WHICH SAMPLES SHOULD BE SUBMITTED WHEN LYMPHOID NEOPLASIA IS SUSPECTED?

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1 WHICH SAMPLES SHOULD BE SUBMITTED WHEN LYMPHOID NEOPLASIA IS SUSPECTED? Which test should be submitted? The answer to this depends on the clinical signs, and the diagnostic question you are asking. If you already have a cytologic or histologic diagnosis of lymphoma and are confident in this diagnosis, the only reason to submit additional testing is for prognostic information based on phenotype, or if treatment is going to be guided by phenotype. If cytology or histology give equivocal results, and/or you are seeking additional prognostic or diagnostic information, use the guidelines below to choose a test, or feel free to call the laboratory at , us at cvmbs [email protected] or go to our website, Species Clinical sign Site Cytology/Histology Best test to submit Dog/Cat Lymphocytosis Peripheral blood Small mature lymphocytes Dog Lymphocytosis Peripheral blood Immature forms/blasts Cat Lymphocytosis Peripheral blood Immature forms/blasts / Immunocytochemistry on blood smear (call laboratory) Dog/Cat Normal WBC Peripheral blood Immature forms/blasts Call laboratory Dog/Cat Lymphadenopathy Lymph node aspirate Confirmed or strongly suspicious for lymphoma Dog/Cat Lymphadenopathy Lymph node aspirate Homogeneous population of small lymphocytes Dog/Cat Lymphadenopathy Lymph node aspirate Heterogeneous or reactive with rare suspicious cells Dog/Cat Organomegaly Aspirate of organ Same rules as lymphadenopathy Dog/Cat Cavity effusion Pleural/peritoneal fluid A large number of abnormal lymphoid cells/large number of small mature cells Dog/Cat Mediastinal mass Mediastinum Suspicious for either lymphoma or thymoma Same rules as lymphadenopathy is the best way to distinguish between these two entities Dog/Cat Cavity effusion Pleural/peritoneal Rare abnormal lymphoid cells fluid Dog/Cat GI Disease Mesenteric lymph Reactive or neoplastic node Dog/Cat CSF CSF Elevated WBC consisting of normal or abnormal lymphocytes Dog Leukopenia Bone marrow Blasts present Dog/Cat Leukopenia Bone marrow Small lymphocytes present (peripheral blood is better if there is lymphocytosis) Cat Leukopenia Bone marrow Blasts present Immunocytochemistry Dog/Cat Leukopenia Bone marrow No clearly neoplastic cells Dog/Cat Hyperglobulinemia Serum Immunofixation Dog/Cat Neutrophilia We cannot diagnose chronic myelogenous leukemia by flow or Dog/Cat Hypercalcemia Testing blood or other aspirates in cases where the only sign is hypercalcemia is generally unrewarding Dog/Cat Screening, no clinical signs We do not recommend either or flow cytometry to be used as a screening test in a healthy animal with no clinical signs

2 SUBMITTING SAMPLES FOR FLOW CYTOMETRY Send all samples overnight with a cold pack for delivery Monday through Friday. Blood and bone marrow can usually be received Saturday (10 am delivery) and will be viable on Monday, but aspirates will not. You will not be charged for the viability test is the sample is dead, but shipping charges will still apply. Do not freeze samples! Blood and bone marrow EDTA tube containing at least 300 ul of sample. Blood samples need to have current (within 2 days) CBC. You can either include a copy of your CBC results, or we can carry out the CBC through CSU. To submit CBC to CSU, please include a second EDTA and fresh blood smear (if a second EDTA tube is impossible to obtain we can split a single tube it is more important to have a properly filled tube for an accurate CBC). Lymph node, mediastinal and other organ aspirates separator). Place 1 ml of saline (.9%, LRS, Norm R) in a red topped tube (plain tube, no additives, no serum Cavity fluid Add 0.1 ml of serum from the patient or another animal of the same species. Aspirate using suction and squirt the contents of the needle and syringe into the saline. Draw up saline through the needle and gently squirt back into tube to obtain more cells. Carry out this process several times if possible the saline should be cloudy. If possible send one tube each EDTA and red top tube containing 400ul or more of fluid. Send EDTA if only enough for one tube. Add a few drops of serum if the total protein is less than 4 mg/dl. Questions? Call us at , cvmbs [email protected] or go to our website,

