0
Editorial |

Platelet Activation Testing for Heparin-Induced Thrombocytopenia Antibodies: A Problem That Needs Fixing? FREE TO VIEW

Theodore E. Warkentin, MD
Author and Funding Information

FINANCIAL/NONFINANCIAL DISCLOSURES: The authors have reported to CHEST the following: T. E. W. reports receiving fees from Instrumentation Laboratory for serving on an advisory board; consulting fees from Aspen Global and W. L. Gore; lecture fees from Instrumentation Laboratory and Pfizer Canada; fees for providing expert testimony in cases regarding thrombocytopenia, coagulopathy, or ischemic limb losses; and royalties from Taylor & Francis Group (Informa) for editing a book on heparin-induced thrombocytopenia. T. E. W. also reports that his institution has received fees from Instrumentation Laboratory, Medtronic Diabetes, and W. L. Gore for research studies, as well as for performing the serotonin release assay.

Department of Pathology and Molecular Medicine, and the Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada

CORRESPONDENCE TO: Theodore E. Warkentin, MD, Room 1-180A, Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences (Hamilton General Site), 237 Barton St E, Hamilton, ON, Canada L8L2X2


Copyright 2016, American College of Chest Physicians. All Rights Reserved.


Chest. 2016;150(3):478-480. doi:10.1016/j.chest.2016.04.001
Text Size: A A A
Published online

Heparin-induced thrombocytopenia (HIT) is an important drug reaction that causes life- and limb-threatening thrombosis. It is considered a “clinical-pathologic” disorder, as the diagnosis is generally made by evaluating various “clinical” criteria (Thrombocytopenia, its Timing of onset, Thrombosis, and absence of oTher explanations, ie, the 4Ts scoring system) and determining whether (“pathologic”) HIT antibodies can be detected in the laboratory. Pathogenic heparin-induced antibodies are of the IgG class and activate platelets via their FcγIIA receptors (platelet IgG receptors), at concentrations of heparin ranging from 0 to 0.5 International Units/mL; characteristically, platelet activation is inhibited at very high heparin concentrations (eg, 100 International Units/mL), as excess heparin disrupts the formation of HIT antigens. HIT antibodies recognize complexes composed of platelet factor 4 (PF4)—a positively charged chemokine found within platelet α-granules—and heparin or certain other polyanions. However, a key problem is that among the many patients who receive heparin and who form anti-PF4/heparin antibodies that are readily detected by enzyme-linked immunosorbent assay (ELISA), only a minority of those antibodies are of the requisite platelet-activating isotype class (IgG), recognize the appropriate antigen site(s), and found in sufficient quantities to trigger platelet activation.

FOR RELATED ARTICLE SEE PAGE 506

In 1986 a test was developed at McMaster University (Hamilton, ON, Canada), the platelet serotonin release assay (SRA), which established conditions whereby platelets (obtained from normal donors) are readily activated by serum (or plasma) from patients with HIT. By “washing” platelets (obtained as platelet-rich plasma [PRP] in acid-citrate dextrose) in a low-pH, calcium-free, apyrase-containing buffer (thereby protecting the platelets from adenosine diphosphate-induced activation during processing) and resuspending the platelets in calcium-containing buffer at physiologic pH, conditions are created that are excellent for detecting pathogenic HIT antibodies, in comparison with classic PRP-based platelet aggregometry. Indeed, the McMaster SRA evolved into something of a “gold standard” for HIT diagnosis, as various studies showed that the probability of developing otherwise unexplained thrombocytopenia was much greater among patients with SRA+ results versus those with ELISA+ (but SRA) results, or control subjects (ELISA/SRA results). Presumably, when performing the SRA, sufficient PF4 is released from platelets during the washing step to allow activation by HIT sera. SRA test sensitivity is enhanced by using platelets from “pedigree” donors (ie, individuals whose platelets react well to HIT sera) and by using “weak-positive” control sera in every experiment (to ensure adequate platelet reactivity).

What happens if PF4 itself, rather than various concentrations of heparin, is added to the reaction mixture when performing a platelet activation assay? This approach was taken by Padmanabhan and colleagues, writing in this issue of CHEST. These workers, from the BloodCenter of Wisconsin in Milwaukee, report a new assay, the PF4-dependent P-selectin expression assay (PEA), for diagnosis of HIT. Platelets are obtained from normal donors (with prostaglandin E1 added to the PRP to reduce platelet activation during handling) and then washed and resuspended in isotonic phosphate-buffered saline-bovine serum albumin (PBS-BSA). However, rather than performing the subsequent reactions with various concentrations of heparin (as in the conventional SRA), these investigators assessed platelet activation (measured by P-selectin expression) in the presence of added PF4, PF4 plus very high concentrations of heparin (inhibiting any HIT antibody-induced platelet activation, as per conventional SRA), as well as in the presence of PBS-BSA buffer alone. These authors then studied their new assay, using samples from 91 patients with relevant clinical information (4Ts score) as well as ELISA results, and compared the results of the PEA with their (Milwaukee) SRA. For patients judged to be “HIT-positive” (based on intermediate 4Ts score and ELISA optical density ≥ 2.0, or high 4Ts score and ELISA optical density ≥ 1.0), they found a higher diagnostic accuracy versus their SRA (area under the curve, 0.92 vs 0.82; P = .02), largely explained by 11 of 16 PEA+/SRA specimens being classified as “HIT-positive.”

