neogenomics dos

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Table of ContentsAnnual Notice to Physicians 4

Licensure and Certification 7

Customer Care 8

Laboratory Locations 9

APvX 10

Client Education 11

Billing Services 12

Client 13

Patient 14

Third Party 15

Medicare 16

Medicaid 20

Specimen Requirements 21

Specimen Requirements & Handling Procedures 22

Specimen Kits 23

Requisitions & Shipping Instructions 28

Test Menu & Descriptions 32

Genetic Pathology Solutions (GPS) 33

Fluorescence in situ Hybridization (FISH) 35

FISH Probes by Disease State 41

Flow Cytometry 42

Flow Cytometry Antibody Library 44

Molecular Genetics 45

Cytogenetics 65

ISH 66

Immunohistochemistry (IHC) 68

IHC Panels 69

Individual Antibodies 76

Special Stains (IHC) 101

IHC Antibody Library 103

Circulating Tumor Cells 104

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Who We AreNeoGenomics is rapidly emerging as one of the nation’s premier cancer genetics laboratories by providing excellence in diagnostic, prognostic, and predictive testing. Comprised of four state of the art facilities with corporate headquarters in Fort Myers, Florida, we offer our expertise in the following technologies: Fluorescence in situ Hybridization, Flow Cytometry, Molecular Genetics, Cytogenetics, Pathology, and Immunohistochemistry.

Our MissionTo improve patient care through exceptional cancer genetic diagnostic services.

Our VisionBy delivering uncompromising quality, exceptional service and innovative solutions, we are becoming America’s premier cancer testing laboratory.

Our Values• Quality

• Integrity

• Customer Focused

• Employee Focused

• Results Focused

• Accountability

• Teamwork

NeoGenomics Laboratories

Annual Notice to Physicians

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To Our Clients In an effort to help laboratories comply with federal laws and regulations, the Office of the Inspector General (OIG) of the Department of Health and Human Services has issued a Compliance Plan for laboratories setting forth certain operational guidelines. The OIG also offers guidance on how laboratory clients can avoid submitting false claims to Medicare (CMS), Medicaid, and other government healthcare programs. Included in the OIG Compliance Plans are recommendations that laboratories should provide annual notification to their clients on pertinent topics. Accordingly, please review the following important information.

Clinical ConsultantTo assist with laboratory testing questions, including ordering and interpretation, please contact our assigned Medical Directors for each facility as follows:

• Florida: Dr. Barbara Chaitin - 239.768.0600 Ext. 2246• Tennessee: Dr. Christopher Mixon - 615.574.6090 Ext. 2101• California: Dr. Sally Agersborg - 949.206.1695 Ext. 2621

New Laboratory Site Locations2011 proved to be a year of growth. To better address the needs of our clients, we have added a laboratory site location in Tampa, Florida effective this January 2012. This is a read-only facility for Cytogenetics and FISH cases. By adding this facility, we will be able to remain consistent with our turnaround times to you our clients.

Medical NecessityFollowing CMS regulations, the hospital requests that a written order and a diagnosis accompany all diagnostic test orders. Tests submitted for Medicare reimbursement must meet program medical necessity requirements or the claim will be denied. For a list of tests for which Medicare has developed specific National Coverage Determinations (NCD) you can access the Center for Medicare and Medicaid Services website at: www.cms.hhs.gov/mcd. Diagnoses provided on these tests will be compared to the NCD. Diagnoses not on the NCD are considered non-covered and the patient may be responsible for payment. If a diagnosis or ICD-9 code does not accompany the order, it may be considered non-covered and the patient may be responsible for payment. The OIG would like us to remind you that laboratory claims submitted for services will only be paid if the service is covered, reasonable, and necessary for the beneficiary, given his or her clinical condition, as defined by Medicare. We are reminding our clients that for every test, medical necessity is necessary for proper reimbursement and that the results we provide to you will be incorporated into the patient’s treatment care plan.

Informed ConsentRecognizing the fact that NeoGenomics Laboratories, Inc. provides genetic testing and that many states require patient education and consent for testing, it is a requirement that our clients fully disclose the necessity and purpose of all testing with their patients as deemed appropriate and document accordingly.




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Requisition RequirementsAside from the necessary reason for the testing (narrative indication for testing or ICD9 diagnosis), NeoGenomics also needs complete patient demographics including full legal name, Social Security Number, DOB, sex of patient and insurance information. What we find extremely helpful is to get a copy of the patient’s insurance card submitted with the requisition. If these required components are missing, it may impact turnaround time for results pending us gathering the missing information.

CPT Codes The CPT codes that are submitted to Centers for Medicare and Medicaid Services (CMS) must be valid at the time the service is provided. NeoGenomics will be using the current year’s CPT codes published at the first of each calendar year by CMS. Our laboratory bills Medicare for tests using the CPT codes listed in the Reference Manual and Fee Schedule.

Medicare Reimbursement Fee ScheduleMedicare reimburses laboratory services based upon their published fee schedule. If you would like a copy of this reimbursement fee schedule, please contact our Billing Department at 866-776-5907 option #2 then #1. Medicaid reimbursement for laboratory services is equal to or less than the amount Medicare reimburses.

Technical Component / Professional Component TestingFederal guidelines require any pathologist who performs the professional component of testing be appropriately trained & credentialed for that specialty. The laboratory releasing these results should also be CLIA Certified or possess state equivalent credentialing. NeoGenomics Laboratories, Inc. does not solicit to potential clients who may not meet these professional and regulatory requirements. Furthermore, it is the responsibility for each client to be knowledgeable of any current individual state licensing requirements for performing professional component interpretations for tests they order.

TC Grandfathering ExemptionPer current Medicare regulations, all Anatomic Pathology Technical Component work performed for Medicare hospital inpatients and outpatients must be billed to Medicare by the hospital unless they meet exemption / “grandfathered” criteria. NeoGenomics Laboratories, Inc. will request signed attestations from each hospital client to indicate grandfather exemption status. Effective 12/22/11, the legislature bodies have extended this grandfathering until 2/28/2012. This means that if the non-exempted client is sending our laboratory technical component testing and the client knows that the tests are for Medicare hospital inpatients or outpatients, the hospital must bill Medicare directly for reimbursement. NeoGenomics will make arrangements to bill the client hospital for the Technical Component testing prior to any testing being conducted. Concordantly, if the hospital is grandfathered, the laboratory is able to bill CMS directly until the end of February unless this exemption is extended. We will continue to follow for any updates.



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Barbara Chaitin, M.DLaboratory Medical DirectorFort Myers, Florida

Christopher Mixon M.D.Laboratory Medical DirectorNashville, Tennessee

Sally Agersborg, M.D.Laboratory Medical DirectorIrvine, California

Advance Beneficiary Notices If there is reason to believe that Medicare will not pay for a test, the patient should be informed by your office. When appropriate, the patient should sign an Advance Beneficiary Notice (ABN) to indicate that he or she is responsible for the cost of the test if Medicare denies payment. This is also indicated on the requisition.

Reflex Laboratory TestsUpon results of an initial laboratory test, our laboratory may perform additional testing which can result in an additional charge as listed on our requisition or directory of services. Best practices in laboratory medicine and the avoidance of performing unnecessary testing help dictate which tests are subject to this practice. An additional fee may be added by every procedure subject to this additional reflex testing. Please contact the laboratory if you have any questions about reflex tests.

Creutzfeldt - Jakob Disease

We would also like to take this opportunity to remind you that our laboratory does not currently offer any testing on tissue or fluid specimens from the central nervous system obtained on patients with a suspected diagnosis of Creutzfeldt - Jakob disease. For any such specimens that may be received by our laboratory, it is our policy to immediately send the specimen directly back to the client from which it was received.

Thank you for your time and attention in these mutual issues. Please feel free to contact us directly if you have any questions.

Sincerely,

NeoGenomics Laboratories 7

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Licensure and Certification

Important: For any compliance or licensing issues please do not hesitate to contact NeoGenomics (866) 776-5907, option 1.

To view all licenses please visit our website at: http://www.neogenomics.com/company-licensures-certifications.htm

Location/Agency Number

–

05D1065194CLIAMedicare Provider

Medicare Provider

Medicare Provider

Rhode Island

Rhode Island

Rhode Island

05D1065194CAP

CAP

7197542Florida 800023287California CLF335014Pennsylvania 030541

LCO00507Maryland 1432

L9228CLIA 10D0998082

7178930California COS800251FloridaNew York

New York

8000171858200

Pennsylvania 030193LCO00484

Maryland 1397

3400014CLIA 44D1004543CAP 7181028Florida 800023286

8333Tennessee 4061Pennsylvania 030259

LCO00485Maryland 1402

Fort Myers, Florida

Irvine, California

Nashville, Tennessee

Licensure and Certification


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Client Services

At NeoGenomics we care deeply about our clients’ patients. This is why we believe it is a necessity that every client is provided with a dedicated Customer Care representative. In order to provide the highest level of customer service, Customer Care Representatives are trained to answer questions regarding test information, specimen requirements, turnaround times, test add-on, and patient results. Customer Care Representatives may also direct client calls immediately to a technical or medical expert as necessary or requested. Clients may contact the performing lab directly at the service locations listed below.

Customer CareTelephone: (866) 776-5907, option 1Fax: (239) 690-4237Email: [email protected] of Operation: Monday – Friday 7:00 AM -10 PM Eastern Saturday 8 AM – 4 PM EasternAfter Hours: 24/7

Specimen Pick-up and CouriersToll Free Telephone: (866) 776-5907, option 1Hours of Operation: 24 hours a day, 7 days a week

Billing ServicesTelephone: (866) 776-5907, option 2Fax: (239) 690-4236

Customer Care


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Laboratory Locations


Fort Myers, Florida

10002 Princess Palm Ave, Suite 228Tampa, FL 33619Phone: (239) 768 - 0600

618 Grassmere Park DriveUnit 20Nashville, TN 37211Phone: (615) 574 - 6090Fax: (615) 574 - 6094

12701 Commonwealth Drive Suite 5Fort Myers, FL 33913Telephone: (239) 768 - 0600Fax: (239) 690 - 4237

5 JennerSuite 100Irvine, CA 92618Phone: (949) 206 -1695Fax: (949) 206 -1865

Irvine, California

Tampa, Florida

Nashville, Tennessee



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APvX

APvX, our web-based laboratory reporting product, offers the convenience, efficiency, and the flexibility of accessing test results any time through a secure Internet connection. Our system is designed to decrease paperwork while increasing workflow by providing a flexible and efficient means for accessing test results. APvX has been developed with the contribution of medical specialists to provide conveniently-easy to use features.

Features & Benefits:• Flexible FISH image capabilities

• More dynamic patient and case searching

• On-line Directory of Services

• Ability to turn reporting features on and off

• User-friendly 24-hour access to results via a secure Internet connection

• Real-time tracking of specimen(s) and result(s)

• Access to historical patient reports

• Simultaneous user access from multiple locations

• Ability to retrieve result reports in a multitude of formats

• Ability to add clinical notes for review and discussion within your practice

• Dynamic report capabilities

• Notifies users when cases are ready to review

• Fast to load macro templates

• Technical assistance

APvX, HL7 & NeoGenomics: Health Level Seven (HL7) is the most successful messaging standard in the healthcare industry, not only in North America, but also around the world. Formed in the United States in 1987, HL7 has the goal of developing an international set of open standards for data format and content that allows different health information systems to easily and effectively communicate with one another.

At NeoGenomics Laboratories we possess the ability to generate HL7 message files for the integration of our Laboratory Information System to a client’s Electronic Medical Record System. We utilize a custom HL7 Integration Engine to extract the data from our LIS system database and convert it into HL7 message files. NeoGenomics Laboratories also has the ability to generate simple CSV files or any other client requested file format to accommodate your interfacing needs. We strive to provide flexible, yet secure connection options for the retrieval of HL7 messages for our clients. Currently, we are able to utilize:

• Secure FTP

• Site to Site VPN tunnels

• HTTPS

• SSH

For more information on APvX, our Laboratory Reporting System, please contact your local Territory Business Manager.

APvX


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NeoUniversity

On-Demand Our OnDemand Training provides self-paced courses to help you stay up-to-date with the latest product demonstrations, FISH signal interpretive training, courses for new assays and technologies, and Alternative Assessments to test your knowledge. Our self-paced learning modules will help you and your organization stay up-to-date with the latest advancements in the world of Pathology.

Our online courses benefit you and your organization:

• Develop your product market knowledge

• Get trained to sign out the Professional Component

• Collaborate with your peers online

• Build a solid foundation in emerging technologies

You can sign up for On-Demand Training at training.neogenomics.com.

Live WebinarsNeoGenomics also hosts periodic training webinars to help you stay abreast of the latest test interpretation methods. Please visit our website at www.neogenomics.com/webinar for the schedule of upcoming webinars.

On Site TrainingFor those of you who prefer an in-person experience, NeoUniversity On-Site will be the right choice. A member of our Medical Staff will join you at your location for NeoFISH & NeoFlow Technical Training. NeoUniversity gives you the opportunity to train and collaborate with NeoGenomics Medical Staff and your peers in an educational environment. This on-site training program occurs multiple times throughout the year and is appropriate for physicians who are interested in providing professional component services for FISH, Flow Cytometry, and Molecular Microdissection. Custom tailored training and curriculum allow for participants to feel confident and prepared to participate in the NeoGenomics TC/PC Program. Ask your local Territory Business Manager for details.

NeoUniversity


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Billing Services

The NeoGenomics Billing Department strives daily to provide our clients and their patients with exceptional customer service. In this section you will find detailed information on:

• Client Billing

• Patient Billing

• Third-party Billing

• Medicare Billing

• Medicaid Billing

Note: To avoid inappropriate billing, we request that each requisition be completed with the appropriate billing party. The NeoGenomics Laboratories Billing Department can be contacted at (866) 776-5907, option 2.

Billing Services


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Client Billing

Clients will be billed monthly or bi-monthly by an itemized invoice that includes the date, patient’s name, accession number, test(s) performed, and the test fees for each specimen completed during the month. Please note that these invoices are payable upon receipt. If you have any questions pertaining to your account, please notify us immediately so that we may resolve them in a timely manner. We can be reached at 866-776-5907; option 2.

Payments should be sent to:

NeoGenomics LaboratoriesPO Box 864403Orlando, FL 32886-4403

Acceptable payment methods are:• Cashier’s check

• Personal check

• Credit card

• Money Order

Any of the above forms of payment must be sent to the above address. Mail or call the Billing Department

When completing a requisition please include:• Patient’s complete name

• Patient’s date of birth

• Patient’s gender

• Medical record (optional)

• List all applicable ICD-9-CM diagnosis codes to their highest level of specificity

• Specimen collection date and time

• Ordering physician’s last and first name

• Treating physician’s last and first name

• Indicate by marking the “Bill To” billing option on the test request form (Client, Pathology Group, etc.)

• Patient’s status: Inpatient, Outpatient, or Non-Patient

Client Billing


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Patient Billing

As an alternative billing method, clients may choose to have their patients billed directly for laboratory services. When direct patient billing is requested, the patient’s complete name, address, and telephone number must be included in the space provided on the test request form. Clients may select either method of billing, subject to the requirements of local law.

When completing a patient billing request mark the “patient” billing option on the requisition and please include:

• Patient’s complete name

• Patient’s address (if “snowbird” please indicate northern address as well)

• Patient’s telephone number



• Patient’s status: inpatient, outpatient, or non-patient


• Responsible party’s name if other than patient

• Responsible party’s complete mailing address, if different from the patient’s



Patient Billing


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Third Party Billing

NeoGenomics Laboratories is a participating provider with Medicare. NeoGenomics Laboratories accepts assignment on all claims submitted to the Medicare and Medicaid programs.

In addition to Medicare and Medicaid, NeoGenomics Laboratories is enrolled with a large number o f private insurance companies and managed care organizations. For those insurance companies and managed care organizations where an agreement does not exist with NeoGenomics Laboratories, we will still file a claim to those carriers. As a service to your patients and in compliance with agreements established with insurance and managed care companies, NeoGenomics Laboratories will bill your patient’s primary insurance or managed care organization directly when provided with complete and accurate billing information.

As a general rule, third-party payers request the information listed below be provided in order to process a claim:




• Patient’s address



• Subscriber’s name if other than patient

• Subscriber’s date of birth

• Complete mailing address of patient or subscriber

• Patient’s relationship to subscriber, identified as: Self, Spouse, Child, and Other




• List all applicable ICD-9-CM diagnosis codes to their highest level of specificity

• Copy of card (front & back)

• Private Insurance: complete name and full address of the insurance company

• Medicare, Medicaid, or insured’s ID member number as it appears on the insurance card

• Group/policy number as it appears on the insurance card

• Employer’s name

Third Party Billing


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Medicare Billing

The Centers for Medicare and Medicaid Services (CMS) is responsible for administering the Medicare and Medicaid programs throughout the United States. In order to ensure that services being paid for by the Medicare program are medically necessary, CMS directed the individual Medicare carriers to identify laboratory tests that require additional medical necessity documentation. As a result, local carriers established a list of tests for which they require documentation of medical necessity before a claim may be paid. These are known as Local Coverage Determinations (LCDs). In addition, CMS itself established national policies over the years and, effective November 25, 2002, implemented the National Coverage Determinations (NCDs). While the NCDs replaced many of the existing local policies, there are still LCDs in effect. An LCD cannot be in direct conflict with any NCD. These are described in more detail below.

Medicare carriers have developed systems using ICD-9-CM diagnosis codes to prevent the payment of claims that they determine are not medically necessary. The patient’s diagnosis and the NCD or the LCD determines medical necessity. It is critical that the ICD-9-CM code(s) used for ordering laboratory services be consistent with the documentation in the patient’s medical records so that the medical necessity information is clear and apparent in the event of a post-payment review. The ICD-9-CM code(s) used must be specific and to the highest level of specificity for a particular patient and the laboratory test(s) ordered by the physician for the particular date of service.

Medicare continues to reimburse these procedures at 80% of the fee schedule amount and requires that the patient be billed for the remaining 20% coinsurance and any deductible amounts. The following information must be provided when NeoGenomics Laboratories is to bill Medicare directly. Indicate with a mark the “Medicare” billing option on the test request form.








• Ordering physician’s last and first name. Medicare requires the name of the physician ordering the test. If the account is set up for a multi-physician group, it is necessary to indicate which physician ordered the test.

• Treating physician’s last & first name.

• Medicare number as it appears on the Medicare card, complete with applicable prefix or suffix; indicate primary or secondary information

• All applicable ICD-9-CM diagnosis codes to their highest level of specificity

• Secondary insurance information if applicable

• Advance Beneficiary Notice (ABN). Patient should sign an ABN form, agreeing to be personally and fully responsible for payment if the physician believes that Medicare is likely to deny payment for the identified service(s).

Medicare Billing

Medicare Allowed at 100%

All Molecular TestingAll Cytogenetics Testing

Medicare Allowed at 80%

All Other Testing not Paid at 100%


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Medicare Billing

Advance Beneficiary NoticeIf reimbursement is denied due to lack of medical necessity documentation, Medicare rules prohibit the laboratory or health care provider from billing the patient unless an Advance Beneficiary Notice (ABN) has been signed and dated by the patient prior to the service. As applicable, an ABN must be completed each time services are ordered. Blanket ABNs are not acceptable to the Medicare program.

The Centers for Medicare and Medicaid Services (CMS) has established a standardized ABN format that must be used. The CMS ABN ensures that the patient understands he/she may be responsible for payment if the test is considered to be medically unnecessary by Medicare. The ABN identifies the laboratory test(s) subject to medical necessity coverage guidelines and gives the reason(s) the test(s) is likely to be denied. The test information on the ABN may be customized to the specific local Medicare carrier policies, identifying it as being subject to an NCD or an LCD.

ComplianceTo comply with these guidelines, physicians should (1) only order tests that are medically necessary for diagnosing or treating their patients; (2) be certain to enter the appropriate and correct ICD-9-CM code in both their patient files and on the test request forms; and (3) always have their patients sign and date an Advance Beneficiary Notice if they believe that the service is likely to be denied, and make sure that patients check an option to indicate their choices regarding the potentially non-covered care described in the body of the ABN.

National Coverage DeterminationsNational Coverage Determinations (NCDs) have been developed to standardize the list of laboratory procedures identified by the local Medicare carriers that require additional medical necessity documentation. The NCDs are consistent for all local Medicare carriers and have established the list of ICD-9-CM codes that are acceptable for each of these procedures. NCDs override and/or replace any local policies established by the local Medicare carriers for these same procedures. If a physician does not submit a supportive ICD-9-CM code for a procedure subject to NCDs, NeoGenomics Laboratories will contact that physician’s office to ask for a copy of a properly executed ABN or to determine whether there are any other applicable diagnoses for that date of service. In a post-payment audit, the requesting physician may still be asked to provide documentation that a procedure(s) was medically necessary for a particular patient’s condition. This is why it is critical for physicians to record appropriate documentation in patient records.


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Medicare Billing

Local Coverage DeterminationsLocal Medicare carriers have the authority to establish policies to limit coverage for laboratory procedures based on the carrier’s view of the medical necessity for that procedure. These local policies, known as Local Coverage Determinations (LCDs), may not in any way conflict with the National Coverage Determinations. The carrier with jurisdiction over the lab that is responsible for performing the service determines the limited coverage procedures for ordering physicians. Again, these procedures and codes vary from carrier to carrier. If a physician does not submit a supportive ICD-CM code for a limited-coverage procedure, NeoGenomics Laboratories will contact that physician’s office to ask for a copy of a properly executed ABN or to determine whether there are any other applicable diagnoses for that date of service. In a post payment audit, the requesting physician may still be asked to provide documentation that a procedure(s) was medically necessary for a particular patient’s condition. This is why it is critical for physicians to record appropriate documentation in patient records. Finally, in some instances, even if a physician thinks a particular test is appropriate for the indicated diagnosis or ICD-9-CM code listed on the test request form, the Medicare carrier with jurisdiction over our laboratory may not accept that code as supporting the medical necessity of the test. As a service to its clients, NeoGenomics Laboratories provides the booklet entitled, Medicare Medical Necessity: Documenting Medicare Medical Necessity of Laboratory Services.

CoverageNeoGenomics Laboratories agrees to accept the Medicare-allowed amount as payment in full for covered services. It is important to understand that assignment does not preclude billing the patient for services denied by Medicare. The following situations may result in a bill to the patient.

Non-Covered ServicesThese services include tests, visits, and procedures that do not meet the requirements of a Medicare benefit category, are statutorily excluded from coverage on grounds other than 1862(a)(1), or are not reasonable and necessary under 1862(a)(1). CMS has determined that it is the provider’s responsibility to inform patients in writing if a service may not be covered. The Medicare program does not cover tests that require but do not have FDA approval. These procedures are referred to as “Investigational use only” and “Research use only” procedures and will be billed to the patient, provided the beneficiary has signed (and NeoGenomics Laboratories has on file) a Medicare Advance Beneficiary Notice.

Not Medically NecessaryServices covered subject to an NCD or LCD, identified as not medically necessary prior to treatment and for which the beneficiary has signed/dated a Medicare Advance Beneficiary Notice that NeoGenomics Laboratories has on file.


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Medicare Billing

Medically Unlikely EditsTo lower the Medicare Fee-for-Service (FFS) paid claims error rate, CMS established units of service edits for Medicare Part B benefit claims referred to as MUEs. Just like the NCCI edit, the MUE edit is an automated prepayment edit that helps prevent inappropriate payments. As the submitted claim is processed by the Medicare claims processing contractors’ systems, the submitted procedures are analyzed to determine if they comply with the MUE policy. An MUE for a HCPCS/CPT code is the maximum units of service under most circumstances that a provider would report for a single beneficiary on a single date of service. MUEs are not meant to establish a Medicare payment policy, but rather improve the accuracy of Medicare payments. MUEs do not exist for all HCPCS/CPT codes.

CMS develops MUEs based on automatic considerations, HCPCS/CPT code descriptors, CPT coding instructions, established CMS policies, nature of service/procedure, nature of an analyte, nature of equipment, and clinical judgment. Prior to implementation of MUEs, national healthcare organizations are offered an opportunity to review and comment about proposed edits. The MUEs do not require Medicare contractors perform manual review or suspend claims. The MUEs only apply to the services specifically listed in the table of MUEs; thus all services will not have MUEs associated with them.

OverutilizationServices performed in excess of the utilization guidelines established under an NCD or LCD.

Medicare Secondary Payer (MSP)Medicare is not considered a primary payer when the beneficiaries ages 65-69 are members of an Employee Group Health Plan. Medicare is the secondary payer for an active individual, under age 65, who is entitled to Medicare on the basis of disability and is covered by a Large Group Health Plan (LGHP=100 or more employees) as a current employee or family member of such employee. Medicare is the secondary payer of claims for medical items and services to the extent that payment has been made or can reasonably be expected to be made for items or services under automobile, no-fault or any liability insurance. Medicare is the secondary payer for beneficiary if beneficiary has established Black Lung medical benefits under Department of Labor or the patient was ineligible on date of service and or patient has no Medicare Part B coverage.Medicare is the secondary payer for beneficiary if beneficiary experiencing End Stage Renal Disease (ESRD). Substantial changes were made to the MSP Disability and ESRD guidelines. The payment status changed for dually entitled individuals if EGHP coverage was a result of current employment prior to the date and the dual entitlement became effective.


