1. Fluids
2. CSF
3. Bone Marrow
4. Peripheral Blood
5. Fine Needle Aspirates
6. Lymph Node Biopsy

Specimen Acceptance Criteria:

1. Flow cytometry is not appropriate in any specimen for Hodgkin's disease. Flow cytometry is not appropriate in blood or bone marrow for anaplastic large cell lymphoma, lymphomatoid granulomatosis (LYG), thymic B cell lymphoma or large cell lymphoma.

2. Pre-approval required for all specimens: Contact Dr. Maryalice Stetler-Stevenson 301-402-1424 or Dr. Constance Yuan 301-496-4709.

3. Pre-scheduling required for all specimens: After approval, all specimens must be pre-scheduled via e-mail nciflowcytometry@mail.nih.gov or call the Flow Lab (301-402-1716) to schedule and to get CSF or bone marrow transport media if needed. CSF or Bone marrow transport media is for flow cytometry B1 B58 specimens only.

4. A specimen cap is in effect . Even after approval, Emergency specimens will take priority over previously scheduled specimens. Emergency specimens are always processed. Only Dr. Stetler-Stevenson can designate a specimen as "Emergency" or "Rush".

5. Specimen labeling must include patient name and unique patient number. If the specimen is unlabeled or mislabeled, the clinician is required to identify specimen in person. Unlabeled or mislabeled specimens will not be processed.

6. All specimens must be ordered in CRIS prior to delivery. Enter Flow Cytometry orders under Anatomic Pathology as far in advance as possible (6 month advance order entry is not unusual for followup protocol specimens). The CRIS order printout in the Flow Cytometry department will be used as a requisition.

Note: CRIS order entry does not replace pre-approval or pre-scheduling. See #2 and #3, above. Specimens will not be accepted without an Active CRIS order. Processing may be initiated upon a verbal request to the pathologist (in #2, above) but processing will not be completed or reported without a requisition.

7. The CRIS order should include the fellow's name and pager number, patient history and clinical question. This information is vital to determine the correct set-up strategy for each specimen.

8. Phlebotomy Collection: Specimens to be collected by phlebotomy will require a second CRIS order. Enter the order as a Research Blood order and include specimen type, collection tube, volume, and STAT delivery to Flow Cytometry (near B-wing elevator) Building 10 Room B1 B58 . If the nurse coordinator or fellow will collect and deliver the specimen directly to flow cytometry, the Research Blood order is not necessary.

9. Specimen rejection: Poor quality specimens are rejected unless the flow cytometry laboratory director approves processing for specimens that are rare; acquired at significant risk or suffering to the patient; or collection that cannot be repeated (i.e. a spleen can not be removed again).

   1. Specimen is examined grossly for clots, hemolysis and other gross characteristics. Viability assessment is performed for most specimens. Criteria for flagging specimen include: clotted specimen due to inadequate heparin, large volumes of bone marrow aspirate indicating heavy peripheral blood contamination, low viability due to excessive age of specimen or poor handling, coagulated or frozen specimen indicating not maintained at appropriate temperature, cell number too low, radioactive specimen, and hemolyzed specimen. These specimens are only processed if cleared by the laboratory director.

   2. Cells derived from blood, bone marrow, fluids and tissue must be viable for flow cytometry testing. Any specimen with fixative added for any length of time i.e. formalin, B5 fixative, alcohol, etc. will be rejected for flow cytometry testing. Fixation is not reversible. In addition, lithium heparin should not be used as it is toxic to cells.

   3. If no cells detected or less than 60% viability, Hematopathology and Cytopathology specimens are returned to these services. For other clotted or non-viable specimens, tests may be performed at discretion of the medical director if specimen is rare, acquired at significant risk or suffering to patient or can not be repeated. Results will be interpreted with caution.

Emergency, Weekend and Evening Specimens:

• Contact the Pathology resident on call.

• Weekend and evening specimens will only be processed in extreme cases where there is a life threatening clinical situation requiring immediate treatment that will be based, at least partially, on Flow Cytometry results.

• Routine weekend and evening specimens can be sent to local reference laboratories at the expense of the clinical protocol.

1. Specimens go to Cytopathology. Note on the requisition that an aliquot should be sent to Flow Cytometry room B1 B58.

1. Before collection, get a Flow CSF transport media tube from the Flow Lab room B1 B58 (near the B-wing elevator) or from the refrigerator in the Intermediate Care Unit Supply Room, room 3-3655 (Soiled Utility) behind the CCR 3 SW North Procedure Unit (badge access required).

2. CSF must be added immediately to the Flow CSF transport media tube at collection. Studies show 50% cell loss within one hour if cells are not added to media for transport.

3. CSF should be delivered STAT to the Flow Cytometry Laboratory, Building 10 Room B1 B58 as cells rapidly disintegrate and are not usable for flow cytometry after 3 hours. Note accurate draw time or the specimen may be rejected.

4. If specimen is drawn because of patient neurologic changes, inform the flow cytometry lab and the results will be processed as a STAT order .

5. If specimens require a morphological diagnosis, send a second specimen to Cytopathology.

1. Notify the CCR Hematology lab that flow immunophenotyping is being performed (301-496-4473). The hematology bm collection tech will bring a BM transport media tube to the specimen collection site and prepare an extra smear for the Flow Cytometry Laboratory. This is in addition to scheduling with the Flow Lab (in 2 above) and entering a CRIS order (in 5 above).

2. Get sterile heparin suitable for injection from the nurse's station.

3. Rinse syringe and needle with sterile heparin, leaving no more than 0.2-0.5 mL in syringe.

4. After aspirate for morphology, reposition needle and, for cellular specimens, slowly aspirate 2-3 mL of bone marrow for flow cytometry. Aspiration of greater than 3mL results in significant dilution of the specimen in peripheral blood. For specimens with low cellularity, reposition and aspirate 3-5mL of bone marrow a second time. Note this on the requisition or the specimen will be rejected.

5. Immediately discharge syringe into transport media tube, cap tube tightly and mix by gentle inversion 5-6 times. Label tube with patient name, unique identifier number and date.

6. Deliver immediately to the Flow Cytometry Laboratory B1B58 (specimens containing hematopoietic neoplasms have a tendency to clot and must be processed immediately). Call for STAT Escort pickup and delivery if you cannot deliver the specimen yourself (301-496-9295).

7. The Active CRIS order should include the fellow's name and pager number, the patient history and clinical question. This information is vital as to the set-up strategy used. If rapid diagnosis and notification of result is required to begin treatment, indicate STAT nature, explain reason and leave name as well as pager number of fellow on evening call.

Peripheral Blood Specimens:

1. Draw at least 10mL blood into sodium heparinized (green) tubes. If the WBC is low, draw extra blood.

2. Deliver specimen and requisition immediately to the Flow Cytometry Laboratory, Building 10 Room B1 B58.

1. Specimens go to Cytopathology. Note on the requisition that an aliquot should be sent to Flow Cytometry room B1 B58. Note specimen site on the requisition.

1. Specimens go to Hematopathology. Note on the requisition that an aliquot be sent to Flow Cytometry room B1 B58. Note specimen site on the requisition.