- No FAQs found
- ABL Kinase Domain Mutation in CML, Cell-based
- ABO Group and Rh Type
- Acid-Fast Bacillus (AFB) Identification, Sequencing and Stain, Paraffin Block
- ADAMTS13 Activity with Reflex to ADAMTS13 Inhibitor
- Alcohol Metabolites, Quantitative, Urine
- Alpha-Globin Common Mutation Analysis
- Alpha-Globin Gene Deletion or Duplication
- Alpha-Globin Gene Sequencing
- Anti-Müllerian Hormone AssessR™
- Anti-PF4 and Serotonin Release Assay (SRA) for Diagnosing Heparin-induced Thrombocytopenia/Thrombosis (HIT/HITT)
- Antiphospholipid Antibodies
- ASCVD Risk Panel with Score
- Autoimmune Epilepsy Evaluation
- Autoimmune Diseases, Tests for
- B-cell and T-cell Clonality Assays by PCR
- B-Type Natriuretic Peptide (BNP)
- BCR-ABL1 Gene Rearrangement, Quantitative PCR
- Beta-Globin Complete
- BRCAvantage®, Ashkenazi Jewish Screen
- BRCAvantage®, Rearrangements
- BRCAvantage™, Comprehensive
- BRCAvantage™, Single Site
- CDH1 Sequencing and Deletion/Duplication
- Clostridium difficile Diagnostic Testing
- C1 Inhibitor, Protein and Functional Tests
- Calreticulin (CALR) Mutation Analysis
- Carbapenem Resistant Enterobacteriaceae Culture Screen
- Cardio IQ Lipoprotein Fractionation, Ion Mobility
- Cervical Cancer, TERC, FISH
- CFvantage® Cystic Fibrosis Expanded Screen
- Chlamydia trachomatis, TMA
- Chlamydia trachomatis/Neisseria gonorrhoeae RNA, TMA
- Chromosomal Microarray, POC, ClariSure®, Oligo-SNP
- Chromosomal Microarray, Postnatal, ClariSure® Oligo-SNP
- Chromosome Analysis and AFP with Reflex to AChE, Fetal Hgb, Amniotic Fluid
- Chromosome Analysis, Amniotic Fluid
- Chromosome Analysis, Blood
- Chromosome Analysis, Blood with Reflex to Postnatal, ClariSure® Oligo-SNP Array
- Chromosome Analysis, Chorionic Villus Sample
- Chromosome Analysis, High Resolution
- Chromosome Analysis, High Resolution with Reflex to Postnatal, ClariSure® Oligo-SNP Array
- Chromosome Analysis, Mosaicism
- Chromosome Analysis, Neonatal Blood
- Chromosome Analysis, Sister Chromatid Exchange
- Chromosome Analysis, Tissue
- Chromosome DEB Assay for Fanconi anemia
- Chronic Lymphocytic Leukemia (CLL) - Diagnostic and Prognostic Testing
- Culture, Fungus
- Culture, Urine, Routine
- Cystic Fibrosis Screen
- Cytomegalovirus (CMV) and Epstein Barr Virus (EBV) PCR
- D-Dimer, Quantitative
- Dementia, Secondary Causes
- Dengue Virus Testing
- Diabetes Risk Panel with Score and Cardio IQ® Diabetes Risk Panel with Score
- Drug Testing, General Toxicology (Blood, Urine, or Serum)
- Drug Toxicology Alcohol Metab, QN, Oral Fluid
- Drug Toxicology Monitoring, Oral Fluid Testing
- Factor V (Leiden) Mutation Analysis
- Familial Mediterranean Fever Mutation Analysis
- First Trimester Screen, hCG
- First Trimester Screen, Hyperglycosylated hCG (h-hCG)
- FISH, Angelman
- FISH, MET Amplification
- FISH, Myeloma, 17p-, rea 14q32 with Reflexes
- FISH, Prader-Willi
- FISH, Prenatal Screen
- No FAQs found
- HCV Genotyping
- Helicobacter pylori (H pylori) Antibody Discontinuation
- Heparin, Anti-Xa
- Hepatitis B Surface Antibody, Quantitative
- Hepatitis B Surface Antigen, Quantitative, Monitoring
- Hepatitis C Antibody with Reflex to HCV RNA, PCR with Reflex to Genotype
- Hepatitis C Viral RNA Genotype 1 NS5A Drug-resistance
- Hepatitis C Viral RNA Genotype 3 NS5A Drug Resistance
- Hepatitis C Viral RNA NS3 Drug Resistance
- Hepatitis C, RNA, Quantitative, PCR
- Hereditary Cancer Panels: MYvantageTM Hereditary Comprehensive Cancer Panel and GIvantageTM Hereditary Colorectal Cancer Panel
- Hereditary Hemochromatosis DNA Mutation Analysis
- Herpes Simplex Virus (HSV) Type-Specific IgG Antibodies
- Herpes Simplex Virus Type 2 (HSV-2) IgG Inhibition, ELISA
- HIV-1 Coreceptor Tropism, Proviral DNA
- HIV-1 Coreceptor Tropism, Ultradeep Sequencing
- HIV-1 Integrase Genotype
