Evaluation of a flow cytometric assay for the confirmation of heparin-induced thrombocytopenia

Abstract 5060 Background Heparin-induced thrombocytopenia and thrombosis (HIT) is an immune-mediated complication that may develop in patients sensitized to heparin. Approximately 5% of patients treated with full dose heparin develop clinical HIT, with about half develop thrombosis that may be associated with severe morbidity and death. However, antibodies may be detected in up to 30% of patients. Thus, current antibody-detection methods carry certain limitations, which pose a serious clinical dilemma in the diagnosis and treatment of HIT. Objectives To compare the gel-particle immuno-assay (PaGIA) for the detection of antibodies against heparin-PF4 complex, with the functional flow cytometric assay (FCA) which determines the capacity of the patient's serum to activate platelets in the presence of heparin, similar in concept to the gold-standard, the radioactive Serotonin-release assay. Methods Sequential samples from patients clinically suspected for HIT were tested by both PaGIA and the FCA (Tomer 1997, 1999). Results 118 samples were tested. Positive: 9 (7.6%) patients tested positive by PaGIA, compared to 19 (16.1%) by the FCA. 7,out of the 9 (77.8%) PaGIA -positive samples were also positive by the FCA (relative sensitivity). Negative: 97 out of 109 gel-negative samples (89.0%) were also negative by the FCA (relative specificity). Thrombosis occurred in 3 of the 9 PaGIA-positive (33%) patients, and in 7 of the 19 FCA-positive (36.8%) patients. Of the 12 PaGIA -negative but FCA-positive patients, 4 (33%) had thrombosis. Death rate was also higher among FCA-positive (n=3) compared to PaGIA-positive (n=1) patients. Of the two PaGIA -positive but FCA-negative patients, one had APLA syndrome (APS) with chronic thrombocytopenia, and one had sepsis with cardiogenic shock and multiorgan failure. ROC-Plot: Overall, the FCA showed significantly higher correlation with the clinical presentation of HIT (4Ts score), compared to the PaGIA (AUC 0.86 vs. 0.62, p<0.001) Conclusion The functional FCA demonstrates superior sensitivity and specificity compared to the antibody- detection PaGIA gel-particle assay. The feasible functional FCA (results in 1.5 hr) might be useful for initial diagnosis of HIT, and particularly for confirmation of HIT in patients with confounding presentation, including negative antibody-detection assay. Disclosures No relevant conflicts of interest to declare.

Download Full-text

A Flow Cytometric Assay of Platelet Activation Marker P-Selectin (CD62P) Distinguishes Heparin-induced Thrombocytopenia (HIT) from HIT with Thrombosis (HITT)

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614371 ◽

1999 ◽

Vol 82 (10) ◽

pp. 1255-1259 ◽

Cited By ~ 38

Author(s):

Wenche Jy ◽

Wei Wei Mao ◽

Lawrence Horstman ◽

Peter Valant ◽

Yeon Ahn

Keyword(s):

Platelet Activation ◽

Flow Cytometric Assay ◽

Laboratory Methods ◽

Fluorescent Intensity ◽

Heparin Induced Thrombocytopenia ◽

Activation Marker ◽

Flow Cytometric ◽

Heparin Administration ◽

Life Threatening ◽

Positive Results

SummaryHeparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios ~2.5 vs. HIT ratios ~1.2; p <0.001). Eleven of 12 HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p < 0.0005). In a case of HIT with silent thrombosis, this assay gave a positive results prior to clinically evident thrombosis. In conclusion, this method distinguishes HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.

Download Full-text

A sensitive and specific functional flow cytometric assay for the diagnosis of heparin‐induced thrombocytopenia

British Journal of Haematology ◽

10.1046/j.1365-2141.1997.2613077.x ◽

1997 ◽

Vol 98 (3) ◽

pp. 648-656 ◽

Cited By ~ 73

Author(s):

Aaron Tomer

Keyword(s):

Flow Cytometric Assay ◽

Heparin Induced Thrombocytopenia ◽

Flow Cytometric

Download Full-text

Determination of heparin-induced thrombocytopenia: A rapid flow cytometric assay for direct demonstration of antibody-mediated platelet activation