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Medicaid Billing

Medicaid is medical assistance for those people who cannot afford their own health care. Medicaid claims can only be filed after all other third-party resources have been exhausted. Patients should be asked at the time of service if there is other coverage, such as Medicare, Medicaid HMO, or private insurance. When applicable, any Medicare, private insurance, or managed care (HMO) information should also be provided. Medicaid is also for persons that have applied for social security disability, but have not met the 18th month waiting period for Medicare eligibility.

When Completing a Medicaid Billing Request:Indicate with a mark the “Medicaid” billing option on the test request form. Each Medicaid agency has its own rules and regulations governing laboratory billing practices. Filing requirements are specific to each state.

Please include:

• Patient’s complete name as it appears on the Medicaid/Medi-Cal card






• Patient’s status: inpatient, outpatient, or nonpatient


• Ordering physician’s signature; necessary only in states where required

• Treating/PCP/PMP/clinician’s first and last name where required

• Provider/authorization number—where required

• List all applicable ICD-9-CM diagnosis codes to their highest level of specificity.

• Complete Medicaid/Medi-Cal number and state as it appears on the Medicaid card. Indicate primary or secondary information.

• If Medicare is primary and an Advance Beneficiary Notice (ABN) is needed, the patient should sign an ABN form when the physician believes that Medicare is likely to deny payment for the identified services.

Note: If Medicaid denies payment for non-covered services or eligibility reasons, the patient may be responsible for the payment. Medicaid is always the last source of payment.

Medicaid Billing

Specimen Requirements


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This section contains logistical details regarding specimen collection and transportation for specimens being sent to NeoGenomics Laboratories. In the following pages we detail Specimen Requirements, components of the Specimen Kits and how to complete a Test Requisition form.

Specimen Requirements & Handling Procedures• Specimen Requirements & Handling Procedures

Specimen Kits • Hematopathology Basic Kit (Tubes Only)

• Hematopathology Plus+ Kit (Tubes, Slides, Formalin)

• IHC/Solid Tumor Kit

• Bladder FISH kit

• Flex-Kit (Miscellaneous)

Test Requisitions and Shipping Instructions• Blue Pathology Requisition

• Red Oncology Requisition

• Green Immunohistochemistry and Solid Tumor Requisition

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DOS Rev • 01.04.13

Hematopathology Basic Kit

Hematopathology Basic KitSpecimen Kit

Kit Components• Dimensions (approx): 2 ¼ H x 5 ¼” W x 4 ½” D

• 1 x UN3373 Biological Substance Category B Marking (Bottom)

• 1 x Foam Insert

• 2 x 6 mL K2-EDTA Lavender Top Tube (Plastic)

• 2 x 6 mL Sodium Heparin Tube, Green Top

• 1 x Absorbent Sheet

• 1 x Biohazard Bag with doc pouch

• 1 x Cool Pack


DOS Rev • 01.04.13

Hematopathology Plus+ Kit

Hematopathology PLUS+ KitSpecimen Kit

Kit Components• Dimensions (approx): 2 ¼ H x 5 ½” W x 6” D


• 1 x Foam Insert

• 2 x 6 mL EDTA Lavender Top Tube (Plastic)

• 2 x 6 mL Sodium Heparin Tube, Green Top

• 2 x Formalin Vial

• 2 x Five Slide Mailer

• 10 x Frosted End / Positively Charged Slides


• 2 x Small biohazard bags or zip locks for formalin jars


• 1 x Cool Pack


DOS Rev • 01.04.13

IHC/Solid Tumor Kit

IHC/Solid Tumor KitSpecimen Kit

Kit Components• Dimensions (approx): 2 ¼ H x 5 ¼” W x 4 ½” D


• 1 x Foam Insert

• 2 x Five Slide Mailer



• 4 x Extra small zip lock bags for blocks

• 1 x Cool Pack


DOS Rev • 01.04.13

BLadder FISH Kit

Bladder FISH KitSpecimen Kit

Kit Components• Dimensions (approx): 4 ¾” H x 4 ¼” W x 4” D

• 1 x UN3373 Biological Substance Category B Marking (Back)

• 1 x Foam Insert

• 1 x Click-seal Container

• 1 x zip lock bag

• 1 x Urine StabilizerTablet


• 1 x ParaFilm strip


• 1 x Cool Pack


DOS Rev • 01.04.13

Miscellaneous Kit

FlexKIT (Miscellaneous) Specimen Kit

Kit Components• Dimensions (approx): 4 ¾” H x 4 ¼” W x 4” D

• 1 x UN3373 Biological Substance Category B Marking (Back)

• 1 x Foam Insert



• 1 x Zip lock Bag

• 1 x Cool Pack

• 1x ParaFilm strip


DOS Rev • 01.04.13

Hematopathology Requisition and Shipping Instructions

Hematopathology Requisition & Shipping Instructions

Requisition

Requisition completed by: Date Ordering Pathologist (please print): Treating Physician (please print):

Specimen ID#: Fixative/Preservative: Archived Specimen: 9 Yes 9 No Collection Date: / / Collection Time: 9 AM 9 PMBody Site: 9 Primary 9 MetastasisIf Metastasis, please list Primary: 9 Bone Marrow: Green Top(s) Purple Top(s) Core Biopsy Clot 9 Peripheral Blood: Green Top(s) Purple Top(s) Other 9 Fresh Tissue (Media Type required): 9 Fluid: CSF Pleural FNA Other 9 Smears: Air Dried Fixed Stained 9 Other:

(Please attach all relevant clinical history and pathology reports)9 New Diagnosis 9 Relapse 9 In Remission

Patient Name:

Date of Birth: MM / DD / YY Sex: 9 Male 9 Female

Medical Record #:

Social Security #:

Reason for Referral:

(Last) (First)

Patient Information

See Attached for Patient Address Information

Diagnosis Code/ICD-9 Code (required): Please document all applicable ICD-9 codes or narrative descriptions for all tests ordered supporting medical necessity which shall be used in patient plan of care.

Specimen Origin (Must Choose 1): Hospital Patient ( in/out ) 9 Non-Hospital Patient

Medicare Patient (Must Choose 1): Yes 9 No

If Hospital Patient and Medicare are marked above, NeoGenomics cannot legally bill

Medicare for technical pathology services. Therefore, the hospital or pathology group must

be billed. Hospital name MUST be present in Client Information section or written below.

9 See Client Information section for hospital name and address.

9 Hospital Name:

Bill to: Hospital 9 Pathology Group 9 Insurance/Medicare 9 Patient

NeoGenomics will bill Medicare for professional interpretation services if Medicare billing

information is attached.

Required: Please attach face sheet and front/back of patient insurance/Medicare card.

Comments:

Hematopathology RequisitionHistomorphology • Flow Cytometry • Cytogenetics • FISH • Molecular Testing

Florida • Tennessee • California

Toll Free: (866)776-5907Press #1 for Customer Service, #2 for Billing

FAX: (239)690-4237

www.neogenomics.com

9 ALK / Lymphoma (2p23)

9 API2/MALT1 t(11;18)

9 BCL6 (3q27)

9 BCR/ABL/ASS t(9;22)

9 CBFB (16q22)

9 CCND1/IgH t(11;14)

9 FGFR1 (8p11)

9 FGFR3/IgH t(4;14)

9 IgH (14q32)

9 IgH/BCL2 t(14;18)

9 IgH/MAF t(14;16)

9 MALT1 (18q21)

9 MLL (11q23)

9 MYC (8q24)

9 MYC/IgH/CEN8 t(8;14)

9 PDGFRa (4q12)

9 PDGFRb t(5;12)

9 PML / RARA t(15;17)

9 p53 (17p13.1)

9 RUNX1/RUX1T1(ETO/AML1) t(8;21)

9 5q-/-5/+5

9 7q-/-7

9 +8

9 13q-/-13

9 20q-

9 Other Global Flow Cytometry (With interpretation) 9 Extended Leukemia/ Lymphoma Panel (31 Markers)

9 Standard Leukemia/Lymphoma Panel (24 Markers)

9 PNH

9 ZAP-70 Lymphoid Panel

9 DNA Content & Cell Cycle Analysis

9 V-Beta T-cell Clonality

9 Additional Global Markers

NeoFlow / Tech-Only Flow Cytometry (No Interpretation)9 Extended Leukemia/ Lymphoma Panel (31 Markers)

9 Standard Leukemia/Lymphoma Panel (24 Markers)

9 ZAP-70 Lymphoid Panel

9 Additional Tech-Only Markers

9 Oncology Chromosome Analysis

9 Reflex to FISH if cytogenetics is normal (reflex FISH panel must be marked)

9 AML: Reflex to FLT3/NPM1 (molecular) when cytogenetics is intermediate risk

9 Other:

Level of Service must be marked.9 Global FISH (With Interpretation) 9 NeoFISH / Tech-Only FISH (No Interpretation)Hematologic FISH Panels

9 AML 9 MDS 9 CLL 9 NHL 9 High Risk MM

9 MM/MGUS

9 Available with Global MM/MGUS panel: Reflex to MM IgH Complex if IgH positive.

9 Available with Tech-Only MM/MGUS panel: Add IgH Complex to run concurrently.

Individual Probes (Please see back for FISH probes by disease state)

Specimen Hold Options (Please see back of requisition for full descriptions)Hold Specimens will result in billed charges to ordering client if testing is not performed. Do not mark a Hold Option when ordering reflex testing.

Flow Cytometry: Cytogenetics: 9 Refrigerate and Hold 9 Culture and Hold 9 Freeze and Hold Molecular Testing: FISH: 9 Freeze and Hold 9 Direct Harvest and Hold for FISH 9 Extract Nucleic Acid and Hold

Testing Services

For a complete list of our test menu please visit www.neogenomics.com • For solid tumor testing, please use the NeoGenomics Immunohistochemistry or Solid Tumor Testing requisitions.

His

tom

orp

holo

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Flo

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me

try

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tics

FISH

Mo

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tics

9 Hematopathology NeoAssist assumes morphology is being performed by ordering

pathologist. NeoGenomics will perform global Flow Cytometry & Cytogenetics automatically. If

deemed medically necessary, additional global testing will be ordered by NeoGenomics

pathologist to include FISH and / or molecular testing. (Please include CBC with requisition.)

9 Global Histomorphology / Full ConsultNeoGenomics pathologist will select testing deemed medically necessary to provide

comprehensive analysis and professional interpretation on all referred material. Please refer

to the back of the requisition for optimal specimen requirements. (Please provide recent CBC

report for global orders)

9 Tech-Only Histomorphology (No Interpretation) Must mark stain(s) below:

9 Iron

9 Kappa / Lambda (ISH)

9 EBER (ISH)

9 Reticulin

9 Wright-Giemsa

9 Other

9 ABL1 Kinase Domain (Gleevec® resistance)

9 B-Cell

9 B-Cell & T-Cell

9 BCL1, t(11;14)

9 BCL2, t(14;18)

9 BCR-ABL1, t(9;22)

9 Reflex to ABL1 Kinase Domain

9 BRAF

9 CEBPA

9 c-KIT

9 DNMT3A

9 FLT3

9 IDH1& IDH2

9 IgVH Mutation

9 inv(16) CBFB-MYH11

9 JAK2 Exon 12-14

9 JAK2 V617F

9 Reflex to JAK2 Exon 12-14

9 KRAS

9 MPL

9 MPN Reflex Panel

9 NPM1

9 NRAS

9 PML-RARA, t(15;17)

9 RUNX1-RUNX1T1 (AML1-ETO), t(8;21)

9 SF3B1

9 T-Cell

9 TP53

9 TPMT SNP

9 WT1

9 Other

9 FlexREPORT: Please add summary report option to this case.

NeoTYPE™ Hematology Profiles:9 NeoTYPE™ CLL Prognostic Profile: SF3B1, IgVH, ZAP-70 (FLOW), CLL FISH Panel

Individual Molecular Tests

Please see back of requisition for full test descriptions, specimen requirements and billing information. PATH REQ • Rev. 09042.12

Shipping Instructions

• Complete Blue Requisition form, making sure all sections are completed in their entirely which includes client, patient, coding, specimen, & billing information, and tests requested. Write patient name and DOB on appropriate number of labels provided with the requisition.

• Place a label on each tube, jar and/or slide. (Each label should have a requisition number, patient name, and DOB). A minimum of two patient identifiers is REQUIRED for each specimen.

• Ensure tube tops and/or slide holders are sealed tightly. Place labeled tubes and/or slide holders into foam insert. Ensure formalin jars are sealed tightly. Place labeled formalin jars separately into small biohazard bag before placing into foam cut-out. Ensure the lid of specimen jar is tightened past the “click” to prevent leakage in transit. Place strip of “Parafilm” around the lid where it meets the jar for additional protection.

• Remove as much air as possible from the biohazard bag and seal it. Place folded test requisition and/or manifest in pocket on side of biohazard bag.

• Place sealed bag with requisition back into box.

• Place cool pack in box, on top of biohazard bag. Do not allow cool pack to be in direct contact with specimen.

• Close box and tuck tabs into place. No tape necessary.


DOS Rev • 01.04.13

Oncology Requisition and Shipping Instructions

Oncology Requisition & Shipping Instructions

Requisition

Date of Birth: MM /DD /YY Sex: M / F(circle)

Medical Record #: Social Security #:

Requisition completed by: Ordering Physician (please print): Treating Physician (please print):

Diagnosis Code/ICD-9 Code(required):

Patient History/ Teatment: (please attach all relevant clinical history)

9 New Diagnosis 9 Relapse 9 In Remission

Patient Name: (Last) (First)

Client Information

Coding Information

Billing Information

Patient Information


RP-03 Rev. 6/11

9 Bone Marrow: Greem Top(s) Purple Top(s) Core Biopsy Clot

9 Peripheral Blood: Green Top(s)_____ Purple Top(s)_____ Other

9 Paraffin Blocks: 9 Fresh Tissue (Media Type required):

9 Fluid: CSF_____ Pleural_____ FNA_____ Other

9 Slides: Stained____ Unstained____

9 Smears: Air Dried____ Fixed_____ Stained_____

9 Urine 9 Bladder Wash 9 Renal Wash

9 Other:

Bill to: 9 Insurance 9 Patient 9 Physician Office *Please attach face sheet and front/back of patient insurance card ONLY if NeoGenomicswill bill patient insurance. Do not include patient insurance information if NeoGenomicswill bill Hospital or Pathology Group.

Patient Status (Choose 1): 9 In-Patient 9 Out-Patient 9 Non-Hospital Patient

See Attached for Patient Billing Information

(Possible ICD-9 codes listed on back of requisition)

Specimen Information

Please document all applicable ICD-9 codes or narrative descriptions for all tests ordered supporting medical necessity which shall be used in patient plan of care. Tests for Medicare patients must be screened to determine if an Advanced Beneficiary Notice (ABN) is required. (Please attach to requisition if required.) An ABN should be provided to the patient if there is a reason to believe Medicare will not pay for the test. Medicare may deny tests due to frequency. Medicare does not generally cover routine screening tests.

9 GPS™ (Genetic Pathology Solutions) may include morphology, flow cytometry,

cytogenetics, FISH, and / or molecular testing if deemed medically necessary. (Please

include recent CBC with requisition.)

Morphology9 Morphologic Evaluation 9 Smear for correlation only

Flow Cytometry (Leukemia and Lymphoma Immunophenotyping)

Standard Panels with Reflex Options Global Stand-Alone Panels

9 Bone Marrow (31 Markers) 9 PNH only

9 Peripheral Blood (28 Markers) 9 DNA Content & Cell Cycle Analysis only

9 Tissue/Fluid (26 Markers) 9 ZAP-70 Lymphoid Panel only

9 Add PNH to panel marked above 9 V-Beta T-cell Clonality

9 Add ZAP-70 to panel marked above

9 Reflex to DNA Content & Cell Cycle Analysis

9 Add V-Beta is abnormal T-cells present

Cytogenetics (specimen type must be noted)

9 Oncology Chromosome Analysis

9 Reflex to FISH if cytogenetics is normal (Reflex panel must be marked below)

9 Other:_____________________________________________________________________

FISH FISH Panels (Please see back for list of included FISH probes)

9 HER2 by FISH: HER2. cen 17 Site: Hours Fixed (req): 9 Bladder Cancer FISH (Bladder Panel)

9 MelanoSITE™ (Melanoma Panel)

9 With Comprehensive Consultation that may include IHC and/or special stains if deemed medically necessary. Available with Global MelanoSITE only.

9 AML 9 MDS 9 CLL 9 NHL 9 High Risk MMw

9 MM/MGUSw

9 Reflex to MM IgH Complex if IgH positive.

Individual Probes (please see back for FISH probes by disease state)9 ALK for NSCLC (2p23) 9 FGFR1 (8p11) 9 PDGFRb t(5;12)

9 ALK for Lymphoma (2p23) 9 IgH (14q32) 9 PML/RARA t(15;17)

9 API2/MALT1 t(11;18) 9 IgH/BCL2 t(14;18) 9 p53 (17p13.1)

9 BCL6 (3q27) 9 MALT1 (18q21) 9 5q-/-5

9 BCR/ABL t(9;22) 9 MLL (11q23) 9 7q-/-7

9 CBFB inv (16) 9 MYC (8q24) 9 +8

9 CCND1(BCL1)/IgH t(11;14) 9 MYC/IgH t(8;14) 9 13q-/-13

9 ETO/AML1 t(8;21) 9 PDGFRa (4q12) 9 20q-

9 Other:_____________________

Molecular Genetics (by PCR or RT-PCR)

Other Tests9 CTC Breast 9 CTC Colorectal 9 CTC Prostate

9 Myeloma Prognostic Risk Signature (MyPRS™)

Comments:___________________________________________________________

______________________________________________________________________

______________________________________________________________________

______________________________________________________________________

Testing Requested

Toll Free: (866)776-5907 Press #1 for Customer Service, #2 for Billing FAX: (239)768-0711

9 BRAF V600E (Colon, Lung) 9 BCL-1 (CCND1) 9 FLT3

9 BRAF V600E/K/D (Melanoma) 9 BCL-2 9 NPM1

9 EGFR 9 B Cell Clonality 9 MPN Reflex Panel

9 KRAS 9 T Cell Clonality 9 JAK2 V617F

9 BCR/ABL (Quant.) 9 PML/RARA 9 JAK2 Exon12

9 BCR/ABL (Qual.) 9 CEBPA 9 MPL W515

9 Other:______________________________________________________________________

Specimen ID#: __________________ Fixative/Preservative:_____________

Body Site: _________________________________ 9 Primary 9 Metastasis

If Metastasis, please list Primary:______________________________________________

Collection Date: _____/______/______ Collection Time: _________am pm

FemaleMale

PM


• Complete Red Requisition form, making sure all sections are completed in their entirely which includes client, patient, coding, specimen, & billing information, and tests requested. Write patient name and DOB on appropriate number of labels provided with the requisition.

• Place a label on each tube, jar and/or slide. (Each label should have a requisition number, patient name, and DOB). A minimum of two patient identifiers is REQUIRED for each specimen.

• Ensure tube tops and/or slide holders are sealed tightly. Place labeled tubes and/or slide holders into foam insert. Ensure formalin jars are sealed tightly. Place labeled formalin jars separately into small biohazard bag before placing into foam cut-out. Ensure the lid of specimen jar is tightened past the “click” to prevent leakage in transit. Place strip of “Parafilm” around the lid where it meets the jar for additional protection.






DOS Rev • 01.04.13

IHC Requisition and Shipping Instructions


• Complete Green Requisition form, making sure all sections are completed in their entirely which includes client, patient, coding, specimen, & billing information, and tests requested. Write patient name and DOB on appropriate number of labels provided with the requisition.

• Place a label on each slide holder and/or block. (Each label should have a requisition number, patient name, and patient DOB). A minimum of two patient identifiers is REQUIRED for each slide holder and/or block.

• Ensure slide holders are closed and sealed tightly. Ensure block cassettes are protected in gauze or individual small sealed bags. Place slides and/or blocks into foam insert.

• Lift foam insert from box and place into biohazard bag along with absorbent sheet.





IHC Requisition & Shipping Instructions

Requisition

Immunohistochemistry RequisitionIHC • Special Stains • ISH • Imaging



FAX: (239)690-4237

www.neogenomics.com

Client Information

Testing Services

IHC

Pa

nels

Coding Information

Billing Information

Adenocarcinoma vs. MesotheliomaBladder vs. Prostate CarcinomaBreast Panel Carcinoma Unknown Primary Site, Female (CUPS-Female) Carcinoma Unknown Primary Site, Male (CUPS-Male) GISTHepatoma/Cholangio vs. Metastatic CarcinomaLung CancerLung vs. Metastatic Breast Carcinoma Lymphoma vs. CarcinomaLymphoma vs. Reactive HyperplasiaMelanoma vs. SqCCAMismatch Repair (MMR) / ColonNeuroendocrine NeoplasmPlasma Cell NeoplasmProstate vs Colon CarcinomaSoft Tissue TumorUndifferentiated Tumor

Global /Tech-Only

Global /Tech-Only

999999999999999999

999999999999999999

999999999

999999999

99

99

Global / Tech-Only

999999999999999999999999999999999999999999999999999

99999999999999999999999999999

9999999999999

99999999999999999999999999999

9999999999999

999999999999999999999999999999999999999999999999999

99999999999999999999999999999999999999999999

99999999999999999999999999999999999999999999

Global / Tech-OnlyGlobal / Tech-Only

Global /Tech-Only

Reflex HER2 FISH options available only with global IHC orders, Tech-Only Clients will use the test add-on process:

9 Reflex to Global HER2 FISH if IHC is ___1+___2+___3+9 Reflex to Tech-Only HER2 FISH if IHC is ___1+___2+___3+

Concurrent HER2 FISH Options available with global and tech-only IHC orders:

9 Global HER2 FISH concurrent with IHC9 Tech-Only HER2 FISH concurrent with IHC

AFBCongo Red/AmyloidGMSIronPAS-HPAS-FungusReticulinTrichromeWright-Giemsa

EBER (ISH)Kappa (ISH) / Lambda (ISH)

Breast Testing Fixation: CAP/ASCO Requirement for Global and Consult Breast Testing

Cold Ischemic time ≤ 1 hour: 9 Yes 9 No

10% neutral buffered formalin: 9 Yes 9 No 9 Unknown

HER2 Fixation duration >6 and <48 hours: 9 Yes 9 No 9 Unknown

ER/PR Fixation duration >6 and <72 hours:* 9 Yes 9 No 9 Unknown* If ER/PR/HER2 is ordered on same block, proper fixation is 6 - 48 hours. If ER/PR is ordered alone, proper fixation is 6 -72 hours.


Requisition completed by: Date

Ordering Pathologist (please print):

Treating Physician (please print):

Specimen ID#: Fixative/Preservative:

Collection Date: / / Collection Time: 9 AM 9 PM

Body Site: 9 Primary 9 Metastasis

If Metastasis, please list Primary:

9 Paraffin Block(s): 9 Fresh Tissue (Media Type required):

9 Slides: Stained Unstained

9 Other:


Patient Name:


Medical Record #:

Social Security #:


(Last) (First)

Patient Information









9 Hospital Name:





Comments:

Please see back of requisition for full test descriptions, specimen requirements and billing information. IHC/ISH REQ • Rev. 09.06.12 For a complete list of our test menu please visit www.neogenomics.com

IHC

Sp

ec

ial S

tain

sIS

HIm

ag

e A

naly

sis

NeoImage: Tech-Only Image Analysis (available on breast stains only). Please mark panel or individual stains below.

9 Breast Panel (ER, PR, Ki-67, HER2, p53)

9 ER 9 PR 9 Ki-67 9 HER2 9 p53


Ind

ivid

ua

l A

nti

bo

die

s

AE1 (Cytokeratin LMW)AE1/AE3 AE1/AE3 (pan-CK)AFP ALK1Amyloid AAmyloid PAnnexin-1BCA225BCL-1 (Cyclin D1)BCL-2BCL-6BerEP4Beta-CateninBG-8CA19-9CA125CalcitoninCaldesmonCalponin-1CalretininCAM 5.2CD1a CD2CD3 (Monoclonal)CD4CD5CD7CD8CD10CD15 CD19CD20 CD21CD22CD23CD30 CD31CD34 CD43CD45 CD56CD57CD61CD68CD79aCD117CD138CDX2CEA (Monoclonal) CEA (Polyclonal)

Chromogranin ACollagen IVCK 5/6CK 7CK 8/18CK 17CK 19CK 20CK 903 (Cytokeratin HMW)CK 903+p63Cytokeratin LMW (AE1)Cytokeratin HMW (CK 903)DesminDOG-1 E-CadherinERA (MOC-31)ERFactor VIIIFactor XIIIaGalectin-3GATA-3GCDFP-15 GFAP Glycophorin AGlypican 3H. PyloriHCGHBME-1HER-2 (IHC)

HHV-8HMB-45HSA Hep Par 1InhibinKappa (IHC) / Lambda (IHC)Ki-67MammaglobinMART-1/Melan AMLH1MOC-31 (ERA)MPOMSH2MSH6

MUC1MUC2MUM1MyoglobinNapsin ANKX 3.1NSEp16p21p53p63p504s (Racemace)Pan-CK (AE1/AE3)Pan-CK+CAM 5.2PankeratinPan-MelanomaPAX-5PAX-8PIN (Prostate)PLAP PMS2PRPSAPSAPRacemace (p504s)RCCS-100SecretagoginSMASMMSmoothelinSurfactant Protein ASynaptophysinTAG 72TdTThyroglobulinTRAPTryptaseTTF-1 TyrosinaseUroplakinVillinVimentinWT-1

9 Full Consult: NeoGenomics pathologist will select testing deemed medically necessary to provide comprehensive analysis and professional interpretation on all referred material. Please refer to the back of the requisition for optimal specimen requirements.