- HIV-1/2 Antigen and Antibodies, Fourth Generation, with Reflexes
- HPV mRNA E6/E7
- Influenza A and B Antigen, Immunoassay
- Influenza Type A and B Antibodies
- Insulin, Intact, LC/MS/MS
- Integrated Screen, Part 1
- Integrated Screen, Part 2
- Intrinsic Factor Blocking Antibody
- No FAQs found
- No FAQs found
- Maternal Serum AFP
- Melanoma, BRAF V600E and V600K Mutation Analysis, THxID®
- Metanephrines, Fractionated, Free, LC/MS/MS, Plasma
- Methylenetetrahydrofolate Reductase (MTHFR), DNA Analysis
- Microalbumin (Urinary Albumin Excretion)
- Pain Management and CYP2D6/CYP2C19
- Pain Management, Naltrexone, Quantitative, Urine
- Partial Thromboplastin Time, Activated (aPTT)
- Penta Screen
- PIK3CA Mutation Analysis
- Platelet Antibody Screen (Indirect)
- PNH with FLAER (High Sensitivity)
- Prothrombin Time with INR
- PTH, Intact and Calcium
- Streptococcus pneumoniae (Pneumococcal) Antibody Tests
- Saccharomyces cerevisiae Antibodies (ASCA) (IgG, IgA)
- Sequential Integrated Screen, Part 1
- Sequential Integrated Screen, Part 2
- Serum Integrated Screen, Part 1
- Serum Integrated Screen, Part 2
- Serum Pregnancy Tests
- Sickle Cell Screen
- Stepwise, Part 1
- Stepwise, Part 2
- SureSwab® Trichomonas vaginalis RNA, Qualitative TMA
- SureSwab®, Candidiasis, PCR
- TP53 Sequencing and Deletion/Duplication
- T4, Free
- Tamoxifen and Metabolites, LC-MS/MS
- Testosterone Testing
- Total Testosterone, LC/MS/MS
- Triple Screen
- No FAQs found
- No FAQs found
- No FAQs found
Anti-PF4 and Serotonin Release Assay (SRA) for Diagnosing Heparin-induced Thrombocytopenia/Thrombosis (HIT/HITT)Test code(s) 414, 14627, 14874, 16284, 16285
This is an outdated version of this FAQ. It was effective 09/17/2013 to 12/16/2016.
The current version is available here.
Question 1. Briefly, what are the anti-PF4 and SRA tests?
The anti-PF4 (ie, heparin-induced platelet antibody) test is an immunologic, enzyme-linked immunosorbent assay (ELISA) that detects IgG antibodies against the platelet factor 4 (PF4)/heparin complex. The assay uses heparin bound to protamine sulfate, and PF4 is supplemented through a platelet lysate.
The SRA test is a functional assay that measures heparin-dependent platelet activation. Patient serum is incubated with donor platelets containing radioactive 14C serotonin and different concentrations of heparin. Antibody present in the patient serum will bind and activate donor platelets, releasing radiolabeled serotonin from the platelet granules. A positive SRA is expected to show >20% release of the 14C serotonin when mixed with patient serum and low dose heparin. In addition, the high dose heparin must reduce the % release obtained with low dose heparin by at least 50% in order to demonstrate that the platelet activation is heparin dependent.
The SRA is considered a better predictor of thrombosis than the anti-PF4 assay.
Question 2. What is the OD result given in the anti-PF4 report and what is its significance?
OD is the optical density; it is an indication of antibody strength. Antibody present in the patient sample binds to the heparin-PF4 coated wells leading to a color-producing reaction. A higher antibody concentration leads to more color production and a higher OD reading. The positive cutoff is ³0.300 OD, and high titer antibodies may yield values up to 3.500 OD.
Question 3. What is the sensitivity of the anti-PF4 and SRA tests?
The anti-PF4 has a sensitivity of >95% and a specificity ranging from 50% to 89%. However, the higher the OD, the greater the probability is for clinical HIT/HITT. Similarly, the specificity increases when the OD is >1.000.1
The SRA has a sensitivity of 88% to 100% and specificity of 89% to 100% for HIT.2
The combined sensitivity of the anti-PF4 and SRA is about 99%.3
Question 4. What is meant by the expression “HIT/HITT in a clinical, pathologic diagnosis?”
