Targeted Gene Regions

Genes interrogated, including relevant transcripts and exons, are listed in alphabetical order.

Clinical Indications

This test is intended for the diagnosis of benign or malignant mesenchymal tumors (sarcomas and their benign mimics) as well as other solid tumors.

Interpretation

The results of this test are to be interpreted in the context of the histological, immunohistochemical, and clinical features of the neoplasm.

Methodology

This test relies on Anchored Multiplex PCR (AMP™) technology to generate scalable, target-enriched libraries for NGS from formalin-fixed, paraffin-embedded tissue sections.

In AMP, unidirectional gene-specific primers (GSPs) are used to enrich libraries for known and unknown mutations. Adapters that contain both molecular barcodes and sample indices enable quantitative multiplex data analysis, read de-duplication, and accurate variant calling. Libraries are sequenced on the Illumina MiSeq instrument, which employs “sequencing by synthesis;” a fluorescence, image-based, reversible-terminator technology to sequence targeted regions of the 59 genes included in the panel.

Sequencing data are analyzed for fusion variant detection using Archer® Analysis bioinformatics tools. Specimen quality control is monitored and recorded by in-house developed software (scripts). Raw sequencing data are de-multiplexed based on a unique index sequence using the Illumina bcl2fastq program. The fastq.gz files are de-duplicated according to the unique molecular barcode present and aligned to the human reference genome hg19. Part of the fusion calling and annotation is performed utilizing the Archer® Quiver™ Fusion Database.

Limitations

This test does not detect missense mutations, insertions, deletions, or copy number changes, and does not distinguish between variants that are inherited versus acquired.

References

1. Archer Dx, FusionPlex Anchored MultiPlex PCR (AMP) technology http://archerdx.com/fusionplex/ [Accessed: July 2018]

2. MiSeq System user Guide, Publication Number 15027617 Rev.0. Illumina, San Diego, CA. 9/2014.

3. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®), Soft Tissue Sarcoma, version 1.2019

4. Taylor BS, Barretina J, Maki RG, Antonescu CR, Singer S, Ladanyi M. Advances in sarcoma genomics and new therapeutic targets. Nat. Rev. Cancer. Jul 14 11(8), 541-57 (2011).

Targeted Gene Regions

Genes interrogated, including relevant transcripts and exons, are listed in alphabetical order.

Clinical Indications

This assay is intended for patients with known or suspected hematologic neoplasms including myelodysplastic syndromes, myeloproliferative neoplasms, acute myeloid leukemia, acute lymphoblastic leukemia, and selected mature lymphoid leukemias.

Interpretation

All variants are classified using Association for Molecular Pathology guidelines for interpretation of somatic variants in cancer.^[8]

Detailed interpretations are provided for each variant, and an overall interpretation of the entire mutational profile summarizes the case findings.

Reported variants include those of strong or potential clinical significance as well as variants of unclear clinical significance.

Known benign polymorphisms are not reported.

Methodology

Nucleic acid extracted from the specimen is subjected to nested multiplex PCR-based target enrichment.

Coding and non-coding regions of targeted genes are amplified and sequenced on an Illumina instrument (San Diego, CA) with paired-end, 150×2 cycle reads.

A customized bioinformatic analytical pipeline is used to map reads to the reference human genome (Genomic Build GRCh37/hg19).

Limitations

This test does not detect structural variants or copy number changes and does not distinguish between variants that are inherited versus acquired.

During internal validation, this test delivered an average of >500X coverage and >98% of targeted regions showed over 100X coverage. The test demonstrated 95.2% sensitivity and 99.9% specificity in identifying single nucleotide variants, small insertions and deletions (indels) (≤10bp) of >5% variant allele fraction (VAF). For the identification of large indels (>10bp) at >5% VAF the test demonstrated 87.5% sensitivity and 99.9% specificity.

Due to the limitations of next-generation sequencing technology, some large insertions may not be detected.