Other:


DOS Rev • 01.04.13

Solid Tumor Requisition and Shipping Instructions


• Complete Grey and Green Requisition form, making sure all sections are completed in their entirely which includes client, patient, coding, specimen, & billing information, and tests requested. Write patient name and DOB on appropriate number of labels provided with the requisition.

• Place a label on each slide holder and/or block. (Each label should have a requisition number, patient name, and patient DOB). A minimum of two patient identifiers is REQUIRED for each slide holder and/or block.

• Ensure slide holders are closed and sealed tightly. Ensure block cassettes are protected in gauze or individual small sealed bags. Place slides and/or blocks into foam insert.

• Lift foam insert from box and place into biohazard bag along with absorbent sheet.





Solid Tumor Requisition & Shipping Instructions

Requisition

Solid Tumor RequisitionPrognostic • Therapeutic • Tumor Profiling



FAX: (239)690-4237

www.neogenomics.com

Client Information

Coding Information

Billing Information*

Breast Testing Fixation: CAP/ASCO Requirement for Global and Consult Breast Testing

Cold Ischemic time ≤ 1 hour: 9 Yes 9 No

10% neutral buffered formalin: 9 Yes 9 No 9 Unknown

HER2 Fixation duration >6 and <48 hours: 9 Yes 9 No 9 Unknown

ER/PR Fixation duration >6 and <72 hours:* 9 Yes 9 No 9 Unknown* If ER/PR/HER2 is ordered on same block, proper fixation is 6 - 48 hours. If ER/PR is ordered alone, proper fixation is 6 -72 hours.


Requisition completed by: Date

Ordering Pathologist (please print):

Treating Physician (please print):

Specimen ID#: Fixative/Preservative:

Collection Date: / / Collection Time: 9 AM 9 PM

Body Site: 9 Primary 9 Metastasis

If Metastasis, please list Primary:

9 Paraffin Block(s): 9 Fresh Tissue (Media Type required):

9 Slides: Stained Unstained

9 Other:


Patient Name:


Medical Record #:

Social Security #:


(Last) (First)

Patient Information









9 Hospital Name:





Comments:

Testing Services

*Please see back of requisition for full test descriptions, specimen requirements and billing information. Solid Tumor Req • Rev. 09.17.12 For a complete list of our test menu please visit www.neogenomics.com/testing-services-test-menu.htm

Test

ing

By

Ca

nc

er

Typ

e

9 Full Consult (Available as global testing only): NeoGenomics pathologist will select testing deemed medically necessary to provide comprehensive analysis and professional interpretation on all referred material. Please refer to the back of the requisition for optimal specimen requirements.


MELANOMA AND THYROID

9 BRAF

9 BRAF

BLADDER CANCER

CUSTOM PANELS

9 Bladder Cancer (FISH)

9 MelanoSITE™ (Melanoma FISH)

9 Reflex to HER2 (FISH) if HER2 (IHC) or within Breast Panel (IHC) is 9 1+9 2+9 3+.

9 Reflex to HER2 (IHC) if HER2 (FISH) is 9 Non-Amplified 9 Equivocal.

9 Breast Panel (IHC)9 HER2 (IHC)

9 HER2 (FISH)

Stand-Alone / Add-On Tests

BREAST CANCER9 HER2 (IHC)

9 HER2 (FISH)

GASTRIC CANCER

9 Reflex to HER2 (IHC) if HER2 (FISH) is non-amplified (ratio <2.0).

9 Reflex to HER2 (FISH) if HER2 (IHC) is 9 1+9 2+9 3+.

9 Reflex to ALK (FISH) if EGFR is negative.

9 ALK (FISH)9 EGFR (Molecular)

LUNG CANCER

9 NeoTYPE Breast Tumor Profile (BRAF, c-KIT, EGFR, PIK3CA, TP53, HER2 FISH)

9 NeoTYPE Colorectal Tumor Profile (BRAF, c-KIT, DNMT3A, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, TP53, MSI)

9 NeoTYPE Gastric Tumor Profile (BRAF, c-KIT, DNMT3A, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, TP53, HER2 FISH)

9 NeoTYPE Lung Tumor Profile (BRAF, c-KIT, DNMT3A, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, TP53, ALK FISH)

9 NeoTYPE Solid Tumor (Other) Profile (BRAF, c-KIT, DNMT3A, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, TP53)

IHC: 9 Global (With Interpretation) 9 Tech-Only (Without Interpretation)

FISH: 9 Global (With Interpretation) 9 Tech-Only (Without Interpretation)

Molecular Microdissection: 9 Global 9 Tech-Only (Estimated percentage tumor within circle (required): %)

Reflex options are available with global test orders only. Tech-Only clients must use the test add-on process. Concurrent test options are available with global AND tech-only test orders.

9 Reflex to MSI (Molecular) if any IHC marker in MMR panel is not expressed.9 Reflex to BRAF (Molecular) if MLH-1 (IHC) is not expressed.

9 Reflex to MMR (IHC) if MSI results unstable.

9 Reflex to BRAF if KRAS is negative.

COLORECTAL CANCER9 Mismatch Repair / MMR (IHC)

9 MSI (Molecular)

9 KRAS (Molecular)

Ne

oTY

PES

NeoTYPE Solid Tumor Profiles (Available as global testing only)

Level of Service Level of Service must be indicated for all primary, reflex, and concurrent IHC, FISH, and Molecular testing marked below. Global testing will be performed unless otherwise noted.

9 BRAF 9 PIK3CA 9 TP53




9 Other

9 Other

9 Other

9 Other

9 Other

9 EGFR (Molecular)9 c-KIT9 EGFR (Molecular)9 c-KIT

9 c-KIT 9 TP539 BRAF

9 NRAS

9 NRAS

9 PIK3CA 9 TP53



9 Other

9 c-KIT

9 c-KIT

9 NRAS 9 PIK3CA9 TP53

9 KRAS

9 KRAS

9 KRAS9 BRAF

Test Menu & Descriptions


DOS Rev • 01.04.13

Service Offerings• Genetic Pathology Solution (GPS)

Methodologies• Fluorescence In Situ Hybridization

• Flow Cytometry

• Molecular Genetics

• Cytogenetics

• Immunohistochemistry

CPT Code DisclaimerThe CPT Codes provided in this section follow American Medical Association (AMA) guidelines and are intended for informational purposes only. It is the sole responsibility of the client to select the appropriate CPT codes and number of units for their patients at all times.



DOS Rev • 01.04.13

Genetic Pathology Solutions

GPS is a medically driven testing program which employs multiple testing methodologies under the direction of a board-certified hematopathologist in order to render a definitive diagnosis. It allows the hematopathologist to initiate one or more of following methodologies: Morphology, Flow Cytometry, Cytogentics/FISH and/or Molecular Genetics. This provides you with:

• Faster results• Elimination of unnecessary testing• Better controlled costs• A higher level of patient care

To order the GPS testing option, simply check the GPS box on the red oncology requisition and ship specimen to our laboratory using the red Hematopathology PLUS+ Kit. Only the appropriate tests will be run and reported individually to the physician. All final results are then provided on a comprehensive report with summary, interpretation, and recommendations.

Program Highlights

Access to Expertise• All cases are managed, read, and signed out by a board-certified hematopathologist.• Component tests are reviewed and completed by certified experts in their respective field.• Expect to be contacted by a hematopathologist to discuss significant diagnoses.• Hematopathologists and cytogenetic professionals are always available for consultation.

Advanced Report Capability• Individual preliminary reports based on each methodology are delivered to the referring hematologist/

oncologist upon completion.• Comprehensive GPS final report summarizes all testing methodologies employed to reach a definitive

diagnosis.• Most informative images from each test methodology are Incorporated into final report.

10-Color Flow CytometryNeoGenomics uses 10-color flow cytometry for improved efficiency and reliability in evaluation and diagnosis of hematopoietic diseases. Some of the advantages over services using fewer fluorochromes are:

• More precise characterization of various cell populations.• Improved detection of low-frequency abnormalities, which is especially important in monitoring minimal

residual disease.• Higher information content per tube conserves small specimens and minimizes chance of incomplete

analyses.• Reduced redundancy in use of antibodies, which helps control costs.• Simpler analysis of the data generated, with shorter reports.



DOS Rev • 01.04.13


Preliminary and Final Result ScheduleTests results can be anticipated on the following days

GPS cases not requiring molecular testing are generally completed by 5 days after receipt.

One Day

X

Two Days Three Days Four Days Five Days Six Days Seven Days

Flow Cytometry

Morphology

FISH

Cytogenetics

Molecular

Final Report

X

X

X

X

X


DOS Rev • 01.04.13

FISH

Fluorescence in situ hybridization (FISH) can help identify subtle or sub-microscopic structural rearrangements, variant chromosomes, and low-frequency abnormalities not readily detectable by classic cytogenetics. FISH incorporates three technologies – cytogenetics, DNA hybridization, and fluorescent microscopy – to provide a uniquely informative combination of resolution and breadth. FISH is also called Molecular Cytogenetics.

FISH tests can be ordered as disease-specific panels or for detection of single abnormalities. For the panels shown below, clients may also choose probes separately on our test requisition.

ALK (2p23) for Lung CancerProbe Set: ALK (2p23) CPT: 88367(x2) Automated or 88368 (x2) Manual Specimen Requirements:

• Paraffin Block: Block plus one H&E slide (circle area of invasive tumor, if tech-only). H&E slide is required. • Slides: 4 positively charged unstained slides

Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turn Around Time: 3-5 days

ALK (2p23) for LymphomaProbe Set: ALK (2p23) CPT: 88367(x2) Automated or 88368 (x2) Manual Specimen Requirements:

• Bone Marrow Aspirate: 1-2mL Sodium Heparin Tube. EDTA tube is acceptable• Peripheral Blood: 2-5mL Sodium Heparin Tube. EDTA tube is acceptable• Fresh, Unfixed Tissue: Tissue in RPMI• Fluids: Equal parts RPMI to specimen volume.• Paraffin Block: Send paraffin block.• Cut Slides: 2 unstained slides cut at 4 microns.

Storage and Transportation: Refrigerate unfixed specimens; do not freeze. For all specimens, use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turn Around Time: Unfixed specimen: 3-5 days. FFPE Specimens: 7 days.

Fluorescence In Situ Hybridization (FISH)

Not all FISH Assays are available from all NeoGenomics sites for NYS specimens. If we are unable to perform due to licensure requirements, the specimen may be sent to a licensed NYS reference laboratory.


DOS Rev • 01.04.13

FISH


AML Panel (Acute Myeloid Leukemia)Probe Set: 5q-/-5/+5, 7q-/-7, +8, MLL (11q23), RUNX1/RUX1T1 (ETO/AML1) t(8;21), PML/RARA t(15;17), CBFB (16q22)CPT: 88367(x13) Automated or 88368(x13) ManualSpecimen Requirements:

• Bone Marrow Aspirate: 1-2mL Sodium Heparin Tube. EDTA tube is acceptable.• Peripheral Blood: 2-5mL Sodium Heparin Tube. EDTA tube is acceptable.• Fresh, Unfixed Tissue: Tissue in RMPI• Fluids: Equal part RPMI to specimen volume.


Bladder Cancer FISHProbe Set: CEN3, CEN7, CEN17, p16CPT: 88121(x1) Automated or 88121(x1) ManualSpecimen Requirements:

• Voided Urine: 50mL with supplied fixative tabletStorage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turn Around Time: 3-5 days

Burkitt LymphomaProbe Set: t(8;14), MYC/IgH, CEN8CPT:88367(x3) Automated or 88368(x3) ManualSpecimen Requirements:

• Bone marrow aspirate: 1-2 mL in sodium heparin tube. EDTA tube is acceptable.• Peripheral blood: 2-5 mL in sodium heparin tube. EDTA tube is acceptable.• Fresh tissue: Tissue in RPMI.• Fluids: Equal parts RPMI to specimen volume.• Paraffin block: Send paraffin block.• Cut slides: 2 unstained slides cut at 4 microns.

Storage and Transportation: Refrigerate unfixed specimens; do not freeze. For all specimens, use cold pack for transport, making sure cold pack is not in direct contact with specimen Turn Around Time: Unfixed specimens: 4 days. FFPE specimens: 7 days


DOS Rev • 01.04.13

FISH


CLL Panel (Chronic Lymphocytic Leukemia)Probe Set: ATM (11q-), p53 (17p13.1), CEN12 (+12), 13q-/-13, CCND1/IgH t(11;14)CPT: 88367(x7) Automated or 88368(x7) ManualSpecimen Requirements:


Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turn Around Time: 3-5 days CML (Chronic Myelogenous Leukemia) Probe Set: BCR/ABL/ASS t(9;22)CPT:88367(x3) Automated or 88368(x3) ManualSpecimen Requirements:



HER2 FISHProbe Set: HER2, cen 17CPT: 88367(x2) Automated or 88368(x2) ManualSpecimen Requirements:

• Paraffin Block: 1 H&E slide (invasive area circled if tech-only), sliced 4-5 microns• Cut Slides: 3 cut slides with 1 H&E slide (circled if tech-only), sliced 4-5 microns



DOS Rev • 01.04.13

FISH


HER2 Non-Breast Probe Set: HER2, cen 17CPT: 88367(x2) Automated or 88368(x2) ManualSpecimen Requirements:

• Tissues must be fixed in formalin for 6-48 hours; zinc fixatives should be avoided. • All tissues are acceptable with the exception of bone, which undergoes decalcification.• Paraffin Block with 1 H&E slide (invasive area circled if tech-only), sliced at 4-5 microns.• Cut slides: 3 cut slides with 1 H&E (circled if tech-only), sliced at 4-5 microns.


High Risk MM Panel *May include plasma cell enrichment*Probe Set: FGFR3/IgH t (4;14), IgH/MAF t(14;16), 13q-/-13, p53 (17p13.1)CPT: 88368(x7) and 88112(*)Specimen Requirements:

• Bone Marrow Aspirate: 2mL Sodium Heparin Tube. EDTA tube is acceptable.Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turn Around Time: 3-5 days

MDS Panel (Myelodysplasia)Probe Set: 5q-/-5/+5, 7q-/-7, +8, 20q-CPT: 88367(x6) Automated or 88368(x6) ManualSpecimen Requirements:

• Bone Marrow Aspirate: 1-2mL Sodium Heparin tube. EDTA is acceptable. (unless Cytogenetics is ordered)• Peripheral Blood: 2-5mL Sodium Heparin tube. EDTA is acceptable. (unless Cytogenetics is ordered)• Fresh, Unfixed Tissue: Tissue in RMPI• Fluids: Equal part RPMI to specimen volume.


MelanoSITE™ (Melanoma)Probe Set: 6p25, cen 6, 6q23, 11q13CPT: 88367(x4) Automated or 88368(x4) ManualSpecimen Requirements:

• 1 H&E slide plus tissue block or 3-5 unstained slides cut at 5 microns. For tech-only, circle area of interest on H&E slide (required).

Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen.Turn Around Time: 3-5 days


DOS Rev • 01.04.13

FISH


MM IgH Complex Panel *May include plasma cell enrichment*Probe Set: CCND1/IgH t(11;14), FGFR3/IgH t(4;14), IgH/MAF t(14;16)CPT: 88367(x6) Automated or 88368(x6) ManualSpecimen Requirements:

• Bone Marrow Aspirate: 1-2mL Sodium Heparin tube. EDTA is acceptable.• Peripheral Blood: 2-5mL Sodium Heparin tube. EDTA is acceptable.• Fresh, Unfixed Tissue: Tissue in RMPI• Fluids: Equal part RPMI to specimen volume.


MM Panel (Multiple Myeloma) *May include plasma cell enrichment*Probe Set: 1q+, +3, +5, +9, 13q-/-13, IgH, and 17q-(p53)CPT: 88367(x11) Automated or 88368(x11) ManualSpecimen Requirements:

• Bone Marrow Aspirate: 1-2mL Sodium Heparin tube. EDTA is acceptable. • Peripheral Blood: 2-5mL Sodium Heparin tube. EDTA is acceptable. • Fresh, Unfixed Tissue: Tissue in RMPI• Fluids: Equal part RPMI to specimen volume.


NHL Panel (Non-Hodgkin’s Lymphoma)Probe Set: ALK for Lymphoma (2p23), BCL6 (3q27), MYC (8q24), CCND1/IgH t(11;14), IgH (14q32), IgH/BCL2 t(14;18), MALT1 (18q21) CPT: 88367(x14) Automated or 88368(x14) ManualSpecimen Requirements:

• Bone Marrow Aspirate: 1-2mL Sodium Heparin Tube. EDTA tube is acceptable• Peripheral Blood: 2-5mL Sodium Heparin Tube. EDTA tube is acceptable• Fresh, Unfixed Tissue: Tissue in RPMI• Fluids: Equal parts RPMI to specimen volume.• Paraffin Block: Send paraffin block• Cut Slide: 12 unstained slides cut at 4 microns.

Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turn Around Time: Unfixed specimen: 3-5 days. FFPE Specimens: 7 days.



DOS Rev • 01.04.13

PDGFRa

Probe Set: PDGFR (4q12)CPT:88367(x3) Automated or 88368(x3) ManualSpecimen Requirements:



t(11;18)Probe Set: t(11;18), API2/MALT1CPT:88367(x2) Automated or 88368(x2) ManualSpecimen Requirements:




DOS Rev • 01.04.13

FISH Probes by Disease State

FISH Probes by Disease StateDisease State/Panel FISH Probe(s)

MM IgH Complex

NSCLC

MDS

Myeloproliferative Neoplasm

NHL

MM-MGUS

Melanoma

Diffuse Large Cell Lymphoma

MALT Lymphoma

High Risk MM

Marginal Zone B-cell Lymphoma

Follicular Lymphoma

Mantle Cell Lymphoma

CLL

CML

Burkitt Lymphoma

Bladder Cancer

Breast Cancer

Anaplasic Large Cell Lymphoma

AML M2

AML M3 (APL)

AML M4

AML

CCND1/IgH t(11;14), FGFR3/IgH t(4;14), IgH/MAF t(14;16)

ALK for NSCLC (2p23)

5q-/-5/+5, 7q-/-7, +8, 20q-

PDGFRa (4q12)

ALK for Lymphoma (2p23), BCL6 (3q27), MYC (8q24), CCND1/IgH t(11;14), IgH (14q32),

IgH/BCL2 t(14;18), MALT1 (18q21)

1q+, +3, +9, +5, 13q-/-13, IgH (14q32), p53 (17p13.1)

RREB1, CEN6, MYB, CCND1

BCL6 (3q27)

AP12/MALT1 t(11;18)

FGFR3/IgH t (4;14), IgH/MAF t(14;16), 13q-/-13, p53 (17p13.1)

MALT1 (18q21)

IgH/BCL2 t(14;18)

CCND1/IgH t(11;14)

ATM (11q-), p53 (17p13.1), CEN12 (+12), 13q-/-13, CCND1/IgH t(11;14)

BCR/ABL/ASS t(9;22)

MYC/IgH t(8;14)

CEN3, CEN7, CEN17, p16

HER2, CEN17

ALK for Lymphoma (2p23)

RUNX1/RUX1T1 (ETO/AML1) t(8;21)

PML/RARA t(15;17)

CBFB (16q22)

5q-/-5/+5, 7q-/-7, +8, MLL (11q23), RUNX1/RUX1T1 (ETO/AML1) t(8;21), PML/RARA t(15;17), CBFB (16q22)

ALL BCR/ABL/ASS t(9;22), MLL (11q23)


DOS Rev • 01.04.13

Flow Cytometry

NeoGenomics uses 10-color flow cytometry for improved efficiency and reliability in evaluation and diagnosis of hematopoietic diseases. Some of the advantages of our 10-color flow cytometry service over those detecting fewer fluorochromes are:

• More precise characterization of various cell populations• Improved detection of low-frequency abnormalities• Higher information content per tube conserves small specimens and minimizes chance of incomplete

analyses• Reduced redundancy in use of antibodies, which helps control costs• Faster signal acquisition for better turnaround time• Simpler analysis of the data generated, with shorter reports

Standard Leukemia/Lymphoma Panel – 24 markers Description: Available as global and tech-only. Markers are CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD13, CD14, CD16, CD19, CD20, CD23, CD33, CD34, CD38, CD45, CD56, CD64, CD117, HLA-DR, kappa, and lambda. Clinical Significance: Useful to aid in diagnosis of leukemia and lymphoma, and for post-treatment follow-up.Specimen Requirements:

• Bone marrow aspirate or peripheral blood: 1-2 mL EDTA preferred. 1-2 mL minimum in sodium heparin tube or ACD (pale yellow/no gel separator) is acceptable. Please provide recent CBC report.

• Bone marrow core: 1-2 cm minimum core length in RPMI. • Fresh, unfixed tissue: 0.2 cm3 minimum in RPMI. • Fluids and FNAs: Equal parts RPMI and specimen.

Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 24 hours CPT: 88184(x1), 88185(x23). Add 88189(x1) for global.

Extended Leukemia/Lymphoma Panel – 31 markers Description: Available as global and tech-only. Markers are CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD23, CD33, CD34, CD38, CD41, CD45, CD56, CD64, CD71, CD117, CD138, CD235a, FMC-7, HLA-DR, kappa, and lambda.Clinical Significance: For diagnosis of leukemia, lymphoma, plasma cell neoplasms, and evaluation of myeloid maturation.Specimen Requirements:



Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 24 hours CPT: 88184(x1), 88185(x30). Add 88189(x1) for global.

Flow Cytometry


DOS Rev • 01.04.13

Flow Cytometry

ZAP-70 Lymphoid Panel Description: Available as global and tech-only. Markers are CD3, CD5, CD19, CD45, and ZAP-70. Clinical Significance: Over-expression of ZAP-70 is a poor prognostic marker in CLL/SLL.Specimen Requirements:



Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 24 hours CPT: 88184, 88185(x4). Add 88187(x1) for global.

High Sensitivity PNH Evaluation Description: Available as global test only. Markers are CD15, CD33, CD45, CD55, CD59, CD235a, and FLAER. Clinical Significance: Useful for diagnosis of PNH and monitoring response to therapy. Small PNH clones may also be identified in patients with aplastic anemia and MDS who may respond to immune modulation therapy. Also identifies patients at increased risk of developing overt PNH. Specimen Requirements:



Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 24 hours CPT: 88184(x1), 88185(x6), 88187(x1).

DNA Content & Cell Cycle Analysis Description: Available as global test only. DNA stain is Draq-5.Clinical Significance: DNA content and cell cycle analysis measures the chromosome content of cancer cells. The prognostic importance of aneuploidy (chromosome number greater or less than the normal diploid number of 46) depends on the tumor type. Specimen Requirements:

• • Bone marrow aspirate or peripheral blood: 1-2 mL EDTA preferred. 1-2 mL minimum in sodium heparin tube or ACD (pale yellow/no gel separator) is acceptable. Please provide recent CBC report.

• • Bone marrow core: 1-2 cm minimum core length in RPMI. • • Fresh, unfixed tissue: 0.2 cm3 minimum in RPMI. • • Fluids and FNAs: Equal parts RPMI and specimen.

Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 24 hours CPT: 88182



DOS Rev • 01.04.13

V-Beta T-Cell Clonality Description: Available as global test only. Markers are VB1, VB2, VB3, VB4, VB5.1, VB5.2, VB5.3, VB7.1, VB7.2, VB8, VB9, VB11, VB12, VB13.1, VB13.2, VB13.6, VB14, VB16, VB17, VB18, VB20, VB21.3, VB22, and VB23 (24 markers). Clinical Significance: Useful in identification of clonal T-cell populations to aid in diagnosis of T-cell lymphoproliferative disorders. Clonal T-cell populations can be identified in some reactive conditions and in some healthy elderly patients. Correlation with morphology, clinical history, and other diagnostic information is necessary for diagnosis. Specimen Requirements:



Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 24 hours CPT: 88184(x1), 88185(x23), 88189(x1).


DOS Rev • 01.04.13

Flow Cytometry Antibody Library

Flow Cytometry Antibody Library

BCL-2

CD1a

CD10

CD103

CD117

CD11b

CD11c

CD13

CD138

CD14

CD15

CD16

CD19

CD2

CD20

CD22

CD23

CD235a (glycophorin a)

CD25

CD3

CD30

CD33

CD34

CD36

CD38

CD4

CD41

CD45

CD5

CD55

CD56

CD57

CD59

CD64

CD7

CD71

CD79a

CD8

FLAER

FMC-7

Gylcophorin a (CD235a)

HLA-DR

Kappa (polyclonal & monoclonal)

Lambda (polyclonal & monoclonal)

MPO

TCR alpha/beta

TCR gamma/delta

TdT (nuclear)

V-Beta T-Cell Clonality

ZAP-70


DOS Rev • 01.04.13

Molecular Genetics

Molecular testing has become the primary tool for precision medicine. NeoGenomics is dedicated to bringing our clients a full complement of clinically relevant diagnostic, predictive, and prognostic tests.

