Test Catalog

Test Id : HITIG

Heparin-PF4 IgG Antibody, Serum

Useful For
Suggests clinical disorders or settings where the test may be helpful

Detection of IgG antibodies directed against heparin/platelet factor 4 complexes that are implicated in the pathogenesis of immune-mediated type II heparin-induced thrombocytopenia, spontaneous heparin platelet-factor 4 IgG antibody, and thrombocytopenia and thrombosis occurring after severe acute respiratory syndrome coronavirus 2 adenovirus vector vaccine

Method Name
A short description of the method used to perform the test

Enzyme-Linked Immunosorbent Assay (ELISA)

NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.

Yes

Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test

Heparin-PF4 IgG Ab (HIT), S

Aliases
Lists additional common names for a test, as an aid in searching

HAT ELISA

Heparin Induced Antibody

Heparin-Dependent Antibody

HIT ELISA

PF4 ELISA

Specimen Type
Describes the specimen type validated for testing

Serum Red

Specimen Required
Defines the optimal specimen required to perform the test and the preferred volume to complete testing

Patient Preparation: Fasting is preferred but not required

Collection Container/Tube: Red top

Submission Container/Tube: Plastic vial

Specimen Volume: 1 mL

 

Specimen Stability Information: Frozen (preferred) 2 years/Refrigerate 7 days

Forms

Specimen Minimum Volume
Defines the amount of sample necessary to provide a clinically relevant result as determined by the Testing Laboratory

0.5 mL

Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected

Gross hemolysis Reject
Gross lipemia Reject

Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included

Specimen Type Temperature Time Special Container
Serum Red Frozen (preferred)
Refrigerated 7 days

Useful For
Suggests clinical disorders or settings where the test may be helpful

Detection of IgG antibodies directed against heparin/platelet factor 4 complexes that are implicated in the pathogenesis of immune-mediated type II heparin-induced thrombocytopenia, spontaneous heparin platelet-factor 4 IgG antibody, and thrombocytopenia and thrombosis occurring after severe acute respiratory syndrome coronavirus 2 adenovirus vector vaccine

Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

There are established and emerging disorders that are collectively termed thrombocytopenia and thrombosis syndromes; the most commonly recognized is heparin induced thrombocytopenia (HIT). Newer associations have also been recognized including adenovirus vector-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine induced thrombocytopenia and HIT-like syndromes that occur in the absence of exposure to heparin (currently termed spontaneous or autoimmune HIT). In this situation, the heparin platelet-factor 4 (PF4) IgG antibody typically develops after surgery or infection.

 

HIT is a serious immune-mediated syndrome (ie, type II HIT or immune HIT) that occurs in 1% to 5% of patients treated with unfractionated heparin and at a lower frequency in patients treated with low-molecular weight heparin.

 

The 4Ts score is a validated scoring system to estimate the pretest clinical probability of HIT. Scores are assigned to the degree and timing of onset of thrombocytopenia, and the presence or absence of thrombosis (arterial or venous) in the absence of other potential explanations for the thrombocytopenia. In HIT, typical onset of thrombocytopenia is between days 5 and 10 of heparin therapy, but thrombocytopenia can arise earlier (less than 5 days after heparin exposure, ie, rapid onset of HIT) or later (greater than 4 weeks after heparin exposure, ie, delayed onset of HIT). The platelet count typically decreases by 40% to 50% from baseline or the postoperative peak (in surgical patients), even though the absolute count may remain normal, and thrombocytopenia resolves within 7 to 14 days of cessation of heparin therapy (unless there is another coexisting cause of thrombocytopenia). Development or progression of (venous or arterial) thrombosis is termed heparin-induced thrombocytopenia with thrombosis syndrome and can occur in 30% to 50% of patients, rarely even following discontinuation of heparin therapy.

