Solid Phase PF4Polyvinylsulfonate Antigen Assay

Several negatively charged substances can cause the cryptic autoepitope within PF4 to become recognizable to HIT antibodies (see Chapters 5-7). Indeed, a commercial assay for HIT using PF4 complexed with polyvinylsulfonate has been developed (Collins et al., 1997; Visentin et al., 2001). Sensitivity and reproducibility were high using polyvinylsulfonate that had been fractionated to a relatively uniform MW (5000 ± 500 Da). Some technical advantages of this assay include the observation that the ratio of PF4/PVS is not critical (cf. PF4-H), with acceptable concentrations of PVS ranging from 0.1 to 100mmol/L for a corresponding concentration of 10 mg/mL PF4. The antigen complex is also stable for long periods.

The manufacturer of the PF4/polyvinylsulfonate EIA (Genetics Testing Institute [GTI], Waukesha, WI) recommends that a confirmatory step be performed, in

TABLE 6 Comparison of Two Commercial Antigen Assays for HIT Antibodies

Asserachrom® (Stago) GTI-PF4

PF4-heparin complex Recombinant PF4

Target antigen Source of PF4

Microwell strips provided

Sample required

Controls supplied

Covers supplied Incubation times and conditions Plate reader settings Detecting antibody system

Reaction stopping solution

Cutoff from negative

Six strips of eight wells, in three nonresealable pouches Plasma (sodium citrate) 200 mmL diluted 1:100 (=2|mL plasma per well) Positive and negative control lyophylate,a calibrating standard

PF4-polyvinyl sulfonate complex PF4 purified from outdated platelets Twelve strips of eight wells (in resealable pouches) Serum or plasma 50 |mL diluted 1:50 (=1 mL serum/plasma per well) Positive and negative control seraa

Multiple provided 37°C (two steps);

then 22°C (~2 h total) 405 or 410 nm Goat anti-IgG/A/M (alkaline phosphatase-conjugated) 3 M sodium hydroxide

>0.4 OD (assumes controls react as expected: pos >1.8, neg >;0.2)

One provided

492 nm

Goat anti-IgG/A/M

(peroxidase-conjugated) 3 M sulfuric acid or 1 M

hydrochloric acid Internal control reagent is used to calibrate aThe Asserachrom® assay provides lyophylized control sera, whereas handling of control sera in the GTI assay is similar to handling of the test sera/plasma.

Abbreviations: EIA, enzyme immunoassay; GTI, Genetics Testing Institute; PF4, platelet factor 4. Source: Warkentin, 2000.

which inhibition by 50% or more in the presence of high heparin (100 IU/mL) is considered supportive of a positive test result. However, this maneuver does not distinguish between clinically relevant and irrelevant anti-PF4-H antibodies, so test specificity may not be meaningfully increased. Furthermore, including this step will either double test costs (by requiring that each assay be performed both in the absence and in the presence of high heparin), or will delay the reporting of a positive test result (in case an algorithm is used in which a tentative positive test result is subsequently "confirmed") (Warkentin and Sheppard, 2006b).

Table 6 compares the two commercial EIAs. In the laboratory in Greifswald, discrepant results between the two assays have been observed in about 15% of patient samples tested.

Some research laboratories perform "in-house" PF4-H EIAs to detect HIT antibodies. An advantage is that an EIA can be used that only detects HIT antibodies of the IgG class (Warkentin et al., 2000, 2005a; Lindhoff-Last et al., 2001; Untch et al., 2002). This improves test specificity, as PF4-H-reactive IgA and IgM class antibodies (which are detected in the two commercial EIAs) are unlikely to cause HIT.

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