ABL1 Kinase Domain Mutation AnalysisDescription: RT-PCR and sequencing of the BCR-ABL1 fusion transcript for qualitative detection of mutations associated with imatinib resistance.Clinical Significance: Testing is recommended in CML with poor initial response to imatinib, relapse, or progression to accelerated/blast phase. Presence and identity of mutation may direct management to alternative drugs or stem cell transplant. Specimen Requirements:

• Bone marrow: 2 mL in EDTA tube• Peripheral Blood: 5 mL in EDTA tube

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred. Turnaround Time: 10 days CPT, AMA 2013: 81401

AML Reflex PanelDescription: Routine cytogenetics with automatic addition of the NeoTYPE™ AML Prognostic Profile when cytogenetics results show intermediate risk including normal cytogenetics, +6, +8, -Y, or del(12p). The AML Prognostic Profile includes concurrent analysis of select exons of the following genes: CEBPA, DNMT3A, FLT3, IDH1, IHD2, NPM1, and WT1.Clinical Significance: Molecular profiling with the NeoTYPE AML Prognostic Profile in AML patients with intermediate-risk cytogenetic abnormalities can improve risk stratification by confirming intermediate risk or reclassifying patients to favorable or unfavorable risk categories. Specimen Requirements:

• Peripheral blood: 2-5 mL in sodium heparin tube and 5 mL in EDTA tube. • Bone marrow: 1-2 mL in sodium heparin tube and 2 mL in EDTA tube..

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 10-12 days CPT, AMA 2013: 88235 or 88237, and 88291. With reflex to molecular testing, add 81245, 81310, 81403(x3), 81479(x2).

Molecular Genetics


DOS Rev • 01.04.13

Molecular Genetics

B-Cell Gene RearrangementDescription: Detection of clonal IgH and Ig kappa gene rearragements by PCR of IgH framework regions 1, 2, 3 and joining regions, Ig kappa FR3 and Jk regions. Clinical Significance: Detects monoclonal B-cell immunoglobulin gene rearrangement. Interpret in clinical context for diagnosis of leukemia, lymphoma, and plasma cell dyscrasia.Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube. • Bone marrow: 2 mL in EDTA tube. • FFPE tissue: 1 H&E slide, plus paraffin block or 5 - 10 unstained slides cut at 5 or more microns. Please use

positively-charged slides and 10% NBF fixative. Do not use zinc fixatives. • Fresh tissue: Two pieces minimum, 0.2 cm3 in RPMI.

Storage and Transportation: Refrigerate fresh tissue until shipping. For all specimens, use cold pack for transport. Make sure cold pack is not in direct contact with specimen. Turnaround Time: 7 days CPT, AMA 2013: 81261, 81264

B-Cell & T-Cell RearrangementDescription: PCR for detection of clonal IgH, Ig kappa, and T-cell receptor gene gamma gene rearragements. Clinical Significance: Detects monoclonal B-cell immunoglobulin gene and T-cell receptor gamma gene rearrangements. Interpret in clinical context for diagnosis of B- and T-cell lymphoproliferative disorders.Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube. • Bone marrow: 2 mL in EDTA tube. • FFPE tissue: 1 H&E slide, plus paraffin block or 5 - 10 unstained slides cut at 5 or more microns. Please use

positively-charged slides and 10% NBF fixative. Do not use zinc fixatives. • Fresh tissue: Two pieces minimum, 0.2 cm3 in RPMI.

Storage and Transportation: Refrigerate fresh tissue until shipping. For all specimens, use cold pack for transport. Make sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81261, 81264, 81342


DOS Rev • 01.04.13

Molecular Genetics

BCL-1 Translocation, t(11;14) Description: Real-time PCR for quantitative detection of t(11;14) BCL1/IgH rearrangements. Analytical sensitivity is approximately 1 tumor cell in 1000 normal cells. Positive results are reported as a ratio between quantities of (11;14) DNA and a normal control gene. This translocation is also known as CCND1/IgH or BCL1/JH. Clinical Significance: Testing may be used to confirm the diagnosis of mantle cell lymphoma, monitor therapy effectiveness, and detect minimal residual disease or relapse. This assay detects rearrangements involving the MTC (major translocation cluster) region. Due to breakpoint variations, this and other PCR-based assays cannot detect all BCL1/IgH translocations that are detected by FISH or cytogenetics. Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube. • Bone marrow: 2 mL in EDTA tube.• FFPE tissue: 1 H&E slide, plus paraffin block or 5-10 unstained slides cut at 5 or more microns. Please use

positively-charged slides and 10% NBF fixative. Do not use zinc fixatives.Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen.Turnaround Time: 5-7 DaysCPT, AMA 2013: 81401

BCL2 Translocation, t(14;18) Description: PCR and fragment analysis for quantitative detection of IGH-BCL2 translocations associated with 70-80% of follicular lymphoma and approximately 20% of diffuse large B-cell lymphoma. Translocations involving the major (MBR), minor (MCR), and 3’ MBR sub-cluster regions of BCL2 are analyzed. Positive results quantify the ratio of mutant BCL2 to internal control DNA. Testing may be performed on plasma to increase sensitivity.Clinical Significance: Useful to confirm diagnosis of follicular lymphoma, monitor therapy effectiveness, and detect minimal residual disease or recurrence. Specimen Requirements:

• Bone marrow: 2 mL in EDTA tube• Peripheral blood: 5 mL in EDTA tube • FFPE tissue: 1 H&E slide, plus paraffin block or 5-10 unstained slides cut at 5 or more microns. Please use

positively-charged slides and 10% NBF fixative. Do not use zinc fixatives.Storage and Transportation: Use cold pack for transporting blood, marrow or block, making sure cold pack is not in direct contact with specimen. Slides can be packed at room temperature.Turnaround Time: 5-7 daysCPT, AMA 2013: 81401


DOS Rev • 01.04.13

Molecular Genetics

BCR-ABL1 Translocation, t(9;22) Description: Real-time RT-PCR for detection of t(9;22) BCR-ABL1 fusion transcripts that result in p190 (E1) or p210 (E13, E14) fusion proteins. Analytical sensitivity is 1 tumor cell in 100,000 normal cells. Log reduction score is reported. Clinical Significance: Useful for diagnosis and monitoring of Philadelphia chromosome-positive cases of CML and ALL. Specimen Requirements:

• Bone marrow: 2 mL EDTA tube• Peripheral blood: 5 mL EDTA tube

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred.Turnaround Time: 5-7 days CPT, AMA 2013: 81206, 81207

BRAF Mutation AnalysisDescription: Bi-directional sequencing of exon 15 of the BRAF gene, which includes qualitative detection of V600 mutations E, K, D, and others, plus other significant exon 15 mutations. For solid tumors, tumor enrichment is performed before extraction.Clinical Significance: Useful in selection of melanoma patients for vemurafenib therapy, for determination of prognosis in thyroid and colon cancers, for predicting response to anti-EGFR therapy in colon cancer, and as aid to diagnosis of hairy cell leukemia. Specimen Requirements:

• FFPE solid tumor tissue: 1 H&E slide, plus paraffin block or 5-10 unstained slides cut at 5 or more microns. Please use positively-charged slides and 10% NBF fixative. Do not use zinc fixatives.

• For tech-only microdissection on solid tumors: Send the H&E slide with area of interest circled, along with block or cut slides as above. Try to enrich to 40% tumor within the circle. Mark on the slide or requisition the estimated percentage tumor in the circle (required).

• Fine needle aspirate (FNA): Requisition must note specimen is FNA. Fresh cells in suspension, unstained air-dried smears (approx. 6-8 slides), or FFPE cell blocks are acceptable if pathologist attaches note verifying sample has >30% tumor or abnormal cells (required). Minimum 10^6 cells.

• Peripheral blood: 5 mL in EDTA tube.• Bone marrow: 2 mL in EDTA tube.

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. All slides can be packed at room temperature. Turnaround Time: 7 daysCPT, AMA 2013: 81210, add 88381 for solid Tumors


DOS Rev • 01.04.13

Molecular Genetics

CEBPA Mutation AnalysisDescription: Bi-directional sequencing of the relevant coding region and fragment analysis for detection of sequence variant and internal tandem duplication mutations. The SNP genotype at rs34529039 is reported. Testing is performed on plasma for increased sensitivity whenever possible. This test can be ordered separately or as part of the NeoTYPE™ AML Prognostic Profile.Clinical Significance: CEBPA mutations are detected in 7-15% of AML patients. Double mutations are associated with good prognosis in patients with intermediate risk and normal cytogenetics who do not have FLT3-ITD mutations. The genotype T at SNP rs34529039 has been associated with shorter event-free survival and time-to relapse in one group of post-stem cell transplant AML patients with intermediate or adverse risk cytogenetics. Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube. • Bone marrow: 2 mL in EDTA tube.

Storage and Transportation: Use cold pack for transport. Make sure cold pack is not in direct contact with specimen Turnaround Time: 7 daysCPT, AMA 2013: 81403

c-KIT Mutation AnalysisDescription: Bi-directional sequencing of KIT exons 8, 9, 11, and 17 for detection of activating mutations including the common mutation D816V. For solid tumors, tumor enrichment is performed before extraction. In hematological disease, testing may be performed on plamsa to increase sensitivity. Clinical Significance: The four tested exons encompass the majority of mutations found in gastrointestinal stromal tumors (GIST), melanoma, core-binding factor AML (CBF-AML), mast cell disease (systemic mastocytosis), and germ cell tumors. Mutation identification is useful for planning TKI therapy and predicting clinical course. Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube.• Bone marrow: 2 mL in EDTA tube.• Fixed cytogenetic cell pellet: Send all available cells suspended in fixative.• FFPE solid tumor tissue: 1 H&E slide, plus paraffin block or 5-10 unstained slides cut at 5 or more microns.

Please use positively-charged slides and 10% NBF fixative. Do not use zinc fixatives.• For tech-only microdissection on solid tumors: Send the H&E slide with area of interest circled, along with

block or cut slides as above. Try to enrich to 40% tumor within the circle. Mark on the slide or requisition the estimated percentage tumor in the circle (required).

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Slides can be packed at room temperature. Turnaround Time: 7 daysCPT, AMA 2013: 81404, add 88381 for solid tumors.


DOS Rev • 01.04.13

Molecular Genetics

CLL Molecular Prognostic PanelDescription: Concurrent analysis of critical regions of the IgVH and SF3B1 genes by RT-PCR and bi-directional sequencing. In IgVH analysis, the mutated VH gene family is identified in positive reports (which have >3% deviation from normal sequence). IgVH mutation may not be detectable in specimens containing <10% clonal B-cells. SF3B1 analysis covers exons 14-17, where at least 90% of the reported mutations are detected. This test detects SF3B1 mutations present at 10-15% or more in a wild-type background.Clinical Significance: Along with FISH, molecular analysis provides critical information for determining prognosis in CLL. IgVH mutation is associated with a favorable prognosis, and SF3B1 mutations are independent predictors of poor outcome. SF3B1 and IgVH analysis may also be ordered individually or as part of the comprehensive NeoTYPE™ CLL Prognostic Profile. Specimen Requirements:

• Peripheral blood (preferred): 2 EDTA tubes, 5 mL each• Bone marrow: Minimum 1 mL in EDTA, 1-3 mL preferred• Fresh lymph node tissue: 0.5 - 1 cm3 in RPMI

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred. Turnaround Time: 10 daysCPT, AMA 2013: 81263, 81479

DNMT3A Mutation Analysis Description: Bi-directional sequencing of exon 26, a mutation hotspot region containing R882 and other mutations. In hematological disease, testing may be performed on plamsa to increase sensitivity. For solid tumors, tumor enrichment is performed before extraction. This test can be ordered separately or as part of the NeoTYPE™ AML Prognostic Profile. Clinical Significance: DNMT3A mutations are found in 20-25% of acute myeloid leukemia and are associated with poor outcome. Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube. • Bone marrow: 2 mL in EDTA tube.• FFPE solid tumor tissue: 1 H&E slide, plus paraffin block or 5-10 unstained slides cut at 5 or more microns.

Please use positively-charged slides and 10% NBF fixative. Do not use zinc fixatives. • For tech-only microdissection on solid tumors: Send the H&E slide with area of interest circled, along with


Storage and Transportation: Use cold pack to transport, making sure cold pack does not touch specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81479, add 88381 for solid tumors


DOS Rev • 01.04.13

Molecular Genetics

EGFR Mutation Analysis Description: Bi-directional sequencing of exons 18-21 of the EGFR gene for detection of EGFR-activating mutations and TKI resistance mutations in these exons. Tumor enrichment is performed before extraction. Clinical Significance: EGFR mutation analysis is recommended in non-small cell lung carcinoma (NSCLC) to detect mutations (commonly L858R and exon 19 deletions) associated with increased sensitivity to EGFR tyrosine kinase inhibitors such as erlotinib. Detection of TKI resistance mutations such as T790M in patients being treated with a TKI is useful for planning alternate treatment. Specimen Requirements:




Storage and Transportation: Use cold pack for transporting block during summer to prevent block from melting. Slides can be packed at room temperature. Turnaround Time: 7 days CPT, AMA 2013: 81253, 88381

FLT3 Mutation AnalysisDescription: Detection of internal tandem duplication and exon 20 tyrosine kinase domain (TKD) mutations using bidirectional sequencing. Positive results identify specific TKD mutations or report ITD results quantitatively as percent abnormal ITD peak. Testing may be performed on plasma to increase sensitivity. This test may be ordered separately or as part of the NeoTYPE™ AML Prognostic Profile. Clinical Significance: Testing for FLT3 and other gene mutations in AML patients with intermediate-risk cytogenetic abnormalities can improve risk stratification. The presence of an FLT3 mutation in a patient with AML implies aggressive disease. Specimen Requirements:

• Bone marrow: 2 mL in EDTA • Peripheral blood: 5 mL EDTA tube

Storage and Transportation: Use cold pack, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81245


DOS Rev • 01.04.13

Molecular Genetics

IDH1 &IDH2 Mutation Analysis Description: Bi-directional sequencing of the exon 4 mutation hotspot regions in both the IDH1 and IHD2 genes. IDH1 and IDH2 are analyzed concurrently. In hematological disease, testing may be performed on plamsa to increase sensitivity. For solid tumors, tumor enrichment is performed before extraction. This test can be ordered separately or as part of the NeoTYPE™ AML Prognostic Profile.Clinical Significance: IDH1 or IDH2 mutations are detected in approximately 15-20% of acute myeloid leukemia (AML) and >70% grade II or III brain gliomas. Patients with AML and mutations are likely to have aggressive disease, while mutations in gliomas are associated with better prognosis. Long-term survival after aggressive tumor resection has been reported for patients with IDH1-positive astrocytomas. Specimen Requirements:

• “Peripheral blood: 5 mL in EDTA tube. • Bone marrow: 2 mL in EDTA tube.• FFPE solid tumor tissue: 1 H&E slide, plus paraffin block or 5-10 unstained slides cut at 5 or more microns.

Please use positively-charged slides and 10% NBF fixative. Do not use zinc fixatives. • For tech-only microdissection on solid tumors: Send the H&E slide with area of interest circled, along with


Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Slides can be packed at room temperature.Turnaround Time: 7 daysCPT, AMA 2013: 81403(2), add 88381 for solid tumors.

IgVH Mutation Analysis Description: RT-PCR and bi-directional sequencing of the variable region of the immunoglobulin heavy chain for detection of mutation from germline sequence. The mutated VH gene family is identified in positive reports (>3% sequence deviation). Mutation may not be detectable in specimens containing <10% clonal B-cells.Clinical Significance: IgVH mutation is a significant prognostic marker in chronic lymphocytic leukemia (CLL). IgVH mutation analysis combined with FISH, ZAP-70, and beta-2 microglobulin measurement provide comprehensive prognostic assessment and may be used to determine the approach to therapy for all CLL patients. Specimen Requirements:


Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred.Turnaround Time: 10 daysCPT, AMA 2013: 81263


DOS Rev • 01.04.13

Molecular Genetics

inv(16), CBFB-MYH11 Translocation Description: Real-time RT-PCR for quantitative detection of the inv(16) CBFB-MYH11 fusion transcript. Positive results are reported as ratio of the amount of fusion transcript with the amount of transcript from a normal control gene. This assay identifies type A fusions, which account for >90%. Analytical sensitivity is 1 tumor cell in 100,000 normal cells. Clinical Significance: TThe inv(16) occurs in about 10% of all acute myeloid leukemia and nearly all cases of AML with eosinophilia, subtype M4eo. The inversion is generally associated with relatively good outcome. This assay is recommended for diagnostic confirmation, for monitoring minimal residual disease, and for detection of relapse. c-KIT mutation testing may be considered for inv(16)-positive AML patients as c-KIT mutations are considered an adverse risk factor in these and other patients with core-binding factor AML. Specimen Requirements:


Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred. Turnaround Time: 5-7 DaysCPT, AMA 2013: 81401

JAK2 V617F Mutation Analysis Description: Detection of the JAK2 V617F mutation. The rare mutation V617I is also detected. Testing is performed on plasma for increased sensitivity whenever possible. V617F testing may be ordered separately, concurrently with full exon 12-14 sequencing, with reflex to exon 12-14 sequencing, or as part of the MPN Reflex Panel. Clinical Significance: The JAK2 V617F mutation is present in approximately 90% of polycythemia vera (PV) cases and approximately 40% of primary myelofibrosis (PMF) or essential thrombocythemia (ET). Mutation analysis helps differentiate reactive conditions from myeloproliferative neoplasms (MPNs). Specimen Requirements:


Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred. Turnaround Time: 5 daysCPT, AMA 2013: JAK2 V617 only: 81270, JAK2 V617F and Exon 12-14 run concurrently or by reflex: 81270, 81403


DOS Rev • 01.04.13

Molecular Genetics

JAK2 Exon 12-14 Mutation AnalysisDescription: TR-PCR and bi-directional sequencing to detect non-V617F mutations in exons 12-14 and most of exon 15, corresponding to the majority of the JAK2 pseudokinase domain. Exon deletion mutations are detectable. Testing is performed on plasma for increased sensitivity whenever possible. V617F analysis is recommended before or concurrently with this test. Exon 12-14 Mutation Analysis may be ordered separately, with concurrent V617F testing, by reflex after negative V617F testing, or as part of the MPN Reflex Panel. Clinical Significance: While the majority of polycythemia vera (PV) patients carry the V617F mutation (~90%), most of those who are negative carry one of over 40 additional JAK2 mutations in exons 12-15. RNA-based testing in this assay allows detection of deletions not detectable by DNA-based tests. Mutation analysis helps differentiate reactive conditions from malignant erythrocytosis. Specimen Requirements:


Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred. Turnaround Time: 5 daysCPT, AMA 2013: JAK2 Exon 12-14 only: 81403, JAK2 V617F and Exon 12-14 run concurrently or by reflex: 81270, 81403

KRAS Mutation AnalysisDescription: Bi-directional sequencing of exons 1 and 2 of the KRAS gene, which includes detection of mutations in codons 12, 13, and 61. Tumor enrichment is performed before extraction. Clinical Significance: Testing is recommended in colorectal cancer as mutations are associated with resistance and shorter overall survival with EGFR-antagonist therapies such as cetuximab or panitumumab. Testing in non-small cell lung cancer may provide prognostic information and predict poor response to EGFR tyrosine kinase inhibitors. Specimen Requirements:




Storage and Transportation: Use cold pack for transporting block during summer to prevent block from melting. Slides can be packed at room temperature. Turnaround Time: 7 daysCPT, AMA 2013: 81275, 88381


DOS Rev • 01.04.13

Molecular Genetics

KRAS & BRAF Mutation AnalysisDescription: Concurrent analysis of KRAS and BRAF gene regions including KRAS exons 1 and 2 (with mutation detection in codons 12, 13, and 61) and BRAF exon 15 to detect V600 mutations and more. Tumor enrichment is performed before extraction. Clinical Significance: KRAS testing is recommended in colorectal cancer as mutations are associated with resistance and shorter overall survival with EGFR-antagonist therapies such as cetuximab or panitumumab. BRAF testing can be useful in determining prognosis and for predicting response to anti-EGFR therapy in colon cancer.Specimen Requirements:

• FFPE solid tumor tissue: 1 H&E slide, plus paraffin block or 10-20 unstained sections cut at 5 or more microns. Please use positively-charged slides and 10% NBF fixative. Do not use zinc fixatives.


Storage and Transportation: Use cold pack for transporting block during summer to prevent block from melting. Slides can be packed at room temperature. Turnaround Time: 7 daysCPT, AMA 2013: 81275, 81210, 88381

Microsatellite Instability Analysis (MSI)Description: PCR and fragment analysis of paired normal and tumor tissue to determine microsatellite instability (MSI) at the standard five NCI-recommended loci. Positive results are reported as MSI-high (at least two markers are unstable) or MSI-low (one marker is unstable). Clinical Significance: MSI analysis and/or MMR IHC may be considered for all new colorectal cancer diagnoses to detect patients at increased risk of carrying germline mutations associated with Lynch Syndrome (HNPCC). Testing is recommended for tumors of patients <50 years of age or meeting other criteria of the Bethesda Guidelines. MSI is also detected in sporadic colorectal cancer and its presence may imply better prognosis.Specimen Requirements:

• Note: An additional patient sample from normal, non-tumor tissue is required for comparison testing in MSI Analysis. Please submit all specimens with one test requisition form.

• Specimen requirements for normal tissue in order of preference are: 1) 5 mL peripheral blood in EDTA tube OR 2) FFPE tissue slides or block containing only non-tumor tissue. Please label these as “normal tissue”. OR 3) In cases where no alternative tissue is available, we can attempt to isolate non-tumor tissue from the tumor specimen submitted. Note “Use tumor sample for normal tissue” on requisition. See requirements below.

• Specimen requirements for tumor tissue: FFPE tissue: Send 1 H&E slide, plus paraffin block or 5 -10 unstained slides cut at 5 or more microns. Please use positively-charged slides and 10% NBF fixative. Do not use zinc fixatives.

• For tech-only microdissection: Send the H&E slide with abnormal areas of interest circled and labeled “tumor”, along with block or cut slides as above. Try to enrich to 40% tumor within the circled tumor area. Mark on the slide and requisition the estimated percentage tumor in the circle (required). If no patient blood specimen was submitted, also circle and label the normal tissue on the corresponding H&E.

Storage and Transportation: Use cold pack for transporting block and/or blood, making sure cold pack is not in direct contact with specimen. Slides can be packed at room temperature. Turnaround Time: 7 daysCPT, AMA 2013: 81301, 88381


DOS Rev • 01.04.13

Molecular Genetics

MPL Mutation Analysis Description: Bi-directional sequencing of exon 10 of the MPL gene to detect all possible mutations at the W515 and S505 codons, and other mutations throughout the exon. Testing is performed on plasma for increased sensitivity whenever possible. This test may be ordered separately or as part of the MPN Panel. Clinical Significance: MPL mutations are detected in up to 5% of patients with JAK2-negative myeloproliferative neoplasms. Mutation analysis helps differentiate reactive conditions from MPNs. Specimen Requirements:


Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred. Turnaround Time: 7 daysCPT, AMA 2013: 81402

MPN Reflex Panel Description: Sequential testing panel including analysis of JAK2 V617F, JAK2 Exon 12-14, and MPL exon 10. Testing proceeds by reflex through the three-step panel until a mutation is identified, when the result is considered informative and no further testing is performed. Testing is performed on plasma for increased sensitivity whenever possible. Tests may also be ordered separately.Clinical Significance: Comprehensive testing to identify mutations associated with the myeloproliferative neoplasms polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Mutation analysis helps differentiate reactive conditions from MPNs. Specimen Requirements:


Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred. Turnaround Time: 10 daysCPT, AMA 2013: 81270, 81402, 81403


DOS Rev • 01.04.13

Molecular Genetics

NeoARRAY™ SNP/Cytogenetic Profile Description: The NeoARRAY SNP/Cytogenetic Profile is available for hematological and solid tumor indications. With the best genome-wide coverage available, this test employs an enhanced SNP microarray with over 2.6 million SNP and non-polymorphic markers for detection of copy number variants (deletions, duplications, and amplifications) and loss of heterozygosity or uniparental disomy (LOH or UPD) in any chromosome. Sensitivity and specificity for detection of copy number variants >400 kb is >99%. Testing may not reliably detect abnormalities present in less than 20% of the cells tested. Balanced rearrangements, including translocations and inversions, are not detectable by this method.Clinical Significance: This test provides genome-wide characterization of chromosomal imbalances at resolution approximately 10-25 times higher than conventional cytogenetics. Testing is appropriate in hematological disorders and solid tumors to obtain a detailed description of breakpoints and unidentified material in abnormal karyotyes, for detecting abnormalities in samples with normal karyotype, and for use after cytogenetics failure due to lack of growth or inadequate metaphases. Microarray testing such as the NeoARRAY is the only test for detection of copy-neutral loss of heterozygosity and uniparental disomy. Array testing is most often used in CLL, MDS, ALL, AML, and solid tumors. Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube.• Bone marrow: 2 mL in EDTA tube.• Fresh tumor tissue: 0.5 – 1 cm3 in RPMI.


