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Solid Tumor Gene Fusion Next-Generation Sequencing Panel

Technical Brief:

Solid Tumor Gene Fusion Next-Generation Sequencing Panel


Test Name

Solid Tumor Gene Fusion Next-Generation Sequencing Panel (SRCNGS) – 59 Genes

Subpanels

Head & Neck Gene Fusion (HDNK) – 30 Genes

NTRK Gene Fusion (NTRK) – 3 Genes

CPT Codes

81445
88381

Methodology

Next-Generation Sequencing

Turnaround Time

14 days

Specimen Requirements

Type:
Formalin-fixed, paraffin-embedded (FFPE) tissue

Volume:
Ten (10) unstained, 4 μM sections of FFPE on charged, unbaked slides

Alternative:
One (1) H&E stained slide with best tumor area circled by a pathologist (minimum of 20% tumor content for best results)

Transport Temperature:
Ambient

Stability

Ambient:
Transport & store at this temperature

Refrigerated:
Unacceptable

Frozen:
Unacceptable

Background Information

Benign and malignant mesenchymal tumors (sarcomas and their mimics) are difficult to diagnose with many benign and malignant entities that differ in their clinical behavior and response to therapy. Many of these tumors harbor gene fusions that are crucial to establishing a definitive diagnosis.

The CC SIGN® Solid Tumor Gene Fusion Next-Generation Sequencing (NGS) Panel is a custom-designed, 59-gene panel, high complexity laboratory-developed test (LDT) designed for targeted sequencing of benign and malignant solid and soft tissue neoplasms. This assay identifies fusion transcripts in targeted regions of RNA from total nucleic acid (TNA) isolated from formalin-fixed, paraffin-embedded (FFPE) tissue specimens.

The test will identify the vast majority of known fusions in benign and malignant mesenchymal tumors, but also has the ability to identify a limitless number of as-of-yet undiscovered gene fusions. This technology only “primes” from one partner of the gene fusion, allowing for the discovery of new gene fusion partners.

Panel Highlights & Available Subpanels

  • Comprehensive detection of gene fusions across 59 targeted genes aids in determining diagnosis, prognosis, and therapeutic options.
  • FFPE tissue removes the need to send fresh or frozen specimens.

Solid Tumor Gene Fusion NGS Panel (SRCNGS) – 59 Genes

  • ALK
  • BCOR
  • BRAF
  • CAMTA1
  • CCNB3
  • CIC
  • CRTC1
  • CSF1
  • EPC1
  • ETV6
  • EWSR1
  • FOS
  • FOSB
  • FOXO1
  • FUS
  • GLI1
  • HMGA2
  • JAZF1
  • MAML2
  • MEAF6
  • MKL2
  • MYB
  • NCOA1
  • NCOA2
  • NCOA3
  • NOTCH1
  • NOTCH2
  • NOTCH3
  • NR4A3
  • NTRK1
  • NTRK2
  • NTRK3
  • NUTM1
  • PAX3
  • PAX7
  • PDGFB
  • PDGFD
  • PGR
  • PHF1
  • PLAG1
  • PRDM10
  • PRKD1
  • RAF1
  • RELA
  • RET
  • ROS1
  • SRF
  • SS18
  • STAT6
  • TAF15
  • TCF12
  • TFE3
  • TFEB
  • TFG
  • TRIM11
  • USP6
  • WWTR1
  • YAP1
  • YWHAE

Subpanel: Head & Neck Gene Fusion (HDNK) – 30 Genes

  • ALK
  • BRAF
  • CAMTA1
  • CRTC1
  • ETV6
  • EWSR1
  • FOS
  • FOSB
  • FOXO1
  • FUS
  • GLI1
  • HMGA2
  • MAML2
  • MKL2
  • MYB
  • NCOA1
  • NR4A3
  • NTRK1
  • NTRK2
  • NTRK3
  • NUTM1
  • PAX3
  • PAX7
  • PLAG1
  • PRKD1
  • RET
  • SS18
  • STAT6
  • TFE3
  • YAP1

Subpanel: NTRK Gene Fusion (NTRK) – 3 Genes

  • NTRK1
  • NTRK2
  • NTRK3

Targeted Gene Regions

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

A

Gene, Transcript, Exons

ABL1
NM_004304
2, 4, 6, 10, 16-23, 25, 26

B

Gene, Transcript, Exons

BCOR
NM_001123385
Exons 3-8, 12, 14, 15

BCOR
NM_017745
Exon 8

BRAF
NM_004333
Exons 1-5, 7-16, 18

C

Gene, Transcript, Exons

CAMTA1
NM_015215
Exons 3, 8-10

CCNB3
NM_033031
Exons 2-6

CIC
NM_015125
Exons 12, 17-20

CRTC1
NM_015321
Exons 1-4

CSF1
NM_000757
Exons 5-8

CSF1
NM_172212
Exon 9

E

Gene, Transcript, Exons

EPC1
NM_025209
Exons 9-11

ETV6
NM_001987
Exons 1-7

EWSR1
NM_005243
Exons 4-14

F

Gene, Transcript, Exons

FOS
NM_005252
Exon 4

FOSB
NM_006732
Exons 1, 2

FOXO1
NM_002015
Exons 1-3

FUS
NM_004960
Exons 3-11, 13, 14

G

Gene, Transcript, Exons

GLI1
NM_005269
Exons 4-7

H

Gene, Transcript, Exons

HMGA2
NM_003483
Exons 1-5

J

Gene, Transcript, Exons

JAZF1
NM_175061
Exons 2-4

M

Gene, Transcript, Exons

MAML2
NM_032427
Exons 2, 3

MEAF6
NM_001270875
Exons 4, 5

MKL2
NM_014048
Exons 11-13

MYB
NM_001130173
Exons 7-9, 11-16

N

Gene, Transcript, Exons

NCOA1
NM_147223
Exons 12-15

NCOA2
NM_006540
Exons 11-16

NCOA3
NM_006534
Exons 2, 13-16

NCOA3
NM_181659
Exon 20

NOTCH1
NM_017617
Exons 2, 4, 24-31

NOTCH2
NM_024408
Exons 5-7, 24-29

NOTCH3
NM_000435
Exons 25-30

NR4A3
NM_006981
Exon 2

NR4A3
NM_173200
Exons 3, 4

NTRK1*
NM_002529
Exons 2, 4, 6, 8, 10-14

NTRK2*
NM_006180
Exons 5, 7, 9, 11-18

NTRK3*
NM_001007156
Exon 15

NTRK3*
NM_002530
Exons 4, 7, 10, 12-16

NUTM1
NM_175741
Exons 2-4, 6

*A specimen positive for a fusion in one of these genes makes the patient a candidate for larotrectinib treatment.
Standalone NTRK testing is also available via the CC-SIGN® NTRK Gene Fusion NGS Panel.