References

1. Papaemmanuil E, Gerstung M, Malcovati L, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013 Nov 21; 122(22):3616-27.

2. Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med. 2011 Jun 30;364(26):2496-506.

3. Döhner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid Leukemia. N Engl J Med. 2015 Sep 17;373(12):1136-52.

4. Nazha A, Zarzour A, Al-Issa K, et al. The complexity of interpreting genomic data in patients with acute myeloid leukemia. Blood Cancer J. 2016 Dec 16;6(12):e510.

5. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016 May 19;127(20):2391-405.

6. “NCCN Guidelines for Treatment of Cancer by Site,” National Comprehensive Cancer Network: https://www.nccn.org/professionals/physician_gls/default.aspx#site.

7. McClure RF, Ewalt MD, Crow J, et al. Clinical Significance of DNA Variants in Chronic Myeloid Neoplasms: A Report of the Association for Molecular Pathology. J Mol Diagn. 2018 Nov;20(6):717-737. DOI: 10.1016/j.jmoldx.2018.07.002. Epub 2018 Aug 20.

8. Li MM, Datto M, Duncavage EJ, et al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017 Jan;19(1):4-23.

Clinical Indications

For patients diagnosed with glioblastoma to determine if a methylated MGMT promoter is present, which is a favorable prognostic indicator for temozolomide treatment. Individuals without a methylated MGMT promoter do not have such a benefit.

MGMT “silence” is the most significant guide for the treatment of glioblastoma. This assay is to validate the methylation status of the MGMT gene.

Methodology

Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. Real-time sequence information is highly suitable for quantification of CpG methylation. We have validated pyrosequencing-based assay in detection of MGMT methylation in paraffin-embedded biopsy tissue specimens. With 10% average methylation as a cutoff, MGMT promoter methylation was detected in glioblastoma, but not detected in non-neoplastic brain tissue. The analytical sensitivity of the assay is 5% of target cells harboring MGMT methylation.

Diagnostic MGMT testing requires sufficient and optimally preserved tumor tissue. The biopsy should be at least 0.5 cm in size and necrosis should be less than 15%. Both frozen and paraffin-embedded tissue are suitable for the pyrosequencing-based MGMT methylation assay.

Interpretation

References

1. Van Meir EG, et al. “Exciting New Advances in NeuroOncology: The Avenue to a Cure for Malignant Glioma”. CA: A Cancer Journal for Clinicians. 60 (3):166–93. doi:10.3322/caac.20069. PMC 2888474. PMID 20445000. 2010.

2. Stupp R, et al. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. N Engl J Med. 352:987-996.

3. Hegi M, et al. MGMT Gene Silencing and Benefit from Temozolomide in Glioblastoma. N Engl J Med. 2005;352;10:997-1003.

4. Chamberlain Marc C, et al. “Early necrosis following concurrent Temodar and radiotherapy in patients with glioblastoma”. Journal of Neuro-Oncology. 82(1):81-3. doi:10.1007/s11060-006-9241-y. PMID 16944309.

5. Vlassenbroeck I, et al. Validation of Real-Time MethylationSpecific PCR to Determine O(6)-Methylguanine-DNA Methyl-transferase Gene Promoter Methylation in Glioma. J. Molecular Diagnostics. 2008;10;4:332-337.

6. Brandes A, et al. MGMT Promoter Methylation Status Can Predict the Incidence and Outcome of Pseudoprogression After Concomitant Radiochemotheraphy in Newly Diagnosed Glioblastoma Patients. J. Clinical Oncology. 2008; 26;13:2192-2197.

Clinical Indications

Quantitative detection of BCR/ABL p210 transcripts (e13a2 or e14a2) in patients with CML.

Methodology

Assay sensitivity was established using RNA extracted from K562 cultured cells suspended in normal buffy coat. This assay successfully detects p210 BCR / ABL transcripts in RNA extracted from one K562 cell in 100,000 peripheral blood leukocytes from a normal individual.