NeoTYPE™ AML Prognostic Profile Description: Bi-directional sequencing and mutation analysis of select exons of the following genes: CEBPA, DNMT3A, FLT3, IDH1, IHD2, NPM1, and WT1. Fragment analysis is performed on select genes for enhanced detection of low levels of insertion/deletion mutations. Testing is performed on plasma for increased sensitivity whenever possible. This Profile may be ordered separately or as part of the AML Reflex Panel. Clinical Significance: Molecular profiling with the NeoTYPE AML Prognostic Profile in AML patients with intermediate-risk cytogenetic abnormalities can improve risk stratification by confirming intermediate risk or reclassifying patients to favorable or unfavorable risk categories. Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube. • Bone marrow: 2 mL in EDTA tube.

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81245, 81310, 81403(3), 81479(2)


DOS Rev • 01.04.13

Molecular Genetics

NeoTYPE™ Breast Tumor Profile Description: Concurrent HER2 FISH and bi-directional sequencing with mutation analysis of the following genes: BRAF, c-KIT, EGFR, PIK3CA, and TP53.Clinical Significance: The NeoTYPE Breast Tumor Profile characterizes primary or metastatic breast tumors of any histological subtype for the most significant genetic changes relevant to therapy decisions, prognosis, and clinical research. It is appropriate for newly diagnosed or relapsing patients, or those with an atypical clinical presentation. Specimen Requirements:

• FFPE solid tumor tissue: 1 H&E slide, plus paraffin block. Please use 10% NBF fixative and do not use zinc fixatives.• Fresh tissue: 1 cm3 fresh tissue that is mostly tumor in RPMI.

Storage and Transportation: Refrigerate fresh tissue in RPMI until shipping. For all specimens, use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81210, 81235, 81404(2), 81405, 88367(2), 88381

NeoTYPE™ CLL Prognostic Profile Description: Concurrent analysis of ZAP-70 by flow cytometry, CLL-related chromosome abnormalities by FISH, and mutations of the SF3B1 and IgVH genes by bi-directional sequencing. Results are issued together in a comprehensive report.Clinical Significance: Accuracy in predicting the course of CLL may be improved by the analysis of multiple prognostic markers. The NeoTYPE CLL Prognostic Profile combines into one test the most significant markers available from FISH, flow cytometry, and molecular analysis, including the newest independent prognostic marker SF3B1 mutations. SF3B1 mutations occur in 10-15% of CLL patients and serve as independent predictors of shortened time to treatment and poorer overall survival in CLL.Specimen Requirements:

• Peripheral blood (perferred): 2 EDTA tubes, 5 mL each• Bone marrow: Minimum 1 mL in EDTA, 1-3 mL preferred• Fresh lymph node tissue: 0.5 - 1 cm3 in RPMI

Storage and Transportation: Refrigerate specimen. Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible. Turnaround Time: 7 daysCPT, AMA 2013: 81263, 81479, 88184, 88185(4), 88187, 88367(7)


DOS Rev • 01.04.13

Molecular Genetics

NeoTYPE™ Colorectal Tumor Profile Description: Concurrent microsatellite instability analysis by PCR/fragment analysis and bi-directional sequencing with mutation analysis of the following genes: BRAF, c-KIT, DNMT3A, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, and TP53. Clinical Significance: The NeoTYPE Colorectal Tumor Profile characterizes primary or metastatic colorectal tumors of any histological subtype for the most significant genetic changes relevant to therapy decisions, prognosis, and clinical research. It is appropriate for newly diagnosed or relapsing patients, or those with an atypical clinical presentation. Specimen Requirements:

• FFPE solid tumor tissue: 1 H&E slide, plus paraffin block. Please use 10% NBF fixative and do not use zinc fixatives.• Fresh tissue: 1 cm3 fresh tissue that is mostly tumor in RPMI.• Note: An additional patient sample from normal, non-tumor tissue is required for comparison testing in MSI

Analysis. Please submit all specimens with one test requisition form.• Specimen requirements for normal tissue in order of preference are: 1) 5 mL peripheral blood in EDTA tube OR

2) FFPE tissue slides or block containing only non-tumor tissue. Please label these as “”normal tissue””. OR 3) In cases where no alternative tissue is available, we can attempt to isolate non-tumor tissue from FFPE tumor specimen submitted. Note “Use tumor sample for normal tissue” on requisition.

Storage and Transportation: Refrigerate fresh tissue in RPMI until shipping. For all specimens, use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81210, 81235, 81275, 81301, 81403(2), 81404(3), 81405, 81479, 88381

NeoTYPE™ Gastric Tumor Profile Description: Concurrent HER2 FISH and bi-directional sequencing with mutation analysis of the following genes: BRAF, c-KIT, DNMT3A, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, and TP53. Clinical Significance: The NeoTYPE Gastric Tumor Profile characterizes primary or metastatic gastric or gastroesophageal tumors of any histological subtype for the most significant genetic changes relevant to therapy decisions, prognosis, and clinical research. It is appropriate for newly diagnosed or relapsing patients, or those with an atypical clinical presentation. Specimen Requirements:


Storage and Transportation: Refrigerate fresh tissue in RPMI until shipping. For all specimens, use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81210, 81235, 81275, 81403(x2), 81404(3), 81405, 81479, 88367(2), 88381



DOS Rev • 01.04.13

NeoTYPE™ Lung Tumor Profile Description: Concurrent ALK FISH and bi-directional sequencing with mutation analysis of the following genes: BRAF, c-KIT, DNMT3A, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, and TP53. Clinical Significance: The NeoTYPE Lung Tumor Profile characterizes primary or metastatic lung tumors of any histological subtype for the most significant genetic changes relevant to therapy decisions, prognosis, and clinical research. It is appropriate for newly diagnosed or relapsing patients, or those with an atypical clinical presentation. Specimen Requirements:


Storage and Transportation: Refrigerate fresh tissue in RPMI until shipping. For all specimens, use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81210, 81235, 81275, 81403(2), 81404(3), 81405, 81479, 88368(2), 88381

NeoTYPE™ Solid Tumor (Other) Profile Description: Concurrent bi-directional sequencing with mutation analysis of the following genes: BRAF, c-KIT, DNMT3A, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, and TP53.Clinical Significance: The NeoTYPE Solid Tumor (Other) Profile characterizes primary or metastatic tumors of any histological subtype for the most significant genetic changes relevant to therapy decisions, prognosis, and clinical research. It is appropriate for newly diagnosed or relapsing patients, or those with an atypical clinical presentation. This test is for tumors arising from various tissues including liver, pancreas, CNS, prostate, kidney, ovary, head and neck, ovary, melanoma, GIST, or others. NeoTYPE Solid Tumor Profiles specific for breast, colorectal, gastric, and lung tumors are available; please see separate listings for descriptions.Specimen Requirements:


Storage and Transportation: Refrigerate fresh tissue in RPMI until shipping. For all specimens, use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81210, 81235, 81275, 81403(2), 81404(3), 81405, 81479, 88381



DOS Rev • 01.04.13

NPM1 Mutation AnalysisDescription: PCR and fragment analysis of exon 12 of the NPM1 gene to detect small insertion mutations specific to AML. Positive results are reported quantitatively as percent abnormal DNA. Testing may be performed on plasma to increase sensitivity. This test may be ordered separately or as part of the NeoTYPE™ AML Prognostic Profile. Clinical Significance: Testing for NPM1 and other gene mutations in AML patients with intermediate-risk cytogenetic abnormalities can improve risk stratification. NPM1 mutations can predict favorable prognosis in AML with normal karyotype. Specimen Requirements:

• Bone marrow: 2 mL in EDTA tube.• Peripheral blood: 5 mL in EDTA tube.

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81310

NRAS Mutation AnalysisDescription: Bi-directional sequencing of NRAS exons 1 and 2 which includes sites of common activating mutations in codons 12, 13, and 61. Clinical Significance: NRAS is highly homologous with KRAS; both are members of the most frequently mutated family of oncogenes. Mutations are found in a wide variety of solid tumors, in advanced systemic mastocytosis, and in myeloid neoplasias. Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube.• Bone marrow: 2 mL in EDTA tube.• FFPE solid tumor tissue: 1 H&E slide, plus paraffin block or 5-10 unstained slides cut at 5 or more microns.



Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81404, add 88381 for solid tumors.



DOS Rev • 01.04.13

PIK3CA Mutation AnalysisDescription: Bi-directional sequencing of PIK3CA exons 1, 9, and 20 which are the most commonly-mutated regions of the gene. Clinical Significance: The PIK3CA gene encodes the p110 alpha catalytic subunit of PI3K enzymes mutations occur in a wide variety of tumors and may have prognostic and therapeutic significance, depending on tumor type. Numerous PI3K-pathway inhibitors are in development. Specimen Requirements:



Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81404, 88381

PML-RARA Translocation, t(15;17)Description: Real-time RT-PCR for quantitative detection of the t(15;17) PML-RARA fusion transcript. Both long and short isoforms of the fusion transcript are detected. Positive results identify the isoform and quantify it as a ratio with the amount of transcript from a normal control gene. Analytical sensitivity is 1 tumor cell in 100,000 normal cells.Clinical Significance: The (15;17) translocation occurs in nearly all cases of acute promyelocytic leukemia (APL, or AML subtype M3). The translocation is associated with a high rate of complete remission due to sensitivity of leukemic cells to all trans-retinoic acid (ATRA). This assay is recommended for diagnostic confirmation and initiation of ATRA therapy, for monitoring minimal residual disease, and for detection of relapse. Specimen Requirements:

• Bone marrow (preferred): 2 mL in EDTA tube.• Peripheral blood (acceptable): 5 mL in EDTA tube

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred. Turnaround Time: 5-7 daysCPT, AMA 2013: 81315



DOS Rev • 01.04.13

RUNX1-RUNX1T1 (AML1-ETO) Translocation, t(8;21)Description: Real-time RT-PCR for quantitative detection of the t(8;21) RUNX1-RUNX1T1 fusion transcript (formerly called AML1-ETO). Analytical sensitivity is 1 tumor cell in 100,000 normal cells. Positive results are reported as a ratio between quantities of (8;21) transcript and a normal control gene.Clinical Significance: The (8;21) translocation occurs in approximately 5% of AML. These cases are usually considered core-binding factor AML (CBF-AML). The translocation is usually associated with a high rate of complete remission and longer overall survival in AML subtype M2. This assay is recommended for diagnostic confirmation of and for monitoring minimal residual disease. c-KIT mutation testing may be considered for t(8;21)-positive AML patients as c-KIT mutations are considered an adverse risk factor in these patients.Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube.• Bone marrow: 2 mL in EDTA tube.• Fixed cytogenetic cell pellet: Send all available cells suspended in fixative.

Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Ship same day as drawn whenever possible; specimens <72 hours old preferred. Turnaround Time: 5-7 daysCPT, AMA 2013: 81401

SF3B1 Mutation AnalysisDescription: RT-PCR and bi-directional sequencing of exons 14-17 of the SF3B1 gene. More than 90% of reported mutations are detected in these exons. This test detects mutations present at 10-15% or more in a wild-type background. Test may be ordered separately or as part of the NeoTYPE CLL Prognostic Profile.Clinical Significance: SF3B1 mutations occur in 10-15% of CLL patients and serve as independent predictors of shortened time to treatment and poorer overall survival in CLL. Mutations are also detected in approximately 28% of MDS and 19% of myelodysplastic/myeloproliferative neoplasms. In MDS/MPN, most mutations were found in refractory anemia with ring sideroblasts (the RARS subtype). Specimen Requirements:



T-Cell Gene RearrangementDescription: Detection of clonal T-cell receptor gamma gene rearrangements by PCR of variable and joining regions. Clinical Significance: Detects monoclonal T-cell receptor gamma gene rearrangement. Interpret in clinical context for diagnosis of T-cell lymphoproliferative disorders. Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube.• Bone marrow: 2 mL in EDTA tube.• FFPE tissue: 1 H&E slide, plus paraffin block or 5-10 unstained slides cut at 5 or more microns. Please use

positively-charged slides and 10% NBF fixative. Do not use zinc fixatives.• Fresh tissue: Two pieces

Storage and Transportation: Refrigerate fresh tissue until shipping. For all specimens, use cold pack for transport. Make sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81342



DOS Rev • 01.04.13

TP53 Mutation AnalysisDescription: Bi-directional sequencing of TP53 exons 4-9.Clinical Significance: TP53 mutations are detected in at least 50% of all adult tumors and are generally associated with a poor prognosis. The TP53 gene encodes the tumor suppressor p53. Germline mutations in TP53 are the cause of Li-Fraumeni Syndrome. Specimen Requirements:

• Peripheral blood: 5 mL in EDTA tube. • Bone marrow: 2 mL in EDTA tube.• FFPE solid tumor tissue: 1 H&E slide, plus paraffin block or 5-10 unstained slides cut at 5 or more microns.



Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81405, add 88381 for solid tumors.

WT1 Mutation AnalysisDescription: Bi-directional sequencing of exons 7 and 9 is performed for detection of sequence variant mutations. Fragment analysis of exon 7 is also performed for enhanced detection of heterozygous insertion/deletion mutations. The SNP genotype at rs16754 is reported. Testing is performed on plasma for increased sensitivity whenever possible. This test may be ordered separately or as part of the NeoTYPE™ AML Prognostic Profile. Clinical Significance: WT1 mutations occur in approximately 12% of acute myeloid leukemia (AML) cases and are associated with poor outcome in cytogenetically normal AML. Within exon 7, the G allele at SNP rs16754 is associated with better prognosis in cytogenetically normal AML, especially in patients with high-risk NPM1/FLT3-ITD mutation status. Specimen Requirements:


Storage and Transportation: Use cold pack for transport, making sure cold pack is not in direct contact with specimen. Turnaround Time: 7 daysCPT, AMA 2013: 81479.

Select Additional Molecular Tests Currently in Deveopment at NeoGenomics• ETV6-RUNX1 (TEL-AML1) Translocation, t(12;21)• TPMT SNP Analysis• UGT1A1 SNP Analysis


DOS Rev • 01.04.13

Cytogenetics

A wide variety of abnormalities can be identified, providing both diagnostic and prognostic information. Acute leukemias, lymphomas and chronic myeloid and lymphoid disorders are examined cytogenetically in order to establish the exact nature of the acquired genetic change. Rearrangements, also known as translocations, inversions, and deletions, can usually be detected under a light microscope. In most leukemias and lymphomas, changes in chromosome number (ploidy) or chromosome structure (rearrangements) are often observed.

Chromosome AnalysisCPT: CPT codes vary per specimen type and number of cells/karyotypes counted. Please note that CPT codes for Cytogenetics can be any combination of the following: 88237, 88239, 88261, 88262, 88263, 88264, 88280, 88285, 88291, 88239Specimen Requirements:

• Bone Marrow Aspirate: 1-2mL Sodium Heparin tube• Peripheral Blood: 2-5mL Sodium Heparin tube• Lymph Node/Tissue Biopsy: 2 pieces minimum, 0.2cm3• Fresh Tissue: Place in RPMI

Storage and Transportation: Refrigerate specimen. Do not freeze. Use cold pack for transport, make sure cold pack is not in direct contact with specimen.Clinical Utility: To detect chromosomal aberrations to help clinicians with diagnostic and prognostic information.Turn Around Time: 3-5 days

Important:• Pediatric tubes should be used for specimens less than 1mL to prevent exposing the specimen • to toxic levels of sodium heparin.• Please ensure equal ratio of specimen to anticoagulant.

Cytogenetics


DOS Rev • 01.04.13

In Situ Hybridization

In situ hybridization targets specific nucleic acids within fixed tissues and cells to indicate a gene’s presence or expression while allowing simultaneous visualization of the tissue’s morphology. NeoGenomics offers chromogenic in situ hybridization (CISH) for analysis of EBER, kappa, and lambda.

EBER by ISH Test Description: In situ hybridization for detection of Epstein-Barr virus-encoded RNA (EBER). Clinical Significance: The Epstein-Barr virus (EBV) probe demonstrates latent EBV infection by hybridizing to abundantly expressed EBER transcripts which are concentrated in the nuclei of latently infected cells. Specimen Requirements

• Cut Slides: 4 cut slides. Sections must be wrinkle and artifact-free. No additives in the water bath. Cut sections at 3-4 microns, and place tissue at the center bottom of a positively charged slide.

• Paraffin block: Formalin-fixed paraffin-embedded tissue. Block should be sent with a cold pack. Block identifiers should be clearly written and match exactly with the specimen ID and the block labeling as noted on the requisition.

Storage and Transportation: Use cold pack for transport. Make sure cold pack is not in direct contact with specimen. Turn around Time: 1 day: tech only. 2 days: with interpretation. 3 days: with consult report. CPT Codes: 88365

Kappa by ISH Test Description: In situ hybridization for detection of kappa light chain mRNA. Testing should be ordered in conjunction with lambda ISH. Positive and negative staining for kappa and lambda are reported and abnormal results are given as percentage ratio. Clinical Significance: Kappa and lambda probes are used to detect antibody producing B-cells in formalin-fixed, paraffin-embedded tissue. Restriction of light chain production to either kappa or lambda (monoclonality) can help distinguish between reactive and neoplastic B-cell proliferations. Specimen Requirements

• Cut Slides: 4 cut slides for each kappa and lambda. Sections must be wrinkle and artifact-free. No additives in the water bath. Cut sections at 3-4 microns, and place tissue at the center bottom of a positively charged slide.



In Situ Hybridization (ISH)


DOS Rev • 01.04.13

In Situ Hybridization

Lambda by ISH

Test Description: In situ hybridization for detection of lambda light chain mRNA. Testing should be ordered in conjunction with kappa ISH. Positive and negative staining for kappa and lambda are reported and abnormal results are given as percentage ratio. Clinical Significance: Lambda and kappa probes are used to detect antibody producing B-cells in formalin-fixed, paraffin-embedded tissue. Restriction of light chain production to either kappa or lambda (monoclonality) can help distinguish between reactive and neoplastic B-cell proliferations. Specimen Requirements

• Cut Slides: 4 cut slides for each kappa and lambda. Sections must be wrinkle and artifact-free. No additives in the water bath. Cut sections at 3-4 microns, and place tissue at the center bottom of a positively charged slide.




DOS Rev • 01.04.13

Immunohistochemisty

Immunohistochemistry is a methodology that detects specific markers (antigens) in tissues. IHC uses a building-block system of antibodies, conjugates, and chromagens (color reagents) to detect these markers. Specific antigens are associated with particular types of cancer. The antibodies used to detect these antigens can be polyclonal or monoclonal, with greater sensitivity achieved using monoclonal antibodies. Immunohistochemistry is widely used for diagnostic and prognostic characterization of carcinoma, lymphoma/leukemia, and sarcoma.

IHC Specimen RequirementsBone Marrow Core Biopsy and/or Aspirate Clot: Clot and core must be sent in separate 10% Neutral Buffered Formalin containers. These should be well processed and contain an adequate amount of sample for IHC evaluation.

Peripheral Blood Smears, Aspirate Smears and Touch Preparations: Smears need to be completely air- dried and labeled with patient name before being placed into slide mailers. Please indicate what type of smear is being sent for testing. Please mark either shipping slide mailer or slide label. (BM=Bone Marrow, TP = Touch Preparations, PB= Peripheral Blood)

Cut Slides: 3 cut slides for each antibody requested, as available, unless otherwise specified. Sections must be wrinkle and artifact free. No additives in the water bath. Cut sections at 3-4 microns, and place tissue at the center bottom of the slide.

Paraffin Block (Preferred for Image Analysis): Formalin-fixed-Paraffin Embedded tissue. Block should be sent with a cool pack. Block identifiers should be clearly written and match exactly to the specimen ID and the block labeling noted on the requisition.

Storage and Transportation: Use cold pack for transport. Make sure cold pack is not in direct contact with specimen.

Turnaround Time: 1 day: tech only. 2 days: with interpretation. 3 days: with consult report.

Most Commonly Used CPT Code: 88342 for IHC, 88360 for quantitative IHC, 88361 for IHC with aid of digital imaging.

Additional CPT Codes:

• 85060 Blood Smear Interpretation• 85097 Bone Marrow Blood Smear Interpretation• 88112 Cytopath, cell enhance tech• 88305 Tissue Exam by Pathologist• 88311 Decalcification Procedure• 88312 Special Stains• 88313 Special Stains• 88321 Microslide Consultation• 88323 Microslide Consultation• 88325 Comprehensive Review of Data• 88342 Immunohistochemistry• 88360 Tumor Immunohistochemistry manual • 88361 Tumor Immunohistochemistry computer assisted

Immunohistochemistry (IHC)


DOS Rev • 01.04.13

Immunohistochemisty

Acute Leukemia IHC PanelDescription: Staining with the following seven antibodies to identify acute leukemia in bone marrow core biopsies: CD34, CD45, TdT, CD117, MPO, Pax-5, CD3. Specimen Requirements:

• Cut Slides: 9 cut slides. Sections must be wrinkle and artifact-free. No additives in the water bath. Cut sections at 3-4 microns, and place tissue at the center bottom of the slide.


• NOTE: Please also send one H&E slide with global testing and consult requests.Storage and Transportation: Use cold pack for transport. Make sure cold pack is not in direct contact with specimen. Turnaround Time: 1 day: tech only. 2 days: with interpretation. 3 days: with consult report. CPT: 88342 for IHC, 88360 for quantitative IHC, 88361 for IHC with aid of digital imaging. Contact NeoGenomics’ Billing Department for units.

Adenocarcinoma vs. Mesothelioma IHC PanelDescription: Staining with the following seven antibodies to differentiate pulmonary adenocarcinoma from malignant mesothelioma, epithelial type: Pan-CK, CEA, MOC-31, BerEP4, TTF1, calretinin, and WT-1. Specimen Requirements:

• Cut Slides: 9 cut slides. Sections must be wrinkle and artifact-free. No additives in the water bath. Cut sections at 3 - 4 microns, and place tissue at the center bottom of the slide.

• Paraffin block: (Preferred for image analysis.) Formalin-fixed paraffin-embedded tissue. Block should be sent with a cold pack. Block identifiers should be clearly written and match exactly with the specimen ID and the block labeling as noted on the requisition.


Bladder vs. Prostate CarcinomaDescription: Staining with the following five antibodies to differentiate bladder from prostate cancer: CK7, CK20, PSA, Ck 903, and p63.Specimen Requirements:

• Cut Slides: 7 cut slides. Sections must be wrinkle and artifact-free. No additives in the water bath. Cut sections at 3 - 4 microns, and place tissue at the center bottom of the slide.



IHC Panels


DOS Rev • 01.04.13

Immunohistochemisty

Breast IHC PanelDescription: Staining with the following four antibodies to characterize breast tumors for therapeutic planning: ER, PR, Ki-67, and HER2. Reflex to HER2 FISH after HER2 IHC is available. Specimen Requirements:

• Cut Slides: 6 slides; send additional slides if FISH reflex option is ordered . Sections must be wrinkle and artifact-free. No additives in the water bath. Cut sections at 3-4 microns, and place tissue at the center bottom of the slide.



