Heparin Induced Thrombocytopenia

Background

For many years, heparin, used intravenously, has been an effective anti-coagulant, preventing clotting in patients at risk for developing disorders of clotting such as deep vein thrombosis. With increasing use, laboratories are increasingly called upon to develop rapid and effective monitoring strategies.

Heparin is a sulfated glycosaminoglycan (GAG) and can naturally be found in human tissues and inflammatory cells such as mast cells. Heparin sulfated is located on the endothelial cells lining the blood vessel wall. Heparin in the serum and heparin sulfate bind antithrombin which is the major inhibitor protein of the coagulation cascade. When binding occurs, the antithrombin protein is altered and this accelerates its inhibition of other serine proteases (factors IXa, Xa, XIa, XIIa, kallikrein, and thrombin).

Heparin can be fractionated into two parts using affinity chromatography with immobolized antithrombin. One fraction, the high-affinity heparin, is responsible for nearly all of the anticoagulant activity. The other fraction, low-affinity heparin, has virtually no anticoagulant activity. The anticoagulant activity is expressed in units relative to an international standard (IU). Low molecular weight heparin (LMWH) is prepared by fractionation.

Patients with this disorder are receiving heparin therapy and develop antibodies to antigens on platelets. This leads to thrombocytopenia which may be profound.

OUTLINE

Disease Associations

Pathogenesis

Laboratory/Radiologic/Other Diagnostic Testing

Prognosis

Treatment

Commonly Used Terms

Internet Links

DISEASE ASSOCIATIONS CHARACTERIZATION

RENAL FAILURE

Heparin-induced skin necrosis in a patient with end-stage renal failure and functional protein S deficiency.

Denton MD, Mauiyyedi S, Bazari H.

Renal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02116, USA.

Am J Nephrol 2001 Jul-Aug;21(4):289-93 Abstract quote

Skin ulceration is a well-characterized thrombotic complication of the heparin-induced thrombocytopenia (HIT) syndrome.

We present the case of a 73-year-old diabetic woman nearing end-stage renal failure who developed extensive upper thigh, abdominal and buttock ulceration following initiation of subcutaneous heparin for prophylaxis against deep vein thrombosis. A preliminary diagnosis of calciphylaxis was made based on the classical distribution and macroscopic appearance of the ulceration in a patient with end-stage renal failure and secondary hyperparathyroidism. However skin biopsy showed complete absence of calcium deposits in the dermal microvasculature. The presence of extensive microthrombi within dermal vessels prompted serologic testing to detect a prothrombotic state. We identified the combined presence of heparin-dependent platelet activating (HIT) antibodies and functional protein S deficiency. To our knowledge this is the first reported case of a dialysis patient presenting with skin ulceration induced by heparin and protein S deficiency.

This case highlights the importance of a skin biopsy and testing for a hypercoaguable state in patients with end-stage renal disease and skin ulceration. We suggest that HIT antibodies should be requested in all dialysis patients presenting with skin ulceration.

PATHOGENESIS CHARACTERIZATION

Antibodies to platelet factor 4 (PF4)
Antibodies bind by their F(ab)'2 region to PF4 after release from intracellular storage

HIT antibodies directed against:
Epitopes within PF4 exposed when heparin-PF4 complexes have formed
Epitopes consisting partly of PF4 and heparin at points where the molecules are in close contact

Investigations of the immunoglobulin subtype transformation of anti-heparin-platelet factor 4 antibodies during treatment with a low-molecular-weight heparin (clivarin) in orthopedic patients.

Ahmad S, Bacher HP, Lassen MR, Hoppensteadt DA, Leitz H, Misselwitz F, Walenga JM, Fareed J.

Loyola University of Chicago, Stritch School of Medicine, Maywood, Ill, USA.

Arch Pathol Lab Med 2003 May;127(5):584-8 Abstract quote
CONTEXT: It is now widely accepted that the pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome is mediated by the generation of a wide array of functional and molecularly heterogeneous anti-heparin-platelet factor 4 (AHPF4) antibodies that may mediate platelet and/or endothelial cell activation/destruction.

