Flow Cytometry

Specimen Ordering

CRIS Order Entry Does Not Replace Pre-Approval or Pre-Scheduling. Specimen processing may be initiated upon a verbal request to the pathologist, but processing will not be completed or reported without a CRIS order.

Go to:  Specimen Scheduling and Delivery

No Specimen Will Be Accepted Without an Active CRIS Order Prior to Delivery.

Enter Flow Cytometry orders for all follow-up orders as far in advance as possible (6-month advance order entry is not unusual for follow-up protocol specimens).  Include the fellow’s name and pager number, patient history and clinical question.

1. Peripheral Blood Flow orders:
   • In CRIS:  Anatomical Pathology / Flow Cytometry, Blood
   • Complete the requested information.
2. Bone Marrow Aspirate Flow orders:
   • In CRIS:  Anatomical Pathology / Flow Cytometry, Bone Marrow
   • Complete the requested information.
3. CSF Flow orders:
   • In CRIS:  Anatomic Pathology / Flow Cytometry, CSF
   • Complete the requested information.
4. Other fluids or non-BM biopsies and aspirates (LN/Tissue/FNA): 
   Non-CSF fluids require Cytopathology triage.  

Enter two (2) CRIS orders with Specimen source / Site indicated for both:  

1. Anatomical Pathology / Cytopathology – Non-GYN

• Special Instructions: “Send aliquot for Flow Cytometry”
• Enter Specimen Type & Anatomic Site of Specimen

2. Anatomical Pathology / Flow Cytometry, Other

• Enter Specimen Type & Site

STAT Requests

If rapid diagnosis and notification of result is required to begin treatment:

• Contact a Flow Cytometry Pathologist to communicate the reason why it is a STAT.
• Only Dr. Wang and the Flow Cytometry Pathologist can approve a STAT status on a specimen.  Triage (301) 480-7179
• Indicate STAT nature in the CRIS order "Special Instructions" section.
• Include name and pager number of fellow on evening call in the "Special Instructions" section.

SPECIMEN LABELING REQUIREMENTS

ALL CRIS orders must match the specimen label information.

Unlabeled, incomplete, incorrect or mismatched specimens missing required label element(s) will not be processed until corrected identification is made IN PERSON.

REQUIRED SPECIMEN LABEL ELEMENTS on all specimen tubes and vials must include each of the following identification elements:

• FULL NAME: Patient first & last name (patient abbreviations or initials are not sufficient)
• UNIQUE #: One or more unique identifiers such as MRN, CRIS, NIH lab accession # or DOB
• DATE: Sample collection date
• SOURCE: Bone marrow aspirate, apheresis product, CSF, FNA, tissue.  EXCLUSION:  Blood.
• Source location: Left or right aspirate, FNA or biopsy site, fluid source (pericardial, pleural, BAL)
• OTHER: List anticoagulants, transport media, stabilizers or other reagents present in sample

Flow Cytometry Specimen Rejection Approval Requests:

Specimen processing of rejected specimens requre flow cytometry pathologist approval.

• Processing poor quality specimens is at the discretion of the Flow Cytometry Head or Deputy Head pathologist.
• Processing approval may include rare specimens; specimens collected at significant risk or suffering to the patient i.e. clotted BM aspirates; or specimens that cannot be recollected i.e. a spleen cannot be removed again.
• Results of poor quality specimens will be interpreted with caution.
• Rejected specimen processing is delayed pending efforts to discuss with the pathologist by one of these methods:
  • Call the triage pathologist (301) 480-7179 or
  • Call direct to Dr. Hao-Wei Wang (301) 480-8043 or
  • E-mail all Flow Cytometry pathologists:  FlowTriageNCI@mail.nih.gov  

Flow Cytometry Specimen Rejection Examples:

1. Unlabeled or mislabeled specimens will not be processed until the specimen label is corrected by knowledgeable staff in person. 
    
2. Specimens arriving after 1 p.m. may be held until the next day (if collected the same day received) or may be rejected if the schedule is full (or if the specimen was collected before the day received).
    
3. Improper collection or handling during transport (often detected during the gross specimen examination):
    
   • Coagulated, hemolyzed or frozen specimen may indicate non-ambient (freeze/heat) temperature conditions.
   • Inappropriate collection containers include lithium heparin tubes or other non-approved anticoagulants.
   • Specimens labeled radioactive or tuberculin hazard are rejected due to unavailable safety equipment in the testing area.
   • Inappropriate volume i.e. BM aspirate volume greater than 5ml (probable peripheral blood contamination unless needle repositioning noted). Note:  BM specimen volume 5 - 10ml are processed without prior pathologist approval; the BM aspirate volume is noted in the final report to interpret with caution.
   • Specimen clotting may be due to inadequate heparin.  Note:  Clotted BM aspirate samples with less than 50% clotting are processed without prior pathologist approval; presence of clots are noted in the final report to interpret with caution. 
      
