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Specimen Collection Guide

Postmortem Section - Procedures for Obtaining Autopsies or Submitting Materials for Examination

Instructions for having an autopsy performed at the NIH Clinical Center by the Postmortem Section of the Laboratory of Pathology

In general, any patient who has been seen at the NIH for any reason is eligible for an autopsy done by the Laboratory of Pathology. In addition, non NIH patients with diseases of significant clinical or basic research interest may have an autopsy done at the NIH if permission is obtained from the Chief of the Postmortem Section and the sponsorship of a principal investigator at the NIH. For an in-patient death or the death of an NIH or non-NIH patient outside the NIH:

1. Upon the death of an in-patient or notification of the death of an NIH patient outside the NIH, obtain a Death Packet from Admissions.

2. If the death falls within the jurisdiction of the Maryland State Medical Examiner, the Medical Examiner must be consulted prior to obtaining permission for autopsy. In general, patients who die from accidental causes, who arrive at the Clinical Center and are pronounced Dead On Arrival, or who die in association with a therapeutic procedure may fall under the jurisdiction of the Medical Examiner.

3. The Death Packet contains all of the necessary forms and instructions for having an autopsy performed on an NIH patient.

4. The NIH follows the State of Maryland's guidelines for consent to post-mortem examination. Permission from next-of-kin must be obtained even if the patient had a stated desire for an autopsy. The following is exerpted from the Annotated Code of Maryland, Section 5-501.

Consent for postmortem examination

(a) In general - Consent for a postmortem examination of a body by a physician is sufficient if the consent is given as provided in this section.

(b) Persons authorized to consent --

 

(1) The consent may be given by any one of the following persons if that person, whether alone or with another, has assumed control of the body for its final disposition:

 

(i) A parent;
(ii) A spouse;
(iii) A child;
(iv) A guardian;
(v) A next of kin; or
(vi) In the absence of these persons, any other person.

(2) If a person does not assume control of a body under paragraph (1) of the subsection, the consent may be given by the State Anatomy Board.

(c) Form of consent--The consent may be in the form of:

 

(1) A written document;
(2) A telegram; or
(3) A recorded telephonic or other recorded message

5. The patient may be transferred to the morgue from the nursing unit to await either autopsy or transfer to the responsible funeral home. The body must have proper identification (toe tag, wrist band) or the autopsy will not be performed.

6. Once permission for autopsy has been obtained, a member of the clinical care team should contact the pathology resident who will be assigned to the case, so that the clinical history and the questions to be answered may be reviewed with the pathology resident. The name and contact information of the pathology resident responsible for a new case may be obtained from the Surgical/Autopsy Pathology office (6-2441) during normal working hours. During weekday evenings and on weekends or holidays the resident on-call is responsible for performing autopsies. The name and contact information may be obtained through the NIH Page Operator.

7. The autopsy will be performed when the body, chart and permission have all arrived in the Laboratory of Pathology. Autopsies are begun routinely from 8:30 AM to 4:00 PM, Monday to Saturday and from 8:30 AM to 2:00 PM on Sundays and Holidays. Permission to perform an autopsy outside these times should be obtained from Dr. David Kleiner, Chief, Postmortem Section, Laboratory of Pathology. He can be reached by page (102-10333), by work phone (301-594-2942) or through the resident on-call.

Submission of Outside Materials for Review by the Postmortem Section

In general, the Postmortem section accepts materials for review or second opinion on patients who have died and have had an autopsy performed elsewhere.

1. The materials submitted may consist of tissue specimens, fixed or frozen, paraffin tissue blocks or stained slides. A cover letter or outside autopsy report identifying the materials should accompany the request for review. Copies of chart information, patient history summaries and laboratory data are helpful and should be made available to the Laboratory of Pathology.

2. The materials and any associated paperwork should be brought to the Surgical/Autopsy Pathology Office (Building 10, Room 2B50, 496-2441). A tissue examination request form should be filled out by the submitting physician. Frozen or Fixed tissue should be brought in sealed containers within an uncontaminated box.

3. The secretaries in the office will assist the submitting physician so that the case is appropriately handled and accessioned. The case is assigned to the resident pathologist on the postmortem service for that month.

4. If surgical or cytopathology materials are submitted with the autopsy materials, they will be separated and submitted to the appropriate section in the Laboratory of Pathology.

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Clinical Cytogenetics Laboratory Information Sheet

Laboratory Director:

 

Diane C. Arthur, M.D.
Phone: (301) 435-3712
FAX: ---(301) 435-3716
E-mail - arthurd@mail.nih.gov
Pager: 102-10334

Laboratory Staff:

Shannon I. Skarshaug, M.S., C.G.(ASCP) – Laboratory Supervisor
Valerie Zgonc, CLSp(CG), M.S. - Cytogenetic Technologist
Veronica Krevinko, M.T.(ASCP) – Cytogenetic Technologist

Laboratory Location: Building 10 - Room 4B50, 4B52

Mailing Address:

NIH/NCI/CCR Laboratory of Pathology
Building 10 - Room 2A33
10 Center Drive
Bethesda, MD 20892-1500

Laboratory Phone: (301) 435-3711

Web Site: http://home.ccr.cancer.gov/lop/Clinical/cytogenetics/default.asp

Test Ordering: Before entering an order for testing in CRIS, we would appreciate a phone call to the laboratory to schedule the testing at least 24 hours in advance of obtaining the sample(s). Please provide the following information when ordering the test:

Patient name, ID number, and date of birth or age
Attending Physician and/or Fellow's name and phone number
Diagnosis and pertinent clinical history
Type of testing to be done
Indication for testing
Date, time, and place the specimen will be collected

Please call the Escort Service when the specimen is collected and request immediate delivery to 10/4B52.

Reporting of Results: When the analysis is complete, a final report will be typed into the SoftPath system for electronic transmission to the CRIS, and a printed report will be sent to the patient's medical record, with a xerox copy to the physician(s). If abnormal or unexpected results are obtained, Dr. Arthur will contact the responsible physician(s) by telephone or electronically.

