Comparative studies in the SRA and IgG-specific anti-PF4/heparin EIA using serially-diluted HIT sera. (A) Known HIT sera (n = 15) diluted from 1 in 5 to 1 in 5120 were tested in the SRA and EIA. (The 1/5 dilution in the SRA—20 μL patient serum to 80 μL washed platelets with heparin added—represents the standard conditions in the SRA.) Typical assay cutoffs are shown with dashed lines. Horizontal lines indicate median values. Compared with standard assay conditions, serial sample dilution generally results in a negative SRA well before a negative EIA result is attained. (B) Individual IgG-specific EIA ODs are shown for the same 15 HIT sera in 3 groupings: SRA⁺ showing maximum OD of any SRA⁺ dilution (leftmost data points); SRA⁺ showing OD of the most dilute yet still SRA⁺ serum dilution (middle data points); and SRA^– showing OD of the first SRA^– dilution (rightmost data points). There is a fourfold serum dilution between the 2 rightmost data sets. No significant differences were observed between the SRA⁺ and SRA^– data sets. EIA-IgG, enzyme-immunoassay (IgG-specific); OD, optical density; PF4, platelet factor 4; SRA, serotonin-release assay; SRA^–, SRA-negative; SRA⁺, SRA-positive.