American Journal of Hematology ◽

10.1002/(sici)1096-8652(199905)61:1<53::aid-ajh10>3.0.co;2-f ◽

1999 ◽

Vol 61 (1) ◽

pp. 53-61 ◽

Cited By ~ 26

Author(s):

Aaron Tomer ◽

Cynthia Masalunga ◽

Thomas C. Abshire

Keyword(s):

Platelet Activation ◽

Flow Cytometric Assay ◽

Heparin Induced Thrombocytopenia ◽

Flow Cytometric ◽

Direct Demonstration ◽

Rapid Flow

Download Full-text

Functional Flow Cytometric Assay for Reliable and Convenient Heparin-Induced Thrombocytopenia Diagnosis in Daily Practice

Biomedicines ◽

10.3390/biomedicines9040332 ◽

2021 ◽

Vol 9 (4) ◽

pp. 332

Author(s):

Brigitte Tardy-Poncet ◽

Aurélie Montmartin ◽

Michele Piot ◽

Martine Alhenc-Gelas ◽

Philippe Nguyen ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Rich Plasma ◽

Daily Practice ◽

Laboratory Diagnosis ◽

Serotonin Release ◽

Confirmatory Test ◽

Flow Cytometric Assay ◽

Heparin Induced Thrombocytopenia ◽

Flow Cytometric ◽

Release Assay

Reliable laboratory diagnosis of heparin-induced thrombocytopenia (HIT) remains a major clinical concern. Immunoassays are highly sensitive, while confirmatory functional tests (based on heparin-dependent platelet activation) lack standardization. We evaluated the diagnostic performance of a functional flow cytometric assay (FCA) based on the detection of heparin-dependent platelet activation with an anti-p-selectin. A total of 288 patients were included (131 HIT-positive and 157 HIT-negative) with a HIT diagnosis established by expert opinion adjudication (EOA) considering clinical data and local laboratory results. The FCA was centrally performed in a single laboratory on platelet-rich plasma, using a very simple four-color fluorometer. The results were standardized according to the Heparin Platelet Activation (HEPLA) index. The serotonin release assay (SRA) was performed in the four French reference laboratories. Based on the final HIT diagnosis established by EOA, the sensitivity and specificity of the FCA were 88 and 95%, respectively, values very similar to those of the SRA (88 and 97%, respectively). This study showed that the FCA, based on easily implementable technology, may be routinely used as a reliable confirmatory test for HIT diagnosis.

Download Full-text

Nuclear oncoprotein expression as a function of lineage, differentiation stage, and proliferative status of normal human hematopoietic cells

Blood ◽

10.1182/blood.v74.5.1517.1517 ◽

1989 ◽

Vol 74 (5) ◽

pp. 1517-1524 ◽

Cited By ~ 55

Author(s):

MB Kastan ◽

KD Stone ◽

CI Civin

Keyword(s):

Progenitor Cells ◽

Control Cell ◽

Hematopoietic Cells ◽

Maturation Stage ◽

Flow Cytometric Assay ◽

Lineage Differentiation ◽

Flow Cytometric ◽

A Cell ◽

Normal Human ◽

Differentiation Stage

Abstract Relative levels of the nuclear oncoproteins c-myb, c-myc, and c-fos were determined in selected subpopulations of normal human bone marrow (BM) cells using a flow cytometric assay which simultaneously detects a cell-surface antigen (as a marker of lineage and stage of maturation) and levels of an intracellular protein. At least two monoclonal antibodies directed against each oncoprotein and specific peptide inhibition controls were used for these determinations. Hematopoietic progenitor cells (CD34+) express the highest levels of c-myb and c-myc, whereas c-fos levels in CD34+ progenitor cells are similar to c-fos levels in mature monocytes and granulocytes. Granulocytes are the only hematopoietic cells examined which do not express detectable levels of c-myb and c-myc. The levels of these oncoproteins in these normal, unstimulated BM cell populations were more closely linked to lineage and maturation stage than to the proliferative status of the given population, as determined by either DNA staining or expression of the cell-cycle specific nuclear protein, Ki67. This flow cytometric assay helps in interpreting the significance of oncoprotein levels in leukemia cells by allowing direct comparisons of a leukemia with the phenotypically similar “normal counterpart control” cell population in normal BM.

Download Full-text