Clinical Cytogenetics Test Menu

General Comments: The Clinical Cytogenetics Laboratory is open from 8 AM to 5:30 PM weekdays. Should an emergency arise after 5:30 PM , or on weekends or holidays, Dr. Arthur can be reached on pager 102-10334.
All specimens for cytogenetic testing should be collected using sterile technique. Specimen containers should be labeled immediately with the patient's name and identification number as a minimum. All specimens should be kept at room temperature and transported to the laboratory as soon as possible, always within 24 hours of collection.
Turnaround times given below are averages. If results are needed sooner for diagnosis/patient management, arrangements can be made by calling Dr. Arthur or the laboratory. If cases are highly complex, or require additional staining techniques, results may take longer.
If you have any questions about specimen requirements or cytogenetic testing, please call
Dr. Arthur at 301-435-3712 or the Clinical Cytogenetics Laboratory at 301-435-3711.

1. Peripheral Blood - Constitutional Karyotype

Description: High-resolution G-banded karyotype analysis for the detection of constitutional numerical or structural chromosome abnormalities.
Specimen Requirements: Peripheral blood collected in a green top (sodium heparin) tube or sodium heparinized syringe.    Volume:  minimum 3 ml for newborns, 5 ml for infants and small children, 7-20 ml for adults.  NOTE:   Larger volumes may be needed from patients who are lymphopenic or immunodeficient.
Turnaround Time:
28 days.

2. Peripheral Blood - Cancer Karyotype

Description: G-banded karyotype analysis for detection of acquired numerical and structural chromosome abnormalities in patients with hematologic malignancies with high numbers of circulating neoplastic cells.
Specimen Requirements: Peripheral blood collected in a green top ( sodium heparin) tube or sodium heparinized syringe. Volumes will vary with the peripheral blast counts; on average 10 ml for children and 20 ml for adults are required.
Turnaround Time: 21 days.

3. Skin - Constitutional Karyotype

Description: G-banded karyotype analysis for the detection of constitutional numerical or structural chromosome abnormalities in patients suspected of having mosaicism or whose blood is suboptimal for cytogenetic testing.
Specimen Requirements: 3-4 mm punch biopsy in a tube containing 1-2 ml sterile tissue culture medium or sterile saline.
Turnaround Time:
28 days.

4. Bone Marrow - Hematologic Disorder/Cancer Karyotype

Description:G-banded karyotype analysis for the detection of acquired numerical and structural chromosome abnormalities in patients with hematologic disorders or malignancies primarily involving the marrow.
Specimen Requirements: 2-3 ml cellular bone marrow aspirated into a syringe containing 0.1 ml sodium heparin (1000 units/ml) per ml of marrow.  If the marrow is not aspirable, a 1.5-2.0 cm trephine core biopsy may be submitted in a tube containing 5 ml sterile tissue culture medium or sterile saline.
Turnaround Time: 21 days.

5. Neoplastic Tissue - Cancer Karyotype

Description:G-banded karyotype analysis of tissues such as lymph nodes, spinal fluid, effusions, or non-hematologic solid tumors for the detection of acquired numerical and structural chromosome abnormalities.
Specimen Requirements: Optimally, a minimum of 40 million viable cells should be submitted; i.e., 1.0 cm3 solid tissue or as much CSF or effusion as possible depending upon the blast counts.  Solid tissue should be submitted in a tube containing 5 ml sterile tissue culture medium or sterile saline.
Turnaround Time: 21 days

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Chromosome Pathology Instructions for Specimen Submission
Download FFPE-FISH Downtime Request Form

  1. In-house Cases
    For specimens collected at the NIH Clinical Center, requesting physicians will enter the request for FFPE FISH in the Clinical Research Information System (CRIS) in the same order used for requesting Surgical Pathology services. The box labeled “Tissue FISH Requested” should be checked and the specific tests should be listed in the ‘Special Instructions’ section at the bottom of the order.

  2. Outside (Submitted) Cases
    If FISH testing is needed on patient blocks or slides submitted from an outside facility for enrollment to a NIH protocol, a CRIS order for “Surgical Pathology – Outside Material”, should be entered. The Tissue FISH Requested box should be checked and specific tests entered into the “Special Instructions” box as for specimens collected at NIH. If a CRIS order cannot be entered because the patient is in pre-admit statues, fill out an “Outside Tissue Examination Form” (available in the Surgical Pathology Office). This form must be filled out completely and the specific FISH tests listed. Submit the CRIS order or Outside Tissue Examination Form along with the slides/blocks and the outside laboratory’s pathology reports. Please include the Outside Case Number, Block ID, and Tissue Source on your request.

  3. Iformation about tissue quality and submission of slides/blocks from other hospitals
    The amount of tissue required for molecular tests on solid tumors is variable and depends upon both specimen size and tumor content of the specimen. When you are requesting specimens from outside hospitals for evaluation at the NIH, a tissue block is preferred, for then we can control how the slides are prepared. However, many institutions will not release paraffin blocks and unstained slides must be requested instead. Please follow the guidelines below for specimen collection and handling:

  • Optimally, tissues should be fixed in formalin for 6-48 hours prior to processing and embedding. Tissue from B-5 or other mercuric fixatives generally does not hybridize well and will not be accepted.
  • Unstained sections should be cut at 5 micron thickness and mounted on TrueBond 380 (preferably) or positively charged slides.
  • For brain sections, two separate sections on a single slide are preferred.
  • Slide should be air (not oven) dried.
  • Slides must be clearly marked with the patient’s IDs.
  • Slides must be tested within 6 weeks of microtomy.
  • Store blocks and slides at room temperature (20-23.5°C) until submission.
  • Submit 4 unstained slides and 1 H&E slide (or 5 unstained slides) for each test.
 

Contact Information

Svetlana Pack, Ph.D
Head, Chromosome Pathology Unit,
Laboratory of Pathology, CCR, NCI, NIH
10 Center Dr., RM 2N115
Bethesda, MD 20892
Phone (301) 451-2723
Fax (301) 480-9488
Email

Zied Abdullaev, Ph.D
Molecular Biologist
Chromosome Pathology Unit,
Laboratory of Pathology, CCR, NCI, NIH
10 Center Dr., RM 2N115
Bethesda, MD 20892
Phone (301) 451-2711
Email

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Flow Cytometry Procedure Manual (Specimen Collections)

  1. FLOW CYTOMETRY OVERVIEW:

LOCATION:  The Flow Cytometry Laboratory is in Building 10, Room B1-B58.  We are located on the B1 level at the end of the B-wing corridor.  Normal work hours are 9:00 a.m. – 5:00 p.m., Monday – Friday excluding holidays and weekends. 