Carcinoma Unknown Primary Site, Female (CUPS IHC Panel - Female )Description: Staining with the following 10 antibodies to determine origin of carcinoma in female patients: CK7, CK20, mammaglobin, ER, TTF1, CEA, CA19-9, S100, synaptophysin, and WT-1. Specimen Requirements:




Carcinoma Unknown Primary Site, Male (CUPS IHC Panel - Male)Description: Staining with the following eight antibodies to determine origin of carcinoma in male patients: CK7, CK20, TTF1, PSA, CEA, CA19-9, S100, and synaptophysin. Specimen Requirements:





DOS Rev • 01.04.13

Immunohistochemisty

GIST IHC PanelDescription: Staining with the following four antibodies to aid in the distinction of gastrointestinal stromal tumors from smooth muscle neoplasms: CD117, DOG-1, CD34, and desmin. Specimen Requirements:




Hepatoma/Cholangio vs. Metastatic Carcinoma IHC PanelDescription: Staining with the following seven antibodies to differentiate hepatic neoplasms from metastases: HSA (HepPar 1), CDX2, CK7, CK20, CAM 5.2, TTF-1, and CEA (polyclonal). Specimen Requirements:




Hodgkin vs. NHL IHC PanelDescription: Staining the following six antibodies to help distinguish between Hodgkin and non-Hodgkin lymphoma : EBER, CD3, CD20, CD79a, CD15, CD30. Specimen Requirements:





DOS Rev • 01.04.13

Immunohistochemisty

Lung Cancer IHC PanelDescription: Staining with the following five antibodies to differentiate primary adenocarcinoma from squamous carcinoma of the lung: chromogranin A, synaptophysin, CK7, p63, and TTF-1. Specimen Requirements:




Lung vs. Metastatic Breast Carcinoma IHC PanelDescription: Staining with the following four antibodies to differentiate lung from metastatic breast carcinoma: TTF1, mammaglobin, GCDFP-15 (BRST-2), and ER. Specimen Requirements:




Lymphoma Phenotype IHC PanelDescription: Staining with the following nine antibodies to help classify the lymphoma by immunophenotyping: CD3, CD20, CD138, CD15, CD30, BCL-2, BCL-6, CD4, CD8. Specimen Requirements:





DOS Rev • 01.04.13

Immunohistochemisty

Lymphoma vs. Carcinoma IHC PanelDescription: Staining with the following two antibodies to distinguish poorly-differentiated carcinoma from lymphoma: CD45 and Pan-CK Specimen Requirements:




Lymphoma vs. Reactive Hyperplasia IHC PanelDescription: Staining with the following four antibodies to aid in the distinction of reactive lymphoid hyperplasia from lymphoma: BCL-2, BCL-6, CD10, and Ki-67. . Specimen Requirements:




Melanoma vs. Squamous Cell Carcinoma IHC PanelDescription: Staining with the following eight antibodies to identify melanoma and differentiate it from squamous cell carcinoma: CD68, Factor XIIIa, CEA (polyclonal), S-100, melanoma cocktail (HMB-45, MART-1/Melan-A, tyrosinase) and Pan-CK+CAM 5.2. Specimen Requirements:





DOS Rev • 01.04.13

Immunohistochemisty

Mismatch Repair Proteins IHC Panel (MMR/Colon Cancer)Description: Staining with the following four antibodies to identify defects in mismatch repair proteins: MLH1, MSH2, MSH6, and PMS2. Specimen Requirements:




Neuroendocrine Neoplasm IHC PanelDescription: Staining with the following six antibodies to to identify neuroendocrine features in tumors: CD56, synaptophysin, chromogranin A, TTF-1, Pan-CK, and CEA (polyclonal). Specimen Requirements:




Plasma Cell Neoplasm IHC PanelDescription: Staining with the following nine antibodies to evaluate plasma cell dyscrasia: CD138, CD79a, EMA, MUM1, CD56, Cyclin D1, CD43, CD20, and CD19. Specimen Requirements:





DOS Rev • 01.04.13

Immunohistochemisty

Prostate vs. Colon Carcinoma IHC PanelDescription: Staining with the following seven antibodies to aid in the differentiation of prostate cancer from colon cancer: CDX2, CK 20, CEA (monoclonal), CA19-9, PLAP, CK 7, and PSA. Specimen Requirements:




Soft Tissue Tumor IHC PanelDescription: Staining with the following seven antibodies to classify soft tissue tumors: Pan-CK, SMA, desmin, S100, CD34, vimentin, and CD68. Specimen Requirements:




Undifferentiated Tumor IHC PanelDescription: Staining with the following four antibodies to help classify tumors as carcinoma, melanoma, lymphoma or sarcoma: Pan-CK, S100, CD45, and vimentin.Specimen Requirements:





DOS Rev • 01.04.13

Immunohistochemisty

AFP Staining Pattern: CytoplasmicDescription: (Alpha-Fetoprotein) AFP is normally synthesized in the liver, intestinal tract, and yolk sac of the fetus. Antibody to AFP has been shown to be useful in detecting hepatocellular carcinomas (HCC) and germ cell neoplasms, especially yolk sac tumors.

ALK1Staining Pattern: Cytoplasmic and NuclearDescription: (Anaplastic Lymphoma Kinase) Recognizes a human p80 protein, identified as a hybrid of the anaplastic lymphoma kinase (ALK) gene and the nucleophosmin (NPM) gene resulting from the t(2;5)(p23;q35) translocation found in 30-50% of CD30+ large cell lymphomas (1). This rabbit monoclonal antibody can be used to detect p80 in these lymphomas and may also be used to detect a recently described subtype of large B cell lymphoma, which expresses the full-length ALK protein (2).

Amyloid ADescription: Amyloid A Component reacts with native and fixed amyloid fibrils. The antibody reacts with amyloid deposits in all tissues including kidney and rectum. Cross-reactivity with serum precursor of protein AA has been observed. The application of both Congo Red and Amyloid A Component in tissues with amyloid deposits has been shown to be superior to Congo Red alone.

Amyloid PDescription: Amyloid P Component reacts with amyloid deposits in all tissues including kidney, rectum and brain. The application of Congo Red, Amyloid P and Amyloid A in tissues with amyloid deposits has been shown to be superior to Congo Red and other histochemical stains.

Annexin A1Description: Annexin (ANXA1) is strongly expressed on the cell membrane and occasionally in the cytoplasm of tumor cells in 97% of samples from patients with hairy cell leukemia. By contrast, B-cell lymphomas other than hairy cell leukemia, including typical splenic lymphoma with villous lymphocytes and variant hairy cell leukemia, are ANXA1-negative. Wang et al. showed that high ANXA1 expression is frequent in esophageal and esophagogastric junction adenocarcinomas, is associated with more advanced pathologic T stage and the presence of distant metastasis, and is an independent prognostic factor for patient survival (Clin Cancer Res. 2006;12[15]:4598-604).

BCA225Description: Anti-BCA-225 antibody recognizes a human breast carcinoma associated glycoprotein BCA-225 (220-225kD). This protein differs in size and distribution from other breast carcinoma antigens. Unlike other antibodies against breast carcinoma antigens, this antibody does not react with benign or malignant colonic, stomach, prostate, liver, pancreas, thyroid, or parotid tissues. Adenocarcinomas of the lung, ovary and endometrium also stain with this antibody.

IHC Individual Antibodies


DOS Rev • 01.04.13

Immunohistochemisty

BCL-1 (Cyclin-D1)

Staining Pattern: NuclearDescription: (Cyclin D1 or PRAD-) BCL-1 is one of the key cell cycle regulators, and functions in association with cdk4 and / or cdk6 by phosphorylating the Rb protein. It is a putative proto-oncogene over expressed in a wide variety of human neoplasms including mantle cell lymphomas (MCL).

BCL-2Staining Pattern: CytoplasmicDescription: Expression of Bcl-2alpha oncoprotein inhibits the programmed cell death (apoptosis). In most follicular lymphomas, neoplastic germinal centers express high levels of Bcl-2alpha protein, whereas the normal or hyperplastic germinal centers are negative.

BCL-6Staining Pattern: NuclearDescription: Bcl-6 proto-oncogene product (a Kruppel-type zinc-finger protein) is mainly expressed in normal germinal center B cells and related lymphomas. Bcl-6 is involved in chromosome rearrangements at 3q27 in non-Hodgkin’s lymphomas and Bcl-6 rearrangements have been detected in 33%-45% of diffuse large B cell lymphomas. Bcl-6 has been detected immunohistochemically in follicular lymphomas, diffuse large B cell lymphomas, Burkitt’s lymphomas and in nodular, lymphocyte predominant Hodgkin’s disease.

BerEP4Description: Ber-EP4 recognizes two glycoproteins of 34 and 49 kDa present on the surface and the cytoplasm of all epithelial cells except the superficial layers of squamous epithelial, hepatocytes and parietal cells. It does not label mesothelial cells and rarely marks mesotheliomas. It shows a broad spectrum of reactivity with human epithelial cells including simple epithelia and basal layers of stratified non-keratinized squamous epithelium and epidermis. Ber-EP4 reportedly distinguishes adenocarcinomas from pleural mesotheliomas.

Beta-CateninDescription: Beta-catenin is a 92 kD protein normally found in the cytoplasm of the cell in the submembranous location. This protein is associated with E-cadherin and may be essential for the function of E-cadherin. Mutations in the beta-catenin gene result in nuclear accumulation of this protein. Nuclear accumulation of this protein has been demonstrated in fibromatosis lesions of the breast, and abdomen and therefore is useful in differentiating this lesion from other spindle cell lesions that may occur in these locations. Nuclear accumulation of beta-catenin has also been demonstrated in colorectal carcinoma.

BG-8Description: Blood group antigens have been examined as potential descriminators between pulmonary adenocarcinoma (PACA) and epithelioid mesotheloma (EM). Lewis-y is the only one of these that appears to have some merit. BG8 is raised from the SK-LU-3 lung cancer line.


DOS Rev • 01.04.13

Immunohistochemisty

CA125

Staining Pattern: CytoplasmicDescription: CA125 determinant is present on a mucin-like glycoprotein of high molecular weight. CA125 has been found on frozen sections of amnion and derivatives of coelomic and mullerian epithelium, including pleura, pericardium and peritoneum. In adult tissues, epithelial cells of fallopian tube, endometrium and endocervix, pancreas, colon, gall bladder, stomach, kidney, apocrine sweat gland and mammary gland.

CA19-9Description: CA19-9, a carbohydrate antigenic determinant identified as a sialylated lacto-N-fucopentose II, is related to the Lewis blood group. The CA19-9 antibody has been shown to label adenocarcinomas of the pancreas, stomach, colon and gall bladder. CA19-9 is also expressed in primary and metastatic ovarian carcinomas.

CalcitoninDescription: Rabbit anti-human calcitonin is a purified immunoglobulin fraction of rabbit serum. Contaminating antibodies have been absorbed by solid-phase absorption. The antibody reacts with human calcitonin and labels C-cells in normal thyroid. It is particularly useful in differentiating medullary carcinoma from papillary and follicular thyroid cancer. When used in conjunction with an anti-thyroglobulin antibody, most medullary carcinomas are positive for calcitonin, and is usually negative for most papillary and follicular types of thyroid cancer.

CaldesmonDescription: Caldesmon is a regulatory protein found in smooth muscle and other tissues which interacts with actin, myosin, tropomyosin, and calmodulin. Anti-caldesmon antibody labels smooth muscle and tumors of smooth muscle, myofibroblastic, and myoepithelial differentiation. Anti-Caldesmon has also been used to differentiate epithelioid mesothelioma from serous papillary carcinoma of the ovary.

CalponinStaining Pattern: CytoplasmicDescription: Calponin, a thin filament associated protein is implicated in the regulation and modulation of smooth muscle contraction. It is capable of binding to actin, calmodulin, troponin C and tropomyosin. Calponin is expressed in smooth muscle and tissues containing significant amounts of smooth muscle. Two isoforms of calponin exist whose molecular weights are 34kDa and 29kDa. Expression of the 29kDa form is primarily restricted to muscle of the urogenital tract. The expression of calponin has also been demonstrated in myoepithelial cells from benign and malignant breast lesions. It stains smooth muscle, myoepithelial cells, myofibroblasts, keratinocytes and nerve fibers. It identifies myoepithelial cells in breast lesions, and helps differentiate breast collagenous spherulosis (positive) from adenoid cystic carcinoma. Adenoid cystic carcinoma in salivary gland tumors is calponin positive.


DOS Rev • 01.04.13

Immunohistochemisty

CalretininStaining Pattern: Nuclear and CytoplasmicDescription: Calretinin is an intracellular calcium-binding protein belonging to the troponin C superfamily characterized by a structural mitif described as the EF-hand domain. The immunohistochemical detection of calretinin in developing cerebellum is restricted to the later stages indicated by weak staining from week 21 of gestation, in Purkinje and basket cells and in neurons of the dentate nucleus. The intensity of staining increases as the cerebellum matures. In tumors, calretinin has been detected in mesotheliomas and some pulmonary adenocarcinomas.

CAM 5.2 CytokeratinStaining Pattern: CytoplasmicDescription: CAM 5.2 will stain most epithelial-derived tissue, including liver, renal tubular epithelium, and hepatocellular and renal cell carcinomas.

CD1a Staining Pattern: MembranousDescription: CD1a is expressed on cortical thymocytes, Langerhans cells, and dendritic cells. It is absent on mature peripheral blood T cells but intracytoplasmic expression is detected on activated T lymphocytes. CD1 proteins have been demonstrated to restrict T-cell response to non-peptide lipid and glycolipid antigens and play a role in non-classical antigen presentation.

CD2Staining Pattern: Cytoplasmic, MembranousDescription: CD2 which is also known as E-rosette receptor, T11 and lymphocyte-function antigen-2 (LFA-2)) is expressed on T cells, thymocytes, and subset of natural killer cells. Human CD2 functions as the receptor for sheep erythrocytes, human CD58 (LFA-3), and CD15s (Sialyl Lewis X). p56lck, p59fyn, CD3eta and CD3epsilon are tyrosine-phosphorylated after CD2 stimulation. CD2 play a role in T cell activation, T- or NK-cell-mediated cytolysis, apoptosis in activated peripheral T cells, and regulation of T cell anergy.

CD3, MonoclonalStaining Pattern: Cytoplasmic, MembranousDescription: This antibody reacts with the intracytoplasmic portion of the CD3 antigen expressed by T cells. It stains human T cells in both the cortex and medulla of the thymus and in peripheral lymphoid tissues. This antibody is suitable for staining normal and neoplastic T cells in formalin-fixed, paraffin-embedded tissues.

CD4Staining Pattern: MembranousDescription: CD4, a single chain transmembrane glycoprotein, is found on a T cell subset (helper/inducer) representing 45% of peripheral blood lymphocytes. It is also present on 80% of thymocytes and at a lower level on monocytes. It is involved in recognition of antigen presented along with MHC class II by APCs. It serves as receptor for HIV.


DOS Rev • 01.04.13

Immunohistochemisty

CD5 Staining Pattern: MembranousDescription: CD5, a transmembrane protein, is found on 95%of thymocytes and 72% of peripheral blood lymphocytes. In lymph nodes, the main reactivity is observed in T cell areas. CD5 is expressed by many T cell leukemia, lymphomas, and activated T cells. Occasionally, CD5 antigen is also expressed on a subset of B cells. Mantle cell lymphomas (same as diffuse centrocytic lymphomas) are CD5+ while the follicle center cell lymphoma are CD5-.

CD7Description: Anti-CD7 is directed against the 40kD transmembrane glycoprotein, CD7, which is present in thymocytes and mature T cells. Anti-CD7 may be used to aid in the identification of T cell lymphomas.

CD8Staining Pattern: MembranousDescription: CD8 molecule consists of two chains, termed alpha and Beta chain, which are expressed as a disulphide-linked alpha/Beta heterdimer or as an alpha/alpha homodimer on T cell subset, thymocytes and NK cells. The majority of CD8+ T cells express CD8 as alpha/Beta heterdimer. CD8 functions as a coreceptor in concert with TCR for binding the MHC class I/peptide complex. The HIV-2 envelope glycoprotein binds CD8 alpha chain (but not Beta chain).

CD10Staining Pattern: Cytoplasmic, MembranousDescription: CD10, also known as Common Acute Lymphocytic Leukemia Antigen (CALLA), is a cell surface enzyme with neutral metalloendopeptidase activity which inactivates a variety of biologically active peptides. CD10 is expressed on the cells of lymphoblastic, Burkitt’s, and follicular germinal center lymphomas, and on cells from patients with chronic myelocytic leukemia (CML). It is also expressed on the surface of normal early lymphoid progenitor cells, immature B cells within adult bone marrow and germinal center B cells within lymphoid tissue. CD10 is also present on breast myoepithelial cells, bile canaliculi, fibroblasts, with especially high expression on the brush border of kidney and gut epithelial cells.

CD15 (Leu-M1)

Staining Pattern: Cytoplasmic, MembranousDescription: (Leu-M1) 3-fucosyl-N-acetyllactosamine (3-FAL) or CD15 or X-hapten plays a role in mediating phagocytosis, bactericidal activity, and chemotaxis. It is present on >95% of granulocytes including neutrophils and eosinophils and to a lesser degree on monocytes. CD15 is also expressed in Reed-Sternberg cells and some epithelial cells. CD15 antibody is very useful in the identification of Hodgkin’s disease. CD15 is occasionally expressed in large cell lymphomas of both B and T phenotypes which otherwise have a quite distinct histological appearance.


DOS Rev • 01.04.13

Immunohistochemisty

CD19Description: CD19 recognizes a 95kD cell surface glycoprotein which is expressed by cells of the B-cell lineage and follicular dendritic cells. CD19 is a co-receptor of CD21and is an important signal transduction molecule which is involved in the regulation of B-lymphocyte development, activation and differentiation. CD19 may provide useful diagnostic information for the study of B-lymphoproliferative disorders

CD20 (L26) Staining Pattern: MembranousDescription: CD20 is a non-Ig differentiation antigen of B cells and the expression of CD20 is restricted to normal and neoplastic B cells, being absent from all other leukocytes and tissues. It acts as calcium channel involved in B cell activation and cell cycle progression.

CD21Staining Pattern: MembraneDescription: CD21 (CR2, C3d receptor and EBV receptor) is expressed strongly on mature B-cells, follicular dendritic cells (FDC) and weakly on immature thymocytes and T-lymphocytes. In B-cell ontogeny, CD21 appears after the pre-B-stage, is maintained during peripheral B-cell development and is lost upon terminal differentiation into plasma cells. Immunohistological analysis of FDC in paraffin sections of NHL with this antibody demonstrates a nodular and usually dense and sharply defined FDC meshwork in follicular lymphomas and a loose, ill-defined FDC of varying size in some diffuse lymphoma types. Precursor B-cell lymphoma (lymphoblastic lymphomas), Burkitt lymphomas, plasmacytomas and hairy cell leukemias constantly lack FDC.

CD22Description: Anti-CD22 is directed against the type 1 integral membrane glycoprotein CD22. Anti-CD22 exhibits a cell membranous and/or cytoplasmic staining pattern and may be used to aid in the identification of B-cell lymphomas.

CD23

Staining Pattern: MembranousDescription: CD23 is a 45kDa glycoprotein, which is present on a subpopulation of freshly isolated peripheral blood and tonsil B cells and strongly expressed on EBV-transformed B lymphoblasts. The CD23 molecule is identical to the low affinity IgE receptor found on B cells. Expression of CD23 has been detected in neoplastic cells from cases of B cell chronic lymphocyctic leukaemia and some cases of centroblastic/centrocytic lymphoma.

CD30 (BerH2)

Staining Pattern: Membranous or paranuclearDescription: CD30, a single chain glycoprotein, is synthesized as a 90kDa precursor which is processed in the Golgi complex into a membrane-bound phosphorylated mature 105/120kDa glycoprotein. The CD30/Ki-1 antigen is expressed by mononuclear Hodgkin and multinucleated Reed-Sternberg cells in Hodgkin’s disease, by the tumor cells of a majority of anaplastic large cell lymphomas, and by a varying proportion of activated T and B cells.


DOS Rev • 01.04.13

Immunohistochemisty

CD31Staining Pattern: Cytoplasmic, MembranousDescription: CD31 is a glycoprotein expressed on endothelial cells and in platelets. It is known to be involved in cell signaling and cell adhesion. Antibody to CD31 is of value in the study of benign and malignant vascular tumors. Staining for CD31 has also been used to measure angiogenesis, which reportedly predicts tumor recurrence.

CD34 (QBEnd/10) Staining Pattern: MembranousDescription: CD34, a single chain transmembrane glycoprotein, is selectively expressed on human lymphoid and myeloid hematopoietic progenitor cells. Staining for CD34 has been used to measure angiogenesis, which reportedly predicts tumor recurrence.

CD43Staining Pattern: Membranous and CytoplasmicDescription: CD43 (leukosialin, sialophorin, or leukocyte sialoglycoprotein) is a cell surface glycoprotein which is expressed on all thymocytes and T-cells. CD43 is involved in activation of T cells, B cells, NK cells, and monocytes.

CD45RODescription: Anti-CD45RO reacts predominantly with the p180 component of the Leukocyte Common Antigen (LCA) antigen family and weakly with the 190 kD and 205 kD isoforms. This reagent reacts with most T lymphocytes, macrophages, and Langerhan’s cells of normal tissue.

CD45 (LCA)Staining Pattern: MembranousDescription: CD45 leucocyte common antigen (LCA) belongs to the family of at least four isoforms of membrane glycoproteins (220, 205, 190, 180kDa) expressed on hematopoietic cell lines but absent on non-hematopoietic cell lines, normal and malignant non-hematopoietic tissues. The intracellular portion of these molecules have protein phosphatase activity and are involved in regulation of transmembrane signals.

CD56 (N-CAM)Staining Pattern: MembranousDescription: Three isoforms of neural cell adhesion molecule (NCAM) are produced by differential splicing of the RNA transcript from a single gene. The 135kDa isoform is the basic molecule which is glycosylated or sialylated to produce the mature species. NCAM (CD56) is reported to express on most neuroectodermal derived cell lines, tissues, and neoplasms such as retinoblastoma, medullblastoma, astrocytoma, and neuroblastoma. It is also expressed on some mesodermally derived tumors such as rhabdomyosarcoma and also on natural killer cells.

CD57Description: Anti-CD57 antibody marks a subset of lymphocytes known as natural killer (NK) cells. Follicular center cell lymphomas often contain many NK cells within the neoplastic follicles. NK-1 also stains neuroendocrine cells and their derived tumors, including carcinoid tumor, and medulloblastoma. NK-1 reportedly also reacts with a variety of cell types in nonlymphoid tissues.


DOS Rev • 01.04.13

Immunohistochemisty

CD61Staining Pattern: CytoplasmicDescription: CD61 (GPIIIa) is a glycoprotein found on megakaryocytes, platelets and their precursors. CD61 antigen plays a role in platelet aggregation and also as a receptor for fibrinogen, fibronectin, von Willebrand factor and vitronectrin.

CD68 (PGM1)

Staining Pattern: Cytoplasmic, MembranousDescription: Anti-CD68 reacts with a 110 kD intracellular glycoprotein associated with the cell membrane of macrophages and some myeloid elements.

CD79aStaining Pattern: MembranousDescription: A disulphide-linked heterodimer, consisting of mb-1 (or CD79a) and B29 (or CD79b) polypeptides, is non-covalently associated with membrane-bound immunoglobulins on B cells to constitute the B cell Ag receptor. CD79a first appears at pre B cell stage and persists until the plasma cell stage where it is found as an intracellular component. CD79a is found in the majority of acute leukemias of precursor B cell type, in B cell lines, B cell lymphomas, and in some myelomas. It is not present in myeloid or T cell lines.

CD117 (c-kit) Staining Pattern: Cytoplasmic, MembranousDescription: Recognizes a protein of 145kDa, which is identified as CD117/p145kit. This rabbit polyclonal antibody does not interfere with the binding of SCF to c-kit. It precipitates both the unoccupied as well as the occupied form of c-kit. The binding of the stem cell factor (SCF) to the c-kit-encoded receptor tyrosine kinase (Type III) stimulates a variety of biochemical responses that culminate in cellular proliferation, migration, or survival. C-kit plays an important role in hematopoiesis, melanogenesis, and gametogenesis.

CD138Staining Pattern: MembranousDescription: CD138 / Syndecan-1 is a transmembrane heparin sulphate proteoglycan which is made up of one core protein and five glycosaminoglycan. CD138 is expected to play a role in cell adhesion. It is expressed on the surface of pre-B cells and plasma cells but is absent from mature B cells. It is a selective marker for B cell lymphoblastic leukemia and lymphoplasmocytoid leukemia. It is lost from the apoptotic myeloma cells; hence is a useful marker for viable myeloma cells.

CDX2Staining Pattern: NuclearDescription: The caudal-related homeodomain protein2 (CDX2) is a transcription factor shown to play a role in the development of small and large intestine in mammals and in the differentiation of intestinal epithelial cells. It has been shown that CDX2 protein detection by IHC coorelates with RNA transcript levels. In studies using 745 cancers from many anatomic sites, colonic adenocarcinomas demonstrated strong staining in 90% of the cases, while adenocarcinomas of the stomach, esophagus and ovary showed extensive staining in only 30% of the cases. Stains of CDX2 and Villin are useful markers in differntial diagnosis of bladder adenocarcinoma and secondary colorectal adenocarcinoma2.


DOS Rev • 01.04.13

Immunohistochemisty

CEA, MonoclonalStaining Pattern: CytopasmicDescription: Carcinoembryonic antigen (CEA), is synthesized during development in the fetal gut, and is re-expressed in increased amounts in intestinal carcinomas and several other tumors. Antibody to CEA is reportedly useful in identifying the origin of various metastatic adenocarcinomas and in distinguishing pulmonary adenocarcinomas (60-70% are CEA+) from pleural mesotheliomas (rarely or weakly CEA+).

CEA, PolyclonalStaining Pattern: CytoplasmicDescription: Carcinoembryonic antigen (CEA), is synthesized during development in the fetal gut, and is re-expressed in increased amounts in intestinal carcinomas and several other tumors. Antibody to CEA is reportedly useful in identifying the origin of various metastatic adenocarcinomas and in distinguishing pulmonary adenocarcinomas (60-70% are CEA+) from pleural mesotheliomas (rarely or weakly CEA+).

Chromogranin AStaining Pattern: CytoplasmicDescription: Chromogranin A (a protein of 439 amino acid which is encoded on chromosome 14) is present in neuroendocrine cells throughout the body, including the neuroendocrine cells of the large and small intestine, adrenal medulla and pancreatic islets. It is an excellent marker for carcinoid tumors, phenochromocytomas, paragangliomas, and other neuroendocrine tumors. Coexpression of chromogranin A and neuron specific enolase (NSE) is common in neuroendocrine neoplasms.