OBJECTIVE: We investigated the differential prevalence and functionality of AHPF4 immunoglobulin subtypes (IgA, IgG, and IgM) in plasmas obtained from orthopedic patients immobilized with Plaster-Cast and treated with clivarin (a low-molecular-weight heparin) in comparison to a placebo for the prophylaxis of deep-vein thrombosis.

DESIGN AND METHODS: Clivarin was administered subcutaneously at a fixed daily dosage of 1750 U without any adjustment or loading dosage. Citrated plasmas were obtained at baseline, at 10 to 14 days, and at postbrace procedure (5-12 weeks). An enzyme-linked immunosorbent assay (ELISA) was used to quantitate the AHPF4 antibody titers. The functionality of the ELISA-positive samples was determined by a 14C-serotonin release assay (SRA).

RESULTS: In the ELISA test, 16 of 1073 samples (1.5%; 6 in clivarin and 10 in placebo groups) were positive for AHPF4 antibodies (mean optical density [OD] = 0.46 +/- 0.02). None of the ELISA-positive samples for AHPF4 antibodies could mediate platelet activation responses as determined by the SRA (0%-3% serotonin release, P >.10, n = 16). Through differential immunoglobulin subtype analysis of the samples positive for (cumulative) AHPF4 antibodies, we determined that their relative prevalence in plasma were as follows: IgM (mean OD = 0.71 +/- 0.13) > IgG (0.31 +/- 0.08) > IgA (0.14 +/- 0.02). Although there was no significant difference in the total antibody titers between clivarin and placebo groups, the antibody subtyping data showed conversion trends (ie, IgA [clivarin to placebo], IgG [placebo to clivarin], and IgM [clivarin to placebo]).

CONCLUSION: These observations indicate that even at reduced dosages, clivarin can shift the immunogenic up-regulation toward the IgG subpopulation; however, the IgG subtype is of a nonfunctional type of AHPF4 antibody and thus may not cause any HIT-related pathogenic responses.

LABORATORY/RADIOLOGIC/
OTHER TESTS
CHARACTERIZATION

NON-SPECIFIC ASSAYS

Global Heparin Assays for unfractionated activity APTT, thrombin clotting time, and activated clotting time

PLATELET COUNT

The Timing of a Positive Test Result for Heparin-Induced Thrombocytopenia Relative to the Platelet Count and Anticoagulant Therapy in 43 Consecutive Cases

Majed A. Refaai, Elizabeth M. Van Cott and Michael Laposata Am J Clin Pathol 2003;119:497-504

SPECIFIC ASSAYS

Clotting antifactor Xa assay

Based upon the heparin-accelerated inhibition of factor Xa
The initial phase of the reaction leads to the neutralized factor Xa proportional to the heparin concentration if antithrombin is present

Thus, the residual factor Xa is measured using a clotting technique

Chromogenic antifactor Xa assay
Similar to the above assay but the residual factor Xa is measured by using a synthetic factor Xa chromogenic peptide substrate

Assays for HIT antibodies
Have utilized assays utilizing PF4 complexed with heparin as a target for the antibodies

Platelet activation assays have serum incubated with test platelets and may measure secretion of radioactive serotonin, previously taken up by the platelets

Clinical significance of a borderline titer in a negative ELISA test for heparin-induced thrombocytopenia.

Refaai MA, Laposata M, Van Cott EM.

Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, USA.
Am J Clin Pathol 2003 Jan;119(1):61-5 Abstract quote
We studied the usefulness of repeated enzyme-linked immunosorbent assay testing for heparin-induced thrombocytopenia (HIT) when the initial test result was negative and sought to determine whether the titer of the initial negative result correlated with the likelihood of obtaining a positive test result in repeated testing.

We divided 150 patients who underwent HIT testing into 3 groups (50 patients each): (1) very low titer negative (0.0%-33.3% of the threshold for a positive test); (2) low titer negative (33.4%-66.6% of the threshold); and (3) high titer negative (66.7%-99.9% of the threshold). Among the patients who underwent a repeat test, 5% (1/20) of group 1 patients, 13% (4/32) of group 2 patients, and 43% (13/30) of group 3 patients tested positive in the repeat test (P = .0026).