SCHEDULING & APPROVAL:  All Flow Cytometry specimens must be pre-approved and scheduled by a pathologist.  See B. Flow Cytometry Specimen Scheduling.

Flow Cytometry CRIS ORDERS:  All Flow Cytometry specimens must have a CRIS order.  Enter orders at the time of approval prior to specimen collection or delivery (see C. Flow Cytometry Specimen CRIS Ordering).
SPECIMEN DELIVERY / CUT-OFF TIME:  Deliver Flow Cytometry specimens STAT at room temperature (20-25C).  Cutoff time for same day processing is 1 p.m. Monday – Friday excluding holidays.  Late delivery, night, weekend, holiday or after-hours specimen processing requires pre-approval by the pathologist.

For late processing approval, contact Dr. Maryalice Stetler-Stevenson 301-402-1424 or Dr. Constance Yuan 301-496-4709.
For Night, Weekend, Holiday delivery, see E.3. Flow Cytometry Specimen Delivery.

  1. FLOW CYTOMETRY SPECIMEN SCHEDULING:

    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. Please note Flow Cytometry Specimen Delivery cutoff time is 1 p.m. Monday – Friday excluding holidays. 
      Flow Cytometry specimens should be scheduled for first morning collection whenever possible.
      Enter a CRIS order for STAT Escort pickup and delivery to Flow Cytometry B1 B58.

    3. Pre-approval and scheduling by a pathologist is required for all specimens:
      Contact via e-mail Dr. Maryalice Stetler-Stevenson 301-402-1424 or Dr. Constance Yuan 301-496-4709.

    4. Enter Flow Cytometry Specimen CRIS Orders immediately after pathologist approval and scheduling (see C. Flow Cytometry Specimen CRIS Orders).
      Specimen triage and scheduling is not complete without CRIS order entry. 
      Specimen processing will not be completed or reported without a CRIS order. 

    5. 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".

  2. FLOW CYTOMETRY SPECIMEN CRIS ORDERS:  

No specimen will be accepted without an Active CRIS order. To request STAT results: If rapid diagnosis and notification of result is required to begin treatment, add the following text to the CRIS Special Instructions section along with the evening on-call fellow name and pager number:  “STAT results requested”. Enter current and follow up Flow Cytometry specimens:  Enter future CRIS orders for follow up Flow Cytometry specimens as far in advance as possible (6 month advance order entry is not unusual for follow up protocol specimens).

  1. CSF CRIS order: After pathologist approval (see B. Scheduling, above), enter CRIS order: Find Patient > Order Entry > select Anatomic Pathology > select Flow Cytometry CSF.

    1. Flow Cytometry CSF order:  Complete order information; add fellow name, pager, patient history and clinical question.
      Flow Cytometry CSF specimens should be scheduled for first morning collection whenever possible. 

    2. Deliver specimen to Room B1-B58 STAT or enter in CRIS > Order Entry > Specimen/Item Transport > Priority: STAT > To Location:  Flow Cytometry B1 B58. 

  2. Peripheral Blood CRIS order: After pathologist approval (see B. Scheduling, above), enter CRIS order: Find Patient > Order Entry > select Anatomic Pathology > select Flow Cytometry Order Set.
    1. Flow Cytometry order:  Complete order information; add fellow name, pager, patient history and clinical question.

    2. Research Blood order:  Add text “Peripheral blood, 20ml sodium heparin green top tube. Deliver STAT to Flow Cytometry Room B1-B58.”  OR deselect Research Blood order for specimens shipped to NIH overnight.
      Flow Cytometry blood specimens should be scheduled for first morning collection whenever possible.

    3. Deliver specimen to Room B1-B58 STAT or enter in CRIS > Order Entry > Specimen/Item Transport > Priority: STAT > To Location:  Flow Cytometry B1 B58. 
  3. Bone Marrow CRIS order: After pathologist approval (see B. Scheduling, above), enter CRIS order: Find Patient > Order Entry > select Anatomic Pathology > select Flow Cytometry Order Set.

    1. Flow Cytometry order:  Complete order information; add fellow name, pager, patient history and clinical question.

    2. Research Blood order:  Deselect Research Blood order.
      Flow Cytometry BM specimens should be scheduled for first morning collection whenever possible. 

    3. Deliver specimen to Room B1-B58 STAT or enter in CRIS > Order Entry > Specimen/Item Transport > Priority: STAT > To Location:  Flow Cytometry B1 B58. 

  4. Other (Non-CSF Fluids, FNAs, tissues) CRIS orders:
    NOTE:  Fluids, FNAs and biopsies require Cytopathology triage.  After pathologist approval (see B. Scheduling, above), Enter TWO orders in CRIS > Find Patient > Order Entry > select Anatomic Pathology :

    1. Cytopathology order:  Include text “Send an aliquot for Flow Cytometry evaluation.
      For further Cytopathology instructions:  http://home.ccr.cancer.gov/lop/Clinical/cytopath/cytopath.asp
    2. Flow Cytometry Order Set
      Flow Cytometry order:  Complete order information; add fellow name, pager, patient history and clinical question.
      Research Blood order:  Deselect Research blood order.

    3. Cytopathology will arrange for delivery of the Flow Cytometry specimen after triage is complete.
  1. FLOW CYTOMETRY SPECIMEN LABELING and COLLECTION:

    1. SPECIMEN LABELING REQUIREMENTS:

      ALL CRIS orders must match the specimen label information. 

      Clinicians are required to identify IN PERSON any specimen with one or more missing, incorrect or mismatched specimen label elements.
      Unlabeled, incorrect or mismatched specimens will not be processed until correction is made.

      ALL SPECIMEN LABELS 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 number, NIH laboratory accession # and/or date of birth

    • DATE:                    Sample collection date

    • SOURCE:             bone marrow, apheresis, CSF, FNA, tissue. 
      Exception:  Blood specimens do not require labeled Source when matched to a CRIS order for blood.

      Additional label information should include the following elements not listed in the CRIS order.