Interpretation

Results are reported as the percentage ratio of fusion gene transcripts to wild-type ABL transcripts (% BCRABL / ABL).

Results are also converted to the International Scale. A BCRABL / ABL value of 0.1% on the International Scale represents a major molecular response by consensus criteria.

References

1. Hughes T, Deininger M, Hochhaus A et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR–ABL transcripts and kinase domain mutations and for expressing results. Blood. 2006;108:28-37.

2. Druker BJ, Guilhot F, O’Brien SG et al. Five-year follow-up of patients receiving imatinib for chronic myelogenous leukemia. N Engl J Med. 2006;355:2408-2417.

3. White HE, Matejschuk P, Rigsby P, et al. Establishment of the first World Health Organization International Genetic Reference Panel for quantification of BCR–ABL mRNA. Blood 2010;116:e111-7.

Clinical Indications

In the pediatric population, many abnormal phenotypes are associated with chromosomal imbalances that can be identified using CMA to detect copy number change (CNC). Thus, whole-genome CMA has replaced chromosome and FISH studies to become the first-tier test for the evaluation of children with unexplained developmental disabilities, intellectual disabilities, dysmorphic features, congenital anomalies, and autism.

Based on numerous published studies, the yield of pathogenic or clinically significant CNC by CMA is approximately 15-20% in a pediatric population, compared with a yield of 3-5% by standard cytogenetic analysis in the same population. Variants of uncertain clinical significance (VOUS), or clinical significance unknown, are found in less than 10% and could play an important role in the clinical diagnosis. To a great extent, parental and family studies can be helpful in the clinical interpretation of these unknowns, as de novo occurrence of the CNC is more likely related to a pathogenic event.

CMA testing for CNC is recommended as a first-line test in the initial postnatal evaluation of individuals with the following:

• Multiple anomalies not specific to a well-delineated genetic syndrome
• Apparently nonsyndromic DD/ID
• Autism spectrum disorders

This is not a recommended test for adult patients with multiple miscarriages or pregnancy losses and no abnormal phenotype. Chromosome analysis is a better test to exclude the possibility of balanced rearrangements that cause pregnancy losses.

Appropriate follow-up is recommended in cases of chromosome imbalance identified by CMA and may include cytogenetic/FISH or other molecular studies of the patient or parents, and clinical genetics consultation. In addition to detecting CNC, the SNP microarray can also be used to diagnose suspected uniparental disomy (UPD) or imprinting disorder, the possible absence of heterozygosity to determine the degree of relatedness by identity-by-descent (autozygosity), and autosomal recessive condition risks. Additional testing may be necessary to confirm disorders associated with the absence of heterozygosity.

SNP testing may be used to identify:

• A chromosomal abnormality or micro-duplication/deletion syndrome with a normal karyotype
• The size of a duplication/deletion involved in an unbalanced translocation
• Triploidy
• Cryptic duplications/deletions in a phenotypically abnormal individual with an apparently balanced karyotype
• Uniparental disomy (UPD)
• Absence of heterozygosity to determine the degree of relatedness by identity-by-descent (autozygosity)

Methodology

The SNP test involves DNA extraction, restriction enzyme digestion, labeling, purification, hybridization, washing, array scanning, analysis, and interpretation. DNA extracted from the patient’s peripheral blood is digested, labeled, and hybridized to the microarray. Following hybridization, the microarray is scanned, and the signal intensities are collected then compared to a reference in order to determine copy number changes and absence of heterozygosity.

Whole-genome SNP microarray testing at Cleveland Clinic utilizes the GGXChip + SNP v1.0 platform, which contains non-polymorphic and polymorphic probes to detect both copy number changes (CNC) and allelic imbalances (SNP probes) within the same array. For CNC analysis, the array is comprised of 107,000 oligonucleotide 60mer probes spaced one probe every 40kb across the backbone of the array, and one probe every 10kb in targeted clinically-significant regions in the genome. For SNP analysis, the array features 60,000 SNP 60mer probes with an effective resolution down to 10Mb. In the backbone regions, the resolution for copy number detection is approximately 120kb and in the targeted regions it is approximately 30kb.