P

Gene, Transcript, Exons

PAX3
NM_181459
Exons 6-8

PAX7
NM_002584
Exons 6-8

PDGFB
NM_002608
Exons 2, 3

PDGFD
NM_025208
Exons 5-7

PGR
NM_000926
Exons 1-3

PHF1
NM_024165
Exons 1, 2, 10-12

PLAG1
NM_002655
Exons 1-4

PRDM10
NM_199437
Exons 12, 13

PRKD1
NM_002742
Exons 10-13

R

Gene, Transcript, Exons

RAF1
NM_002880
Exons 4-12

RELA
NM_021975
Exons 3 , 4, 11

RET
NM_020630
Exons 2, 4, 6, 11, 15, 16

RET
NM_020975
Exons 8-14

ROS1
NM_002944
2, 4, 7, 31-38

S

Gene, Transcript, Exons

SRF
NM_003131
Exons 2-4

SS18
NM_001007559
Exons 2-6, 8-11

SS18
NM_005637
Exons 2, 3

STAT6
NM_001178078
Exons 1-7, 15-20

T

Gene, Transcript, Exons

TAF15
NM_139215
Exons 5-7

TCF12
NM_207036
Exons 4-6

TFE3
NM_006521
Exons 2-8

TFEB
NM_007162
Exons 1-4, 9

TFG
NM_006070
Exons 3-7

TRIM11
NM_145214
Exons 2, 3

U

Gene, Transcript, Exons

USP6
NM_004505
Exons 1-3

W

Gene, Transcript, Exons

WWTR1
NM_015472
Exons 3,

Y

Gene, Transcript, Exons

YAP1
NM_001130145
Exons 1-9

YWHAE
NM_006761
Exon 5

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).

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Hematologic Neoplasm Next Generation Sequencing Panel

Technical Brief:

Hematologic Neoplasm Next-Generation Sequencing Panel


Test Name

Hematologic Neoplasm Next Generation Sequencing Panel – 63 genes
Bone Marrow: HNMNGS   Peripheral Blood: HNPNGS

Subpanels

Myeloid Panel – 50 genes
Bone Marrow: MYNGSM   Peripheral Blood: MYNGSP

Acute Lymphoblastic Leukemia (ALL) Panel – 26 genes
Bone Marrow: ALLBM   Peripheral Blood: ALLPB

Chronic Lymphoproliferative Disorders (LPD) Panel – 7 genes
Bone Marrow: LPMNGS   Peripheral Blood: LPPNGS

Myeloproliferative Neoplasms Panel – 3 genes
Bone Marrow: MPNM   Peripheral Blood: MPNP

CPT Codes

81455

Methodology

Next-Generation Sequencing

Turnaround Time

10 days (upon specimen receipt)

Specimen Requirements

Type:
Bone marrow aspirate

Volume:
2 mL

Type:
Peripheral blood

Volume:
4 mL

Do not freeze specimens.

Stability

Ambient:
48 hours

Refrigerated:
7 days

Frozen:
Unacceptable

Background Information

Recurrent mutations are found in numerous hematologic neoplasms including myelodysplastic syndromes, myeloproliferative neoplasms, acute myeloid leukemia, acute lymphoblastic leukemia, and selected mature lymphoid leukemias.[1-4] The identification of such mutations provides pathologists and clinicians with useful data that may assist in the diagnosis, classification, prognostic evaluation, and therapeutic management of these malignancies. Mutational data in these disorders has been incorporated into the current diagnostic criteria of the World Health Organization Classification of Hematopoietic and Lymphoid Tissues, and into practice guidelines from the National Comprehensive Cancer Network.[5,6]

Cleveland Clinic Laboratories offers a next-generation sequencing panel that analyzes the clinically relevant regions of 62 genes known to be mutated in hematologic neoplasms. This test, performed on peripheral blood or bone marrow aspirate, identifies single nucleotide variants, insertions, and deletions in the targeted genes. Whole-genome copy number analysis may also be obtained by concurrently ordering Cancer Chromosome Microarray + SNP testing.

Smaller subpanels are available for focused disease testing.

Refer to the CC-SIGN® Hematologic Neoplasm Next Generation Sequencing Panel overview for more information.

Panel Highlights & Available Subpanels

Hematologic Neoplasm Next Generation Sequencing Panel – 63 genes

Analyzes the clinically relevant regions of 63 genes known to be mutated in hematologic neoplasms.