Thus, nearly half of patients with initially negative HIT test results had positive results in the repeat test if the negative titer was 66.7% or more of the threshold. If laboratories report the HIT titer, rather than just negative or positive, the titer might help clinicians predict which patients have HIT despite a negative initial test, and the overall sensitivity for diagnosing HIT might be improved.

Development of a High-Pressure Liquid Chromatography Method for Diagnosis of Heparin-Induced Thrombocytopenia

Sandra Koch, MD,1 Job Harenberg, MD,1 Michael Ödel,2 Heinrich Schmidt-Gayk, MD,2 Stefan Walch, MD,2 and Uwe Budde, MD
Am J Clin Pathol 2002;117:927-934 Abstract quote

Owing to the disadvantage of radioactivity of the carbon 14 serotonin release assay and the time-consuming procedure of the enzyme immunoassay, we developed a high-pressure liquid chromatography (HPLC) method to detect serotonin released from donor platelets in the presence of heparins and serum samples from patients with heparin-induced thrombocytopenia (HIT).

Samples were analyzed from 60 healthy control subjects, 19 patients with HIT, and 20 patients without HIT after incubation with heparin, low-molecular-weight heparin (LMWH), and danaparoid. Serotonin release was measured from platelets, 300 × 103/µL, by HPLC.
Serotonin eluted as a single peak from the HPLC column. Serum samples from patients with HIT released 5.5 to 352.5 and 6.6 to 1,533.3 ng/mL of serotonin from platelets in the presence of 0.2 IU/mL of heparin and LMWH, respectively. In the presence of 0 IU/mL of heparin, LMWH, danaparoid, and control samples, less than 2.5 ng/mL of serotonin were released.

The HPLC method permits a rapid, sensitive, and quantitative determination of serotonin released from donor platelets for laboratory confirmation of HIT.

PROGNOSIS CHARACTERIZATION

ELISA TESTING

Clinical significance of a borderline titer in a negative ELISA test for heparin-induced thrombocytopenia.

Refaai MA, Laposata M, Van Cott EM.

Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, USA.

Am J Clin Pathol 2003 Jan;119(1):61-5 Abstract quote
We studied the usefulness of repeated enzyme-linked immunosorbent assay testing for heparin-induced thrombocytopenia (HIT) when the initial test result was negative and sought to determine whether the titer of the initial negative result correlated with the likelihood of obtaining a positive test result in repeated testing.

We divided 150 patients who underwent HIT testing into 3 groups (50 patients each): (1) very low titer negative (0.0%-33.3% of the threshold for a positive test); (2) low titer negative (33.4%-66.6% of the threshold); and (3) high titer negative (66.7%-99.9% of the threshold). Among the patients who underwent a repeat test, 5% (1/20) of group 1 patients, 13% (4/32) of group 2 patients, and 43% (13/30) of group 3 patients tested positive in the repeat test (P = .0026).

Thus, nearly half of patients with initially negative HIT test results had positive results in the repeat test if the negative titer was 66.7% or more of the threshold. If laboratories report the HIT titer, rather than just negative or positive, the titer might help clinicians predict which patients have HIT despite a negative initial test, and the overall sensitivity for diagnosing HIT might be improved.

Am Clin Lab 2001;20:35-38.
Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Twentieth Edition. WB Saunders. 2001.
Rosai J. Ackerman's Surgical Pathology. Eight Edition. Mosby 1996.
Sternberg S. Diagnostic Surgical Pathology. Third Edition. Lipincott Williams and Wilkins 1999.
Robbins Pathologic Basis of Disease. Sixth Edition. WB Saunders 1999.
DeMay RM. The Art and Science of Cytopathology. Volume 1 and 2. ASCP Press. 1996.
Weedon D. Weedon's Skin Pathology Second Edition. Churchill Livingstone. 2002
Fitzpatrick's Dermatology in General Medicine. 5th Edition. McGraw-Hill. 1999.
Weiss SW and Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors. Fourth Edition. Mosby 2001.