3 SUBMITTING SAMPLES FOR Samples on slides can be kept indefinitely and do not require special storage or shipping. Samples in suspension (blood, cavity fluid, CSF) should be sent with a cold pack using express mail. These samples do not have to be sent for Friday delivery. Blood and bone marrow EDTA tube containing at least 200 ul of sample. Lymph node and other organ aspirates Aspirate onto slides, or into an EDTA tube with NO added fluid (i.e. don t add saline). If you aspirate onto slides and want to confirm that you have a good sample, the slides can be stained and still used for. Cavity fluid Send fluid in EDTA, or on a slide, either a smear or cytospin. If you aspirate onto slides and want to confirm that you have a good sample, the slides can be stained and still used for. CSF Multiple cytospin preps are the best sample (stained or unstained). An EDTA tube can also be sent. In order for the sample to be diagnostic we estimate you need 50,000 cells. Therefore if the CSF has a white count of 100, we need 0.5 mls of fluid, or the cells from 0.5 mls spun onto slides. A note about non diagnostic samples. Non diagnostic samples are obtained when there is too little DNA, or when there is an inhibitor of the PCR reaction. In the case of non diagnostic samples, we will repeat the assay under conditions that eliminate the effects of the inhibitor 1, but if that is not successful we will assume there was insufficient DNA. You will be charged for non diagnostic samples even if we are unable to obtain a result, so please be sure your samples are cellular. When possible, we try to contact the submitting clinic if we think the sample isn t going to be diagnostic, but this isn t always feasible. 1 You re probably wondering why we don t use these conditions all the time? There are several ways to manage inhibitors in PCR reactions, and they are all very expensive. Since we only rarely obtain non diagnostic samples, we keep costs down by only using these methods when necessary. Questions? Call us at , cvmbs [email protected] or go to our website,

4 SUBMITTING SAMPLES FOR C KIT MUTATION ANALYSIS AND MAST CELL PANEL Mutation analysis only cytology sample unstained. The best sample is an aspirate or impression smear of a biopsy of the mass on a slide, stained or Sample must have 10% mast cells in order to detect the mutation, so lymph node aspirates draining the mass are generally not useful. You can submit a stained slide if you would like to estimate the number of mast cells before submitting. *Remember*, Diff Quick stains don t always pick up mast cell granules. Mutation analysis only paraffin embedded sample (from a biopsy) Request that the laboratory to which you submitted your biopsy cut 5 8 curls or shavings from the paraffin embedded section and send these to our laboratory. No special shipping or handling requirements apply. Please fill out our submission form (link) and (cvmbs [email protected]) or fax ( ) this to us so we have sample information when the curls arrive. Full mast cell panel If mass removal has not yet been performed: Prior to, or immediately after removing the mass, please make several aspirates on slides. These will be used for the c kit mutation analysis. Once the aspirates have been obtained, proceed with routine biopsy submission. Aspirates must be obtained from fresh tissue prior to formalin exposure as even formalin fumes can damage DNA for PCR analysis. Pack the formalin vial with two sealable (ziplock type) bags and have the slide in a different sealed bag. If mass removal and biopsy submission have already been performed: Request that the laboratory who has the initial biopsy send the blocks to the CSU Veterinary Diagnostic Laboratory (shipping address at the end of this document). If it is not possible to send blocks, please request the original lab send at least 6 unstained sections on positively charged slides, as well as 5 8 curls as described above. If more than one block exists on the case, please be sure the sections are obtained from the block which contains the bulk of the mast cell tumor. Please fill out the submission form and the completed form to [email protected] Questions about the mutation analysis alone? Call us at or at [email protected]. Questions about the c kit panel? Call the Molecular Pathology Laboratory ( ) or [email protected].