In our validation, the resolution for AOH was detected at approximately 1.5Mb, but the resolution is dependent on SNP probe coverage. For clinical purposes, AOH greater than 10Mb will be reported; however, in chromosomes associated with imprinting disorders, smaller changes will be evaluated further and may be reported. For a complete list of clinically-recognized regions of the genome and imprinted chromosomes please visit www.signaturegenomics.com.⁶

Interpretation

A written summary of the microarray test is provided in the Test Overview. Gains and losses are reported based on genomic content in line with ACMG guidelines for microarray interpretation. Copy number variations (CNV) or CNC devoid of relevant gene content or reported as common findings in the general population may not be reported.

A copy number change of uncertain clinical significance may be detected and will be reported per ACMG guidelines in one of three subcategories:³

• Uncertain clinical significance, likely pathogenic;
• Uncertain clinical significance, likely benign; or
• Uncertain clinical significance, no classification.

While most copy number changes observed by chromosomal microarray testing can readily be characterized as pathogenic or benign, there are limited data available to support the definitive classification of a subset into either of these categories. In these situations, a number of considerations are taken into account to help interpret results including the size and gene content of the imbalance, whether the change is a deletion or duplication, the inheritance pattern, and the clinical and/or developmental history of a transmitting parent. The continual discovery of novel copy number variations and published clinical reports means that the interpretation of any given copy number change may evolve with increased scientific understanding. The detection of excessive homozygosity may suggest the need for additional laboratory testing to confirm uniparental disomy or to test for mutations in genes associated with autosomal recessive disorders consistent with the patient’s clinical presentation that are present in regions of homozygosity.

Limitations

This platform is not optimized to detect low-level mosaicism and uniparental disomy of the heterodisomy type.

This platform will not detect balanced alterations (reciprocal translocations, Robertsonian translocations, inversions, and balanced insertions), point mutations, or imbalances of regions not represented on the microarray.

The failure to detect evidence of uniparental disomy does not exclude the clinical diagnosis of an imprinting associated disorder. UPD may be of the heterodisomy type, which is not detected by the array, and mechanisms other than UPD can cause the disorder.⁹

Similar to the copy number-only CMA, the CMA copy number + SNP cannot detect balanced rearrangements and may not be capable of detecting low-level mosaicism. It also does not
detect point mutations, small deletions or insertions below the resolution of the assay, or other types of mutations, such as epigenetic changes.

Finally, test results are sometimes of uncertain clinical significance, and studies of additional family members may be required to assist with interpretation.

References

1. Miller DT et al. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet. 2010;86:749-764.

2. Shaffer LG et al. Working Group of the Laboratory Quality Assurance Committee of the American College of Medical Genetics. Genetics in Medicine. 2007;9:654-662.

3. Kearney HM et al. American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants. Genetics in Medicine. 2011;13(7);680-52.

4. Manning M et al. Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities. Genetics in Medicine. 2010;12:742-745.

5. Stavropoulos J and Shago M. CCMG guidelines for genomic microarray testing. Approved by the board of directors, Canadian College of Medical Geneticists. 2010.

6. SignatureChip OS Disorders Tested. Clinically recognized regions of the genome assayed by the SignatureChip OS(v4)/Signature PrenatalChip OS(v4). Signature Genomics from PerkinElmer, Spokane, WA 99207 USA. (www.signaturegenomics.com).

7. Kearney HM et al. Diagnostic implications of excessive homozygosity detected by SNP-based microarrays: consanguinity, uniparental disomy, and recessive single-gene mutations. Clin Lab Med. 2011;31(4):595-613.

