Matthew Crawford (Presenter)
Lab Corp
Authorship: Matthew L. Crawford, Yvonne Z. Wright, Russell P. Grant
Laboratory Corporation of America Holdings, 1447 York Ct., Burlington, NC, 27215
| Short Abstract Heparin-induced thrombocytopenia (HIT) is an antibody-mediated complication of heparin therapy caused by immunization against platelet factor 4 complexed with heparin. The serotonin release assay using washed-platelets is a functional assay and considered the gold standard, however, it’s highly complex and requires a radioactive doping of a donor platelet solution. We’ve developed and validated a method which incorporates the measurement of endogenous release of serotonin created by patient sera without the need for exogenous doping or radioactive labeling. 94% clinical concordance proves that by LC-MS/MS analysis of endogenous serotonin release could be an equivalent measure of HIT without the added complexity. |
Long Abstract
Heparin-induced thrombocytopenia (HIT) is a potentially catastrophic, antibody-mediated complication of heparin therapy caused by immunization against platelet factor 4 (PF4) complexed with heparin or other polyanions (1-6). HIT antibodies bind to PF4/heparin complexes on the platelet surface resulting in platelet activation, ultimately leading to a platelet count decrease that can be accompanied by life-threatening thrombosis. The laboratory investigation of HIT is challenging and requires correlation between clinical symptoms and laboratory assays. The most common assay performed is a serologic assay that detects the presence of HIT antibodies without regard for their functional ability. However, these assays suffer from low specificity and frequently give positive results in the absence of clinical HIT. Functional assays that measure platelet activation by HIT antibodies in the presence of heparin are considered gold standard diagnostic laboratory tests due to their ability to detect the patient’s underlying procoagulable state in those with true HIT (7). However, functional assays are complex to perform, often requiring washed-platelets that are not widely available due to their instability. The serotonin release assay (SRA) using washed-platelets is a well-established functional HIT assay that is both highly sensitive and specific for HIT (1-6).
Previously the serotonin release assay in the clinical lab was performed as described by Sheridan et al (8). It measures a release of radioactively labeled serotonin which has been doped into fresh donor platelets. This requires special regulatory requirement which adds to the complex nature of the test. Our goal was to facilitate a measurement using LC-MS/MS that did not require exogenous doping or radiolabelling while reflecting, as best possible, the true biology occurring in a patient undergoing this adverse event.
Many challenges arose while developing this assay which utilizes a cellular based biological reagent (donor platelets solution). Numerous avenues of variability exist within this assay; from the complex nature of the biological reaction occurring ex-vivo to the performance variability of the biological reagent prepared on a daily basis. Experiments were performed to understand the relationship between release of endogenous serotonin and an exogenous form of serotonin doped into a platelet solution. Exhaustive efforts were made to screen a large cohort (n > 100) of possible platelet donors to understand and minimize the inherent variability of the donor platelet reagent.
The resulting assay was thoroughly validated and controlled for both measurement and biological variation. It included evaluations of matrix effects, sensitivity, selectivity, stability, inaccuracy, imprecision, carryover, and linearity. Verification of the reporting cut-off was conducted by demonstrating correlation and interpretive concordance of patient results with the existing send-out laboratory. The new assay was shown to be specific for the analysis of each analyte in serum. No significant interference was observed in the calibrator matrix and the assay specificity for the analyte was demonstrated to be unaffected by the stable isotope-labeled internal standards. Carryover following ULOQ was shown to give a response less than that observed in the LLOQ, as were drug and analyte interferences at physiologically significant amounts of the potentially interfering substances screened. Moreover, two distinct SRM transitions for detection of the analyte were compared and observed to provide equivalent quantitative measurements. SRM ion ratios for each analyte were evaluated across multiple batches in order to provide acceptance criteria for future data reduction and reporting. Matrix effects evaluated by sample mixing, and spike and recovery were acceptable in the new assay. Matrix affects in lipemic, icteric, and hemolyzed samples were not acceptable. The assay was shown to be reproducible and accurate for the measurement of the serotonin in calibrators and quality controls. The stability of the analyte in calibrators, quality controls, and patient samples were extensively studied. Additionally platelet solution, ACD whole blood (for platelet prep), and microtiter plate supernatant stability was shown. Post-processing (i.e., following complete extraction procedure) stability was demonstrated for batches stored on the bench-top (15-30C) for 4 hours in either acetonitrile or ethyl acetate and in the autosampler coolstack (10 C) for 72 hours. Calibrator reproducibility was shown across 5 separate analytical runs.
Moreover, correlation to the existing gold standard assay yielded 94% clinical agreement, proving that by harnessing the sensitivity and selectivity intrinsic to LC-MS/MS analysis, measurement of endogenous serotonin release could be an equivalent measure of HIT without the added complexity of radiolabelling and platelet doping. The inherent high throughput capabilities of the LC-MS/MS analysis allows for the validated assay to be used in conjunction with an in-house enzyme immunoassay to provide screening and confirmation capabilities with results to clinicians with 24-48hr turnaround time versus 7-10 days historically.
References & Acknowledgements:
1.Greinacher A. Heparin-induced thrombocytopenia. J Thromb Haemost. 2009;7 Suppl 1:9-12
2.Linkins LA, Dans AL, Moores LK et al. Treatment and prevention of heparin-induced thrombocytopenia: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest. 2012 Feb;141(2 Suppl):e495S-530S.
3.Warkentin TE. Think of HIT. Hematology Am Soc Hematol Educ Program. 2006:408-14.
4.Warkentin TE, Sheppard JA. Testing for heparin-induced thrombocytopenia antibodies. Transfus Med Rev. 2006 Oct;20(4):259-72.
5.Francis JL. A critical evaluation of assays for detecting antibodies to the heparin-PF4 complex . Semin Thromb Hemost. 2004;30(3):359-368.
6.Cuker A. Heparin-induced thrombocytopenia: present and future. J Thromb Thrombolysis. 2011;31(3):353-66.
7.Sadik AG, Jennings DL, Nemerovski CW, et al. Impact of Platelet Function ASsays on the Cost of Treating Suspected Heparin-Induced Thrombocytopenia. Journal of Pharmacology Practice. 2014; DOI:10. 1177/0897190014522065.
8.Watson H, Davidson, Keeling D. Guidelines on the diagnosis and management of heparin-induced thrombocytopenia: second edition. British J Haem. 2012; DOI:10.1111/bjh.12059
9.Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin induced thrombocytopenia. Blood, 1986 67: 27-30.
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