  • ABL1
  • ASXL1
  • BCOR
  • BCORL1
  • BRAF
  • CALR
  • CBL
  • CDKN2A
  • CEBPA
  • CSF3R
  • CUX1
  • DDX41
  • DNMT3A
  • EED
  • ETNK1
  • ETV6
  • EZH2
  • FBXW7
  • FLT3
  • GATA1
  • GATA2
  • GNAS
  • IDH1
  • IDH2
  • IKZF1
  • JAK2
  • JAK3
  • KDM6A
  • KIT
  • KMT2A
  • KRAS
  • LUC7L2 (C7orf55)
  • MPL
  • MYD88
  • NF1
  • NOTCH1
  • NPM1
  • NRAS
  • PAX5
  • PHF6
  • PIGA
  • PPM1D
  • PRPF8
  • PTEN
  • PTPN11
  • RAD21
  • RIT1
  • RUNX1
  • SETBP1
  • SF3B1
  • SH2B3
  • SMC1A
  • SMC3
  • SRSF2
  • STAG2
  • STAT3
  • STAT5B
  • SUZ12
  • TET2
  • TP53
  • U2AF1
  • WT1
  • ZRSR2

Subpanel: Myeloid Neoplasm Next Generation Sequencing Panel – 50 genes

Examines 50 genes mutated in myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemia.  This panel includes all 34 genes recommended by the Association for Molecular Pathology for analysis of chronic myeloid neoplasms.[7]

  • ABL1
  • ASXL1
  • BCOR
  • BCORL1
  • CALR
  • CBL
  • CEBPA
  • CSF3R
  • CUX1
  • DDX41
  • DNMT3A
  • EED
  • ETNK1
  • ETV6
  • EZH2
  • FLT3
  • GATA1
  • GATA2
  • IDH1
  • IDH2
  • JAK2
  • KIT
  • KMT2A
  • KRAS
  • MPL
  • NF1
  • NPM1
  • NRAS
  • PHF6
  • PIGA
  • PPMID
  • PTEN
  • PTPN11
  • RAD2
  • RUNX1
  • SETBP1
  • SF3B1
  • SH2B3
  • SMC1A
  • SMC3
  • SRSF2
  • STAG2
  • STAT3
  • STAT5B
  • SUZ12
  • TET2
  • TP53
  • U2AF1
  • WT1
  • ZRSR2

Subpanel: Acute Lymphoblastic Leukemia Panel – 26 genes

Includes 26 genes recurrently mutated in lymphoblastic leukemias.

  • ABL1
  • CBL
  • CDKN2A
  • EED
  • ETV6
  • EZH2
  • FBXW7
  • FLT3
  • IKZF1
  • JAK2
  • JAK3
  • KDM6A
  • KMT2A
  • KRAS
  • NOTCH1
  • NRAS
  • PAX5
  • PHF6
  • PTEN
  • RUNX1
  • SH2B3
  • STAT5B
  • SUZ12
  • TET2
  • TP53
  • WT1

Subpanel: Chronic Lymphoproliferative Disorders Panel – 7 genes

Targets seven genes mutated in mature lymphoid leukemias, including chronic lymphocytic leukemia, lymphoplasmacytic leukemia, hairy cell leukemia, and large granular lymphocyte leukemias.

  • BRAF
  • MYD88
  • NOTCH1
  • SF3B1
  • STAT3
  • STST5B
  • TP53

Subpanel: Myeloproliferative Neoplasms Panel – 3 genes

Detects mutations associated with myeloproliferative neoplasms.

  • CALR
  • JAK2
  • MPL

Targeted Gene Regions

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

A

Gene, Transcript, Exons

ABL1
NM_005157.5
Exons 4-6

ASXL1
NM_15338.5
Exons 10-13

B

Gene, Transcript, Exons

BCOR
NM_17745.5
Exons 2-15

BCORL1
NM_021946.4
Exons 1-12

BRAF
NM_004333.4
Exon 15

C

Gene, Transcript, Exons

CALR
NM_004343.3
Exon 9

CBL
NM_005188.3
Exons 8, 9

CDKN2A
NM_000077.4
Exons 1, 2

CDKN2A
NM_058195.3
Exon 1

CEBPA
NM_004364.4
Exon 1

CSF3R
NM_000760.3
Exons 14-17

CUX1
NM_001202543.1
Exons 15-24

CUX1
NM_001913.4
Exons 1-23

D

Gene, Transcript, Exons

DDX41
NM_016222.3
Exons 1-17

DNMT3A
NM_022552.4
Exons 2-23

E

Gene, Transcript, Exons

EED
NM_003797.4
Exons 1-12

ETNK1
NM_018638.4
Exon 3

ETV6
NM_001987.4
Exons 1-8

EZH2
NM_004456.4
Exons 2-20

F

Gene, Transcript, Exons

FBXW7
NM_018315.4
Exons 7-11

FLT3
NM_004119.2
Exons 14-17, 19-20

G

Gene, Transcript, Exons

GATA1
NM_002049.3
Exons 2, 4

GATA2
NM_032638.4
Exons 2-6

GNAS
NM_000516.5
Exons 8-11

I

Gene, Transcript, Exons

IDH1
NM_005896.3
Exon 4

IDH2
NM_002168.3
Exon 4

IKZF1
NM_006060.5
Exons 2, 3, 5-7

J

Gene, Transcript, Exons

JAK2
NM_004972.3
Exons 12-16

JAK3
NM_000215.3
Exons 11-18

K

Gene, Transcript, Exons

KDM6A
NM_021140.3
Exons 1-29

KIT
NM_000222.2
Exons 2, 8-11, 13, 17

KMT2A
NM_005933.3
Exons 1-36

KRAS
NM_004985.4
Exons 2-4

L

Gene, Transcript, Exons

LUC7L2 (C7orf55)
NM_001244585.1
Exons 2-11

M

Gene, Transcript, Exons

MPL
NM_005373.2
Exons 10-11

MYD88
NM_002468.4
Exon 5

N

Gene, Transcript, Exons

NF1
NM_000267.3
Exons 1-57

NF1
NM_001042492.2
Exon 31

NOTCH1
NM_17617.4
Exons 26, 27, 34

NPM1
NM_002520.6
Exons 8-11

NRAS
NM_002524.4
Exons 2-4

P

Gene, Transcript, Exons

PAX5
NM_016734.2
Exons 1-10

PHF6
NM_001015877.1
Exons 2-10

PIGA
NM_002641.3
Exons 2-6

PPM1D
NM_003620.3
Exons 1-6

PRPF8
NM_006445.3
Exons 2-43

PTEN
NM_000314.6
Exons 1-9

PTPN11
NM_002834.3
Exons 3, 4, 12, 13

R

Gene, Transcript, Exons

RAD21
NM_006265.2
Exons 2-14

RIT1
NM_006912.5
Exon 5

RUNX1
NM_001754.4
Exons 2-9

RUNX1
NM_001122607.1
Exon 5

S

Gene, Transcript, Exons

SETBP1
NM_015559.2
Exon 4*

* Exon is only partially analyzed from genomic coordinates chr18:42531679-42532175.