8. Papenhausen P et al. UPD detection using homozygosity profiling with a SNP genotyping microarray. Am J Med Genet A. 2011;155A(4):757-68. ACMG guidelines.

9. Tucker, T et al. Uniparental disomy: can SNP array data be used for diagnosis? Genetics in Medicine. 2012;14:753-756.

Clinical Indications

Laboratory testing with PCR is routinely used as an aid in the diagnosis of HSV infections for patients with meningitis and meningoencephalitis. Despite its prevalence, HSV remains an underdiagnosed disease, and early diagnosis and detection results in improved patient care.

This test should not be used to screen patients not exhibiting signs or symptoms supportive of an HSV infection.

Limitations

Although HSV PCR represents a significant advance in the diagnosis of HSV CNS disease, a negative result does not eliminate the possibility of HSV infection.

In some patients, HSV DNA may only be present in cerebrospinal fluid for three to four weeks after the initial presentation of symptoms, and DNA levels may be undetectable over a period of time. Although false-positive results may occur, they are rare. Repeat testing should be considered for patients with a low likelihood of HSV infection if a false-positive reaction is suspected.

Methodology

A polymerase chain reaction (PCR) test on spinal fluid can detect the genetic material (DNA) of the HSV virus. This assay also differentiates the HSV-1 and HSV-2 subtypes.

Nucleic acid amplification assays, such as PCR, are the best methods for testing spinal fluid for the presence of HSV when HSV meningitis or meningoencephalitis is suspected.

The PCR test detects viral DNA in specimens and is quicker, safer, and more sensitive and specific than earlier methods. Cleveland Clinic Laboratories uses rapid-cycle PCR, which is considered the gold standard for detection of HSV in CSF specimens.

References

1. Genital Herpes: Centers for Disease Control and Prevention. Retrieved 7/18/06. Available from http://www.cdc.gov/std/healthcomm/fact_sheets.htm.

2. Herpes simplex virus infections of the central nervous system. Kimberlin DW. Semin Pediatr Infect Dis. 2003. Apr;14(2):83-9.

Clinical Indications

This array can be used for diagnostic testing of hematological malignancies for myeloid and lymphoid disease. Common indications include, but are not limited to, acute and chronic leukemia, myelodysplastic syndrome (MDS), and myeloproliferative disorders. The assay can assess prognosis and monitor disease progression and response to therapy (loss/gain of genomic DNA; loss of heterozygosity (LOH)).

Methodology

Designed by the Cancer Cytogenomics Microarray Consortium (CCMC), the Agilent SurePrint G3 Cancer CGH + SNP 4x180K Microarray Kit is a cancer-specific microarray designed to target genomic regions associated with cancer. This array has the ability to detect genome-wide copy number variants (CNV) while simultaneously detecting LOH.

The array contains 20,000 cancer-associated 60-mer CGH probes covering more than 500 cancer-related genes (1 probe per 0.5-1Kb) in addition to 60,000 single nucleotide polymorphism (SNP) probes and backbone probes evenly distributed across the genome.

Interpretation

A written summary and interpretation of the microarray findings are provided in the Test Overview:

1. Normal.

2. Pathogenic.

3. Unclear clinical significance, likely pathogenic.

4. Unclear clinical significance, not otherwise specified.

5. Unclear clinical significance, likely benign.

Limitations

Microarray analysis cannot detect balanced rearrangements like translocations, inversions, and insertions that may be important for diagnosis and prognosis of hematological disorders.

In addition:
• Low-level mosaicism may not be detected.
• May not be appropriate for individuals with expected lower levels of malignant cells.
• Not recommended for minimal residual disease

The assay does not detect:
• Balanced chromosomal rearrangements.
• Base pair mutations and small deletions/duplications.
• Positional information for chromosome rearrangements.
• Low-level mosaicism (small clones).

Therefore, microarray findings should be interpreted together with other concurrent test results, such as flow cytometry, morphology, FISH, chromosome analysis, and other studies
as appropriate.