    • CONTENTS:         Reagents or anticoagulants such as sodium heparin, transport media, stabilizers

    • Source Specifics:  Source specifics i.e. left or right aspirate, biopsy or FNA site, fluid source (pericardial, pleural, BAL)

    1.  SPECIMEN COLLECTION REQUIREMENTS for Flow Cytometry:

      1. Peripheral Blood collection:  After pathologist approval (see B. Scheduling) and order entry (C.1. CRIS Blood orders):

        1. 10 - 20ml blood in sodium heparin green top vacutainer tubes.
          For Absolute Lymph # less than 1.0 K / uL, collect an additional 10ml.

        2. Deliver room temperature (no ice, no cold packs).

        3. Deliver specimen to Room B1-B58 STAT before 1 p.m. or enter in CRIS > Order Entry > Specimen/Item Transport > Priority: STAT > To Location:  Flow Cytometry B1 B58

      2. Bone Marrow aspirate collection: After pathologist approval (see B. Scheduling) and order entry (C.2. CRIS BM orders):

        1. Notify the CCR Hematology lab that Flow Cytometry immunophenotyping is being performed (301-496-4473). The hematology bm collection tech will bring a 10ml sodium heparin green top vacutainer tube to the specimen collection site and prepare an extra smear for the Flow Cytometry Laboratory.

        2. Pre-prepare collection tubes as follows:

          1. Prepare 6ml syringe and needle with 0.5ml sterile 1,000 units/ml sodium heparin from the nurse's station.

          2. Attach patient label to a10ml sodium heparin green top vacutainer tube.

          3. Record “Bone Marrow” and today’s date on the patient label.

          4. Remove the stopper from the vacutainer tube.

        3. Aspirate the specimen for flow cytometry:

          1. REPOSITION NEEDLE after aspirate for morphology (and cytogenetics if requested).

          2. Slowly aspirate 3-5 mL of bone marrow for flow Cytometry.

          3. The fellow or assisting nurse must immediately dispense the 3 - 5ml BM aspirate into the labeled 10ml sodium heparin green top vacutainer tube, recap the tube and invert 5 times to mix well.

          4. Verify vacutainer specimen label includes “Bone Marrow”, patient name, number and today’s date.

        4. Deliver specimen at room temperature to B1-B58 STAT before 1 p.m. or enter in CRIS > Order Entry > Specimen/Item Transport > Priority: STAT > To Location:  Flow Cytometry B1 B58. 

      3. Cerebral Spinal Fluid (CSF) collection:  After pathologist approval (see B. Scheduling) and order entry (C.3. CRIS CSF orders):

        1. CSF collection requires a CSF Transport media tube.

        2. Before collection, pick up a Flow Cytometry CSF transport media tube (green label, pink reagent in tube):

          1. CCR 3SW North Procedure Unit or
          2. Flow Cytometry Lab room B1-B58 or call the lab (301-402-1716)

        3. Write “CSF” and today’s date on a patient label with name and unique identifier number.

        4. Attach label to Flow Cytometry CSF transport media tube.

        5. Slowly aspirate 3ml or more CSF for flow Cytometry.

        6. The fellow or assisting nurse must immediately dispense the CSF into the labeled CSF transport tube, cap the tube and invert 5 times to mix well.

        7. Verify the CSF tube specimen label includes “CSF”, patient name, number and today’s date.

        8. Deliver specimen at room temperature to B1-B58 STAT before 1 p.m. or enter in CRIS > Order Entry > Specimen/Item Transport > Priority: STAT > To Location:  Flow Cytometry B1 B58. 

      4. Non-CSF Fluid, Fine Needle Aspirate or LN / Tissue Biopsy collection: After pathologist approval (see B. Scheduling) and order entry (C.4. CRIS orders):

        1. All fluids, FNAs and biopsies require triage through Cytopathology.

        2. Cytopathology will arrange for specimen delivery to the Flow Cytometry lab after triage.

        3. For further Cytopathology specimen collection instructions:  http://home.ccr.cancer.gov/lop/Clinical/cytopath/cytopath.asp

          Note:  Enter TWO CRIS orders (C. 4., above); add text “Send an aliquot for Flow Cytometry evaluation.” to the Cytopathology CRIS order.

          IMPORTANT!! Do not add fixatives to the Flow Cytometry aliquot i.e. formalin, B5 fixative, alcohol, etc. Fixation will cause cell death and is not reversible.

  2. FLOW CYTOMETRY SPECIMEN DELIVERY:

    1. ROUTINE DELIVERY (before 1 p.m.) Monday – Friday regular workday, all specimens:

      1. Collection date must be recorded on specimen label.

      2. Deliver specimen STAT to Flow Cytometry Lab Room B1-B58 before 1 p.m. or
        Enter in CRIS > Order Entry > Specimen/Item Transport > Priority: STAT > To Location:  Flow Cytometry B1 B58. 

      3. Do not use ice, do not use cold packs, do not use drop box.

        Exception:  Deliver frozen specimens STAT on dry ice.

    2. LATE DELIVERY (after 1 p.m.) Monday – Friday regular workday, all specimens:

      1. Specimens arriving after 1 p.m. will not be processed without pathologist pre-approval.

      2. For late processing approval, e-mail Dr. Maryalice Stetler-Stevenson 301-402-1424 or Dr. Constance Yuan 301-496-4709.

    3. NIGHT, WEEKEND, HOLIDAY DELIVERY:

    BLOOD or BM SPECIMENS

    1. Blood/BM Night delivery (after 5 p.m.) Sunday – Thursday:
      Collection date must be recorded on specimen label.
      Deliver room temperature (20-25C) to Drop Box outside Flow Cytometry Room B1 B58

    2. Blood/BM Sunday or holiday delivery (before 1 p.m.) excluding Friday holidays:
      Collection date must be recorded on specimen label.
      Deliver room temperature (20-25C) to Drop Box outside Flow Cytometry Room B1 B58

    3. Blood/BM Saturday, Friday after 5 p.m. or Friday Holiday delivery:
      Specimen will be too old for processing on the next regular work day (more than 24 hours old). 
      Discard the specimen and schedule specimen recollection (see B. Specimen Scheduling, above).

