1.3a - Anatomic Pathology - Cytology Laboratory General Information

ANATOMIC PATHOLOGY -- CYTOLOGY LABORATORY
Cytopathologist:
Marie Lithgow, MD (x36073)
John Lee, MD (x35994)
Merry Beth Spradling, MD (x35020)
Huihong Xu, MD (x 36692)
Cytotechnologists:
Extension x35184 (cytology diagnostic lab); x35945(cytology prep lab)
Rachael Silvia SCT, ASCP
Heather Shone SCT, ASCP
Sandra Resendes CT, ASCP

Cytology prep lab & specimen drop-off:
Bldg. 3, Rm. 1A116 - open Monday-Friday 8:00AM to 4:30PM.
Extension 35945
Cytology diagnostic lab:
Bldg. 3, Rm. 1C108 - open Monday-Friday 8:00AM to 4:30PM.
Extension 35184

1. General information and requirements
Cytology specimens are stained according to a modified Papanicolaou stain (Pap stain). Except for specimens submitted as direct smear slides, all fresh and fixed specimens sent to Cytology are prepared as cell monolayers for microscopic examination using the Thin Prep 2000 Processor, and stained with the Pap stain.
Maximum precautions are necessary to avoid exposure to potentially infectious specimens when delivering patient specimens to the laboratory. Specimens must be placed in appropriate leak-proof containers (depending on the type of specimen collected) and placed inside "Biohazardous Specimen" bags.
 A properly completed Cytology Requisition (Handwritten or Electronic*), must accompany all specimens with the following information:

1. Patients full name and full social security.
2. Ordering provider's name, pager and/or phone number (Note: this must be legible).
3. Date the specimen was obtained.
4. Specimen type.
5. Pertinent clinical history, working diagnosis and/or indications for cytological examination.
6. State clearly if leukemia/lymphoma is suspected.
7. Specific requests such as "rush" or "STAT" must be clearly stated.
   1. Rush reading will not be done if requested after 2:00 pm to ensure same day processing and interpretation.
   2. For emergency cases, the Cytology laboratory will attempt to process a rush or STAT specimen received after 2:00pm, provided that the request is made by the attending of the clinical service and discussed  with the cytopathologist on service and technical staff on duty.

*An Electronic Cytology Requisition can be used in the CPRS system by clicking on “ORDERS”; “MAIN LAB MENU”; “CYTOPATHOLOGY REQUEST FORM” (Found under “All Locations” on the Main LAB Menu). Follow the prompts to fill out the form. A hard copy of the form MUST be printed and sent with the specimen.
A properly completed specimen label must be placed on the specimen container with the following information:

1. Patient’s full name
2. Patient’s full social security number
3. Collection date
4. Specimen site

2. Specimen delivery and pick up

1. Specimens should be delivered directly to the lab at the West Roxbury Campus during normal operating hours. Those taken after hours and on weekends should be placed in the Baxter refrigerator (with the glass door) on the bottom shelf labeled “Pathology”, in the main laboratory near the Send-Out section.
2. Specimens (JP) should be delivered directly to the send-out desk on the 2^nd floor lab area, B wing, of the Boston campus for transport to West Roxbury Cytology. Those taken after hours or on the weekends should be placed in the appropriately designated refrigerator, the 2^nd floor, B wing.
3. Specimens (BRK) should be delivered directly to Brockton laboratory (Bldg 3, first floor, room A-123) during lab hours for transport to West Roxbury Cytology. After hours and weekend samples should be delivered to Urgent Care for transport to West Roxbury Cytology.
4. All fresh fluid specimens must be delivered to the laboratory promptly for optimum cellular preservation and diagnostic interpretation. Fluid specimens in syringes must have needle removed before submitting them to the laboratory.

3. Submission of specimens

Cytology fixative (CytoLyt) and other cytology supplies should be obtained from Rm. 1A116, Building 3, ext. 35945 or 35184. Please call in advance. Supplies can also be obtained from the Send Out department located in the main lab.
Please note: Specimens may be rejected if certain conditions of specimen handling and requisitioning are not met. Conditions include but are not limited to: lack of proper identification (full name and full social security number) on the specimen container and/or requisition, failure to adequately fill out the cytology requisition form, spilled fluid specimen, broken specimen container or slides, unlabeled direct smear slides, and improperly collected/fixed specimens.

          A.  Voided Urine/ other GU specimens
The ideal urine specimen is 50-100mls of clean-catch, midstream morning urine placed in a properly labeled 100ml capacity screw-top container. 24 hour urine collection is inappropriate and will not be accepted. If delay in delivery is anticipated, add equal volume of cytology fixative (CytoLyt) and refrigerate. Urine specimens intended for both urinalysis and cytology should be processed independently. The sample can either be split or collected separately (Note: Cytology is not responsible for urinalysis).

