Serotonin‐release assay‐negative heparin‐induced thrombocytopenia

Abstract

Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Pathogenic HIT antibodies can be detected by the serotonin-release assay (SRA), a platelet activation test. We have regarded the SRA performed in our medical community ("McMaster" SRA) as having high sensitivity and specificity. Recently, the concept of "SRAnegative HIT" has been proposed for enzyme-immunoassay (EIA)-positive/SRAnegative patients with a HIT-compatible clinical picture, who test positive in a PF4-enhanced platelet activation assay. After identifying an index case of SRAnegative HIT, we estimated the frequency of this condition by performing the "PF4-SRA" (modified SRA using high concentrations of added PF4 rather than heparin) in EIA-positive patients from a cohort study evaluating clinical and laboratory diagnosis of HIT. We defined SRA-negative HIT as patients meeting three criteria: clinical picture compatible with HIT (4Ts ≥ 4 points); EIA-positive (≥1.00 units); and PF4-SRA-positive. Among 430 patients, 35 were EIA-positive/SRA-positive and 27 were EIA-positive/SRA-negative. Among these 27 SRA-negative patients, three were found to have subthreshold levels of platelet-activating antibodies by PF4-SRA, of whom one met clinical criteria for SRA-negative HIT. Thus, based on identifying one patient with SRA-negative HIT within a cohort study that found 35 SRA-positive HIT patients, we estimate the sensitivity of the McMaster SRA for diagnosis of HIT to be 35/36 (97.2%; 95% CI, 85.8-99.9%). Although the McMaster SRA is highly sensitive for HIT, occasional SRA-negative but EIA-positive patients strongly suspected of having HIT can have this diagnosis supported by a PF4-enhanced activation assay such as the PF4-SRA.

Challenges in Detecting Clinically Relevant Heparin-Induced Thrombocytopenia Antibodies

“…However, more recent studies 21 58 from our group do support the concept of SRA-negative HIT. In one study, we systematically evaluated 27 EIA-positive/SRA-negative samples by PF4-SRA; we found one patient with plausible SRA-negative HIT on both clinical and laboratory grounds.…”

“…Our laboratory has independently shown that the sensitivity of our SRA could also be enhanced by measuring patient serum-dependent serotonin release in the presence of high concentrations of PF4 (50–100 μg/mL), rather than heparin, in a modified assay we called the “PF4-SRA.” 21 57 58 In our first report describing the PF4-SRA, 57 we found that the additional patients identified as having platelet-activating antibodies did not appear to have a clinical picture suggestive of HIT. In other words, it seemed plausible that (conventional) SRA-negative blood samples that tested positive in the PF4-SRA might be similar to SRA-negative/EIA-positive blood samples, in that the patients most likely did not have HIT.…”

Response to The challenges of diagnosing heparin‐induced thrombocytopenia in patients with COVID‐19

“…We agree that false‐positive enzyme immunoassay (EIA) detection of anti–platelet factor 4 (PF4)/heparin antibodies could explain the results we observed in patients 2 and 3, 2 and this has been the conventional interpretation when functional testing (such as the serotonin‐release assay [SRA]) returns negative. We suggested that a false‐negative SRA result could have explained our findings, as opposed to the contention by May et al that we concluded they were falsely positive, to broaden our discussion about SRA‐negative HIT, a relatively new and evolving clinical condition 3‐6 …”

COVID-19 versus HIT hypercoagulability

“…Given the importance of HIT ascertainment in this novel patient population, we recommend referral of EIA-positive or LIA-positive blood samples for testing by functional (platelet activation) assay, such as the SRA. However, given emerging data regarding occasional false-negative SRA results [ 114 ], we also suggest that SRA-negative blood samples (but with strong clinical suspicion of HIT corroborated by strong-positive EIA or LIA) be referred to a laboratory with experience in performing newer PF4-dependent platelet activation assays, such as the PF4-SRA [ 114 ], PF4/heparin-SRA [ 115 ], or the P-selectin expression assay [ 116 ].…”