Off-the-shelf cryopreserved platelets for the detection of HIT and VITT antibodies

Abstract

Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.

Graphical abstract

Figure 1.

HIT antibodies activate PF4-treated cryopreserved platelets, including 1-year-old platelets, and TSP1 release correlates with serotonin release. (A) TRAs were performed with 3 platelet-activating HIT (closed circles) and 3 NC (open circles) samples using 20 lots of cryopreserved platelets. Each data point represents the mean of technical triplicates. (B) TSP1 release from 7 of the 20 cryopreserved platelet lots stored for >4 weeks at −80°C (4W) were compared with activation of platelets stored for ≤1 week at −80°C (baseline [B]). Four-week-old platelets ranged in storage age from 29 to 36 days, whereas baseline platelets ranged in storage age from 1 to 7 days. Each data point represents the mean of technical triplicates. (C) PF4-dependent platelet activation induced by a HIT (closed circle) and NC sample (open circle) with 3 cryopreserved platelet lots stored at −80°C for 12 months. Each data point represents the mean of technical triplicates. (D) Platelet activation induced by TRAP. Data are from technical triplicates with mean ± SD displayed. Groups are compared using two-tailed, unpaired t test with Welch’s correction. ∗∗P < .005. Studies were performed at Retham Technologies (Wauwatosa, WI) on cryopreserved platelets that were not subject to shipping. NC, normal control.

Figure 2.

The PF4-TRA is highly sensitive and specific for the detection of platelet-activating HIT and VITT antibodies. (A) PEA and SRA results for the cohort of HIT-suspected patient samples tested in the PF4-TRA are shown on the x-axis and y-axis, respectively. Red dotted lines represent positive cutoff values (19% P-selectin expression for the PEA and 20% serotonin release for the SRA). (B) PF4-TRA results for 34 HIT-suspected patient samples and 4 VITT patient samples are shown. ELISA-positive/PEA-positive samples, ELISA-positive/PEA-negative samples, and ELISA-negative/PEA negative samples are indicated by closed circles, closed triangles, and open triangles, respectively. Each data point represents the mean of technical duplicates. An ordinary 1-way analysis of variance was used for comparisons, and means are shown as dotted lines. (C) TRA results for OD-matched HIT ELISA-positive/PEA-positive patient samples (closed circles) vs HIT ELISA-positive/PEA-negative patient samples (closed triangles) are shown. Each data point represents the mean of technical duplicates. A 2-tailed, unpaired Student t test was used to compare OD values and TSP1 fold changes. (D) PEA and (E) SRA results were correlated with TSP1 release for the HIT-suspected patient samples tested in the PF4-TRA. Linear regression analysis (black solid line) with 95% CI (dashed lines) are presented. The fold increase in TSP1 released from platelets incubated with the HIT/VITT cohort samples relative to release seen with a normal serum control is depicted on the y-axis for (B), (C), (D), and (E). ∗∗∗∗P < .0001. IgG, immunoglobulin G; SpHIT, spontaneous HIT.

Figure 3.

The heparin-TRA is highly sensitive and specific for detection of platelet-activating HIT, but not VITT antibodies. (A) PEA and SRA results for the cohort of HIT-suspected patient samples tested in the heparin-TRA are shown on the x-axis and y-axis, respectively. Red dotted lines represent positive cutoff values (19% P-selectin expression for the PEA and 20% serotonin release for the SRA). (B) Heparin-TRA results are displayed for 29 HIT-suspected patient samples and 4 VITT patients. ELISA-positive/PEA-positive samples, ELISA-positive/PEA-negative samples, and ELISA-negative/PEA-negative samples are indicated by closed circles, closed triangles, and open triangles, respectively. Each data point represents the mean of technical duplicates. Ordinary 1-way analysis of variance was used for comparisons, and means are shown as dashed lines. (C) Results from the TRA performed in the presence of high concentrations of UFH (100 U/mL) are presented. ELISA-positive/PEA-positive samples, ELISA-positive/PEA-negative samples, and ELISA-negative/PEA-negative samples are indicated by closed circles, closed triangles, and open triangles, respectively. Each data point represents the mean of technical duplicates. Means are shown as dashed lines. (D) PEA and (E) SRA results were correlated with TSP1 release in the heparin-TRA for HIT-suspected patient samples (excluding the heparin-noninhibitable samples, P1 and P2). Linear regression analysis (black solid line) with 95% CI (dashed lines) are presented. The fold increase in TSP1 released from platelets incubated with the HIT/VITT cohort samples relative to release seen with a normal serum control is depicted on the y-axis for (B), (C), (D), and (E). ∗∗∗∗P < .0001. ns, not significant; SpHIT, spontaneous HIT; UFH, unfractionated heparin.

Figure 4.

TSP1 release in the PF4-TRA and heparin-TRA were well correlated, and cryopreserved platelets support FcᵞRIIa-mediated platelet activation. (A) Ten HIT-suspected patient samples with sufficient volumes to test in both the PF4-TRA and heparin-TRA stimulated similar levels of TSP1 release in both the PF4-TRA and heparin-TRA (R² = 0.724; P = .0018). Data are from technical duplicates, and correlation was determined by linear regression analysis (black solid line) with 95% CI (dashed lines) displayed. (B) TSP1 release induced by HIT and VITT samples from cryopreserved platelets incubated with monoclonal antibody IV.3 or IC in the PF4-TRA are shown. Data are from technical duplicates with mean ± SD displayed. ∗∗P < .005; ∗∗∗P < .001. IC, isotype control; ns, not significant.