., 1991). Figure 1 summarizes washed platelet assays for HIT.
Preparation of Platelets for Washed Platelet Assays
1. Collect 8.4 volumes of blood from a normal donor into 1.6 volumes of acid-citrate-dextrose (ACD).
Comment. Aspirin-free normal blood donors whose platelets are known to respond well to HIT sera should be selected, as there is considerable heterogeneity to platelet activation by HIT sera among platelets obtained from different normal individuals (Warkentin et al., 1994). In Hamilton, platelets from two donors are
TABLE 1 Two Clinicopathologic Syndromes: HIT and Antiphospholipid Syndrome
Laboratory features Structure of the major antigen
Nonspecific nature of anionic component of antigen (cross-reactivity)
Sequestered location of antigen Functional assay
Inhibition of functional assay
Antigen assay Relative sensitivity of assays for antibodies Relative specificity of assays for disease state
"Cryptic" autoepitope on PF4 expressed when complexed with heparin
Variable reactivity when heparin substituted by LMWH, danaparoid, pentosan polysulfate, and others PF4 within platelet
«-granules Heparin-dependent activation of platelets by patient serum or plasma Inhibition by high-dose heparin PF4-H-EIA Antigen > functional
Functional > antigen
"Cryptic" autoepitope on b2-GPI expressed when bound to anionic phospholipid (Pengo et al., 1995) Variable reactivity when cardiolipina substituted by phosphatidylserineb or other molecules (e.g., irradiated plastic) Anionic phospholipids of inner leaflet of bilipid membranes Prolongation of phospholipid-dependent coagulation assay by patient's plasma Inhibition of lupus anticoagulant assays by excess phospholipid Anticardiolipin EIA Antigen > functional
Functional > antigen
Note: Information on relative sensitivity and specificity of functional and antigens assays for HIT and antiphospholipid antibody syndrome are provided elsewhere (Visentin et al., 1994; Ginsberg et al., 1995; Berube et al., 1998; Warkentin et al., 2000).
aCardiolipin is found primarily in the inner leaflet of the mitochondrial membrane.
bPhosphatidylserine is located in the inner leaflet of platelet membranes; thus, antiphospholipid antibodies with antiphosphatidylserine activity could be relatively more important in the pathogenesis of thrombocytopenia. Abbreviations: EIA, enzyme immunoassay; ß2-GPI, beta2-glycoprotein I; LMWH, low molecular weight heparin; PF4-H, platelet factor 4-heparin; HIT, heparin-induced thrombocytopenia.
combined. In Greifswald, platelets from four different donors selected randomly are prepared and tested individually. ABO blood group discrepancies do not affect the results of these assays (Greinacher et al., 1991).
2. Perform differential centrifugation to obtain ACD-anticoagulated platelet-rich plasma (PRP).
Comment. Low-speed centrifugation prepares ACD-anticoagulated PRP. Additional ACD (111 pL/mL PRP) is added (Greifswald) to ensure that the pH of the PRP is sufficiently low (<6.5) to prevent platelet aggregation that otherwise would occur during platelet pelleting: the platelet release reaction is triggered by close platelet contact in low calcium concentrations at physiological pH (Kinlough-Rathbone et al., 1983). If the serotonin release method is used, the PRP is incubated at 37° C for 30 min with [14C]serotonin (0.1 pCi/mL of PRP added from a stock solution of 50 pCi/mL of [14C]serotonin) (Lee et al., 1996).
3. Wash the platelets by pelleting them from PRP, then gently resuspend the platelets in calcium- and magnesium-free Tyrode's buffer, pH 6.3, containing glucose (5.6mmol/L) and apyrase (2.5U/mL).
Comment. Tyrode's buffer consists of physiological concentrations of sodium chloride (NaCl, 137mmol/L), potassium chloride (2.7mmol/L), calcium chloride
TABLE 2 Classification of Laboratory Tests for HIT
Platelet activation (functional) assays Washed platelet assays
Serotonin release assay (SRA): quantitation of 14C-radiolabeled serotonin released from dense granules of activated platelets (Sheridan et al., 1986); chemical and chromatographic detection of serotonin also described (Fouassier et al., 2006) Heparin-induced platelet activation (HIPA) test: visual assessment of platelet aggregation
(Greinacher et al., 1991; Eichler et al., 1999) ATP release detected by luminography (Stewart et al., 1995)
Platelet microparticle assay: quantitation of platelet-derived microparticles by flow cytometry (Leeetal., 1996) Platelets in citrated platelet-rich plasma (c-PRP)
Platelet aggregation test (PAT): assessment of platelet aggregation using conventional aggregometry (Fratantoni et al., 1975; Chong et al., 1993a) Annexin V-binding assay: quantitation by flow cytometry of annexin V binding to anionic phospholipids expressed by activated platelets (Tomer, 1997; Tomer et al., 1999) Serotonin release detected by flow cytometry (Gobbi et al., 2003) Antigen assays (PF4-dependent) Enzyme immunoassays (EIAs)
Target antigen: PF4-H complexes (Amiral etal., 1992) Target antigen: PF4-polyvinylsulfonate complexes (Visentin et al., 2001) Fluid-phase immunoassay (Newman et al., 1998)
Rapid assays (based on PF4- or PF4/heparin-coated particle agglutination) Particle gel immunoassay (PaGIA) (Meyer et al., 1999) Particle immunofiltration assay (PIFA)
(CaCl2, 2 mmol/L), magnesium chloride (MgCl2, 1.0mmol/L), and sodium dihy-drogen phosphate (NaH2PO4, 3.3 mmol/L); however, calcium-free and magnesium-free Tyrode's is used in this wash step to avoid activating the coagulation factors and platelets. The low pH prevents platelets from aggregating during pelleting. Apyrase is an enzyme that degrades adenine nucleotides (i.e., accumulation of the ADP from the platelets is prevented). Azide-free bovine serum albumin (3.5 mg/mL) and hirudin (1 U/mL) are included in the wash buffer in Greifswald, but not Hamilton, although HEPES (5 mmol/L) is added to this buffer in Hamilton. Following resuspension, the platelets are incubated for 15 min at 37°C (Greifswald).
4. Pellet the washed platelets as before, and then gently resuspend the platelets into calcium- and magnesium-containing Tyrode's buffer, pH 7.4, without apyrase or hirudin.
Comment. Following resuspension, the platelets should "rest" for 45 min at 37°C (Greifswald). The final resuspension buffer (Tyrode's buffer at physiological pH) contains calcium (2 mmol/L) and magnesium (1 mmol/L Hamilton; 2 mmol/L Greifswald). The platelet count is adjusted to a minimum of 300 x 109/L; thus, after addition of washed platelets (75 mL) to the microtiter wells containing test serum (20 mL) and heparin-buffer (5 mL in Hamilton, 10 mL in Greifswald), the final platelet concentration will be at least 215 X 109/L. Apyrase must not be included in this buffer, as the ADP released during assessment of HIT-induced platelet activation is an important potentiator of platelet Fc receptor-mediated platelet activation (Polgâr et al., 1998).
ACD-anticoagulated whole blood (pH<6.5) Centrifuge for platelet-rich plasma (PRP)
I Incubate with 14C-serotonln for 30 min at 37"C y (Serotonin-release assay, SRA)
Centrifuge to pellet platelets i
Resuspend in calcium-free Tyrode's buffer with apyrase (pH = 6.2) Centrifuge to pellet platelets
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