SF3B1
NM_012433.3
Exons 13-16

SH2B3
NM_005475.2
Exon 2

SMC1A
NM_006306.3
Exons 1-25

SMC3
NM_005445.3
Exons 1-29

SRSF2
NM_003016.4
Exons 1, 2

STAG2
NM_00104279.2
Exons 3-35

STAT3
NM_003150.3
Exons 20, 21

STAT5B
NM_012448.3
Exons 16-18

SUZ12
NM_015355.3
Exons 1-16

T

Gene, Transcript, Exons

TET2
NM_001127208.2
Exons 3-11

TP53
NM_000546.5
Exons 2-11

U

Gene, Transcript, Exons

U2AF1
NM_006758.2
Exons 2, 6

W

Gene, Transcript, Exons

WT1
NM_000378.4
Exons 1-9

Z

Gene, Transcript, Exons

ZRSR2
NM_005089.3
Exons 1-11

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.

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MGMT Pyrosequencing Methylation Assay for Glioblastoma

Technical Brief

MGMT Pyrosequencing Methylation Assay for Glioblastoma


Test Name

MGMT Methylation (MGMT)

CPT Codes

81287

G0452

Methodology

Polymerase Chain Reaction (PCR)

Pyrosequencing

Turnaround Time

7 days

Specimen Requirements

Type:
Formalin-fixed, paraffin-embedded block

Volume:
1 block

Transport Temperature:
Ambient

Minimal biopsy size 0.5 x 0.5 x 0.5 cm with mainly tumor tissue. Necrosis area should be less than 15%.

Cut 12 charged, unbaked slides, 4um thick. Pre- and post- H&E slides must be examined. Tumor area will be microdissected from USS.

Alternative Specimen

Type:
Formalin-fixed, paraffin-embedded block

Volume:
1 block

Transport Temperature:
Ambient

Minimal biopsy size 0.5 x 0.5 x 0.5 cm with mainly tumor tissue. Necrosis area should be less than 15%.

Cut 8 sections, 10 um thick and place in microcentrifuge tube. Pre- and post- H&E slides must be examined. Tumor area will be microdissected from USS.

Stability

Ambient:
Acceptable

Refrigerated:
Unacceptable

Frozen:
Unacceptable

Reference Range

Positive for MGMT methylation: ≥ of 10% of any CpG island or average of all 5 CpG islands.

Negative for MGMT methylation: >10% of methylation or average of all CpG islands analyzed.

Background Information

Glioblastoma is the most common and most aggressive malignant primary brain tumor. While occurring in only two to three cases per 100,000 people in North America, glioblastoma represents 52% of all functional tissue brain tumor cases and 20% of all intracranial tumors. Prognosis for those diagnosed with glioblastoma is poor, with a median survival time of about 14 months.1

Patients with glioblastoma can be treated with alkylating agents, such as Temador® (temozolomide). Epigenetic silencing of the MGMT (O6-methylguanine-DNA methyltransferase) DNA-repair gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with glioblastoma who receive temozolomide.2,3

Temozolomide kills tumor cells by producing cross-links between DNA strands and inhibiting DNA replication. The most common alkylation site is the O6 position of guanine. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that reverses such DNA alkylation and confers chemoresistance by repairing DNA damage. Temozolomide seems to work by sensitizing the tumor cells to radiation.4

Recent clinical studies confirm that the presence of MGMT promoter methylation in tumor samples corresponds to an increased likelihood that tumor cells would be responsive to temozolomide.3,4,5,6 If the promotor was methylated, temozolomide was more effective. It is estimated that approximately 40 to 50% of glioblastoma tumors exhibit MGMT gene methylation, which correlates significantly with reduced DNA damage repair induced by alkylating agents and significantly enhanced chemosensitivity.4

According to recent clinical trials, glioblastoma patients with MGMT methylation respond to temozolomide two to three times better than those lacking of MGMT methylation. Prolonged overall and progression-free survival at 24 months was 80% for those with MGMT methylation vs. 20% for those lacking MGMT methylation.

Diagnostic MGMT testing requires sufficient and optimally preserved tumor tissue. Cleveland Clinic’s MGMT methylation assay is a new, quantitative MSP test to detect the methylation status of brain tissue that has undergone thorough clinical evaluation. Our protocol calls for a minimal tissue sample size of ½-centimeter, which is much smaller than other laboratories that typically require at least a minimum 1-centimeter sample size. The best results are obtained with cryopreserved tumor specimens.

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

Positive for MGMT methylation:
Equal or greater than 10% of methylation in any CpG island or in average of all CpG islands analyzed.

Negative for MGMT methylation:
Less than 10% of any CpG island or in average of all CpG islands analyzed.

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.

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BCR / ABL p210 Quantitative RT-PCR Assay

Technical Brief

BCR / ABL p210 Quantitative RT-PCR Assay with International Scale Reporting for Minimal Residual Disease in Chronic Myeloid Leukemia


Test Name

BCR / ABL p210 RT-PCR, quantitative (BCRPCR)

CPT Code

81206
G0452

Methodology

Reverse Transcription/Polymerase Chain Reaction (RT/PCR)

Turnaround Time

5 – 7 days

Specimen Requirements

Clients should ship specimens as “Priority Overnight” to ensure specimen stability.

Do not ship on Fridays or the day preceding a holiday.

Specimen Type:
Whole blood

Volume:
10 mL

Minimum Volume:
4 mL

Collection Container:
Lavender BD Hemogard™ K2EDTA Tube

Transport Temperature:
Ambient

Clearly indicate specimen type on the label.