4. Specimens with very low cellular content due to cell loss or disease conditions.
    
5. Fixed Specimen / Specimen in Fixative: 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. 
    
6. Low Viability / Non-Viable cells:  Viability less than 60% may be rejected. Cells derived from blood, bone marrow, fluids and tissue must be viable for flow cytometry testing. Cell death may be due to excessive age of specimen, poor handling, or patient therapy.

Specimen Handling of Rejected Specimens:

1. The Flow Cytometry laboratory director or alternate will contact the clinician for recollection of rejected specimens.
2. The rejected specimen will be accessioned and finalized. 
3. In addition, the clinician will be notified and the specimen will be held at room temperature for retrieval by the sending service i.e. Hematopathology, Cytopathology or clinical group for alternate testing.  
4. Specimens remaining in the Flow Cytometry lab will be discarded the following work week.

1. Flow Cytometry testing is not appropriate for the following differential diagnosis specimens:

• Not appropriate for any specimen for Hodgkin lymphoma.
• Not ideal for anaplastic large cell lymphoma, lymphomatoid granulomatosis (LYG), thymic B cell lymphoma or large cell lymphoma.

2. Pre-approval / Scheduling Is Required for All Specimens.

• Contact the on-call Triage Flow Cytometry pathologist by calling the Triage phone line: (301) 480-7179; or

• Contact all Flow Cytometry pathologists by email: FlowTriageNCI@mail.nih.gov; or

   Go to Specimen Ordering and Labeling (see above).

3. Pre-approval / Scheduling 1:00 p.m. Specimen Delivery Cut-Off Time, Monday - Friday.

• Schedule flow cytometry specimen collection for the first morning collection / STAT Escort pickup and delivery to Flow Cytometry Lab, Room 3S240 / 3S238.
• Specimens received before 1:00 p.m. will be processed the same day.
• Specimens arriving after 1:00 p.m. will not be processed without pathologist pre-approval (see 2. above)

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 a Flow Cytometry pathologist can designate a specimen as "Emergency" or "Rush" after direct contact (see 2. above).

5. Emergency, Weekend, Holiday and Evening Specimen Delivery.

Blood specimens collected 24 hours before a workday will be processed the next day (i.e., on Sunday before a Monday or on a holiday before a workday). Deliver to the Flow Cytometry Drop Box located in 3North corridor near 3N248.  Blood specimens collected more than 24 hours before a workday will not be processed; recollect.

After Working Hours:

Please Contact the Page Operator (301) 496-1211 to reach the Pathology Resident on Call

Weekend and evening specimens will only be processed in extreme cases when a life-threatening clinical situation requires immediate treatment based, at least partially, on flow cytometry results.

For EMERGENCY weekend or holiday delivery or processing of CSF, Fluid, or BM specimens, call the NIH Page Operator (301) 496-1211 to contact the Pathology Resident On-Call.

Non-Emergency routine weekend and evening specimens sent to local reference laboratories will be billed to the clinical protocol.  

IMPORTANT!! Flow Cytometry aliquot must be FIXATIVE-FREE i.e. no formalin, no B5 fixative, no alcohol, etc. Fixation will cause cell death and is not reversible.

Specimen delivery cutoff time is 1 p.m. Monday – Friday workdays (no weekends or holidays).

After Working Hours:

Please Contact the Page Operator (301) 496-1211 to reach the Pathology Resident on Call

Deliver to Room 3S240 / 3S238 Magnuson Center:  Blood, bone marrow, CSF, FNAs, fluid or other cell suspensions at room temperature (20 - 25C). Deliver frozen cells on dry ice.  (Do NOT place any specimen type other than blood or bone marrow in the flow cytometry drop box.)

Peripheral Blood Sodium Heparin vacutainers, Collection & Delivery

• Enter “Flow Cytometry, Blood” order in CRIS
• 18ml blood in three (3) 6ml sodium heparin green top vacutainer tubes.
• For Absolute Lymph count < 1.0 K / uL, collect an additional 20-30ml.
• Print labels using positive patient ID procedures (mobile SoftID station and printed Collection Label).
• Attach Collection Label (patient full name, unique identifier, date, “Peripheral Blood" or code FLOWB).
• Deliver STAT or call Escort STAT pickup and delivery to 3S240 / 3S238 at room temperature, no cold packs.