    CSF SPECIMENS

    1. CSF Night delivery (after 5 p.m.) Monday – Thursday:
      Collection date must be recorded on specimen label.
      CLEARLY label bag:  Flow Cytometry, Room B1 B58
      Deliver to refrigerator outside cytology preparation laboratory 2A21 or
      Store refrigerated on the unit for STAT morning delivery the next regular workday.

    2. CSF Weekend or holiday delivery:
      Collection date must be recorded on specimen label.
      CLEARLY label bag:  Flow Cytometry, Room B1 B58
      Deliver to refrigerator outside cytology preparation laboratory 2A21 or
      Store refrigerated on the unit for STAT morning delivery the next regular workday.

      Note:  CSF specimens collected on weekends or holidays may be rejected by pathologist as too old to process.

    3. CSF Saturday or Friday Holiday delivery:
      Specimen will be too old for processing on the next regular work day (more than 24 hours old). 
      Discard the specimen and schedule specimen recollection (see B. Specimen Scheduling, above).

    Non-CSF FLUID, FNA, TISSUE OTHER

    1. Contact the Pathology Resident On-Call: (301) 496-1211 (NIH Page Operator).
      For Cytopathology instructions:  http://home.ccr.cancer.gov/lop/Clinical/cytopath/cytopath.asp

    2. CLEARLY label specimen bag:  Flow Cytometry, Room B1 B58

    3. Deliver bag CLEARLY labeled Flow Cytometry to refrigerator outside cytology preparation laboratory 2A21 or
      Store bag CLEARLY labeled Flow Cytometry to refrigerator on the unit for STAT delivery the next regular workday.

    4. Cytopathology will arrange for specimen delivery to the Flow Cytometry lab after triage.

      IMPORTANT!! Do not add fixatives to the Flow Cytometry aliquot i.e. formalin, B5 fixative, alcohol, etc. Fixation will cause cell death and is not reversible.


    1. EMERGENCY SPECIMEN delivery

      1. Send routine weekend and evening specimens to local reference laboratories billed to the clinical protocol.

      2.  In extreme cases when a life threatening clinical situation requires immediate treatment based, at least partially, on Flow Cytometry results, specimen processing requires pathologist approval.

      3. Emergency specimens will take priority over previously scheduled specimens. Emergency specimens are always processed. Only Dr. Stetler-Stevenson can designate a specimen as "Emergency".  Contact by e-mail Dr. Maryalice Stetler-Stevenson 301-402-1424 or through the Pathology Resident On-Call:  : (301) 496-1211 (NIH Page Operator).

      4. After Emergency specimen designation by Dr. Maryalice Stetler Stevenson, indicate the reason for Emergency designation in the CRIS Special Instructions section along with the evening on-call fellow name and pager number and the text “Emergency STAT results requested”.

  3. FLOW CYTOMETRY SPECIMEN REJECTION:

    1. Specimens arriving after 1 p.m. will not be processed without pathologist pre-approval. For late processing approval, e-mail Dr. Maryalice Stetler-Stevenson 301-402-1424 or Dr. Constance Yuan 301-496-4709.

    2. Unlabeled or mislabeled specimens will not be processed unless the clinician or collecting personnel can identify the specimen in person.

    3. Poor quality specimens will be rejected (see examples below).  However, poor quality specimens may be processed at the discretion of the Flow Cytometry laboratory director for rare specimens; specimens collected at significant risk or suffering to the patient; or specimens that cannot be recollected i.e. a spleen cannot be removed again.  Results of poor quality specimens will be interpreted with caution.

    4. The Flow Cytometry laboratory director or alternate will contact the clinician for recollection of rejected specimens. The rejected specimen will be accessioned and finalized.

    5. The rejected specimen will be held at room temperature for retrieval by the sending servicefor alternate testing  i.e. Hematopathology, Cytopathology or clinical group.  Specimens remaining in the Flow Cytometry lab will be discarded the following work week.

Poor quality specimen examples include:

  • Damage or improper handling detected by gross examination of Specimen i.e. clotting may be due to inadequate heparin. Coagulated, hemolyzed or frozen specimen may indicate non-ambient temperature conditions.

  • Inappropriate collection containers include lithium heparin tubes (toxic to cells) or other non-approved anticoagulants.

  • Specimens labeled radioactive or tuberculin hazard are rejected due to unavailable safety equipment in the testing area.

  • Inappropriate specimen volume:  Bone marrow aspirate volume greater than 5ml indicates probable peripheral blood contamination unless needle repositioning is noted.

  • Specimens with very low cellular content may be rejected.

  • 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.

  • 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, fixation or patient therapy.

     

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Hematopathology Section Specimen Collection Manual

The Hematopathology Section of the Laboratory of Pathology, NCI, offers specialized testing to assist in the diagnosis of lymphoproliferative disorders and immunodeficiency states. Testing is done in collaboration with the Immunohistochemistry Service of the Specialized Diagnostic Unit, and the Flow Cytometry Service. Specimen Collection and Handling

Specimen Collection and Handling

NIH Biopsy Specimens

Tissue biopsy specimens should be submitted to the Hematopathology Laboratory, Building 10, Room 2N110, within two hours of surgical removal.

Specimens should be submitted unfixed, in a sterile container, suspended in sterile normal buffered saline or tissue culture media (RPMI). The container should be labeled with the patient’s name, NIH identification number, and the identity of the tissue biopsy site. The specimen should be adequate in size to permit preparation of routinely fixed and processed material for histological examination, as well as frozen section histochemistry and/or flow cytometry.

A CRIS Requisition for Anatomic Pathology Services must be completed for all submitted specimens, according to NIH guidelines. Under "Special Instructions", indicate the need for Lymphocyte Surface Marker studies and state the NIH protocol and need for evaluation of particular antigens, if required; e.g. please evaluate for expression of CD20 or CD25.

In order to facilitate expeditious processing of fresh tissue specimens, biopsies may be delivered in person to the Hematopathology Section Laboratory by NIH clinical staff or the NIH Tissue Procurement Nurse prior to completion of the CRIS requisition. In such instances, the NIH protocol and need for special studies may be communicated verbally. The CRIS requisition should be completed as soon as the operative procedure is completed, within 1 hour.