The chromosomal SNP Array for hematology may or may not detect low-level mosaicism; therefore, this test is not recommended for detecting minimal residual disease.

Clinical Indications

Patients with a family history of elevated Lp(a) and/or a family history of premature cardiovascular disease that is not explained by high LDL or low HDL.

Also used for heart disease patients with a normal lipid profile and mildly elevated cholesterol and/or low-density lipoprotein cholesterol (LDL-C), as it is believed that an elevated Lp(a) may worsen other heart and vascular disease processes.

An elevated Lp(a) may suggest the need for more aggressive treatment of LDL and other, more treatable risk factors down to acceptable levels.

Limitations

For the most accurate results, wait at least two months after a heart attack, surgery, stroke, infection, injury, or pregnancy to check blood level.

In general, lipids should not be measured right after excessive alcohol intake, with severely uncontrolled diabetes, or during rapid weight loss.

Methodology

Lipoprotein (a) in serum is quantitatively measured on the IMMAGE 800 Immunochemistry system by rate nephelometry. Antibody to human Lp(a) is brought into contact with Lp(a) in a sample.

The IMMAGE 800 Test measures the rate of increase in light scattered from particles suspended in solution as a result of complexes formed during an antigen-antibody reaction.

The increase in light scatter resulting from the antigen-antibody reaction is converted to a peak rate signal, which is a function of the sample Lp(a) concentration. Following calibration, the peak rate signal for a particular assay is automatically converted to concentration units by the analyzer.

References

1. IMMAGE 800 Immunochemistry System Operations Manual, Instructions #A11403, March 2004, Beckman Coulter Instruments, Inc., Fullerton, CA 92834-3100.

2. Beckman Coulter IMMAGE 800 Immunochemistry System, Chemistry Information Manual, Beckman Coulter Instructions #962248, March 2000, Beckman Coulter Instruments, Inc., Fullerton, CA 92834-3100.

3. Tietz, NW. Specimen Collection and Processing: Sources of Biological Variation. Textbook of Clinical Chemistry. WB Saunders, Philadelphia, PA. 1986;478-518.

4. National Committee for Clinical Laboratory Standards. Procedures for the Handling and Processing of Blood Specimens, Approved Guideline. NCCLS publication H18-A, Villanova, PA.1990.

5. Schreiner, JP, Heiss G, Tyroler HA, Morrisett JD, Davis CD, Smith R. Race and Gender Differences in the Association of Lp(a) with Carotid Artery Wall Thickness: The Atherosclerosis Risk in Communities (ARIC) Study. Arterioscler Thromb Vasc Biol. 1996;16:471-478.

6. National Committee for Clinical Laboratory Standards, How to Define, Determine, and Utilize Reference Intervals in the Clinical Laboratory: Proposed Guideline. NCCLS publication C28-P, Villanova, PA. 1990.

7. Tietz, NW. Clinical Guide to Laboratory Tests. 2nd ed, WB Saunders, Philadelphia, PA. 1990.

8. Henry JB, ed. Clinical Diagnosis and Management by Laboratory Methods. 17th edition. 1984.

9. Statland, Bernard E. Clinical Decision Levels for Lab Tests. Medical Economic Book, Oradel, New Jersey.1983.

10. Tietz, NW, ed. Fundamentals of Clinical Chemistry. 3rd Edition, WB Saunders, Philadelphia, PA.1987.

11. National Committee for Clinical Laboratory Standards. Method Comparison and Bias Estimation Using Patient Samples: Tentative Guideline. NCCLS publication EP9-T, Villanova, PA. 1993.

12. National Committee for Clinical Laboratory Standards. Precision Performance of Clinical Chemistry Devices: Tentative Guideline, 2nd Edition. NCCLS publication EP5-T2, Villanova, PA. 1992.