1. Ureter Brushings - Place brush in CytoLyt fixative and agitate gently. Submit the container with brush to the cytology lab.
   
   B.  Protocol for ordering FISH testing (UroVysion) for urine cytology
   1. Preferred specimen: voided urine
   2. The patient must have a previous diagnosis of urothelial cell carcinoma and/or hematuria to be a billable test.
   3. At least 80 mls of urine must be submitted. The UroVysion FISH test requires 50 mls of urine and 30 mls will be used for cytology.
   4. Prior to FISH testing, cytologic testing will be performed. If the cytology results are positive or negative, a FISH test will not be performed. If the cytology results are reported as atypical or suspicious, and the clinician has requested a FISH test, then UroVysion FISH will be performed.
   5. A properly completed requisition requesting UroVysion FISH and cytology must accompany all specimens. The requisition must include the clinical history of “hematuria” and/or “previous history of urothelial cell carcinoma (UCC)”.
   6. All specimens intended for UroVysion FISH must be preserved with CytoLyt (provided by cytology) upon collection. Mix urine with Cytolyt 50% in a 1:1 solution. CytoLyt preservative must be added to the specimen as soon as possible for optimal results. It is recommended that specimens be processed within 72 hours of collection. If urine is not shipped immediately after collection, then refrigerate (do not freeze).
   7. Specimens received without a properly completed requisition or without CytoLyt preservative will not be processed.
   8. After the urine cytology is processed and reported, the sample will be sent to Quest for UroVysion FISH by the VA Boston Healthcare System Pathology and Laboratory Medicine Service West Roxbury Division. Other VISN I sites performing their own urine cytology testing will send Urovysion FISH test requests directly to Quest Diagnostics.. Turn-around time for the report will be 2-3 weeks.
   9. The results of the UroVysion FISH test will be reported as a supplemental report to the original cytology report. (Urovysion FISH is not a stand-alone test. At no time will the Urovysion FISH test be performed on urine specimens without a cytologic diagnosis on the concurrent urine specimen).

   C.  Sputum specimens
   An early morning deep cough specimen obtained before breakfast is preferred. Ask the patient to rinse mouth before coughing. Collect a single fresh cough specimen in a cup (with a tight fitting lid) for cytology. If the specimen is collected at home, the patient should be instructed to refrigerate the specimen until it can be delivered to the medical center. The service collecting the sputum should add CytoLyt preservative to the sputum after it is obtained. (obtain supply of fixative from room 1A116, bldg. 3 at WR or 2^nd floor send-out desk at Boston). Cap container tightly and bring or send to room 1A116 bldg. 3, at the West Roxbury campus or the 2^nd floor send-out desk, B wing at the Jamaica Plain campus, or Bldg 3 first floor A-123 at the Brockton campus, with completed requisition form. Refrigeration of fixed sputum specimens is not necessary but is preferred if delivery is significantly delayed. Unfixed sputum specimen must be refrigerated. A sputum cytology series consists of one specimen collected on each of three or five consecutive days. Please deliver each specimen as it is obtained, and label each specimen with the proper date. Do not save specimens to deliver all at once. Multiple specimens received on the same day and not individually labeled with a different date will be combined as one specimen.

           D.  Body Cavity and other (synovial, hydrocele, cyst) fluids

For VA Boston sites:
All fluids should be collected in containers without fixative. 50 to 100 mls is a satisfactory volume; do not send amounts in excess of 1 liter. In cases where lesser amounts are available, they will be examined but results may not be optimal. Refrigerate if specimen cannot be delivered promptly. Deliver immediately if lymphoma is suspected.

For other VISN1 Laboratory sites (the following pertains to the laboratory only):
If the fresh specimen will not arrive at the West Roxbury Cytology Laboratory within 24 hours, then add an equal volume of CytoLyt fixative to the sample.  If the sample is too large to accommodate this volume, then a well-mixed aliquot of the specimen with an equal volume of fixative may be utilized (50-100 ml of sample is optimal); it is not practical to process large volumes of fluid, and large volumes do not increase diagnostic sensitivity (Ref. 1, 2).  Excess fresh fluid should be kept refrigerated at the original sending site for additional evaluation if needed for 2 weeks after the case is finalized.
 
References:

1. The Art and Science of Cytopathology. DeMayR., page 270, ASCP, 2012.
2. www.aruplab.com, CORP-APPEND-0249M, REV1, February 2017, page 2; accessed 11/15/2017.