Physicians are encouraged to consult with the staff of the Hematopathology Section regarding any specialized testing required. (Dr. Elaine S. Jaffe, 496-0183, or Dr. Stefania Pittaluga, 402-0297). Questions regarding delivery of specimens may also be directed to Medical Technologist, Theresa Davies-Hill, 496-1567. The Hematopathology Fellow on Service may be reached through the Section Office, 496-0183. The Hematopathology Fellow will assist in the preparation of specimens for analysis. Therefore, special requests should be discussed with the Hematopathology Fellow prior to submission of the tissue specimen.

  1. Results of Immunohistochemistry will be considered in the context of the routine histological findings, and included in surgical pathology report.

Processing of Outside Fresh Lymph Node Biopsy
Specimens for Immunohistochemistry for Protocol Evaluation

Under certain circumstances, biopsies may be performed outside of the NIH and submitted for surface marker analysis for NIH protocol evaluation. Fresh or snap frozen tissue specimens should be accompanied by routinely processed slides and/or paraffin blocks for routine histological evaluation, in concert with special studies.

If the biopsy is performed in the Washington, D.C. area, it may be submitted in sterile saline or tissue culture media, according to the guidelines outlined above for NIH biopsy specimens. The tissue should be delivered to the Hematopathology Section, Building 10, Room 2B42, within 6 hours of removal. If it cannot be submitted fresh within the time limit, it may be snap frozen according to the procedure listed below.

1. Intact lymph node (or other involved site) should be sectioned at 2-3 mm intervals.

 

For immunohistochemical confirmation of phenotype in a patient with an established diagnosis, needle core biopsies may be used if an excisional biopsy cannot be performed. LP/NCI must have the original diagnostic biopsies for review, if needle biopsies are to be used for protocol evaluation.

2. A representative cross section of the lymph node should be snap frozen in OCT embedding compound (using a cryomold, if possible).

3. Freezing should be performed in liquid nitrogen bath or freezing bath of dry ice and 2-methyl butane (temperature -70 °C or less).

4. Wrap frozen block in aluminum foil and place inside zip lock bag. Label specimen with patient's name and institutional surgical pathology number. Hold at -70° C until shipping.

5. Ship specimen by overnight express on dry ice. Pack securely in styrofoam shipping container containing 15-20 lbs of dry ice.

6. In addition to snap frozen specimen, submit:

  1. H&E stained slides of fixed, paraffin embedded tissue to be retained by NCI.

  2. Representative paraffin block or 12 paraffin embedded recut slides on charged slides for immunohistochemistry/ in situ hybridization.

  3. A copy of the outside surgical pathology report for all specimens submitted.

In order to avoid delay in performing immunohistochemical studies, please submit the above materials at the same time as the frozen tissue. We cannot perform immunohistochemical studies until we have reviewed the routine H&E stained slides of the submitted material.

7. Notify NIH staff physician in advance of shipping, and indicate that someone will be available to receive tissues. .

8. Complete an NIH Tissue Examination Form and submit with all materials to the Hematopathology Section Office, 10/ 2B42.

Drafted : November 8, 1996

Revised Procedure: April 6, 2007

Hematopathology Consultations

The staff of the Hematopathology Section provide consultation in selected cases of diagnostic difficulty. The Section is not a routine reference laboratory, and does not accept cases for routine immunophenotyping. The following is a list of instructions regarding cases to be submitted in consultation. We ask that each case be accompanied by a cover letter from a physician involved in the patient’s care (Pathologist or Clinician), and a copy or copies of the outside pathology report(s):

The cover letter should provide:

 

_____ 1. Pertinent clinical information (Brief clinical history)

_____ 2. The reason for the consultation

_____ 3. Specific questions to be answered

_____ 4. The referring pathologists working diagnosis or differential diagnosis

In addition, please submit representative H&E stained slides along with the paraffin block. For PCR studies to detect clonal Ig or T-cell receptor gene rearrangements, we require a formalin (not B5) fixed block. If a paraffin block cannot be submitted, please submit at least 12 unstained slides on charged slides.

 The Hematopathology Section will retain representative H&E stained slides on all cases submitted in consultation. It is preferable to submit recut slides, rather than originals, as we cannot take responsibility to return the original slides after our review. Outside special stains and immunohistochemical slides will be returned to the contributing laboratory. In selected cases if recuts cannot be prepared due to limitations in the biopsy material, original slides will be returned.

 Please direct the materials to

Dr. Elaine S. Jaffe
Building 10, Room 2B42
10 Center Drive , NIH, MSC-1500
Bethesda , MD 20892-1500
Office Phone: 301-496-0183
Office FAX 301-402-2415
Email

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Specimen Collection and Handling For The Molecular Diagnostics Unit of the Laboratory of Pathology/NCI

Contact Info: Tina Pham - 301-435-2632 / Trinh Pham 301-435-5234 / Winnifred Navarro 301-435-2627 / Liqiang Xi 301-594-1525

The Molecular Diagnostics Unit offers PCR-based molecular testing designed to assist in the diagnosis of both solid tumors and lymphoproliferative disorders.

I. Submission of IN-HOUSE samples:  

Specimens delivered to Rm. 2N116A in Bldg. 10 will be accepted no later than 4:30 pm.  

When submitting patient samples to the molecular diagnostics unit, the clinician or requesting physician must inform the Molecular Diagnostics Unit prior to the submission. This notification can be accomplished by entering a CRIS order at least 24 hr prior to submission of the sample. The CRIS order is listed under the heading of Anatomic Pathology, sub-heading, Molecular Pathology. The information submitted in the order must include the type of specimen and the requested tests. Pertinent information regarding the clinical history, name, age, gender, and preliminary diagnosis as well as the name and phone number of the clinician should also be included.

(1). BLOOD - 5 cc of whole blood is collected in light blue citrate tubes. The tubes are wrapped in plastic bags, (at room temperature) and sent immediately to the Molecular Diagnostics Unit.

(2). BONE MARROW ASPIRATES, CSF, AND OTHER TISSUES  

(a). Bone Marrow Aspirates -

  • Draw 2-3 cc in plain syringe with no anticoagulant. DO NOT USE HEPARINIZED SYRINGE

  • IMMEDIATELY place sample into the EDTA tube (lavender top) and mix gently to prevent clotting

(b). CSF - samples are placed in a sterile, capped plastic tube, wrapped in a plastic bag and then placed in a second bag or cup with wet ice. At no time should the ice and the tube have direct contact.  