Bone Marrow aspirate Heparinized Syringe, Collection & Delivery

See also BM Aspirate collection instructions

• Go to scheduling.com to schedule BM aspirate collection.
• Enter “Flow Cytometry, Blood” order in CRIS.
• Before collection, get sodium heparinized syringe from the Pharmacy for BM aspirate collection.
• The fellow or assisting nurse must aspirate 3 - 5ml BM aspirate IMMEDIATELY into the heparinized syringe.
  Tip: Aspirate a small air bubble into syringe, cap syringe and use bubble to mix well by inverting 5 times.
• Print labels using positive patient ID procedures (mobile SoftID station and printed Collection Label).
• Attach Collection Label (patient full name, unique identifier, date, “BONE MARROW" or code FLOWM).
• Deliver STAT or call Escort STAT pickup and delivery to 3S240 /3S238 at room temperature, no cold packs.

Cerebral Spinal Fluid (CSF) Transport media, Collection & Delivery

• Enter “Flow Cytometry, CSF” order in CRIS.

Before collection, get Flow CSF transport media tube:

• 15ml conical tube:  Pink solution, blue cap, green label

• Pick up from CCR 3SW North Procedure Unit or 3SE South Day Hospital; or

• Flow Cytometry Lab 3S240 / 3S238, Magnuson Center (call ahead 301-480-7179 or 301-480-8074).

• The fellow or assisting nurse must dispense 3 - 4ml CSF IMMEDIATELY into the CSF transport media.

• Cap the tube and invert 5 times to mix well. 
  IMPORTANT:  Label / Deliver specimen STAT.  Half of cells are lost within hours without media.

• Collect using positive patient ID procedures (mobile SoftID station and printed Collection Label).
• Attach Collection Label (patient full name, unique identifier, date, “CSF” or code FLOWC).
• Deliver STAT or call Escort STAT pickup and delivery to 3S240 / 3S238 at room temperature, no cold packs.

Non-CSF Fluid, Fine Needle Aspirate or LN / Tissue Biopsy Triage in Cytopathology

See also Flow Cytometry, Other

1. All non-CSF fluids, FNAs and LN/tissue biopsies:

• Enter “Flow Cytometry, Other” order in CRIS noting the specimen and source.
• Deliver specimen labeled with Flow Cytometry label to Flow Cytometry 3S240 / 3S238.

IMPORTANT!! Flow Cytometry aliquot must be FIXATIVE-FREE i.e. no formalin, no B5 fixative, no alcohol, etc. Fixation will cause cell death and is not reversible.

2. Specimen volume < 10mL

          Before collection, get Flow CSF transport media tube:

• 15ml conical tube: Pink solution, blue cap, green label
• Pick up from CCR 3SW North Procedure Unit or 3SE South Day Hospital; or
• Flow Cytometry Lab 3S240, Manguson Center (call ahead 301-480-7179 or 301-480-8074).
• The fellow or assisting nurse must dispense 3 - 4ml of collected specimen IMMEDIATELY into the CSF transport media.
• Cap the tube and invert 5 times to mix well. 
  IMPORTANT:  Label / Deliver specimen STAT or call Escort STAT pickup and delivery to LP Flow Cytometry.  Half of cells are lost within hours without media.
• Print labels using positive patient ID procedures (mobile SoftID station and printed Collection Label).
• Attach Collection Label (patient full name, unique identifier, date, “<source>” or code FLOWO).
• Deliver STAT or call Escort STAT pickup and delivery to 3S240 / 3S238 at room temperature, no cold packs.

3. Specimen volume > 10mL

          Before collection, get plain container with sufficient volume for the specimen to be submitted:

• The fellow or assisting nurse must dispense the collected specimen into the plain container.
• Cap the container and seal for transport.
  IMPORTANT:  Label / Deliver specimen STAT or call Escort STAT pickup and delivery to LP Flow Cytometry.  Half of cells are lost within hours without media.
• Print labels using positive patient ID procedures (mobile SoftID station and printed Collection Label).
• Attach Collection Label (patient full name, unique identifier, date, “<source>” and code FLOWO).
• Deliver STAT or call Escort STAT pickup and delivery to 3S240 / 3S238 at room temperature, no cold packs.