Alternative Specimen

Specimen Type:
Bone marrow

Volume:
5 mL

Minimum Volume:
2 mL

Collection Container:
Lavender BD Hemogard™ K2EDTA Tube

Transport Temperature:
Ambient

Clearly indicate specimen type on the label.

Stability

Ambient:
48 hours

Refrigerated:
7 days

Ambient:
Unacceptable

Reference Range

BCR ABL p210 transcripts not detected

Additional Information

Background Information

A translocation between chromosomes 9 and 22, resulting in the formation of a BCR / ABL fusion transcript, has long been recognized as a hallmark of chronic myeloid leukemia (CML). Although the breakpoints in BCR and ABL are variable, in >95% of cases of CML, the translocation results in production of the p210 isoform of the fusion protein (e13a2 or e14a2 fusion genes).

Modern therapy for CML, including tyrosine kinase inhibitors, has resulted in effective therapeutic options for CML patients. With this advance in treatment has come the need for effective monitoring for the presence of minimal residual disease (MRD) and the ability to recognize disease progression at an early stage. Techniques such as fluorescence in situ
hybridization (FISH) and metaphase cytogenetics provide valuable information at the time of initial diagnosis of CML and metaphase cytogenetics is helpful for identifying the emergence of additional chromosomal abnormalities, however, neither of these techniques are sufficiently sensitive to monitor MRD.[1],[2] For this reason, a BCR ABL p210 quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed, validated, and implemented in the Department of Molecular Pathology.

To facilitate comparison of quantitative RT-PCR results between laboratories and platforms, International Scale reference materials were established by the World Health Organization.[3] Results of this assay are now reported on the International Scale.

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 BCRABL 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 BCRABL mRNA. Blood 2010;116:e111-7.

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Whole Genome Single Nucleotide Polymorphism Microarray Testing

Technical Brief

Whole Genome Single Nucleotide Polymorphism (SNP) Microarray Testing


Test Name

Chromosomal Microarray SNP, Constitutional (CRMSNP)

CPT Code

81229

Methodology

Genomic Oligonucleotide-SNP Microarray

Turnaround Time

10 – 14 days

Additional time may be required for reflex testing, if necessary.

Specimen Requirements

Type:
Whole blood

Volume:
4 mL

Minimum Volume:
1 mL

Specimen Container:
Lavender BD Hemogard™ K2EDTA Tube

Transport Temperature:
Ambient

If aliquoting is necessary, sterile aliquot tubes must be used.

Especially in case of neonates and difficult-to-draw cases, every attempt will be made to run the array with limited specimen.

Stability

Ambient: 
48 hours

Refrigerated:
72 hours

Frozen:
Unacceptable

Reference Range

Refer to report

Background Information

Evidence-based approaches, coupled with technological advancements in the field of genetics, are changing how the human genome is analyzed and interpreted for diagnostic purposes. Chromosomal microarray analysis (CMA) is a recommended first-tier test for diagnosing unexplained intellectual disabilities, dysmorphic features, congenital anomalies, and autism.1-5 This technique compares relative fluorescent signal intensities of two different genomes, a test, and a reference, that compete for hybridization to DNA sequences representing the whole human genome in order to detect a gain or loss of genetic material. However, CMA does not detect allelic imbalances resulting in absence of heterozygosity (AOH).

Advances in the field of genetics and diagnostic medicine have resulted in the refinement of the existing CMA platform by incorporating single nucleotide polymorphism (SNP) markers into the existing non-polymorphic markers used to detect gains and losses of DNA. The analysis of SNP data provides information about the allelic imbalances associated with AOH. Identifying regions of excessive homozygosity on a single chromosome could suggest uniparental disomy (UPD), which may warrant further clinical and laboratory investigation when observed on chromosomes with known imprinting disorders associated with UPD. In addition, the detection of excessive homozygosity on multiple chromosomes may suggest consanguinity and, therefore, could be useful in determining candidate genes for further testing for autosomal recessive disorders.

The Agilent 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. Data for the SNP markers are displayed to demonstrate either homozygosity (AA or BB) or heterozygosity (AB) at every SNP locus. AOH is indicative of hemizygosity if the corresponding CMA data shows a deletion of the region. If the CMA data is normal, AOH signifies homozygosity.

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.

Uniparental Disomy

Uniparental disomy (UPD), the presence of two copies of a chromosome or chromosomal region from a single parent, is clinically-relevant when it involves loci that undergo genomic imprinting.

There are two types of UPD:

  • Isodisomy, in which the two parental copies are identical; and
  • Heterodisomy, in which the parental copies are derived from both parental homologs and are, therefore, not identical.

SNP microarrays will detect isodisomy but not heterodisomy, since only isodisomy or segmental isodisomy results in homozygosity.

UPD is the most likely explanation for an absence of heterozygosity that is restricted to a single chromosome, especially if the region is very large. Several chromosome regions are imprinted and lead to an abnormal phenotype in the presence of UPD derived from a particular parent (Figure 1). UPD in other regions of the genome, even if covering a large region, is generally not considered to be a pathogenic finding in and of itself (see recessive disease discussion below).

Figure 1: Uniparental Disomy 15

Identity by Descent (IBD) and Consanguinity

The observation of multiple AOH regions, also known as long continuous stretches of homozygosity (LCSH), present on multiple chromosomes is generally assumed to reflect inheritance of these regions by descent from a common ancestor. This type of homozygosity is referred to as “identity by descent.” A single or a few small isolated stretches can be the result of a founder effect in an isolated population. The presence of especially long stretches on multiple chromosomes suggests the possibility of a more direct biological relationship between the parents (i.e., parental consanguinity) (Figure 2). A consanguineous relationship refers to the sharing of a common ancestor and the term consanguinity is generally used when individuals are second cousins or closer.

However, there are other explanations for a relatively high level of identity by descent. For example, a high overall level of homozygosity can result from unusual recombination or segregation patterns during meiosis. It may also be observed for a distantly related couple who have multiple common ancestors. The latter circumstance may occur, for example, in individuals from an isolated population that arose recently from a small founding group or in populations where cousin marriages are common. For the above reasons, the SNP data themselves are not diagnostic of a specific degree of parental relatedness.7-8 Therefore, SNP data must be interpreted in the context of additional family and social history information and the clinician must determine whether it is appropriate to pursue the question of parental consanguinity for individual families.