            E.  Cerebrospinal fluids (CSF)
Any volume can be processed for diagnosis. If specimen can be delivered immediately, or if lymphoma is suspected, add no fixative. If there is a delay in delivery, add an equal volume of cytology fixative (CytoLyt)
Note: IF THERE IS A CLINICAL DIFFERENTIAL DIAGNOSIS THAT INCLUDES CREUTZFELDT- JAKOB DISEASE (CJD), NOTIFY THE CYTOLOGY LAB AND LABEL THE CYTOLOGY REQUISITION AND THE SPECIMEN CONTAINER WITH “CJD”.
(Avoid collection of CSF on Friday afternoons since the Cytology lab is closed on weekends).
 
             F.  Fine Needle Aspirations
Call the cytology laboratory (ext. 35184 or 35946) to arrange time for rapid on-site evaluation (ROSE, i.e.adequacy assessment), if needed with 24-hour notice. If personnel from cytology are not available to assist at the procedure, place specimen and needle rinses into CytoLyt.  If leukemia/lymphoma is suspected, indicate on the requisition and place additional passes in a tube with RPMI for Flow Cytometry evaluation. All samples must be labeled with the patient’s full name and full social security number. Deliver the samples to the laboratory promptly with completed requisition.
   
  a. Instructions for ThyroSeq on Thyroid Fine Needle Aspirations :
 1. Place patient label on the ThyroSeq Preserve vial.

1. In Interventional Radiology, the ThyroSeq vials are located in the box labeled “CBL Path” with the foam insert.
2. IR will note the expiration date on the vials and will notify the Cytology lab if additional vials are needed.
3. Storage of ThyroSeq Preserve vials prior to specimen collection:

             a. ThyroSeq Preserve vials prior to specimen is light sensitive, always store vials in a dark place (inside te collection kit box or dark  baggie) and at room temperature(+15 to +25^oC) until the expiration date.

 2. Express a full second FNA pass into the ThyroSeq Preserve vial (1.5 mL tube).The solution in the 1.5 ml tube will become turbid and will darken when sufficient cellularity is achieved. DO NOT SEND THE NEEDLE.
 3. In addition, for better representation of large-sized nodules, wash the needle form all other passes taken from the nodule and express them into the SAME THYROSEQ vial. However, be sure that the solution is not retained in teh needle or syringe and the final volume in the vial remains the same.
 4. After collection, secure  the tube cap and invert several times to mix the collected sample.
 5. Send labeled ThyroSeq vial with labeled routine thyroid FNA materials to the Cytology lab (i.e. CytoLyt tube and Cytology requisition form).
 6. After specimen collection , the Thyoseq Preserve vial may be stored for:

1. No longer than 3 hours at room temperature (+15 to +25°C).
2. No longer than 24 hours at +2 to +8°C, i.e. in a refrigerator.

 7. Please notify the Cytology lab (857-203-5184) if there be a delay in specimen transport in excess of the above storage requirement times.

1. For ThyroSeq specimens will not be recieved in the Cytology lab by 4 pm on Friday afternoons, please contact the Cytology lab in advance to make arrangements for weekend storage.

 8. Samples will be held in Cytology (for 1 year in the freezer) and upon clinician request sent to CBL Path for ThyroSeq testing.
 9. Turnaround time for the results from time of sendout is estimated at 10-14 business days.
 10. The results will be faxed to the Cytology lab and an addendum will be issued to the Cytopathology report.
          
         G.  Washings and Brushings
Washings of any site should be delivered unfixed, immediately to room 1A116. If delivery is delayed, add an equal volume of CytoLyt fixative to specimen and refrigerate. Brushings of any site should be handled by removing any protective sleeve and dropping the brush immediately in CytoLyt fixative, followed by gentle shaking of the brush in the fixative. Leave the brush in the fixative container and deliver to room 1A116, bldg. 3 at the West Roxbury campus or 2^nd floor send-out desk at the Jamaica Plain campus or Bldg 3 first floor A-123 at the Brockton campus as soon as possible. Fixative and specimen tubes pre-filled with fixative can be obtained from room 1A116, bldg.

           H.  Gyn specimens
Thinprep Gyn specimens: Obtain vials with PreservCyt fixative and separate ThinPrep Gyn cytobrush collection device from room 1A116. Label vial with the patient's full name and full social security number. After obtaining sample using cytobrush, gently agitate brush in PreservCyt solution, remove brush, and re-cap vial. Send vial with the appropriate requisition to the cytology laboratory for processing. Please note: history should include the patient's age, date of last menstrual period, presence of IUD and history of hormonal therapy if any, in addition to the routine information required. Please also note if a cytobrush was used and note if the patient patient is at high risk for Squamous Intraepithelial Lesion (SIL). Avoid taking Gyn samples during menstruation.