But is the PEA really an improvement over the SRA? After all, the SRA is not a standardized assay, but rather one with technical-methodologic differences among competing reference laboratories. For example, the McMaster group (to which I belong) has also found that addition of PF4 will increase the sensitivity of the (McMaster) SRA to detecting a subgroup of anti-PF4/heparin antibodies that are inherently platelet-activating, but that test negative in the conventional SRA. Indeed, approximately one-third of ELISA+/SRA sera have low levels of platelet-activating antibodies detected by the modified “PF4-SRA.” However, in contrast to the Milwaukee group, we did not view these additional patients as likely having HIT, based on case review. In fact, the McMaster group in recent years has recommended use of an even higher threshold of serotonin release to define clinically significant HIT antibodies in the conventional SRA (from 20% to 50% serotonin release),, as it is our experience that weakly positive SRA results usually are found in patients in whom non-HIT explanations for thrombocytopenia appear most likely. In other words, the McMaster SRA already has high sensitivity for diagnosing HIT, and efforts to enhance its sensitivity further (such as by adding PF4) could tip the balance toward loss of diagnostic specificity without an offsetting improvement in detecting “true” patients with HIT. However, the SRA is a technically demanding and nonstandardized assay, so differences in performance are expected among laboratories. Such differences in interlaboratory assay performance could lead one laboratory to conclude that the addition of PF4 to the activation assay might increase sensitivity for detecting patients with clinical HIT, some of whom may have been missed because of lower sensitivity of their in-house SRA. But without direct laboratory-to-laboratory assay comparisons, it is difficult to make strong conclusions, and so it remains uncertain whether a new platelet activation test is needed.

The article by Padmanabhan and colleagues contributes to the important concept that among the panoply of anti-PF4/polyanion antibodies generated by patients treated with heparin, there are those that are inherently platelet-activating and those that are not. Logically, a patient with only weakly activating anti-PF4/heparin antibodies might not really have HIT as the explanation for his or her clinical picture. These concepts would suggest that the laboratory perhaps should not simply report the results of any platelet activation test—whether conventional SRA, SRA-PF4, or PEA—as positive or negative, but rather provide the actual quantitative results obtained, as this will assist the experienced clinician in discerning to what extent the laboratory results support or refute a diagnosis of HIT. After all, isn’t judging the interconnectedness of the clinical and laboratory data the essence of a clinical-pathologic disorder such as HIT?

References

Warkentin T.E. .Chong B.H. .Greinacher A. . Heparin-induced thrombocytopenia: towards consensus. Thromb Haemost. 1998;79:1-7 [PubMed]journal. [PubMed]
 
Warkentin T.E. .Heddle N.M. . Laboratory diagnosis of immune heparin-induced thrombocytopenia. Curr Hematol Rep. 2003;2:148-157 [PubMed]journal. [PubMed]
 
Warkentin T.E. .Arnold D.M. .Nazi I. .Kelton J.G. . The platelet serotonin-release assay. Am J Hematol. 2015;90:564-572 [PubMed]journal. [CrossRef] [PubMed]
 
Warkentin T.E. .Sheppard J.I. .Moore J.C. .Moore K.M. .Sigouin C.S. .Kelton J.G. . Laboratory testing for the antibodies that cause heparin-induced thrombocytopenia: how much class do we need? J Lab Clin Med. 2005;146:341-346 [PubMed]journal. [CrossRef] [PubMed]
 
Sheridan D. .Carter C. .Kelton J.G. . A diagnostic test for heparin-induced thrombocytopenia. Blood. 1986;67:27-30 [PubMed]journal. [PubMed]
 
Warkentin T.E. . How I diagnose and manage HIT. Hematology Am Soc Hematol Educ Program. 2011;2011:143-149 [PubMed]journal. [PubMed]
 
Warkentin T.E. .Hayward C.P.M. .Smith C.A. .Kelly P.M. .Kelton J.G. . Determinants of donor platelet variability when testing for heparin-induced thrombocytopenia. J Lab Clin Med. 1992;120:371-379 [PubMed]journal. [PubMed]
 
