A Multiplex Flow Cytometry Bead Assay For Simultaneous Detection Of IgG, IgA, IgM Heparin-Dependent Antibodies

Heparin-Induced Thrombocytopenia (HIT) is an adverse reaction to heparin, in which affected patients produce platelet activating antibodies specific for complexes of platelet-factor 4 (PF4) and heparin (HPF4) resulting in a prothrombotic condition that in severe cases can be life-threatening. HIT is not rare, since as many as 600K people/year are affected. It is therefore important that a quick and accurate diagnosis of HIT be made, which requires both compatible clinical findings and serologic evidence of a heparin dependent antibody. First line testing consists of immunologic assays that detect IgG, IgA and IgM antibodies targeting PF4 in a complex with heparin or another polyanion. The most widely used assay is the PF4 ELISA, in which HPF4 bound to the wells of a microtiter plate is the target antigen. The ELISA has high sensitivity (near 100%) for antibody detection but a low positive predictive value for HIT diagnosis. Therefore, samples giving positive ELISA results are often retested using a functional assay like the serotonin release assay (SRA) that is more specific for HIT diagnosis. However, the SRA is difficult to perform and interpret and is not widely available. Further, the ELISA is time-consuming, costly, and most versions cannot distinguish IgG, IgA, IgM (IgG/A/M) antibodies. Knowledge of the antibody isotype can be useful when ELISA and SRA results are discrepant. For instance, strong ELISA results due to clinically insignificant IgM antibodies would not be detected by SRA, and additional testing to confirm IgM antibody isotype can lead to a delay in HIT diagnosis, and in some cases continuation of high cost treatments with direct thrombin inhibitors (DTI). Moreover, there is evidence that some IgA and IgM antibodies may cause HIT.

Despite the high sensitivity of the ELISA and high specificity of the SRA, the shortcomings mentioned for these tests highlight the need for improved methods to support a diagnosis of HIT. We report development of a multiplex flow cytometry bead assay (MFBA) for simultaneous detection of IgG/A/M heparin-dependent antibodies that allows higher throughput and has sensitivity and specificity comparable to the PF4 ELISA.

For the MFBA, polystyrene beads, to which chemically depolymerized heparin is covalently linked are incubated with purified platelet PF4 to produce HPF4-bead targets. Lyophilized HPF4 beads are stable for at least 3 months. HPF4-beads are incubated with serum, washed, and incubated with anti-human -IgG, -IgA, and –IgM, each with different fluorescent labels for detection of HIT antibodies by flow cytometry. A total of 164 patient samples could be tested by a single technologist in 8 hours. Serum samples from 300 patients with suspected HIT and previously tested by SRA were tested by both MFBA and an in-house PF4 ELISA including high dose heparin (100 U/L) confirmation step. Studies were approved by institutional IRB. MFBA results showed good sensitivity (87% IgG, 63% IgA, 36% IgM) for IgG, good specificity (96% IgG, 91% IgA, 93% IgM) for all isotypes when compared to the ELISA, and good sensitivity/specificity (93%/91%) compared to SRA. The area under the curve (AUC) calculated for the receiver operating characteristic graphs compared to the SRA were excellent (0.9-1.0) for both MFBA (AUC = 0.95) and PF4 ELISA (AUC = 0.98). All samples positive by MFBA inhibited with high dose heparin suggesting that this extra step is not required, and 3 samples that tested IgG positive in both MFBA and SRA gave indeterminate results (not inhibited with high dose heparin) in ELISA.

Overall, these data show that the MFBA has accuracy, sensitivity, and specificity for detection of IgG antibodies comparable to the PF4 ELISA and the “gold standard” SRA with the additional advantage of simultaneously distinguishing IgG/A/M antibody isotypes, which will lead to reduced confirmation testing. Sensitivity of MFBA for detection of IgA and IgM antibodies was significantly lower compared to ELISA, but this reflects the assay design intended to increase specificity of the MFBA for these two isotypes. In addition to its multiplex capabilities, the MFBA allows for higher sample throughput and shortened turn-around times that will contribute to overall improved patient care.