             I.  Miscellaneous (i.e. Breast/nipple secretion)
Place specimen in CytoLyt and deliver to the laboratory with requisition.

4. Borrowing slides from the laboratory
Glass slides which have been signed out by the pathologist may be borrowed for a maximum of one day. The slides of specimens which have not been signed-out may not leave the laboratory. The borrower of slides must sign out such material from either the cytology laboratory or from the histology/cytology preparatory laboratory. Delinquency in returning borrowed material may result in loss of borrowing privilege.

5. Obtaining outside review of signed-out cytology cases
The physician requesting an outside review of a signed-out cytology case must do so in writing, providing the patient's name and full social security number, accession number of the case(s) to be reviewed, the outside pathologist to whom the slides should be sent, as well as his/her complete office or hospital address. The cytology laboratory will forward the slides to the consultant.

6. Reporting of cytology results
The Cytopathology division of the VISN 1 Network Consolidated Laboratory located at the West Roxbury VAMC includes a statement of adequacy and a diagnosis.

•  Non-Gyn specimen reporting

 "No malignant cells identified": the specimen is cytologically benign but may show changes associated with inflammation, reactive atypia, chemotherapy etc.      
"Morphologic atypia/Atypia": nuclear changes are present which are of uncertain significance. The changes could be caused by an inflammatory or reactive process, or could represent a neoplastic condition. A severe atypia diagnosis may include a request for repeat or other follow-up.
"Suspicious for malignancy": severe cytologic changes which may represent a malignant process but are seen in too few cells or in too poorly preserved cells to warrant a definitive diagnosis of malignancy.
"Positive for malignancy": the specimen is diagnostic of malignancy; the type of malignancy is indicated whenever possible.
"Inadequate": the specimen may be too poorly preserved for diagnosis or may not be representative of the site being sampled.
"Non diagnostic": the cells may be too necrotic or obscured by inflammation or debris for an adequate evaluation of the sample, or features necessary for diagnosis may be lacking.

• Gyn specimen reporting

Gyn specimens are reported according to the 2014 Bethesda System, Third edition, which includes a statement regarding adequacy of specimen and a diagnostic category

Bethesda System 2014 (in use from January 1, 2017)
Specimen adequacy:
Satisfactory for evaluation
(Describe presence or absence of endocervical/transformation zone components and any other quality indicators, e.g., partially obscuring blood, inflammation, lack of history etc)
Unsatisfactory for evaluation
(Describe reason, explain if specimen is rejected/not processed or if processed and examined but unsatisfactory for evaluation because of (explain reason)
General diagnostic categorization:
Negative for Intraepithelial Lesion  or Malignancy
Epithelial Cell Abnormality: See interpretation/results, specify "squamous" or "glandular" as appropriate
Other: See interpretation/results (e.g. endometrial cells in a woman of or over 45 years of age)
Interpretation/Results:
Negative for IEL or malignancy: there is no cellular evidence of neoplasia
Organisms (Trichomonas vaginalis, Shift in vaginal flora suggestive of bacterial vaginosis, bacteria morphologically consistent with Actinomyces spp, Fungal organisms morphologically consistent with Candida spp, cellular changes consistent with Herpes simplex virus, cellular changes consistent with cytomegalovirus) are mentioned if present.
Other non neoplastic findings (reactive cellular changes, glandular cells status post hysterectomy, and atrophy are mentioned if present (optional).
Other: endometrial cells present in a woman of or older than 45 years of age
Epithelial Cell Abnormality
Squamous cell
Atypical squamous cells
-of uncertain significance (ASCUS)
-cannot exclude High Grade Squamous Intraepithelial Lesion (ASC-H))
Low Grade Intraepithelial Lesion (LSIL), encompassing: HPV/mild dysplasia/CIN I
High Grade Squamous Intraepithelial Lesion (HSIL), encompassing moderate and severe dysplasia, CIS/CIN II, and CIN III
-with features suspicious for invasion if invasion is suspected

  Squamous Cell Carcinoma

Glandular cell
Atypical
-endocervical cells (NOS or specify in comment)
-endometrial cells (NOS or specify in comment)
-glandular cells (NOS or specify in comment)
Atypical
-endocervical cells, favor neoplastic
-endometrial ce;;s, favor neoplastic
Endocervical Adenocarcinoma in-situ
Adenocarcinoma
-endocervical
-endometrial
-extrauterine
-Not otherwise specified
Other malignant neoplasms, specify
Adjunctive Testing: Reflex HPV is performed on ASCUS results. Exception: To decline reflex HPV testing for patients age 21-24 years old, please ensure this request is clearly stated on the printed requisition form; otherwise reflex HPV will be performed on ASCUS.
 

Cytology General Information