 

Other Syndromes of Thrombocytopenia and Thrombosis:

There are an increasing number of reports of patients who develop thrombocytopenia and thrombosis after surgery, particularly after orthopedic surgery and after selected infections. The clinical course and laboratory characteristics of this group of patients are similar to the classical HIT occurring with heparin exposure except perhaps development of high titer antibodies against heparin/PF4 complexes. An emerging recognition is the development of thrombocytopenia and thrombosis occurring 3 to 4 weeks after adenovirus vector SARS-CoV-2 exposure. The clinical course is also similar to immune HIT.

 

Laboratory Characteristics of HIT:

HIT is caused, in at least 90% of cases, by antibodies to antigen complexes of heparinoid (heparin or similar glycosaminoglycans) and PF4. PF4 is a platelet-specific heparin-binding protein that is abundant in platelet alpha granules from which it is secreted following platelet stimulation. A reservoir of PF4 normally accumulates on vascular endothelium. Following heparin administration, immunogenic complexes of PF4 and heparin can provide an antigenic stimulus for antibody development in some patients. Antibodies bound to platelets that display complexes of PF4/heparin antigen can activate platelets via interaction of the Fc immunoglobulin tail of the IgG antibody with platelet Fc gamma IIa receptors, leading to perpetuation of the pathologic process that can cause platelet-rich thrombi in some cases.

 

Functional assays for HIT antibody detection rely on antibody-mediated heparin-dependent platelet activation. The endpoint of platelet activation may be platelet aggregation, or platelet secretion of serotonin or adenosine triphosphate using patient serum or plasma supplemented with heparin and platelets from carefully normal selected donors. The sensitivity of functional assays for HIT ranges from 50% to 60% for heparin-dependent platelet aggregation assays, to 70% to 80% for serotonin release assays. The specificity of positive functional tests for HIT diagnosis is believed to be high (> or =90%). However, because of their complexity, functional tests for detecting HIT antibodies are not widely available.

 

Enzyme-linked immunosorbent assays (ELISA) are available to detect HIT type 2 (HIT-II) antibodies and are based on the detection of human IgG antibodies that react with solid phase antigen complexes of heparinoid and human PF4 (H/PF4) complexes. The ELISA for H/PF4 antibodies is very sensitive for antibody detection but relatively nonspecific for clinical HIT diagnosis.

 

Routine screening of all patients prior to, during, or following heparin use is currently not recommended. A positive H/PF4 ELISA result has relatively low and uncertain predictive value for the development of clinical HIT-II.

 

Clinical Picture of Immune HIT or HIT-like Syndromes:

HIT in patients not previously exposed to heparin:

1) Decrease in platelet count (thrombocytopenia) of 50% from baseline or postoperative peak.

2) Onset of thrombocytopenia beginning approximately 5 to 10 days after initiation of heparin. This may or may not be associated with new or progressive thrombosis in patients treated with heparin.

 

Patients previously exposed to heparin (especially within the preceding 100 days): in addition to the above findings, the onset of thrombocytopenia could occur within 24 to 48 hours after reexposure to heparin.

 

Spontaneous or Autoimmune HIT:

Patients typically present a week to 10 days after surgery or viral infections with symptoms of thrombosis (venous thromboembolism) or abdominal pain (suggesting adrenal infarction) and thrombocytopenia.

 

Vaccine Induced Thrombocytopenia and Thrombosis:

Patients typically present 4 days to 4 weeks after receiving the vaccine. Symptoms may include new onset of severe headache (suggesting cerebral venous sinus thrombosis), abdominal pain (suggesting mesenteric/portal vein thrombosis), or venous/arterial thromboembolism.

Reference Values
Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

HIT ELISA:

<0.400

 

HIT Interpretation:

Negative

Interpretation
Provides information to assist in interpretation of the test results

Results are reported as:

1) Heparin-induced thrombocytopenia (HIT) enzyme-linked immunosorbent assay (ELISA) optical density (OD)

2) Heparin inhibition (%)

3) Interpretation.