HIT/HITT is a diagnosis that requires both clinical judgment and laboratory corroboration, ie, a clinical, pathologic diagnosis.
Thrombocytopenia in a hospitalized patient is very common and may be due to a vast number of causes (eg, consumption, dilutional effect from fluids or transfusions, drugs, sepsis, splenic sequestration, etc.). For patients exposed to heparin, HIT/HITT is an important consideration given the life-threatening potential. Applying a standardized and objective approach to the clinical assessment can help determine the pretest probability of HIT/HITT. The most common and widely accepted scoring model is called the 4T’s (http://asheducationbook.hematologylibrary.org/content/2003/1/497/F3.expansion.html). Another diagnostic challenge is that antibodies may be identified in patients with little or no clinical symptomatology.
Question 5. What is the significance of a positive anti-PF4 with a negative SRA?
A published algorithm that incorporates the clinical assessment suggests that patients with a moderate pretest probability and the laboratory results above are “indeterminate” for HIT/HITT.4
This scenario may represent a true antibody response, but one that lacks sufficient titer or avidity to activate platelets in vitro. The fact that the antibodies are measurable only with the anti-PF4 ELISA may also be indicative of the assay’s greater sensitivity. While this explanation has a good scientific foundation, some patients may still have clinically significant antibodies, thereby the rationale for the “indeterminate for HIT/HITT diagnosis.” False-positive anti-PF4 ELISA results have also been attributed to cross-reactivity to antiphospholipid antibodies.5
Question 6. Are the tests offered as a reflex or panel?
Quest Diagnostics offers both tests individually, together as a panel, and as a reflex (a positive heparin-induced platelet antibody reflexes to SRA).
A cautionary note when ordering the panel or reflex – if the result interface (connection from Quest Diagnostics to your computer system) does not accept partial results, the anti-PF4 ELISA results will not be received until the SRA is reported. Confirming the status of your interface with an IT specialist is recommended. Alternatively, physicians may view all results irrespective of this issue using the Care 360 product.
Question 7. Is the anti-PF4 ELISA IgG-specific?
Yes. Only IgG antibodies activate platelets, thus IgM and IgA classes are of minor clinical relevance.6, 7 Furthermore, the British Society for Haematology recommends that only the IgG isotype be tested.8
Question 8. Does Quest Diagnostics perform the heparin neutralization step in the anti-PF4 ELISA?
Quest Diagnostics does not perform the heparin neutralization procedure, sometimes referred to as a confirmatory step. It is our experience that virtually every positive sample is neutralized, and therefore we concluded that it offered no significant insight beyond that of the OD value. A retrospective study also reported that some patients with high ELISA OD values were misclassified by yielding a confirm negative result despite presence of clinical HIT/HITT.9
Question 9. How long does the patient have to be off heparin before testing?
Given the short half-life (30 minutes to 2 hours) of unfractionated heparin, waiting a minimum of 2 hours is suggested.
Question 10. For the SRA, what is the significance of an indeterminate or low positive result?
A SRA result is considered indeterminate when there is a >20% release of 14C serotonin in the patient serum/low dose heparin mixture and an absence of inhibition with high-dose unfractionated heparin (UFH). There may be other non-heparin–dependent antibodies present (ie, Class I HLA antibodies or platelet receptor specific antibodies) that may cause platelet activation.
A low-positive SRA is repeated using platelets from a different donor to demonstrate reproducibility. At this point, specific conclusions about a low-positive result cannot be made. It is recommended that the result be considered in the context of all the data at hand, specifically the clinical assessment and anti-PF4 result.
- Zwicker JI, et al. J Thromb Haemost. 2004;2:2133–2137.
- Ortel TL. Hematology Am Soc Hematol Educ Program. 2009;225-232.
- Linkins L, et al. Chest. 2012;141(suppl):e495S-e530S.
- Arepally GM, Ortel TL. N Engl J Med. 2006;355(8):809-817.
- Pausner R, et al. J Thromb Haemost. 2009;7(7):1070-1074.
- Warkentin TE. Hematology Am Soc Hematol Educ Program. 2011;2011:143-149.
- Greinacher A, et al. J Thromb Haemost. 2007;5:1666-1673.
- Watson H, et al. Br J Haemotol. 2012;159:528-540.
- Whitlatch NL, et al. Thromb Haemost. 2008;100:678–684.
Document: FAQS.06 Revision: 1