Figure 2: Multiple AOH regions were identified, consistent with parental relatedness or identity by descent.

Autosomal Recessive Disease Risk

Regardless of whether AOH results from uniparental disomy (UPD) or identity by descent, homozygosity anywhere in the genome raises the possibility of recessive conditions.

Our reports alert the physician to the increased possibility of these conditions for regions of homozygosity greater than 10Mb. The referring physician can use this information in conjunction with clinical features and family history to determine whether mutation testing of individual genes is warranted.

Triploidy

Triploidy can be seen prenatally and appears at an appreciable frequency in miscarriages, but it is extremely rare postnatally.

The three types of triploidy are 69,XXX; 69,XXY; and 69,XYY. With aCGH, 69,XXY and 69,XYY triploidy can be detected, but not 69,XXX. The SNP Microarray allows for the detection of all types of triploidy due to the capability of detecting four genotypes (AAA, AAB, ABB, and BBB) rather than the normal three genotypes (AA, AB, and BB).

Genetic Counseling

A referral to a clinical genetics professional is often appropriate for individuals and families undergoing whole-genome microarray testing. This may be valuable both before and after testing.

Families should be aware of the possibility of a result of uncertain clinical significance and the need for parental blood samples to help interpret the change. Families should also understand that findings of AOH may require additional testing of the proband before a diagnosis can be made.

Clinical geneticists can guide testing strategies and further evaluate the patient in light of the test results. In some cases, it may be important to discuss the potential for discovery of parental consanguinity. Genetic counseling can also elicit a thorough family and social history, which can be critical in the interpretation of the SNP array results.

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.

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Detection and Typing of HSV-1 and HSV-2 in Cerebrospinal Fluid (CSF) by Polymerase Chain Reaction

Technical Brief

Detection and Typing of HSV-1 and HSV-2 in Cerebrospinal Fluid (CSF) by Polymerase Chain Reaction (PCR)


Test Name

Herpes Simplex Virus by PCR, CSF (HSPCRC)

CPT Codes

87529 (x2)

Methodology

Polymerase Chain Reaction (PCR)

Turnaround Time

1 – 3 days

Specimen Requirements

Type:
Cerebrospinal fluid (CSF)

Specimen Container:
Sterile container

Transport Temperature:
Refrigerated

If aliquoting is necessary, sterile aliquot tubes must be used.

Stability

Ambient: 
7 days

Refrigerated:
1 month

Frozen:
Unacceptable

Background Information

The herpes simplex virus (HSV) is a common sexually transmitted disease virus that infects the skin, lips, oral cavity, eyes, genital tract, and central nervous system. HSV disease can range from the usual mild illness, indistinguishable in most patients, to sporadic, severe, and life-threatening disease in infants.

Infections with HSV-1 and HSV-2 can differ somewhat in their clinical manifestations and severity. HSV-1 is more frequently associated with orolabial infection (cold sores), whereas HSV-2 primarily is more frequently associated with urogenital infections in adults. Although there are differences in the frequency of infections at particular sites, in reality, both viral subtypes can cause either of these diseases.1

HSV also can cause viral central nervous system (CNS) disease. For example, HSV-1 is the most common cause of sporadic encephalitis that, if left untreated, has a high mortality rate. HSV-2 is a cause of meningitis, which occurs most frequently among young adults. Both HSV subtypes are also a cause of neonatal disease acquired during exposure of the infant to HSV-infected secretions during birth.

Early and rapid diagnosis of HSV meningitis/meningoencephalitis is important to direct therapy and minimize the suffering in affected patients. It also helps to reduce empiric antibacterial therapy and the likelihood of side effects from unnecessary therapy. In the past, diagnosis relied on brain biopsy. Fortunately, new molecular diagnostic tests, such as rapid-cycle PCR on cerebrospinal fluid, have virtually replaced the need for brain biopsy.2

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.

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Chromosomal Single Nucleotide Polymorphism (SNP) Microarray for Hematology on Bone Marrow

Technical Brief

Chromosomal Single Nucleotide Polymorphism (SNP) Microarray for Hematology on Bone Marrow


Test Name

Bone Marrow Cancer Chromosome Microarray + SNP (BMHSNP)

CPT Code

81406

Methodology

Genomic microarray

Turnaround Time

14 days

Specimen Requirements

Type:
Bone marrow

Volume:
4 mL

Specimen Container:
Lavender BD Hemogard™ K2EDTA Tube

Transport Temperature:
Ambient

Collect the specimen Monday–Friday only.

Type:
Bone marrow

Volume:
4 mL

Transport Temperature:
Ambient

Collect the specimen Monday–Friday only.

Specimen Collection & Transport

Pathology Consultation Kits

For hassle-free specimen shipping, our laboratories can provide clients with Bone Marrow Biopsy Pathology Consultation Kits.

Each kit includes the following:

– (1) White CCL box (6.5 x 6 x 2.75 inches)
– (2) Slide holders
– (2) 10% neutral buffered formalin
– (2) Lavender K2EDTA tubes
– (2) Green Sodium Heparin tubes
– (1) Foam insert
– (1) Specimen bag
– (1) Absorbent sheet
– Shipping Label and Bag

Stability

Ambient:
48 hours

Refrigerated:
Unacceptable

Frozen:
Unacceptable

Background Information

Chromosomal microarrays are applied for the detection of genomic and allelic imbalances in hematological malignancies and solid tumors. Chromosomal microarray (CMA) and single-nucleotide polymorphism array (SNP-A) provide a combination of karyotyping and FISH with whole-genome scan and a targeted approach at a higher resolution.