Collagen Type IVDescription: Collagen Type IV is the major component of the basal lamina, so antibodies to this molecule confirm its presence and reveal the morphological appearance of the structure. Normal tissue stains with this antibody in a fashion consistent with the sites of mesenchymal elements and epithelial basal laminae. Anti-Collagen IV can also be useful in the classification of soft tissue tumors; schwannomas, leiomyomas, and their well-differentiated, malignant counterparts usually immunoreact with this antibody. The vascular nature of neoplasms, hemangiopericytoma, angiosarcoma, and epithelioid hemangioendothelioma can be revealed by this antibody with greater reliability than non-specific stains (e.g. silver reticulum).

Cyclin D1 (BCL-1)Description: BCL-1 is one of the key cell cycle regulators, and functions in association with cdk4 and / or cdk6 by phosphorylating the Rb protein. It is a putative proto-oncogene over expressed in a wide variety of human neoplasms including mantle cell lymphomas (MCL).


DOS Rev • 01.04.13

Immunohistochemisty

Cytokeratins 5/6Staining Pattern: CytoplasmicDescription: Twenty human keratins are divided into acidic (pI <5.7) and basic (pI >6.0) subfamilies. Members of the acidic and basic subfamilies are found together in pairs. The composition of keratin pairs varies with the epithelial cell type, stage of differentiation, cellular growth environment, and disease state. Many studies have shown the usefulness of keratins as markers in cancer research and tumor identification.

Cytokeratin 7Staining Pattern: CytoplasmicDescription: Cytokeratin 7 is a basic cytokeratin which is found in most glandular and transitional epithelia but not in the stratified squamous epithelia. Keratin 7 is expressed in the epithelial cells of ovary, lung, and breast but not of colon, prostate, or gastrointestinal tract. Antibody to cytokeratin is useful in distinguishing ovarian carcinomas (keratin 7+) from colon carcinomas (keratin 7-).

Cytokeratin 8/18Description: Anti-cytokeratin 8 & 18 is a cocktail of two mouse monoclonal antibodies. Cytokeratins 8 & 18 can be found in most simple epithelium, e.g. thyroid, female breast, gastrointestinal tract, and respiratory tract. Adenocarcinomas and most non-keratinizing squamous carcinomas will stain, but keratinizing squamous carcinomas will not. This cocktail is used when attempting to demonstrate the presence of Paget cells; there is very little keratin 18 in the normal epidermis so this will only stain Paget cells. This approach facilitates the interpretation using immunostains and is more sensitive than mucin histochemistry.

Cytokeratin 17Staining Pattern: CytoplasmicDescription: The type I keratin 17 (K17) shows a peculiar localization in human epithelial appendages including hair follicles, which undergo a growth cycle throughout adult life. Additionally K17 is induced, along with K6 and K16, early after acute injury to human skin. Predominant expression of K17 and the frequent expression of K8 and K19, with little K6/K16 and K1/K10 expression are the characteristic features of basal cell carcinomas (BCC), suggesting that BCC is differentiated towards undifferentiated follicular epithelia, most probably hair bulge cells.

Cytokeratin 19Staining Pattern: CytoplasmicDescription: Keratin 19 is a member of type I acidic subfamily of intermediate filaments. It is expressed in various different human tissues except in liver. Keratin 19 is not expressed in hepatocytes, therefore, antibody to keratin 19 is useful in the identification of liver metastasis.


DOS Rev • 01.04.13

Immunohistochemisty

Cytokeratin 20Staining Pattern: CytoplasmicDescription: Keratin 20 / cytokeratin20 (CK20) is a Type-I keratin which is primarily expressed in gastric and intestinal epithelium, urothelium, and Merkel-cells. CK20 is expressed in adenocarcinomas of the colon, stomach, pancreas and the bile system. CK20 is also present in mucinous ovarian tumors, transitional-cell and Merkel-cell carcinomas. Notably, the squamous cell carcinomas and adenocarcinomas of the breast, lung, and endometrium, non-mucinous tumors of the ovary, and small cell carcinomas lack in CK20.

Cytokeratin 903 HMW (34ßE12) (CK34)Staining Pattern: CytoplasmicDescription: In normal cells, Ab-3 labels squamous, ductal and other complex epithelia. It reacts with benign small-acinir lesions of the prostate and does not react with hepatocytes, pancreatic acinar cells, proximal renal tubes or endometrial glands. In tumor cells, this antibody is reactive with both squamous and ductal neoplasms and variably with those derived from simple epithelium.

Cytokeratin LMW (AE1) Staining Pattern: CytoplasmicDescription: Twenty human keratins are resolved with two-dimensional gel electrophoresis into acidic (pI <5.7) and basic (pI >6.0) subfamilies. The acidic keratins have molecular weights of 56.5, 55, 51, 50, 50’, 48, 46, 45, and 40kDa. The basic keratins have molecular weights of 65-67, 64, 59, 58, 56 and 52kDa. Members of acidic and basic subfamilies are found together in pairs. The composition of keratin pairs varies with cell type, differentiation status and environment. Many studies have shown the usefulness of keratins as markers in cancer research and tumor diagnosis.

D2-40 (Podoplanin)Staining Pattern: Cytoplasmic (lymphatic epithelium)Description: Cyclin D2 is a G1 cyclin required for G1-phase progression and is a strong candidate for a proto-oncogene. cyclin D2 can phosphorylate pRB when associated with cdk4 and/or cdk6.

Desmin Staining Pattern: CytoplasmicDescription: Desmin is an intermediate filament protein of both smooth and striated muscles. Antibody to desmin reacts with striated (skeletal and cardiac) as well as smooth muscle cells. In skeletal and cardiac muscles, the staining is confined to the Z-bands giving a characteristic striated appearance. Anti-desmin antibody is useful in identification of tumors of myogenic origin.

DOG-1Description: DOG1 is used as an aid in the identification and diagnosis of gastrointestinal stromal tumors (GIST) within the context of an antibody panel, the patient’s clinical history, and other diagnostic tests evaluated by a qualified pathologist.


DOS Rev • 01.04.13

Immunohistochemisty

E-CadherinStaining Pattern: MembranousDescription: E-cadherin (uvomorulin, cell-CAM120/80) is a calcium dependent cell adhesion molecule expressed predominately in epithelial tissues. It plays an important role in the growth and development of cells via the mechanisms of control of tissue architecture and the maintenance of tissue integrity. Numerous studies have demonstrated that reduction and/or loss of E-cadherin expression in carcinomas correlates positively with the potential of these tumors for invasion and metastasis.

EMA Staining Pattern: Cytoplasmic, MembranousDescription: Epithelial membrane antigen (EMA), is a mucin-like glycoprotein. Antibody to EMA has been shown useful as a pan-epithelial marker for detecting early metastatic loci of carcinoma in the bone marrow or liver.

ERStaining Pattern: NuclearDescription: ER exists in two types: alpha and Beta. They are similar in structure. The ligand binding domain and the DNA binding domain are highly conserved in these proteins while the N-terminal transactivating domain is diverged considerably. Five isoforms of the hER beta gene, designated hER beta 1-5 have been identified. ER-Beta binds to estrogen and activates genes through direct interaction with estrogen specific gene elements (ERE’s). ER-Beta RNA has been detected in human thymus, spleen, ovary and testis and in rat ovary and prostrate. This tissue distribution overlaps but is not identical to that of ER - alphaRNA.

Factor VIII Staining Pattern: CytoplasmicDescription: Factor VIII related antigen or von Willebrand factor is a multimeric glycoprotein. It has functional binding domains to platelet glycoprotein Ib, glycoprotein IIb/IIIa, collagen and heparin. von Willebrand factor is synthesized by endothelial cells and stored in the Weibel-Palade granules. It mediates platelet adhesion to injured vessel walls and serves as a carrier and stabilizer for coagulation factor VIII. von Willebrand factor is one of the most useful markers to identify endothelial (or megakaryocytic) lineage of neoplasms. As not all endothelial cells synthesize / store) this molecule, about 30% of tumors of vascular origin fail to stain for factor VIII related antigen, regardless of whether they are benign or malignant. Staining for factor VIII related antigen has also been used to measure angiogenesis, an indicator of tumor recurrence.

Factor XIIIaStaining Pattern: CytoplasmicDescription: Factor XIII in both reduced and non-reduced forms. It does not react with human Factor XIII B-chain or human Factor XII. Factor XIII is a Beta-globulin found in plasma and is composed of two subunits. Factor XIII-A is the catalytic subunit and is a dimer of M.W. 160kDa. Factor XIII is present in plasma as an alpha2Beta2 heterodimer (M.W. 320kDa); whereas in platelets, only the alpha2 unit exists. Factor XIIIa is a dermal dendrocyte marker and shows variable reaction with these types of tumors. It can be used for histiocytic phenotyping and has been reported to mark capillary hemangiomas and tumors of the central nervous system. Factor XIII has also been used with CD34 to differentiate between dermatofibroma and dermatofibrosarcoma protuberans.


DOS Rev • 01.04.13

Immunohistochemisty

Galectin-3Description: Galectin-3 is a 31 kD beta-galactosidase binding lectin. It has been associated with binding to the basement membrane glycoprotein laminin. Anti-galectin-3 has been demonstrated to be valuable in differentiating between benign and malignant thyroid neoplasms in both histologic sections and fine needle aspiration biopsy material. Anti-galectin-3 antibody has also been useful in identifying anaplastic large cell lymphoma.

GATA-3Description: GATA-3 orchestrates gene expression profiles during embryogenesis of a variety of human tissues, including hematopoietic cells, skin, kidney, mammary gland, and the central nervous system. GATA-3 appears to control a set of genes involved in the differentiation and proliferation of breast cancer. The expression of GATA-3 has a strong association with the expression of estrogen receptor-alpha (ER) in breast cancer, and there is mounting evidence that GATA-3 can be used as a clinical marker to determine response to hormonal therapy and to refine the prognosis of breast cancer patients. GATA-3 has also been shown to be a novel marker for bladder cancer

GCDFP-15 (BRST-2)Staining Pattern: CytoplasmicDescription: (GCDFP-15) Gross cystic disease of the breast is benign premenopausal disorder in which cysts are a predominant pathological lesion. These cysts appear to be formed from excessive apocrine cystic secretions. This fluid is composed of several glycoproteins including a unique 15kDa monomer protein, Gross Cystic Disease Fluid Protein-15 (GCDFP - 15). Cytosolic analysis of normal tissue specimens from all major organs has demonstrated GCDFP15 in apocrine epithelia, lacrimal, ceruminous and Moll’s glands and in numerous serous cells of the submandibular, tracheal, bronchial, sublingual and minor salivary glands. GCDFP15 and prostate specific antigen are co-expressed in androgen receptor-positive breast tumours.

GFAP Staining Pattern: CytoplasmicDescription: This antibody reacts with human GFAP and has been solid phase absorbed with human and cow serum. Anti-GFAP stains astrocytes and some groups of ependymal cells and their corresponding tumors. In the peripheral nervous system, Schwann cells, enteric glial cells and satellite cells are stained. Weak staining of axons has been observed which is caused by cross-reaction with neurofilament. It is useful for distinguishing neoplasms of astrocytic origin from other neoplasms in the central nervous system. Negative staining has been observed with lymphatic tissue, muscle, gastrointestinal tract, liver, kidney, pancreas and bladder.

Glycophorin ADescription: Glycophorin A is a sialoglycoprotein present on human red blood cells and their precursors. Anti-Glycophorin A has been used to characterize erythroid cell development and in the diagnosis of erythroid leukemias.


DOS Rev • 01.04.13

Immunohistochemisty

Glypican 3Description: Anti-Glypican 3 (GC33) Mouse Monoclonal Primary Antibody is directed against the heparan sulfate proteoglycan, glypican 3. This antibody may be used to aid in the differentiation of hepatocellular carcinoma from normal liver or benign lesions.

HCGDescription: Human chorionic gonadotropin (hCG) is a protein secreted in large quantities by normal trophoblasts; the antibody detects cells and tumors of trophoblastic origin such as choriocarcinoma. Large cell carcinoma and adenocarcinoma of the lung demonstrate hCG positivity in 90% and 60% of cases respectively. 20% of squamous cell lung carcinomas are positive for hCG. hCG expression by non-trophoblastic tumors may indicate aggressive behavior since it has been observed that hCG may play a role in the host response to a given tumor.

Helicobacter pyloriDescription: Anti-H. pylori antibody reacts with a spiral bacillus bacteria located on the surface of the pyloric and stomach mucosa. Studies have shown that H. pylori plays an important role in the etiology of chronic gastritis and the development of peptic ulcer disease.

Hep Par 1Description: Staining Pattern: CytoplasmicDescription: Anti-Hepatocyte Specific Antigen recognizes both benign and malignant liver derived tumors such as hepatoblastoma, hepatocellular carcinoma and hepatic adenoma. It recognizes both adult and fetal liver tissue. The typical pattern is a granular cytoplasmic staining. This antibody is useful in differentiating hepatocellular carcinomas from adenocarcinomas, either primary or metastatic. This antibody also can be used in differential diagnostic separation of hepatoblastoma versus other small round cell tumors.

HER-2/neu (4B5) Ventana Pathway®Staining Pattern: MembranousDescription: This antibody is intended for in vitro diagnostic use. Ventana Medical Systems, Inc’s (Ventana) PATHWAY anti-HER-2/neu (4B5)Rabbit Monoclonal Primary Antibody (PATHWAY HER-2 (4B5)) is a rabbit monoclonal antibody intended for laboratory use for the semi-quantitative detection of HER-2 antigen in sections of formalin-fixed, paraffin-embedded normal and neoplastic tissue on a Ventana automated immunohistochemistry slide staining device. It is indicated as an aid in the assessment of breast cancer patients for whom Herceptin treatment is considered.


DOS Rev • 01.04.13

Immunohistochemisty

HMBE-1Description: Anti-HBME-1 has been demonstrated to label mesothelial cells, both benign and malignant (malignant mesothelioma) and thus has been used in distinguishing mesothelioma from adenocarcinomas of various origins. More recently this antibody has been used to distinguish thyroid carcinomas (both follicular and papillary) from benign thyroid lesions.

HSA (Hepatocyte Specific Antigen - Hep Par 1)Staining Pattern: CytoplasmicDescription: Anti-Hepatocyte Specific Antigen recognizes both benign and malignant liver derived tumors such as hepatoblastoma, hepatocellular carcinoma and hepatic adenoma. It recognizes both adult and fetal liver tissue. The typical pattern is a granular cytoplasmic staining. This antibody is useful in differentiating hepatocellular carcinomas from adenocarcinomas, either primary or metastatic. This antibody also can be used in differential diagnostic separation of hepatoblastoma versus other small round cell tumors.

Human Herpes Virus 8 (HHV8) Staining Pattern: NuclearDescription: HHV 8 is the likely etiological agent of Kaposi’s sarcoma (KS). HHV 8 encodes a latent nuclear antigen (LNA), which is the product of the viral gene of 73. LNA is capable of forming a complex with retinoblastoma susceptibility gene product, which may be related to its oncogenic activity.

HMB-45Staining Pattern: CytoplasmicDescription: By immunohistochemistry, Ab-1 specifically recognizes a protein in melanocytes and melanomas. Intradermal nevi, normal adult melanocytes, and non-melanocytic cells are negative. It does not stain tumor cells of epithelial, lymphoid, glial, or mesenchymal origin.

InhibinStaining Pattern: CytoplasmicDescription: Inhibin is a dimeric glycoprotein hormone from TGF-beta family made up of alpha and beta subunits. It inhibits the production of follicle-stimulating hormone from pituitary while activin stimulates the production of FSH. It is suggested that inhibin may act as a gonadal tumor suppressor.

Kappa Staining Pattern: CytoplasmicDescription: Antibody to the kappa light chain of immunoglobulin is reportedly useful in the identification of leukemias, plasmacytomas, and certain non-Hodgkin’s lymphomas. Demonstration of clonality in lymphoid infiltrates indicates that the infiltrate is clonal and therefore malignant.


DOS Rev • 01.04.13

Immunohistochemisty

Ki-67Staining Pattern: NuclearDescription: Ki-67 is a nuclear protein, which is expressed in the proliferating cells. Ki-67 is preferentially expressed during late G1-, S-, M-, and G2-phases of the cell cycle, while cells in the G0 (quiescent) phase are negative for this protein.

Lambda Staining Pattern: CytoplasmicDescription: Antibody to the lambda light chain of immunoglobulin is reportedly useful in the identification of leukemias, plasmacytomas, and certain non-Hodgkin’s lymphomas. Demonstration of clonality in lymphoid infiltrates indicates that the infiltrate is clonal and therefore malignant.

LCA (CD45)Description: CD45 belongs to the family of at least four isoforms of membrane glycoproteins (220, 205, 190, 180kDa) expressed on hematopoietic cell lines but absent on non-hematopoietic cell lines, normal and malignant non-hematopoietic tissues. The intracellular portion of these molecules have protein phosphatase activity and are involved in regulation of transmembrane signals

MammaglobinDescription: Mammaglobin encodes a 10 kDa glycoprotein and is distantly related to a family of epithelial secretory proteins that includes rat estramustine-binding protein/prostatein and human Clara cell 10 kDa protein (CC10)/uteroglobin. Mammaglobin, a mammary-specific member of the uteroglobin family, is known to be overexpressed in human breast cancer. Studies suggest that mammaglobin is one of the first relatively mammary-specific and mammary-sensitive markers (85%). Mammaglobin may be valuable used in a panel with GCDFP-15 and ER in evaluating tumors of unknown primary sites.

Mart1/Melan A (A103)Staining Pattern: CytoplasmicDescription: Melan-A (MART-1, Melanoma Antigen Recognized by T-cells 1), is a differentiation antigen that is expressed in 100% of melanocytes, most melanomas and 50-60% of melanoma cell lines. Melan A recognizes a subcellular fraction found in melanosomes. Melan-A is a useful addition to melanoma panels since it is specific for melanocytic lesions. Both HMB-45 and Melan-A are coexpressed in the majority of melanomas, as well as uniquely expressed in certain cases. Studies have shown that Melan-A is more sensitive than HMB-45 when labeling metastatic melanomas. Melan-A antibody labels the tumor cells of a subset of adrenocortical carcinomas and sex cord tumors of the gonads.

MLH1Description: MLH1 is a mismatch repair gene that is deficient in a high proportion of patients with microsatellite instability (MSI-H). This finding is associated with the autosomal dominant condition Hereditary Non-Polyposis Colon Cancer (HNPCC). The anti-MLH1 antibody is useful in screening patients and families for this condition. Colon cancers that are microsatellite unstable have a better prognosis than their microsatellite stable counterparts.


DOS Rev • 01.04.13

Immunohistochemisty

Moc-31Staining Pattern: MembranousDescription: Also known as Epithelial specific Antigen/Ep-CAM, consists of two 34 and39 kDa glycoproteins. These glycoproteins are located on the cell membrane surface and in the cytoplasm of virtually all epithelial cells with the exception of most squamous epithelia, hepatocytes, renal proximal tubular cells, gastric parietal cells and myoepithelial cells. MOC-31 is used in a panel of antibodies as a negative marker for mesothelioma; and a negative stain for MOC-31 has been shown to exclude lung adenocarcinoma. MOC-31 is useful in differentiating tumors of unknown origin in liver cancers and distinguishing cholangiocarcinoma (+) from hepatocellular carcinomas (-). MOC-31 may be advantageous in the demonstration of epithelial cell differentiation in cases where anti-cytokeratins are not clearly positive or in cases where a false positivity for cytokeratin cannot be excluded, such as in submesothelial cells.

MPO Staining Pattern: CytoplasmicDescription: Myeloperoxidase is an important enzyme used by granulocytes during phagocytic lysis of foreign particles engulfed. In normal tissues and in a variety of myeloproliferative disorders myeloid cells of both neutrophilic and eosinophilic types, at all stages of maturation, exhibit strong cytoplasmic reactivity for MPO. Erythroid precursors, megakaryocytes, lymphoid cells, mast cells, and plasma cells are nonreactive. MPO is not observed in the neoplastic cells of a wide variety of epithelial tumors and sarcomas. MPO is useful in differentiating between myeloid and lymphoid leukemias.

MSAStaining Pattern: CytoplasmicDescription: This antibody is specific for alpha- and gamma-actins of smooth muscle and reacts with myocardium and skeletal muscle, arterial wall muscle fibers, smooth muscle of the G.I. tract, myometrium, prostatic stroma and bladder wall. This antibody should be useful as a muscle marker in normal and uncharacterized tissues.

MSH2Description: MSH2 is a mismatch repair gene which is deficient in a high proportion of patients with microsatellite instability (MSI-H). This finding is associated with the autosomal dominant condition Hereditary Non-Polyposis Colon Cancer (HNPCC). The anti-MSH2 antibody is useful in screening patients and families for this condition. Colon cancers that are microsatellite unstable have a better prognosis than their microsatellite stable counterparts.

MSH6Description: Anti-MSH6 is used to qualitatively identify human DNA mismatch repair (MMR) protein MSH6, expressed in the nucleus of normal proliferating cells. Deficient or low levels of MSH6 are associated with colorectal and other cancers.


DOS Rev • 01.04.13

Immunohistochemisty

MUC1Description: Anti-MUC1 is directed against the membrane bound, glycosylated phosphoprotein MUC1. This antibody may be used to aid in the identification of normal and neoplastic MUC1 expressing cells.

MUC2Description: The heterogeneous pattern of mucin expression, including the expression of the intestinal mucin MUC2, may provide new insights into the differentiation pathways of gastric carcinoma. MUC2 is specifically expressed in goblet cells of the small intestine & colon. Expression is rare outside of the GI tract except for mucinous carcinoma of the breast and clear cell-type carcinoma of the ovary.

MUM-1Staining Pattern: Nuclear, CytoplasmicDescription: Multiple myeloma oncogene-1 (MUM-1) is a 50 kDa protein encoded by the MUM-1 gene. IRF4 / MUM-1 is expressed in the nuclei and cytoplasm of plasma cells and a small percentage of germinal center (GC) B cells located in the light zone. This antibody labels MUM-1 protein in centrocytes and their progeny, plasma cells, activated T cells and a wide spectrum of hematolymphoid neoplasms derived from these cells. Therefore, this antibody can be used as a powerful tool for the identification and the subclassification of lymphoid malignancies.

MyoglobinDescription: Immunostaining with anti-myoglobin is useful for the identification of tumors of muscle origin. Since myoglobin is found exclusively in skeletal and cardiac muscle and is not present in any other cells of the human body, it may be used to distinguish rhabdomyosarcoma from other soft tissue tumors. Anti-myoglobin staining is also useful when demonstrating rhabdomyoblastic differentiation in other tumors, e.g. neurogenic sarcomas and malignant mixed mesodermal tumors of the uterus and ovary.

Napsin ADescription: Immunohistochemical studies have revealed high expression levels of napsin A in human lung and kidney but low expression in spleen. Napsin A is expressed in type II pneumocytes and in adenocarcinomas of lung. The high specificity expression of napsin A in adenocarcinomas of lung is useful to distinguish primary lung adenocarcinomas from adenocarcinomas of other organs.

NSE Staining Pattern: CytoplasmicDescription: Enolases are homo- or heterodimers of the three subunits: alpha (46kDa), beta (44kDa), and gamma (46kDa). The alpha-subunit is expressed in most tissues and the beta-subunit only in muscle. The gamma-subunit is expressed primarily in neurons, in normal and in neoplastic neuroendocrine cells. Coexpression of NSE and chromogranin A is common in neuroendocrine neoplasms.


DOS Rev • 01.04.13

Immunohistochemisty

PAX-5 Staining Pattern: NuclearDescription: PAX-5 is a B-cell specific activator protein (BSAP). In early stages of B-cell development, PAX-5 influences the expression of several B-cell specific genes, such as CD19 and CD20. PAX-5 is expressed primarily in pro-, pre-, and mature B-cells, but not in plasma cells. There is an excellent correlation between CD20 and Pax-5 expression; however anti-Pax-5 exceeds the specificity and sensitivity of L26 (CD20) because of its earlier expression in B-cell differentiation and its ability to detect all committed B-cells, including classic Hodgkin’s lymphoma. It is very specific to B-cell lineage and does not stain T-cells. In essence, PAX-5 may be a superior pan B-cell marker to CD20.

PAX-8Description: PAX-8 antibody is used an aid to determine if the disease can be classified as renal cell carcinoma, thyroid carcinoma, or ovarian non-mucinous carcinoma.

p16Description: p16 is used for detection of the p16INK4a protein on FFPE tissue sections prepared from cervical biopsies. Positive staining is characterized as diffuse, continuous staining of cells of the basal and parabasal cell layers of the squamous cervical epithelium, with or without staining of cells of intermediate to superficial cell layers. This pattern is representative of overexpression of the p16INK4a protein within the cervical epithelium. Negative staining is demonstrated by either focal staining or no staining of the cervical epithelium.

p21Description: p21 is cyclin-dependent kinase inhibitor 1A (p21, Cip1), also known as CDKN1A. p21 acts as an inhibitor of the cell cycle during G1 phase and is tightly controlled by the tumor suppressor protein p53. Normal cells generally display a rather intense nuclear p21 expression. Loss of p21 expression has been associated with poor prognosis in several carcinomas (gastric carcinoma, non-small cell lung carcinoma, thyroid carcinoma).

p53Staining Pattern: NuclearDescription: p53 is a tumor suppressor gene expressed in a wide variety of tissue types and is involved in regulating cell growth, replication, and apoptosis. It binds to mdm2, SV40 T antigen and human papilloma virus E6 protein p53 senses DNA damage and possibly facilitating repair. Mutation involving p53 is found in a wide variety of malignant tumors, including breast, ovarian, bladder, colon, lung, and melanoma.

p63Staining Pattern: NuclearDescription: The p63 gene, a homologue of the tumor-suppressor p53, is highly expressed in the basal or progenitor layers of many epithelial tissues. P63 shows remarkable structural similarity to p53 and to the related p73 gene. Unlike p53, the p63 gene encodes multiple isotypes with remarkably divergent abilities to transactivate p53 reporter genes and induce apoptosis.