If fresh CSF is not available, cytospins of the CSF that are, air-dried, and Diff-Quick stained may be submitted in lieu of fresh samples.

II. Submission of Samples from OUTSIDE of the NIH:

Notification of submission of samples from outside the NIH must to be made as a consultation to the Laboratory of Pathology. The Molecular Diagnostics Unit is not a reference laboratory.

(1). BLOOD - 5 cc of whole blood is collected in light blue citrate tubes. The tubes are wrapped in plastic bags and "bubble wrap" to prevent breakage. The wrapped tubes are then placed into a Styrofoam box with "cold packs" (i.e., "picnic-type" frozen ice packs) and transported overnight by courier to the address given below.

(2). BONE MARROW ASPIRATES -

  • Draw 2-3 cc in plain syringe with no anticoagulant. DO NOT USE HEPARINIZED SYRINGE

  • IMMEDIATELY place sample into the EDTA tube (lavender top) and mix gently to prevent clotting

The sample is wrapped in a plastic bag and "bubble wrap," placed into a Styrofoam box with "cold packs" for shipping by overnight delivery by courier to the address given below. Do not freeze.

(3). Submitted FFPE tissues All FFPE biopsy tissues performed for molecular diagnostic testing should be submitted routinely through surgical pathology. The surgical pathology staff is responsible for triaging the case for all specialized testing, including molecular diagnostic assays. It is essential that separate CRIS orders for both surgical pathology examination, and molecular diagnostic testing are submitted, and the required tests are listed on both CRIS forms. If the patient does not have an active MRN, then it is essential that a tissue examination sheet be completed in the Surgical Pathology main office (Building 10/2B50), and all required assays are listed on this form. Click the links to see specific detailed submission guidelines for Neuro-oncology and Thoracic Oncology .

Blood and bone marrow samples should be sent directly to: Molecular Diagnostics Unit, Lab of Pathology/NCI/NIH 9000 Rockville Pike, Bldg. 10/ Room 2N116A, Bethesda, MD. 20892

All other tissues samples should be brought to the main Surgical Pathology office (Building 10/2B50).

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SPECIMEN COLLECTION

 

CONTACT INFORMATION

Intra-Operative Consult (Frozen Section) Resident: 102-10375 (8:30 - 5PM)

Hot Seat Resident: 102-10358 (8:30 to 5:00 PM)

Pathology Resident On-Call (before 8:30 AM, after 5 PM, weekends, holidays): (301) 496-1211 (NIH Page Operator)

Surgical Pathology Office: (301) 496-2441

Clinical Laboratory Manager: (301) 594-9532

Tissue Procurement Nurse: (301) 402-3106 or page 102-11902

Hematopathology Laboratory (301) 496-1567

Hematopathology Office (301) 496-0183

Histology Laboratory (301) 496-3126

Frozen Section Laboratory (301) 496-1110


INTRAOPERATIVE CONSULTS (FROZEN SECTIONS)

Page the Intra-Operative Consult (Frozen Section) Resident: 102-10375 for assistance. The Hot Seat Resident may be paged at 102-10358, as a back-up. Please provide the patient information and Operating Room Number when calling.

Please page the Pathology Resident On-Call if it is after 5 PM or on weekends or holidays by calling the NIH Page Operator at (301) 496-1211.

FRESH SPECIMENS FROM OPERATING ROOM

  • All fresh specimens collected in the Operating Room should be delivered to the Frozen Section room (10/2C533) with the proper identification labels. Each specimen must be recorded in the log book at the time of drop-off. All paperwork should be placed with the specimen or with the specimen log book. All specimens must be placed in the locked refrigerator labeled "For Surgical Pathology Specimens."
  • NOTE: If specimen is place in refrigerator after 4 PM or on weekends and holidays, please page the Pathology Resident on-call, through the NIH page operator at (301) 496-1211. The Resident may instruct you to place the specimen in 10% formalin (see instructions below).

    PREVENT SPECIMEN AUTOLYSIS:

  • To prevent autolysis of fresh samples (without fixative) or drying out too quickly, it is i mportant to keep them cold (2°C to 6°C).
  • It is usually best to place a fresh specimen directly in the specimen container and keep cool in the refrigerator as soon as it is collected. Exceptions are noted below.
  •  

    -To prevent small samples, such as needle core biopsies from drying out too quickly, place in Teflon pad and wet with cold (2°C to 6°C) saline, (pH 7.0 -7.4).

    -Small curettage collections may be wetted with a very small volume cold (2°C to 6°C) saline, (pH 7.0 -7.4). , if necessary, if there is no blood of body fluid present from the collection process.

    - NEVER USE WATER OR WARM SALINE!

  • IF TRANSPORT OF FRESH SPECIMEN IS DELAYED, place the specimen container in a specimen bag (double zip lock bag) and place on ice. Do NOT allow the container or specimen to come in contact with ice, because as it melts, the water may enter the specimen container.
  • NOTES:

    1) For small biopsies, such as those collected during endoscopy, cystoscopy, and GYN procedures, please place specimens in 10% formalin fixative as soon as possible and affix a “ 10% formalin warning label”. For questions about fixation, please see below or page the HOT SEAT resident.

    2) For Hematopathology specimens (lymph node biopsies) please contact the Hematopathology at (301) 496-1567 or the Hematopathology office at (301) 496-0183. Lymph node biopsies should be delivered to the Hematopathology Section directly for fresh tissue procurement.

    DO NOT USE FIXATIVE ON LYMPH NODE SPECIMENS, UNLESS DIRECTED BY HEMATOPATHOLOGY.

    For more information on Hematopathology specimens, please see: http://home.ccr.cancer.gov/LOP/Clinical/hematopath/hem_spec.asp

    FIXED SPECIMENS

  • Specimens obtained in the clinics should be fixed in 10% formalin unless otherwise specified and brought to the Surgical Pathology Service, room 2A22 with a corresponding requisition form. Specimens without requisition forms will not be processed until proper identification of the specimen is received in the laboratory.
  • Containers should be properly identified with labels showing, name, medical record number, clinic location, type of specimen & site of biopsy (e.g., skin biopsy, left upper thigh).
  • Containers with fixative should have a label identifying the fixative (10% formalin warning label).
  • The amount of Formalin should be at least TWICE the volume of the specimen, but preferably TWENTY times the volume.
  • SPECIMEN LABELS (ALL SPECIMENS)

    • All specimens must be labeled with name of the patient, medical record number, date of birth, unit number or OR room #, description ( site description, specimen number). The information on the labels MUST match the paperwork and CRIS order. Please PRINT the name of the submitting physician on the label.
     