 

Typical patterns of results and interpretations are depicted in the following table. Interpretive comments will also accompany test reports, when indicated.

 

 

HIT ELISA OD

Heparin inhibition

Interpretation

Normal range

<0.400

Not done

Negative

Positive

> or =0.400

> or =50%

Positive

Equivocal

> or =0.400

<50%

Equivocal

 

A negative result of testing for human platelet factor 4 (H/PF4) antibodies has about a 90% negative predictive value for exclusion of clinical type II HIT (HIT-II).

 

As up to 10% of patients with clinical HIT may have a negative H/PF4 antibody ELISA result, a negative H/PF4 antibody ELISA result does not exclude the diagnosis of HIT when clinical suspicion remains high. A functional assay for HIT antibodies (eg, heparin-dependent platelet aggregation or serotonin release assay) may be helpful in these circumstances. Call 800-533-1710 for ordering information.

 

A positive result is indicative of the presence of H/PF4 complex antibodies. However, this test's specificity is as low as 20% to 50% for clinical diagnosis of HIT, depending on the patient population studied. For example, up to 50% of surgical patients and up to 20% of medical patients treated with heparin may develop H/PF4 antibodies as measured by ELISA, and only a small proportion (1%-5%) develop clinical HIT. Accordingly, this test does not confirm the diagnosis of HIT-II. The diagnosis must be made in conjunction with clinical findings, including evaluation for other potential causes of thrombocytopenia.

 

The presence of H/PF4 antibodies likely increases the risk of clinical HIT, with risk probably partly dependent on associated medical and surgical conditions, but currently there is little data about relative risk of HIT in various populations with positive tests for H/PF4 antibodies.

Cautions
Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

Heparin-induced thrombocytopenia is a clinical diagnosis that is complemented by laboratory testing for antibodies to human platelet factor 4 (H/PF4) complexes and/or functional assays like the serotonin release assay. Assay results provide information on the presence or absence of H/PF4 antibodies, which are implicated in the pathogenesis of type II heparin-induced thrombocytopenia (HIT-II) with or without thrombosis. However, results of the H/PF4 antibody assay must be interpreted in conjunction with clinical findings (4T score) and other pertinent tests to evaluate other causes of thrombocytopenia (eg, sepsis, intravascular coagulation and fibrinolysis, thrombotic thrombocytopenic purpura, post-transfusion purpura, malignancy, drug-induced thrombocytopenia, autoimmune thrombocytopenia) or to confirm the findings of this assay.

 

Some low-titer, low-avidity antibodies and some antibodies that recognize sites on H/PF4 complex may not be detected using this assay.

 

Some patients may have naturally occurring antibodies to heparin PF4 (no evidence of heparin dependence) of no known significance with respect to pathogenesis of HIT-II.

Supportive Data

The IgG human platelet factor 4 (H/PF4) enzyme-linked immunosorbent assay (ELISA) was compared with both the IgGAM H/PF4 ELISA (GTI Immucor) and the heparin-dependent platelet serotonin release assay (SRA) (Quest Diagnostics and Wisconsin Blood Center) in 208 patients. Assuming the SRA as the gold standard, the data were analyzed to determine the sensitivity of the ELISA assays for a positive SRA. Of the 208 patients tested, 49 had a positive SRA. With the IgGAM H/PF4 ELISA, 47/49 were positive (sensitivity 96%); with the IgG H/PF4 ELISA, 45 were positive (sensitivity 92%). Of those that tested negative with the SRA (n=159), 67 (42%) tested positive with the IgGAM H/PF4 ELISA, 37 (23%) tested positive with the IgG H/PF4 ELISA. (Mayo validation data)

 

In order to determine possible cross-reactivity between the target antigen and antibodies other than heparin-associated antibodies, 68 samples containing a variety of antibodies that included known antibodies to platelet alloantigens, platelet autoantibodies, antibodies to HLA class I and antirheumatoid factor were tested in this assay and none were found to cross react with the target antigen immobilized in the microwells.