13. Wild SH, Fortmann SP, Marcovina Sm. A Prospective Case-Control Study of Lipoprotein(a) Levels and Apo(a) Size and Risk of Coronary Heart Disease in Stanford Five-City Project Participants. Arterioscler Thromb Vasc Biol. 1997;17:239-245.

Clinical Indications

Suspicion for HIT in patients exposed to UFH or low molecular-weight heparin (LMWH).

Interpretation

Testing for HIT encompasses both immunologic (Anti-PF4 IgG ELISA) & functional assays (heparin-induced platelet aggression [HIPA] and serotonin release [SRA]).

Limitations

A positive reaction obtained by the anti-PF4 IgG antibody screening ELISA does not confirm the diagnosis of HIT; a functional assay must be performed to confirm HIT.

The presence of immune complexes or other immunoglobulin aggregates in the patient sample may cause nonspecific binding in the ELISA and produce false positives.

The platelet functional assays are not as sensitive as the anti-PF4 IgG antibody ELISA, but have higher specificity and, therefore, should be used only as confirmatory tests.

Non-heparin dependent antibodies, such as anti-HLA, can cause platelet activation independent of heparin/PF4 immune complexes, rendering the functional assays indeterminate.

Methodology

Laboratory testing for HIT is a two-stage process. Testing begins with a high-sensitivity ELISA screen for anti-PF4 IgG antibodies.

The anti-PF4 IgG assay (GTI Diagnostics, Waukesha, WI) is performed by a solid-phase ELISA method. The antibody in the patient’s specimen will bind to microwells coated with PF4 complexed with polyvinyl sulfonate. After serial addition of alkaline phosphatase labeled anti-human IgG and the substrate p-nitrophenyl phosphate, the optical density (O.D.) of developed color is measured in a spectrophotometer. The anti-PF4 IgG assay is performed with the patient’s specimen with and without additional soluble heparin (final heparin concentration of 100 U/mL) as well as positive and negative controls.

If the O.D. is less than 0.4, it is considered a negative result.

If the anti-PF4 IgG result is positive with the O.D. greater than 0.4, % inhibition will be calculated as follows:

Patient specimen with heparin – Negative control
[1- (___________________________ )] x 100 = % Inhibition
Patient specimen without heparin – Negative control

The inhibition of a positive reaction by 50% or more (by calculation) in the presence of excess heparin is considered confirmatory for the presence of specific antibodies that react with PF4: heparin.

Heparin-induced platelet aggregation testing (HIPA) is performed by mixing donor platelet-rich plasma (providing the platelets) with patient plasma (providing the antibodies). Low-dose UFH (0.1-0.5 U/ml) is added. If antibodies are present, immune complexes form. The antibody/PF4/heparin complexes activate the platelet FcγRIIa and the platelets aggregate (> 30% aggregation indicates a positive confirmatory result). As an additional control for specificity, high-dose UFH (100 U/ml) is also tested. The excess heparin prevents the formation of immune complexes and should not induce platelet aggregation. If aggregation is seen with the high-dose heparin, the test is considered indeterminate due to non-specific cross reactivity. This test has a higher specificity for pathologic HIT than the screening assay, but the sensitivity is only 50-80%.

The serotonin release assay (SRA) (sent out) and other washed platelet assays offer more sensitive confirmatory testing (80-90%). These tests are performed in a similar manner to the HIPA, relying on donor platelets and patient plasma or serum. With the SRA, a > 20% release of serotonin in the presence of low-dose UHF is considered positive. If there is > 20% serotonin release in the presence of high-dose UFH, the test is considered indeterminate. If the screening test is positive with a high pretest probability, and the HIPA is negative, performing the SRA may be helpful.

In patients suspected of developing a HIT-like syndrome while on low molecular weight heparin (LMWH), confirmatory testing is performed with LMWH instead of UFH.

References

1. Kelton JG, Warkentin TE. Heparin-induced thrombocytopenia: a historical perspective. Blood. 2008 Oct 1;112(7):2607-16.