Cleveland Clinic Laboratories uses a cancer-specific microarray designed by the Cancer Cytogenomics Microarray Consortium (CCMC) that targets genomic regions associated with cancer. This array can be used for diagnostic testing of hematological malignancies. Common indications include, but are not limited to, acute and chronic leukemia, myelodysplastic syndrome (MDS), and myeloproliferative disease.

This array has the ability to detect genome-wide copy number variants (CNV) with simultaneous detection of loss of heterozygosity (LOH), also known as copy neutral LOH (cnLOH). The array contains approximately 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. This analysis can interrogate the whole genome for recurrent and novel clinically-relevant copy number variation (CNV) at an increased resolution over FISH or chromosome analysis.

Chromosomal microarray aids in diagnosis, prognosis, and therapeutic management by identifying gains, losses, or LOH in hematological disorders. It is also helpful in monitoring disease progression. Chromosome analysis or karyotyping for detection of genetic abnormalities is hindered by suboptimal cell growth or chromosome morphology, and clonal or subtle unbalanced chromosomal abnormalities may be missed. FISH provides an improved rate of detection of clonal abnormalities when compared to karyotyping, but only for the targeted region.

Chromosome or FISH testing cannot detect copy-neutral events that are associated with hematological disorders, which are often due to mutations and subsequent selection of mutant tumor-suppressor genes or oncogenes. However, SNP microarray detects submicroscopic chromosomal variants involving gains or losses in chromosomes across the genome in addition to LOH.

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.

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Lipoprotein (a) in Serum

Technical Brief

Lipoprotein (a) in Serum


Test Name

Lipoprotein (a) (LPA)

CPT Codes

83695

Clinical Information

Evaluation of coronary artery disease risk associated with elevations of the atherogenic lipoprotein (a).

Methodology

Nephelometry (NEPH)

Turnaround Time

1 – 2 days

Specimen Requirements

Type:
Serum

Volume:
1 mL

Minimum Volume:
0.5 mL

Specimen Container:
Gold BD Hemogard™ Serum Separation Tubes (SST)™

Transport Temperature:
Refrigerated

Allow specimen to clot completely at room temperature. Separate serum from cells within 2 hours of collection.

Stability

Ambient:
8 hours after separation from cells

Refrigerated:
14 days after separation from cells

Frozen:
30 months after separation from cells

Patient Preparation

Patients should fast for at least 12 hours before blood is drawn.

Reference Range

0-40 mg/dL

Background Information

Lipoprotein (a) [Lp(a)] is a spherical lipid particle that is genetically determined and remains at relatively constant levels over an individual’s lifetime. It contains two crosslinked proteins as part of its structure: apolipoprotein(a) covalently bound to apolipoprotein B-100. Lp(a) is important as a serum marker for coronary artery disease independent of diet and lipid levels. Elevated Lp(a) levels are associated with increased risk and severity of atherosclerosis, coronary heart disease, and stroke.

Similar to LDL-cholesterol, Lp(a) is synthesized in the liver. Although Lp(a) shows some homology to LDL-cholesterol in structure, Lp(a) differs from LDL in molecular weight, electrophoretic mobility, and protein/lipid ratio. Physiologic circulating levels of Lp(a) do not appear to be regulated by the same mechanisms of LDL-cholesterol. Likewise, cholesterol feeding does not appear to increase levels of Lp(a) in plasma, although it does increase levels of LDL cholesterol. Most pharmacologic agents that have an effect on lowering LDL-cholesterol levels have little effect on levels of Lp(a), thus also indicating regulation under different metabolic control.

The causes of high Lp(a) are kidney disease and certain family (genetic) lipid disorders.

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.

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Testing for Heparin-Induced Thrombocytopenia

Technical Brief:

Testing for Heparin-Induced Thrombocytopenia


Test Name

Anti-Platelet Factor 4 (PLATF4)

All positive specimens (OD>= 0.400) will have confirmatory platelet aggregation testing (PLHEPP or PLLMWP) ordered and charged.

CPT Codes

86022 (x2)

Methodology

Enzyme-Linked Immunosorbent Assay (ELISA)

Turnaround Time

1 day

Specimen Requirements

Volume:
2 mL

Minimum Volume:
1 mL

Specimen Type:
Plasma

Collection Container:
Light Blue Sodium Citrate Coagulation Tube

Transport Temperature:
Froze

3.2% sodium citrate is the preferred anticoagulant recommended by CLSI.

Stability 

Ambient: 
4 hours

Refrigerated: 
Unacceptable

Frozen: 
2 months

Background Information

Heparin-induced thrombocytopenia (HIT) is a clinically significant immune-mediated disorder, characterized by antibodies forming immune complexes with the chemokine platelet factor 4 (PF4) bound to unfractionated heparin (UFH), leading to paradoxical thrombosis.1 The IgG immune complexes engage Fc-gamma receptor IIa (FcγRIIa) expressed on platelets and possibly leukocytes,2 initiating a signal transduction cascade resulting in cellular activation. Activated platelets may potentiate thrombin generation, form thrombi, and induce a prothrombotic state involving both the venous and arterial systems.3 In the process, platelets are consumed, which leads to the observed thrombocytopenia. It is critical that HIT is recognized early so that patients can be alternatively anticoagulated and avoid further exposure to heparin.

Based on the findings of a consensus report,4 HIT is regarded as a clinicopathologic syndrome, requiring both clinical features and laboratory detection of the pathologic antibodies. Clinically, HIT needs to be differentiated from other potential causes of thrombocytopenia, particularly the well-characterized non-immune heparin-associated thrombocytopenia (HAT).