Clinical Reference
Recommendations for in-depth reading of a clinical nature

1. Husseinzadeh HD, Gimotty PA, Pishko AM, Buckley M, Warkentin TE, Cuker A: Diagnostic accuracy of IgG-specific versus polyspecific enzyme-linked immunoassays in heparin-induced thrombocytopenia: a systematic review and meta-analysis. J Thromb Haemost. 2017 Jun;15(6):1203–1212. doi: 10.1111/jth.13692

2.Warkentin TE, Greinacher A, eds: Heparin Induced Thrombocytopenia. Marcel Dekker; 2000:400

3.Warkentin TE, Sheppard JI, Moore JC, Sigouin CS, Kelton JG. Quantitative interpretation of optical density measurements using PF4-dependent enzyme-immunoassays. J Thromb Haemost. 2008 Aug;6(8):1304-1312. doi: 10.1111/j.1538-7836.2008.03025.x

4.Trossaert M, Gaillard A, Commin PL, Amiral J, Vissac AM, Fressinaud E: High incidence of anti-heparin/platelet factor 4 antibodies after cardiopulmonary bypass surgery. Br J Haematol. 1998 Jun;101(4):653-655. doi: 10.1046/j.1365-2141.1998.00750.x

Method Description
Describes how the test is performed and provides a method-specific reference

Enzyme-linked immunosorbent assay (ELISA) testing is performed on the Janus G3 integrated liquid handling system and BioTek plate reader using the Immucor GTI Diagnostics, Inc PF4 IgG assay test kit.

 

Patient serum is incubated in microwells precoated with an antigen complex of platelet factor 4 (PF4) and polyanionic heparinoid substitute (polyvinyl sulfonate; PVS). If antibodies to this complex are present in the patient serum, they will bind to the PF4:PVS antigen complex; all other antibodies are washed away. An anti-IgG reagent (conjugate) is added to the wells, incubated, and washed to remove any unbound conjugate. p-Nitrophenylphosphate is added and incubated; color is generated when bound conjugate cleaves a chromogenic phosphate substrate. The reaction is stopped and the absorbance measured using a spectrophotometer at 405 nm.

 

Addition of excess heparin (100 U/mL) to patient serum prior to testing inhibits the reaction between heparin-dependent antibodies and the PF4:PVS complex, and decreases absorbance (reactivity). This procedure is used to confirm a positive screening result is caused by heparin-dependent antibodies. Results are calculated as the percent heparin inhibition of the reactivity of the antibody.(Collins JL, Aster RH, Moghaddam M, et al: Diagnostic testing for heparin-induced thrombocytopenia [HIT]: An enhanced platelet factor 4 complex enzyme linked immunosorbent assay [PF4 ELISA]. Blood 1997 [Suppl 1] 90:461a; package insert: PF4 IgG assay. Immucor GTI Diagnostics, Inc; Rev D, 05/2015)

PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information

No

Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.

Monday through Sunday

Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

1 to 3 days

Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

7 days

Performing Laboratory Location
Indicates the location of the laboratory that performs the test

Rochester

Fees
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Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.

This test has been modified from the manufacturer's instructions. Its performance characteristics were determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information
Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.

CPT codes are provided by the performing laboratory.

86022

LOINC® Information
Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the order and results codes of this test. LOINC values are provided by the performing laboratory.

Test Id Test Order Name Order LOINC Value
HITIG Heparin-PF4 IgG Ab (HIT), S In Process
Result Id Test Result Name Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
21468 Heparin Inhibition 73817-9
21469 HIT Interpretation 73819-5
21470 HIT Comment 73816-1
46915 HIT ELISA 73818-7

Test Setup Resources

Setup Files
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Excel | Pdf

Sample Reports
Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports
International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports

Test Update Resources

Change Type Effective Date
Test Status - Test Resumed 2021-11-19
Test Status - Test Down 2021-11-02