Commonly Used Terms

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Internet Links

Last Updated 5/10/2003

Disease Associations
Pathogenesis
Laboratory/Radiologic/Other Diagnostic Testing
Prognosis
Treatment
Commonly Used Terms
Internet Links

LABORATORY/RADIOLOGIC/ OTHER TESTS	CHARACTERIZATION
NON-SPECIFIC ASSAYS
Global Heparin Assays for unfractionated activity	APTT, thrombin clotting time, and activated clotting time
PLATELET COUNT
The Timing of a Positive Test Result for Heparin-Induced Thrombocytopenia Relative to the Platelet Count and Anticoagulant Therapy in 43 Consecutive Cases Majed A. Refaai, Elizabeth M. Van Cott and Michael Laposata	Am J Clin Pathol 2003;119:497-504
SPECIFIC ASSAYS
Clotting antifactor Xa assay	Based upon the heparin-accelerated inhibition of factor Xa The initial phase of the reaction leads to the neutralized factor Xa proportional to the heparin concentration if antithrombin is present Thus, the residual factor Xa is measured using a clotting technique
Chromogenic antifactor Xa assay	Similar to the above assay but the residual factor Xa is measured by using a synthetic factor Xa chromogenic peptide substrate
Assays for HIT antibodies	Have utilized assays utilizing PF4 complexed with heparin as a target for the antibodies Platelet activation assays have serum incubated with test platelets and may measure secretion of radioactive serotonin, previously taken up by the platelets
Clinical significance of a borderline titer in a negative ELISA test for heparin-induced thrombocytopenia. Refaai MA, Laposata M, Van Cott EM. Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, USA.	Am J Clin Pathol 2003 Jan;119(1):61-5 Abstract quote We studied the usefulness of repeated enzyme-linked immunosorbent assay testing for heparin-induced thrombocytopenia (HIT) when the initial test result was negative and sought to determine whether the titer of the initial negative result correlated with the likelihood of obtaining a positive test result in repeated testing. We divided 150 patients who underwent HIT testing into 3 groups (50 patients each): (1) very low titer negative (0.0%-33.3% of the threshold for a positive test); (2) low titer negative (33.4%-66.6% of the threshold); and (3) high titer negative (66.7%-99.9% of the threshold). Among the patients who underwent a repeat test, 5% (1/20) of group 1 patients, 13% (4/32) of group 2 patients, and 43% (13/30) of group 3 patients tested positive in the repeat test (P = .0026). Thus, nearly half of patients with initially negative HIT test results had positive results in the repeat test if the negative titer was 66.7% or more of the threshold. If laboratories report the HIT titer, rather than just negative or positive, the titer might help clinicians predict which patients have HIT despite a negative initial test, and the overall sensitivity for diagnosing HIT might be improved.
Development of a High-Pressure Liquid Chromatography Method for Diagnosis of Heparin-Induced Thrombocytopenia Sandra Koch, MD,1 Job Harenberg, MD,1 Michael Ödel,2 Heinrich Schmidt-Gayk, MD,2 Stefan Walch, MD,2 and Uwe Budde, MD	Am J Clin Pathol 2002;117:927-934 Abstract quote Owing to the disadvantage of radioactivity of the carbon 14 serotonin release assay and the time-consuming procedure of the enzyme immunoassay, we developed a high-pressure liquid chromatography (HPLC) method to detect serotonin released from donor platelets in the presence of heparins and serum samples from patients with heparin-induced thrombocytopenia (HIT). Samples were analyzed from 60 healthy control subjects, 19 patients with HIT, and 20 patients without HIT after incubation with heparin, low-molecular-weight heparin (LMWH), and danaparoid. Serotonin release was measured from platelets, 300 × 103/µL, by HPLC. Serotonin eluted as a single peak from the HPLC column. Serum samples from patients with HIT released 5.5 to 352.5 and 6.6 to 1,533.3 ng/mL of serotonin from platelets in the presence of 0.2 IU/mL of heparin and LMWH, respectively. In the presence of 0 IU/mL of heparin, LMWH, danaparoid, and control samples, less than 2.5 ng/mL of serotonin were released. The HPLC method permits a rapid, sensitive, and quantitative determination of serotonin released from donor platelets for laboratory confirmation of HIT.