A useful clinical scoring system, referred to as the “4Ts,” is helpful in determining whether a patient fits into the “high probability,” “intermediate probability,” or “low probability” category.5

The criteria below should be assessed to determine the pretest probability of HIT:

4Ts Scoring System for Assessing Clinical Risk of HIT

Score each category, then add all points to determine maximum score. (Maximum possible score = 8 points)

Thrombocytopenia

0 points = <30% fall or nadir <10×109/L

1 point = 30-50% fall or nadir 10-19×109/L

2 points = >50% fall and nadir 20-100×109/L

Timing of Platelet Count Fall

0 points = <4 days without recent exposure

1 point = Consistent with day 5-10 (but not clear), or >10 days, or ≤1 day with heparin 30-100 days prior

2 points = Day 5-10 or ≤1 day if recent heparin (within 30 days)

Thrombosis

0 points = None

1 point = Progressive, recurrent, or silent thromboses

2 points = Proven thrombosis, skin necrosis, or acute systemic reaction with heparin bolus

Other Causes for Thrombocytopenia

0 points = Definite

1 point = Possible

2 points = None evident

Pretest Probability

<3 points = Low

4-5 points = Intermediate

6-8 points = High

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]).

Positive Anti-PF4 IgG ELISA Result

When the anti-PF4 IgG antibody ELISA and platelet functional assay (heparin-induced platelet aggregation testing [HIPA] or serotonin release assay [SRA]) are both positive, the diagnosis of HIT is supported by laboratory findings.

If the anti-PF4 IgG antibody ELISA is positive or equivalent, but the platelet functional assays (see Methodology) are both negative, the diagnosis of HIT is less likely. However, clinical judgement is important since the confirmatory tests are not as sensitive for detecting HIT as the ELISA. Reassessing the pretest probability is suggested. If clinical suspicion remains high, retesting may be justified in case the antibody titer was too low to induce immune complex-mediated platelet activation in the initial work-up.

If the anti-PF4 IgG antibody ELISA is positive, but the HIPA is negative, ordering the SRA is suggested if clinical suspicion remains high. The SRA has a higher sensitivity than the HIPA.

Negative Anti-PF4 IgG ELISA Result

If the anti-PF4 IgG antibody ELISA is negative, HIT is very unlikely and further testing is unnecessary. In rare situations where clinical necessity dictates confirmatory testing despite a negative anti-PF4 result, the SRA is the recommended confirmatory assay since it has a higher sensitivity than the HIPA.

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.

No Antibodies Detected:

If the O.D. < 0.4, a diagnosis of HIT is unlikely (negative predictive value 97-99%), and no further testing is necessary.

Heparin Dependent Anti-PF4 IgG Antibodies are Present:

If the O.D. > 0.4 and % inhibition with soluble heparin is > 50%, the assay is positive. The ordering clinician is immediately notified of the result.

The HIPA platelet functional assay (with higher specificity) is then performed to confirm the diagnosis of HIT. Recommendations to order the SRA will be made.

Heparin-Dependent Anti-PF4 IgG Antibodies Equivocal:

If the O.D. > 0.4 and % inhibition with soluble heparin is < 50%, the assay is equivocal.

Platelet functional assays (with higher specificity) are performed to further evaluate.

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.

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Stool Culture for Yersinia

Technical Brief

Stool Culture for Yersinia


Test Name

Yersinia Culture (YERCUL)

CPT Codes

87046

Methodology

Culture

Turnaround Time

5 days

Specimen Requirements

Specimen Type:
Stool

Volume:
5 mL

Collection Container:
Para-Pak™ C&S (Culture & Sensitivity) Stool Transport System

Transport Temperature:
Refrigerated

Stability

Ambient:
2 hours

Refrigerated:
Preserved – 24 hours
Unpreserved – 2 hours

Frozen:
Unacceptable

Background Information

Bacterial, parasitic, and viral agents can cause infectious gastroenteritis. Approximately 48 million people become ill and 128,000 require hospitalization from foodborne diseases in the United States each year.1 Enterohemorrhagic Escherichia coli (EHEC) is one of the top five causes of foodborne illness requiring hospitalization in the United States.1 Most clinical laboratories that perform enteric cultures on stool samples routinely include media and methods to rule out Campylobacter spp., Salmonella spp., Shigella spp., and EHEC.

Shigella, which accounts for less disease prevalence in the U.S. (~14,000 cases/year), is highly infectious and its detection in all stool samples is important to prevent further disease from spreading person to person. Yersinia enterocolitica can also cause foodborne disease, but at a much lower incidence; consequently, most laboratories do not routinely look for this pathogen. The incidence of Y. enterocolitica, as reported by CDC Food Net activities, is about one culture-confirmed case per 100,000 population per year. This compares to the > 1 million cases of Salmonella and > 800,000 cases of Campylobacter reported annually to CDC in the U.S.2 Blood transfusion reactions have occurred from blood products contaminated with Y. enterocolitica, however, gastroenteritis is not usually a part of the resultant bacteremia.

Y. enterocolitica can cause gastroenteritis in an individual who has consumed contaminated food or water. Y. enterocolitica has been isolated from raw meats, such as beef, lamb, pork and chicken, but can also be found in cooked, pre-packaged deli meats. Consumption of raw or improperly cooked pork is the main source of gastroenteritis in humans, with the specific association with improperly prepared and handled pork chitterlings.3,4,5,6 Drinking unpasteurized milk, untreated water, or coming into contact with infected animals also can be the source of infection.

The severity of the disease is related to the specific serotype of Y. enterocolitica as well as load of organism consumed. The range of diseases associated with Y. enterocolitica is self-limited gastroenteritis to terminal ileitis, to mesenteric lymphadenitis that is often mistaken as appendicitis. Occasionally, skin rash and joint pains can accompany Yersinia gastroenteritis.1,3

Children are more commonly diagnosed with Y. enterocolitica than adults. Patients with gastroenteritis associated with Yersinia are more likely to present with fever, diarrhea, and abdominal pain that can last for seven days. A carrier state of the organism can ensue for up to several months. Septicemia can result if organisms migrate out of the gastrointestinal tract via the lymphatics and find their way to lymph nodes. Persons at the highest risk for systemic disease are the elderly and immunocompromised populations. In particular, persons with underlying metabolic diseases that are associated with iron overload (hemochromatosis), cancer, liver disease, and steroid therapy are at the highest risk of more serious Y. enterocolitica disease. A case of ileal perforation post gastroenteritis has been reported with a review of other surgical complications of Yersinia gastroenteritis.6

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.