DOS Rev • 01.04.13

Immunohistochemisty

P504sStaining Pattern: CytoplasmicDescription: P504s is an enzyme in the ß-oxidation of branched-chain fatty acids. Expression of P504s protein is found in prostatic adenocarcinoma but not in benign prostatic tissue. It has also been found to stain premalignant lesions of the prostate: high-grade prostatic intraepithelial neoplasia (PIN) and atypical adenomatous hyperplasia. P504s can be used as a positive marker for PIN. It may be useful to confirm the diagnosis of small foci of prostate carcinoma in needle biopsies. P504s stains the majority prostate cancer, however, P504s has been shown to stain many other types of carcinoma such as hepatoma, breast carcinoma, pancreatic islet tumor and desmoplastic small round cell tumor.

Pan Keratin (AE1/AE3/ PCK26)Staining Pattern: CyotplasmicDescription: AE1/AE3 recognizes the acidic and basic (Type I and II) subfamilies of cytokeratins. The cocktail of these two antibodies can be used to detect most human epithelia. The acidic cytokeratins have molecular weights of 56.5, 55, 51, 50, 50, 48 46, 45, and 40 kDa. The basic cytokeratins have molecular weights of 65-67, 64, 59, 58, 56 and 52 kDa. This pan cytokeratin antibody has proved useful as a screener for the majority of human carcinomas.

Pan-Cytokeratin Plus (AE1/AE3 + CK 8/18)Staining Pattern: CytoplasmicDescription: AE1/AE3 recognizes acidic and basic subfamilies of cytokeratins. The cocktail of these two antibodies can be used to detect most human epithelia. The acidic cytokeratins have molecular weights of 56.5, 55, 51, 50, 50, 48 46, 45, and 40 kDa. The basic cytokeratins have molecular weights of 65-67, 64, 59, 58, 56 and 52 kDa. Clone 5D3 recognizes cytokeratin (CK) 8 and 18 intermediate filament proteins. These are 52.5 kDa and 45 kDa respectively. In normal tissues, 5D3 recognizes all simple and glandular epithelium. In the past, AE1/AE3 has had problems marking certain tissues types and adenocarcinomas. The addition of CK 8/18 remedies some of these problems. For example a study of twenty-eight lipid cell (steroid cell) tumors of the ovary were studied by immunohistochemistry; 46% were positive for Cytokeratin 8/18 antibody, 37% were positive with the Cytokeratin cocktail AE1/AE3.

Pan-MelanomaDescription: The combination of HMB45, MART-1 Cocktail and Tyrosinase make this quadruple antibody cocktail a first-order pan melanoma screener, and may prove to be a valuable marker for melanoma metastasis in sentinel lymph nodes. The HMB45 clone reacts with a neuraminidase-sensitive oligosaccharide side chain of a glycoconjugate present in immature melanosomes. The HMB45-reactive antigen is present in cutaneous melanocytes, prenatal and infantile retinal pigment epithelium and melanoma cells. It is also thought to be oncofetal in nature. This antibody has been shown to label the majority of melanomas. The MART-1/Melan A recognizes a protein of 18kDa, identified as MART-1 (Melanoma Antigen Recognized by T cells 1) or Melan-A. Melan-A is a useful addition to melanoma panels which is specific to melanocytic lesions. Studies have also shown that MART-1 is more sensitive than HMB45 when labeling metastatic melanomas. Tyrosinase is a key enzyme involved in the initial stages of melanin biosynthesis. Studies have shown Tyrosinase to be a more sensitive marker when compared to HMB45 and MART-1. It has also been shown to label a higher percentage of desmoplastic melanomas than HMB45.


DOS Rev • 01.04.13

Immunohistochemisty

PIN

Staining Pattern: Cytoplasmic, NuclearDescription: In normal epithelia, HMW Cytokeratins (CK5 and CK14) stain basal epithelia in the prostate gland. p63 is detected in prostate basal epithelial nuclei in normal prostate, however, it is negative in malignant tumors of the prostate gland. Thus p63 is useful as a differential marker for benign and malignant tumors of prostate gland and can be useful as a negative marker. Expression of P504S protein is found in prostatic adenocarcinoma, but not in benign prostatic tissue. It has also been found to stain premalignant lesions of the prostate: high-grade prostatic intraepithelial neoplasia (PIN) and atypical adenomatous hyperplasia. P504S can be used as a positive marker for PIN. It will be useful to confirm the diagnosis of small focus of prostate carcinoma in needle biopsies. The combination of HMW CKs + p63 + P504S may be extremely useful for diagnosing prostatic intraepithelial neoplasia, especially in difficult cases and in cases with limited tissues. The HMW CKs and p63 stain normal (negative marker) and benign prostate glands, and the P504S stains cytoplasm in prostate adenocarcinoma and atypical adenomatous hyperplasia.

PLAP

Staining Pattern: CytoplasmicDescription: Reacts with a membrane-bound isoenzyme (Regan and Nagao type) of Placental Alkaline Phosphatase (PLAP) occurring in the placenta during the 3rd trimester of gestation. This antibody is highly specific to PLAP and shows no cross-reaction with other isoenzymes of alkaline phosphatases. It is useful in the identification of testicular germ cell tumors. Unlike germ cell tumors, PLAP-positive somatic cell tumors uniformly express epithelial membrane antigen (EMA).

PMS2Description: The gene product of PMS2 forms a heterodimer with MLH1 that interacts with MSH2 bound to mismatched bases in DNA. PMS2 functions as one of the major DNA mismatch repair genes along with MSH2, MLH1 and MSH6. Mutations in these genes are associated with hereditary nonpolyposis colon cancer (HNPCC), one of the most common hereditary diseases in man. Immunohistochemistry studies have further determined that the microsatellite instability phenotype in endometrial carcinoma is linked to defects in the MLH1/PMS2 gene.

PRStaining Pattern: NuclearDescription: The progesterone receptor is an estrogen-regulated protein. It has been proposed that expression of PR determination indicates a responsive estrogen receptor (ER) pathway, and therefore, may predict likely response to endocrine therapy in human breast cancer. A number of studies have shown that PR determination provides supplementary information to ER, both in predicting response to endocrine therapy and estimating survival. PR has proved superior to ER as a prognostic indicator in some studies.

PSAStaining Pattern: CytoplasmicDescription: PSA is a chymotrypsin-like serine protease (kallikrein family) produced by the prostate epithelium. PSA is used to confirm prostatic acinar cell origin in primary and metastatic carcinomas and to rule out non-prostatic carcinoma mimics. This antibody is to be used for paraffin-embedded tissue only and is not to be used in serum testing.


DOS Rev • 01.04.13

Immunohistochemisty

PSAPStaining Pattern: CytoplasmicDescription: Prostatic acid phosphatase (PSAP) is one of the two antigenic markers of prostatic carcinoma, the other being prostate specific antigen. It belongs to the kallikrein family of serine proteases and is suggested to act as a hydrolase to split phospharyl choline in semen and as a transferase.

RCC (Renal Cell Carcinoma)Staining Pattern: Cytoplasmic, MembranousDescription: In a normal kidney, gp200 is localized along the brush border of the pars convoluta and pars recta segments of the proximal tubule, as well as focally along the luminal surface of Bowman’s capsule adjoining the outgoing proximal tubule. Of other normal tissues examined, the gp200 is also localized along the luminal surfaces of breast lobules and ducts, the luminal surface of the epididymal tubular epithelium, within the cytoplasm of parathyroid parenchymal cells, and focally within the colloid of thyroid follicles. Thirty-one other normal tissues do not express similar or cross-reacting antigens. Reportedly, gp200 is expressed by 93% of primary and 84% of metastatic renal cell carcinomas.

S-100Staining Pattern: CytoplasmicDescription: S100 recognizes proteins of 21-24kDa, identified as the A and B subunits of S100 protein. S100 belongs to the family of calcium binding proteins such as calmodulin and troponin C. S100A is composed of an alpha and beta chain whereas S100B is composed of two beta chains. Antibody to S100 stains Schwannomas, ependymomas, astrogliomas, and almost all benign and malignant melanomas and their metastases. S100 protein is also expressed in the antigen presenting cells such as the Langerhan’s cells in skin and interdigitating reticulum cells in the paracortex of lymph nodes. The diagnosis of Histocytosis X is confirmed by S100 staining. S100 PAb is excellent for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues. S100 protein is highly soluble and may be eluted from frozen tissue during staining.

SecretagoginDescription: Secretagogin is found in prostatic adenocarcinomas as opposed to adenocarcinomas from other organs. Reports have demonstrated SCGN staining in most normal neuroendocrine tissues, except for the adrenal cortex. In other studies, SCGN staining was positive in most small cell lung cancers, showing expression more frequently than neuron specific enolase (NSE), chromogranin or synaptophysin. Secretagogin has been demonstrated in a subset of brain tumors by immunohistochemistry. SCGN was detected in singular neurons of the frontal and parietal neocortex, in basket and stellate cells of the cerebellar cortex, and in secretory neurons of the anterior part of the pituitary gland. SCGN is a novel neuroendocrine marker and should be useful in routine surgical pathology.

SMA (Smooth Muscle Actin) Staining Pattern: CytoplasticDescription: This antibody stains smooth muscle cells in artery vessel walls, gut wall, and myometrium. Myoepithelial cells in breast and salivary gland are also stained. It reacts with tumors arising from smooth muscles and myoepithelial cells.



DOS Rev • 01.04.13

SMM (Smooth Muscle Myosin)Staining Pattern: CytoplasmicDescription: Smooth Muscle Myosin is a cytoplasmic structural protein, which is a major component of the contractile apparatus in smooth muscle cells. Expression of smooth muscle myosin is developmently regulated, appearing early in smooth muscle development, and is specific for smooth muscle development.

SmoothelinDescription: Smoothelin is a constituent of the smooth muscle cell cytoskeleton protein exclusively found in differentiated smooth muscle cells (SMC). Cells with SMC-like characteristics, such as myofibroblasts, myoepithelial cells, skeletal and cardiac muscle, do not contain smoothelin. Distinguishing bladder muscularis mucosae (MM) from muscularis propria (MP) muscle bundles is crucial for accurate staging of bladder carcinoma. Strong smoothelin expression is nearly exclusively observed in muscularis propria. Therefore, the staining pattern of MP (strongly positive) and MM (negative or weakly positive) is useful for staging bladder urothelial carcinoma. Anti-smoothelin immunostaining can be helpful in differentiating benign (+) from malignant smooth muscle tumors (-), and other mimics(-).

Surfactant Protein ADescription: Lung surfactant protein-A (SP-A) is a major phospholipid-associated glycoprotein in surfactant and is a member of the C-type lectin superfamily that inhibits lipid secretion and enhances the uptake of phospholipid by alveolar type II cells. Levels of SP-A in amniotic fluid are reported to reflect the degree of fetal lung maturity and inadequate levels of surfactant at birth, a frequent occurrence in premature infants, results in respiratory failure. In individuals with lung adenocarcinomas, high concentrations of SP-A have been reported in pleural effusions except in poorly differentiated lung adenocarcinomas where a significant decrease of SP-A immunoreactivity has been reported.

SynaptophysinStaining Pattern: CytoplasmicDescription: This antibody recognizes a protein of 38kDa, identified as synaptophysin. It labels normal neuroendocrine cells of human adrenal medulla, carotid body, skin, pituitary gland, thyroid, lung, pancreas, gastrointestinal mucosa, Paneth’s cells in the gastrointestinal tract and of gastric parietal cells. Neurons in the brain, spinal cord, and retina are also labeled. In combination with anti-chromogranin A and anti-NSE, Ab to synaptophysin is very useful in the identification of normal neuroendocrine cells and neuroendocrine neoplasms.

TAG 72Description: TAG-72 is a high molecular weight glycoprotein that is present in human adenocarcinomas and in lesser amounts, non-neoplastic tissues. It has also been found to be useful to distinguish between mesothelioma and adenocarcinoma, however, false positive reactions can occur so results must be interpreted with the utmost caution.



DOS Rev • 01.04.13

TdTStaining Pattern: NuclearDescription: Terminal Deoxynucleotidyl Transferase (TdT). TdT is a DNA polymerase located in the cell nucleus which catalyses the polymerization of deoxynucleotides at the 3’ hydroxyl ends of oligo or polydeoxynucleotide initiators and functions without a template. TdT is considered to be a highly specific marker for the diagnosis and classification of acute lymphoblastic lymphoma/leuksemias. The determination of TdT expression is most valuable when it is different to differentiate histologically between lymphoblastic lymphoma and Burkitt’s lymphoma.

ThyroglobulinStaining Pattern: CytoplasmicDescription: This antibody recognizes a glycoprotein of 330kDa, identified as thyroglobulin. Antibody to thyroglobulin has been shown to be useful in positive identification of thyroid carcinomas of the papillary and follicular types. BIOCAREs cocktail of 2H11 and 6E1 antibodies stains thyroglobulin in follicular epithelial cells as well as colloid tissue. Demonstration of thyroglobulin in a metastatic lesion establishes the thyroid origin of the tumor. Adenocarcinomas of non-thyroidal origin are not reactive.

TRAcPDescription: Anti-TRAcP antibody labels the cells of hairy cell leukemia (HCL) with a high degree of sensitivity and specificity. Other cells stained with this antibody are tissue macrophages and osteoclasts, which also express abundant TRAcP activity.

TryptaseDescription: Tryptases constitute a subfamily of trypsin-like proteinases, stored in the mast cell secretory granules and basophils. Upon cellular activation, these enzymes are released into the extracellular environment. Anti-tryptase is a good marker for mast cells, basophils, and their derivatives.

TTF-1Staining Pattern: NuclearDescription: TTF-1(Thyroid transcription factor-1) is a member of the NKx2 family of homeodomain transcription factors. It is expressed in epithelial cells of the thyroid gland and the lung. Nuclei from liver, stomach, pancreas, small intestine, colon, kidney, breast, skin, testes, pituitary, prostate, and adrenal glands are unreactive. TTF-1 is detected in primary lung adenocarcinomas and small cell carcinomas and is absent in colon and breast carcinomas. Staining with TTF-1 antibody is useful for distinguishing between tumors of lung and non-lung origin.

TyrosinaseDescription: Tryptases constitute a subfamily of trypsin-like proteinases, stored in the mast cell secretory granules and basophils. Upon cellular activation, these enzymes are released into the extracellular environment. Anti-tryptase is a good marker for mast cells, basophils, and their derivatives.



DOS Rev • 01.04.13

UroplakinStaining Pattern: CytoplasmicDescription: Uroplakins (UPs) are a family of transmembrane proteins (UPs Ia, Ib, II and III) that are specific differentiation products of urothelial cells. In non-neoplastic urothelium, UPIII is expressed in the luminal surface plasmalemma of superficial (umbrella) cells. UPIII detects 53 - 66% urothelial carcinomas, whereas many non-urothelial carcinomas were UPIII-negative. Recent studies of UP gene expression in normal urothelium and bladder cancer specimens found that UP expression was absent after malignant transformation. Thus, UP expression might reflect the malignant potential of urothelial cancer cells as well as being cytodifferential markers of urothelial cells.

VillinDescription: Villin is a 95kD glycoprotein of microvilli associated with rootlet formation in gastrointestinalmucosal epithelium. Anti-villin labels the brush border area in the gastrointestinal mucosalepithelium. This antibody has been useful in differentiating gastrointestinal adenocarcinoma, neuroendocrine carcinomas and ovarian adeno-carcinomas from adenocarcinomas from other organs. Also labeled by this antibody are Merkel cells of the skin.

VimentinStaining Pattern: CytoplasmicDescription: Vimentin is the main intermediate filament protein in mesenchymal cells and is therefore of value in the differential diagnosis of undifferentiated neoplasms.

WT1Description: WT1 is a suppressor gene located on chromosome 11p13 . Wilms’ Tumor Protein (WT1) has been identified in proliferative mesothelial cells, malignant mesothelioma, ovarian cystadenocarcinoma, gonadoblastoma, nephroblastoma and desmoplastic small round cell tumor. Lung adenocarcinomas rarely stain positive with this antibody.


DOS Rev • 01.04.13

IHC Special Stains

AFB (Acid-Fast Bacteria)CPT: 88312Stain Procedure: Ziehl-Neelsen Method for Acid-Fast Bacteria StainDescription: Ziehl-Neelsen AFB Stain is used to detect the presence of acid-fast mycobacteria in tissue sections. Acid-fast techniques are of value in the detection of mycobacteria, rod-shaped organisms that sometimes exhibit filamentous (fungus-like) growth. The most significant disease-prodcting mycobacteria are Mycobacterium tuberculosis and Mycobacteriam leprae.

Congo Red/AmyloidCPT: 88313Stain Procedure: Alkaline Congo Red StainDescription: The Congo Red Special Stain demonstrates amyloid in tissue sections. Amyloid is predominantly a fibrillar protein that deposits in tissue under certain pathologic conditions. Following Congo Red Staining, bright apple-greed birefringence exhibited under polarized light is considered specific for amyloid.

GMSCPT: 88312Stain Procedure: Grocott Methenamine-Silver Nitrate Fungus StainDescription: GMS Fungus stain is used to demonstrate fungal organisms in tissue sections.

IronCPT: 88313Stain Procedure: Prussian Blue Stain for Ferric IronDescription: Prussian Blue Stain is used for the detection of ferric (FE3+) iron in tissues. Ferric iron is normally found in small amounts in the bone marrow and the spleen. Abnormally large deposits may be seen in hemochromatosis and hemosiderosis.

PAS - FungusCPT: 88312Stain Procedure: Hotchkiss-McManus Periodic Acid Schiff Reaction for FungiDescription: The principle of this stain is similar to that described in the PAS with/without. The Polysacchrides present in fungal cell walls react with Schiff reagent to demonstrate the fungi.

PAS with/without DiastaseCPT: 88313(x2) (with and without Diastase), can also be ordered individuallyStain Procedure: Periodic Acid Schiff Reaction with and/or without Diastase DigestionDescription: The PAS Stain with Diastase Digestion demonstrates glycogen in tissue sections. PAS Stain is for the demonstration of polysaccharides, neutral mucosubstances, and basement membranes. These two procedures are usually stained simultaneously to demonstrate the glycogen that is dissolved away in comparison to the untreated slide.

IHC Special Stains


DOS Rev • 01.04.13

IHC Special Stains

ReticulinCPT: 88313Stain Procedure: Modified Gomori Stain for Reticular FibersDescription: The demonstration of reticular fibers in tissue sections can be important in the differential diagnosis of certain types of tumors. A change from the normal reticular fiber pattern, as is seen in some liver diseases, is also an important diagnostic finding.

TrichromeCPT: 88313Stain Procedure: Masson Trichrome StainDescription: Trichrome stains are frequently used to differentiate between collagen and smooth muscle in tumors and to identify increases in collagenous tissue in diseases such as cirrhosis of the liver.

IHC Stains Coming Soon:• Adenovirus• Bartonella Henselae• CD123• CD38 • CDX2/CK7 Double Stain• CEACAM5• Cytokeratin OSCAR• Cytomegalovirus• DBA.44• ERCC1• HPV • HSV I• HSV II• IGA• IGG• IGM• Mesothelin• MITF• Myo D1• PAX2• Survivin• Thrombomodulin (TM)• Thymidylate Synthase (TS)• Thyroid Stimulating Hormone


DOS Rev • 01.04.13

Immunohistochemisty Antibody Library

Immunohistochemistry Antibody Library

AFP

CDX2

MUC1

MUC2

MUM1

Myoglobin

Napsin A

NKX 3.1

NSE (Neuron Specific Enolase)

MSH6

MSH2

ALK1

Amyloid A

Amyloid P

BCA225P

BCL-1 (Cyclin D1)

BCL-2

PAX-8

p16

p21

p53

p63

p504s

BCL-6

CA125

CA19-9

BerEP4

Beta-Catenin

BG-8

Calponin - 1

Caldesmon

Calcitonin

Pan-Melanoma

PAX-5

Calretinin

Pan Keratin (AE1/AE3/PCK26)

Pan-Cytokeratin Plus (AE1/AE3 + CK 8818)

CD1a

CD2

PLAP

PMS2

PR (Progesterone Receptor)

PSA (Prostate Specific Antigen)

PSAP (Prostatic Acid Phosphatase)

RCC (Renal Cell Carcinoma)

S-100

Secretagogin

Smooth Muscle Actin

CD4

Desmin

D2-40 (Podoplanin)

CD5

CD8

CD7

E-Cadherin

EMA (Epirhelial Membrane Antigen)

ER (Estrogen Receptor)

Factor VIII

Factor XIIIa

Galectin-3

GCDFP-15 (BRST-2)

GFAP (Glial Fibrillary Acidic Protein)

Desmoglein 3

DOG-1

CD10

Synaptophysin

TAG 72

TdT

Thyroglobulin

CD23

AFB (Acid-Fast Bacertia)

WT1

SPECIAL STAINS

Tryptase

TRAP

CD30 (BerH2)

HMB-45 (Melanosome)

HHV-9 (Human Herps Virus-8)

HBME-1

Helicobater Pylori

Glycophorin A

Glypican 3

HCG

HER-2/neu (4B5) Ventana Pathway®

TTF - 1

Tyrosinase

Uroplakin

HSA Hep Par 1

Vimentin

Villin

Congo Red/Amyloid

Inhibin

Reticulin

GMS

Iron

Kappa lg Ligh Chain

Wright-Giemsa

Lambda lg Light Chain

Trichrome

CD117 (c-kit)

PAS with/without Diastase

CD138

CEA, Monoclonal

CEA, Polyclonal

Chromogranin A

Cytokeratin 5/6

Collagen Type IV

Cytokeratin 7

Cytokeratin 17

Cytokeratin 8/18

Cytokeratin 19

Cytokeratin 20

Cytokeratin 903 HMW (34βE12)

Cytokeratin 903 + p63 cocktail

PIN-4 Triple Stain

CD3, Monoclonal

CD43

CD45 (LCA)

CD45RO

CD56 (N-CAM)

CD57

CD61

CD68 (PGM1)

CD79a

CD31

CD34 (QBEnd/10)

Cytokeratin LMW (AE-1)

Cytokeratin CAM 5.2

Surfactant Protein A

Smooth Muscle Myosin

Smoothelin

CD15 (Leu-m1)

CD20 (L26)

CD19

CD21

Ki - 67

Mart1/Melan A (A103)

MammalobinA (A103)

Moc - 31

MLH1

MSA (Muscle Specific Actin)

MPO (Myeloperoxidase)

PAS - Fungus


DOS Rev • 01.04.13

Circulating Tumor Cells

The Circulating Tumor Cell test is a simple blood test that captures and assesses circulating tumor cells to monitor the progress of patients with Metastatic Breast, Colorectal or Prostate cancer. The CTC test makes it possible to determine if tumor cells of epithelial origin are circulating in a patient’s bloodstream.

How should the CTC test be used?1. Submit 10 mL of peripheral blood in CellSave Preservative Tube.

2. Establish a baseline.

3. Treat Patient.

4. Submit specimen every 4 – 6 weeks or prior to next round of therapy to monitor patient’s progress.

5. Evaluate and Consider Options.

Why is a CTC test helpful?CTCs are a strong, independent predictor of progression-free survival (PFS) and overall survival (OS). Monitoring CTCs helps clinicians monitor patients early in the course of therapy, evaluate response, and make critical treatment decisions. The CTC test represents a marked improvement over the current standard of care for determining disease progression and response to treatment.

• Analytical sensitivity: 1 CTC/7.5 mL whole blood.

• Analytical specificity: 99.7%.

CTC Specimen Requirements• Samples can only be collected in CellSave* tubes. The preservative is essential for the stability of

circulating tumor cells, if present.

• Draw CellSave tube prior to the administration of therapy.

• Fill one tube with 10 mL of peripheral blood and immediately mix by gently inverting the tube 6-8 times to prevent clotting.

• Store at room temperature (15-30°C) until shipping. Do not freeze or refrigerate specimen.

• Protect sample from extreme temperature during shipping/transport with cool pack.

• Ship promptly. Samples must be processed within 96 hours of collection.

• Patients on Doxorubicin (Adriamycin) must wait at least seven (7) days after administration to draw sample for CTC test.

NeoGenomics Laboratories will provide CTC collection kit (with Cell Save tube) and requisitions for test ordering and shipping.

Circulating Tumor Cells (CTC)

neogenomics dos

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