  • All RADIOACTIVE OR INFECTIOUS SPECIMENS, should be properly labeled and bagged.
  • PAPERWORK

  • Operating Room Specimen Record must be delivered by 4 PM. If patient is still undergoing surgery, please submit specimen record ASAP, but no later than 9 AM on the morning after surgery.

  • Research Specimens: Request & Certification for Research Procurement of Human Biological Materials (NIH-2803-1) This form must be delivered with research tissue; see section below regarding Research Procurement.

  • REQUISITION FORMS (CRIS ORDERS FOR ALL CLINICAL SPECIMENS)

  • CRIS requisition forms will print in 2A22: These forms must have the following information:
  • Request number

    Name of patient, Medical Record number, DOB, Age, Sex, Unit Number

    Intitial diagnosis, Protocol number, Institute

    Name of Physician & Name of Physician entering the request

    Specimen & site of biopsy (e.g., skin biopsy, left upper thigh).

    • CRIS orders must be placed by the Surgeon or Surgical Fellow as soon as possible, and no later than 5 PM on the day of surgery. If the CRIS is down, you must submit a CRIS Downtime Requisition for Tissue Exam/Cytology Exam. The form is available at here.

    • The specimen description in the CRIS must match the Operating Room Specimen Record and the specimen label as accurately as possible.

     

    SPECIMENS WILL NOT BE ACCEPTED OR PROCESSED IN THE LABORATORY UNLESS PROPERLY LABELLED AND WITH THE CORRESPONDING REQUISITION FORM (CRIS ORDER). Specimens without a CRIS order will not be processed until proper identification of the specimen is received in the laboratory. In urgent situation, the pathologist may place the specimen in fixative while waiting for the order. Unlabelled or mislabeled specimens will not be processed unless properly identified, resolved to the pathologist satisfaction, and documented in the CC Occurrence Reporting System.

    ATTEMPTS WILL BE MADE INMEDIATELY TO CONTACT THE RESPONSIBLE PHYSICIAN AND/OR PRIMARY CARE DOCTOR. IN URGENT SITUATIONS, THE CHIEF OF SURGERY BRANCH AND/OR INSTITUTE DIRECTOR MAY BE NOTIFIED.

    THE CHIEF OF THE SURGICAL PATHOLOGY SECTION WILL BE NOTIFIED INMEDIATELY.

    RESEARCH PROCUREMENT

    When a patient has been scheduled for a surgical procedure or a biopsy
    during which specimens will be collected for research the research team
    or designated clinical staff should initiate the form, "Request &
    Certification for Research Procurement of Human Biological Materials"
    (NIH-2803-1) and obtain the signature of the PI/AI.  For inpatients, the
    form is placed in the inpatient chart.  For outpatients, the form can be
    sent to the PACU on the day of and prior to the procedure taking place.
    We know there will be times when proactive planning will not work and a
    patient is taken to the procedure area without the required Form NIH
    2803-1.  You can bring the completed form to the PACU or the OR Front
    Desk and the staff will make sure it is delivered to the right hands.
    Research material will not be released unless the Request &
    Certification for Research Procurement of Human Biological Materials
    (Medical Records form NIH-2803-1) is complete and signed. Refer to
    Medical Administrative Series M01-2 for more details.

    If the specimen is time sensitive the research team should call the resident before the procedure takes place to coordinate quick release. If a critical research specimen requires prompt transport to the lab, the pathologist can be notified prospectively to make appropriate arrangements.

    Most specimens removed during a surgical procedure must be reviewed grossly by an NCI pathologist before being released for research to insure that sufficient diagnostic material required for the clinical care of the patient has been submitted for clinical pathology evaluation. This includes any specimens that are divided in the Operating Room, but does not include the following exceptions:

    • Protocol driven needle core biopsies or other small biopsies.
    • Samples transported to other CLIA certified labs for division and test reporting of clinical results (e.g., bone marrow biopsies, muscle biopsies. ocular specimens, if they are sent to a CLIA certified lab that issues a diagnostic report).
    • Samples which are transported to areas for division by LP Pathologists outside the operating room (e.g., TIL specimens, UOB nephrectomy & prostate specimens, lymph node biopsies divided by Hematopathology).
    The OR team will call the Hot Seat resident (page 102-10358) to review
    the specimen in either the surgical suite or frozen section room.
    Shortly thereafter, the OR staff will notify the person designated on
    Form NIH 2803-1 when the specimen is procured and/or ready for release
    and give the location for pick up.
    • If the Hot Seat is not available, the intra-operative consult resident (page 102-10375) will serve as a back up in emergency situations and the on-call resident will assist after hours and holidays (NIH pager operator, 6-1211). 
    • The OR should also tell the pathologist and research team the OR suite # and phone number from which it can be obtained, if necessary.        
    • If the research team arrives before the pathologist, please be patient and do not take the specimen until the pathologist reviews and releases it. 
    • In some cases, the pathologist will review the specimen in the OR, but most research specimens will exit the operating room suite via the Frozen Section Laboratory (room 10/2C533) and be reviewed in that location.       
    • Exempt samples most likely will not be passed through the Frozen Section Lab, since the lab is usually locked when not in use.

    • While the OR is staffed until 11 PM Monday thru Friday, the doors do lock after 6 PM.  If you are picking up a specimen after 6:00 p.m., please use the phone located outside the PACU to call the Main OR number (301-496-5646) to advise them of your arrival.

     

    For more information see:

    RESERVATION OF MATERIAL All specimens will be kept, properly fixed in formalin for a period of a month in the Gross Surgical Pathology room (2A22). Then the specimens will be moved to the Autopsy room where they will be preserved for another month before discarding them. Processed (paraffin embedded) from clinical specimens are not discarded. OTHER TYPES SPECIMENS FOR LABORATORY OF PATHOLOGY

     

    Last Updated 2/11/2014 1:46:09 PM

     
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