5 SUBMITTING SAMPLES FOR IMMUNOCYTOCHEMISTRY (CD3, PAX5 AND CD18) Aspirates, blood and bone marrow Submit a minimum of 3 air dried slides together with the submission form. The slides can be stained or unstained. can also be performed on these slides after ICC has been run if desired. The standard panel for ICC is CD3, PAX5 and CD18, but other stains are available, and individual stains can be carried out rather than the panel. For these special requests, please contact the Molecular Pathology Laboratory at or [email protected].

6 TESTING AND REPORTING SCHEDULES Please note that this schedule is subject to change, so check back if timing is critical for your patient. Sample received Set up Reported Mon Wed Thurs Fri Weekend Thurs Sat (10 am) Mon Tues Wed Sample received Set up Reported Mon Mon or Tues Wed Tues Tues or Wed Wed Wed Wed or Thurs Weekend Thurs Thurs or Fri Weekend Fri Fri Weekend samples are set up on the day they are received or the next day. Our reporting schedule is as described above (Wednesday and the weekend) but when possible we try to report flow cytometry results as they are obtained. You are always welcome to call the laboratory if you need verbal results before the report can be generated. c kit mutation testing Cytology sample received Set up Reported Thurs Wed Thurs Fri Weekend Paraffin sample received Set up Reported Wed Tuesday Wed Fri Weekend or if part of the MCT panel, results are Reported through the Molecular Pathology Laboratory c kit panel and ICC testing These assays are performed through the Molecular Diagnostics Laboratory. Contact Brad Charles ( ) for information about sample schedule. C kit mutation analysis is carried out on Fridays and reported over the weekends if the test is submitted as a stand alone assay. If the test is part of the mast cell tumor panel, reporting will come from the Molecular Pathology Laboratory (phone , [email protected]). If mutation analysis is requested on biopsies in paraffin embedded sections, the samples must be received by Wednesday to be set up on Friday (these samples take considerably longer to work with than non formalin fixed specimens).

7 SHIPPING INSTRUCTIONS Fed Ex 1. Pack your specimens as usual. If your specimen needs a coolant, use a frozen ice pack but do not use dry ice. 2. Call Federal Express toll free at or GO FEDEX to request a pick up. Some remote areas have pick ups earlier in the day to insure next day delivery so be sure to ask when your specimens should be ready for pick up. 3. Call us for our Fed Ex number (we can t post it on line). When you have the number, call Fed ex and tell the customer service agent you are sending your specimens and that you will need a diagnostic specimen pack and airbill for your sample (if necessary). Be sure to request Standard Overnight service and check off "Recipient Box" for Payment. Also be sure and record the weight on the form. 4. The cost will be added to your accession's test charges and will be reflected on your sales service invoice and statement. 5. You may request either Saturday pick up (for Monday delivery) or Saturday delivery (with Friday pick up). Samples picked up Friday without a special request of Saturday deliver will arrive on Monday morning. Please be aware that there will be an additional $10.00 charge added to your sales service invoice for either Saturday pick up or delivery. If it is necessary to send flow cytometry samples over the weekend (we discourage this), they must be delivered on Saturday so they can be refrigerated. 6. Lab packs are available at no charge by calling GO FEDEX and pre printed airbills are available by calling the CSU Diagnostic Lab at The SHIPPING ADDRESS for Fed Ex/UPS/DHL is: CSU Dlab 300 West Drake Road Fort Collins, CO UPS/DHL We do not have an account number for these two services. Otherwise follow instructions for Fed Ex, including the shipping address. Regular mail If you are sending samples by regular mail, use the mailing address below. Pre filled mailers are available from the CSU Diagnostic Laboratory by calling The SHIPPING ADDRESS for regular mail is: Colorado State University Veterinary Diagnostic Laboratory 200 West Lake Street 1644 Campus Delivery Fort Collins, CO 80526