2. Xiao Z, Visentin GP, Dayananda KM, Neelamegham S. Immune complexes formed following the binding of antiplatelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion. Blood. 2008 Aug 15;112(4):1091-100.

3. Walenga JM, Jeske WP, Messmore HL. Mechanisms of venous and arterial thrombosis in heparin-induced thrombocytopenia. J Thromb Thrombolysis. 2000 Nov;10 Suppl 1:13-20.

4. Warkentin TE, Chong BH, Greinacher A. Heparin-induced thrombocytopenia: towards consensus. Thromb Haemost. 1998;79:1-7.

5. Warkentin TE, Heddle NM. Laboratory Diagnosis of Immune Heparin-Induced Thrombocytopenia. Current Hematology Reports. 2003;2:148-157.

Clinical Indications

A request for Yersinia culture from stool should be made if a patient is suspected of having gastroenteritis associated with Yersinia, for example, when there has been exposure to undercooked pork chitterlings, or when routine stool cultures are negative for Salmonella and Campylobacter. In addition, patients presenting with diarrhea and associated symptoms of appendicitis and/or septicemia should be considered for a Yersinia culture request.

This test should be done as an adjunct to the routine culture and not in place of it.

Children, the elderly, and immunocompromised patients are the more likely patients at risk for Yersinia gastroenteritis.

Interpretation

Yersinia enterocolitica can grow on most routine laboratory media, including Maconkey’s agar, but grows more slowly than other members of the normal GI flora Enterobacteriaceae. Cefsulodin-irgasan-novobiocin (CIN) agar will be planted in addition to a Maconkey’s agar when a request is made for isolation of Y. enterocolitica.

Most results should be available within 48-72 hours after collection and processing of the stool samples.

Growth and identification, specifically of Y. enterocolitica, will be considered a positive result; no growth as a negative result.

Serotyping of the Y. enterocolitica is not performed routinely in clinical laboratories and is not widely available in reference laboratories.

Most cases of Yersinia gastroenteritis are self-limited and do not require treatment; however, if there is concomitant systemic disease and/or if the patient is immunocompromised, susceptibility testing can be performed.

Limitations

Overgrowth with normal flora GI bacteria and/or other GI pathogens may limit the growth of Y. enterocolitica.

Culture of stool samples after beginning treatment may limit detection.

Methodology

Y. enterocolitica is a gram-negative bacterium that can be isolated in culture from stool specimens.

Stool should be submitted to the laboratory within two hours of collection or transported in Cary Blair transport media and refrigerated if there will be delays. The order should be placed for a Yersinia culture when the stool is submitted.

Cultures are performed seven days per week.

References

1. http://www.cdc.gov/foodborneburden/2011-foodborneestimates.html. CDC website.

2. Long C, Jones TF, Vugia DJ, Scheftel J, Strockbine N, Ryan P, Shiferaw B, Tauxe RV, Gould LH. Yersinia pseudotuberculosis and Y. enterocolitica infections, FoodNet, 1996-2007. Emerg Infect Dis. 2010;16:566-7.

3. Wanger, A. Chapter 44. Yersinia. In Murray PR et al. (eds). Manual of Clinical Microbiology 9th ed. ASM Press: Washington, 2007.

4. Fosse J, Seegers H, Magras C. Prevalence and risk factors for bacterial foodborne zoonotic hazards in slaughter pigs: a review. Zoonoses Public Health. 2009;56:429-54.

5. Centers for Disease Control and Prevention. Yersinia enterocolitica gastroenteritis among infants exposed to chitterlings—Chicago, Illinois, 2002. MMWR Morb Mortal Wkly Rep. 2003;52:956-8.

6. De Berardis B, Torresini G, Brucchi M, Marinelli S, Mattucci S, Schietroma M, Vecchio L, Carlei F. Yersinia enterocolitica intestinal infection with ileum perforation: report of a clinical observation. Ata Biomed. 2004;75:77-81.