Padmanabhan A. .Jones C.G. .Curtis B.R. .et al A novel PF4-dependent platelet activation assay identifies patients likely to have heparin-induced thrombocytopenia/thrombosis. Chest. 2016;150:506-515 [PubMed]journal
 
Nazi I. .Arnold D.M. .Warkentin T.E. .Smith J.W. .Staibano P. .Kelton J.G. . Distinguishing between anti-platelet factor 4/heparin antibodies that can and cannot cause heparin-induced thrombocytopenia. J Thromb Haemost. 2015;13:1900-1907 [PubMed]journal. [CrossRef] [PubMed]
 
Warkentin T.E. .Sheppard J.I. .Heels-Ansdell D. .et al Heparin-induced thrombocytopenia in medical surgical critical illness. Chest. 2013;144:848-858 [PubMed]journal. [CrossRef] [PubMed]
 
Linkins L.A. .Bates S.M. .Lee A.Y.Y. .Heddle N.M. .Wang G. .Warkentin T.E. . Combination of 4Ts score and PF4/H-PaGIA for diagnosis and management of heparin-induced thrombocytopenia: prospective cohort study. Blood. 2015;126:597-603 [PubMed]journal. [CrossRef] [PubMed]
 

Figures

Tables

References

Warkentin T.E. .Chong B.H. .Greinacher A. . Heparin-induced thrombocytopenia: towards consensus. Thromb Haemost. 1998;79:1-7 [PubMed]journal. [PubMed]
 
Warkentin T.E. .Heddle N.M. . Laboratory diagnosis of immune heparin-induced thrombocytopenia. Curr Hematol Rep. 2003;2:148-157 [PubMed]journal. [PubMed]
 
Warkentin T.E. .Arnold D.M. .Nazi I. .Kelton J.G. . The platelet serotonin-release assay. Am J Hematol. 2015;90:564-572 [PubMed]journal. [CrossRef] [PubMed]
 
Warkentin T.E. .Sheppard J.I. .Moore J.C. .Moore K.M. .Sigouin C.S. .Kelton J.G. . Laboratory testing for the antibodies that cause heparin-induced thrombocytopenia: how much class do we need? J Lab Clin Med. 2005;146:341-346 [PubMed]journal. [CrossRef] [PubMed]
 
Sheridan D. .Carter C. .Kelton J.G. . A diagnostic test for heparin-induced thrombocytopenia. Blood. 1986;67:27-30 [PubMed]journal. [PubMed]
 
Warkentin T.E. . How I diagnose and manage HIT. Hematology Am Soc Hematol Educ Program. 2011;2011:143-149 [PubMed]journal. [PubMed]
 
Warkentin T.E. .Hayward C.P.M. .Smith C.A. .Kelly P.M. .Kelton J.G. . Determinants of donor platelet variability when testing for heparin-induced thrombocytopenia. J Lab Clin Med. 1992;120:371-379 [PubMed]journal. [PubMed]
 
Padmanabhan A. .Jones C.G. .Curtis B.R. .et al A novel PF4-dependent platelet activation assay identifies patients likely to have heparin-induced thrombocytopenia/thrombosis. Chest. 2016;150:506-515 [PubMed]journal
 
Nazi I. .Arnold D.M. .Warkentin T.E. .Smith J.W. .Staibano P. .Kelton J.G. . Distinguishing between anti-platelet factor 4/heparin antibodies that can and cannot cause heparin-induced thrombocytopenia. J Thromb Haemost. 2015;13:1900-1907 [PubMed]journal. [CrossRef] [PubMed]
 
Warkentin T.E. .Sheppard J.I. .Heels-Ansdell D. .et al Heparin-induced thrombocytopenia in medical surgical critical illness. Chest. 2013;144:848-858 [PubMed]journal. [CrossRef] [PubMed]
 
Linkins L.A. .Bates S.M. .Lee A.Y.Y. .Heddle N.M. .Wang G. .Warkentin T.E. . Combination of 4Ts score and PF4/H-PaGIA for diagnosis and management of heparin-induced thrombocytopenia: prospective cohort study. Blood. 2015;126:597-603 [PubMed]journal. [CrossRef] [PubMed]
 
NOTE:
Citing articles are presented as examples only. In non-demo SCM6 implementation, integration with CrossRef’s "Cited By" API will populate this tab (http://www.crossref.org/citedby.html).

Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging & repositioning the boxes below.

Find Similar Articles
CHEST Journal Articles
PubMed Articles
  • CHEST Journal
    Print ISSN: 0012-3692
    Online ISSN: 1931-3543