Diagnostic Cytopathology Laboratory
Ductal carcinoma of breast diagnosed with fine needle aspiration (22 gauge) biopsy (left), showing unequivocal invasion in CellientTM cell block (middle), and validated overexpression of Her2/neu by immunohistochemistry (right).
GYN Pap Testing for Cervical Cancer
Detailed Specimen Collection Instructions
Supervisor: Marypatricia Scott
Andrew Fischer, MD
Ali Akalin, MD
Gamze Ayata, MD
Ediz Cosar, MD
Armando Fraire, MD
Zhong Jiang, MD
Dina Kandil, MD
Christopher Owens, M.D.
Xioafei Wang, MD
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To use the smallest possible biopsy in order to make safer, less painful, and faster definitive diagnoses.
Screen for cervical cancer using the most sensitive and cost-effective techniques.
Develop new technologies for using small biopsies in diagnosis.
Advance cancer research through study of the molecular basis of diagnostic changes in cell structure.
Educate medical students, residents and fellows, and cancer researchers about cytopathology diagnosis.
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The UMASS Diagnostic Cytopathology Laboratory includes a staff of 10 Cytopathologists and 10 cytotechnologists dedicated to detecting and definitively diagnosing cancers and other diseases using the smallest possible biopsy or cell sample. An estimated 100,000 cytology samples are anticipated in 2010. Fine needle aspiration (FNA) biopsies can be performed on a stat basis by the Cytopathologists, or patients can be scheduled to the Cytopathology FNA clinic at the University campus. Cases prepared at outside laboratories can be referred to the Laboratory’s nationally recognized Cytopathologists for confirmation of diagnoses, or for consultation. The Laboratory is equipped with the state-of-the-art Hologic Imaging System for Pap tests and is one of a handful of Laboratories in New England with rapid CellientTM processing of microbiopsies for histologic examination and immunohistochemistry.
Through cooperation with the adjacent Diagnostic Molecular Oncology Laboratory, the Immunohistochemistry Laboratory, and Hematopathology, Cytology samples can be tested for high risk HPV types, HPV genotyping, flow cytometry, and a full range of immunohistochemistry and molecular testing.
The Diagnostic Cytopathology Laboratory Quality Assurance Policy exceeds governmental CLIA mandates. The staff actively consult with each other and the nationally recognized sub-specialist UMASS Surgical Pathologists, providing a diagnostic precision at the highest national level. The Staff Cytopathologists contribute many publications annually, relating to basic and applied cancer research, and improving diagnosis through technology innovations.
Staff Cytopathologists consult each other on a fine needle aspiration biopsy. From left to right: Drs. Andrew Fischer, Ali Akalin, Armando Fraire, Dina Kandil, and Zhong Jiang
Pap Testing for Cervical Cancer
The Laboratory will process an estimated 90,000 gynecologic Pap Tests in 2010. Click for Detailed Specimen Collection Instructions. The Laboratory uses the Hologic Imaging system for ThinPrep Pap tests. Clinicians receive a single integrated report with results of requested screening for high risk human papilloma virus (HPV). HPV 16/18 type specific genotyping can also be ordered, and is reported as an addendum to the Pap test result. Guidelines for HPV testing are available at http://www.asccp.org/edu/hpv_testing.shtml
The Laboratory correlates GYN Pap test results with subsequent biopsies, provides clinicians with a list of results of their patient’s pap test, flags abnormal pap tests that have not been followed-up, and conducts a monthly colposcopy conference with clinicians. Consultation with the staff Cytopathologists about Pap Test results and clinical guidelines is encouraged.
The Laboratory also diagnoses colposcopic endocervical brushings, a sensitive and relatively painless procedure that can replace the need for more aggressive endocervical curettage in patients with abnormal pap test results.
ThinPrepTM Pap Tests with Low grade squamous intraepithelial lesion (left), high grade squamous intraepithelial lesion (middle),
and endocervical adenocarcinoma as seen in a CellientTM –type cell block prepared from residual material from a Pap test vial (right).
The Laboratory processes an estimated 10,000 non-gynecologic cytology samples annually, from essentially any body site. Click here for Detailed Specimen Collection Instructions. Cytology screening for bladder cancer includes the combination of sensitive and specific morphologic analysis together with targeted reflex testing for chromosomal abnormalities (FISH) if ordered by clinicians. Other body cavity fluids and lavage samples can yield diagnoses of infectious or neoplastic diseases, including lymphoma (see images below). Brushing samples from the pulmonary system, biliary system, GI tract and other sites also allow definitive diagnostic classification of neoplasms and some infectious processes.
Aggressive T-Cell (gamma-delta) lymphoma in a pleural fluid (left), immunostained for CD3 in a cell block (right).
Bronchial brushing samples with large cell carcinoma (left), and small cell carcinoma as seen in the CellientTM cell block (right).
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Fine needle aspiration (FNA) biopsies are minimally invasive, safe, rapid, and definitive means of diagnosing tumors, including lymphomas and soft tissue tumors (see below). The Laboratory diagnoses about 2500 FNAs per year. The CellientTM Automated Cell Block System (http://www.cellientsystem.com) was developed and patented by The University of Massachusetts, and it allows efficient and rapid processing of micro-sized FNA samples for histologic sectioning. CellientTM processing allows histologic diagnosis to complement conventional preparations, and provides a platform for a full range of immunohistochemistry and molecular diagnostic techniques—allowing FNA samples to often provide as much diagnostic information as more invasive core biopsies. Publications from UMASS have shown the ability of CellientTM cell blocks to allow definitive diagnosis of invasion in FNA samples of breast cancers (see image at the home page).
The staff Cytopathologists perform FNA biopsies of palpable or superficial swellings/lumps at the request of clinicians. FNAs can be scheduled, or they can be performed on a Stat basis (see Contact Information) for inpatients or outpatients in clinician’s offices. There are many advantages of having a Cytopathologist perform the FNA: The Cytopathologists are highly experienced in working with the patients to acquire and process diagnostic FNA samples, providing local anaesthesia if needed. FNA samples are also efficiently triaged for microbiology studies or flow-cytometry as clinically indicated. The Cytopathologists assess the adequacy of the biopsy and in about two thirds of cases can provide a definitive immediate diagnosis, thereby reducing patient anxiety, saving the patient the waiting time for scheduling more invasive biopsies, and speeding up subsequent work-ups. Unless otherwise requested, results of immediate diagnoses are conveyed to the requesting clinicians rather than directly to the patients.
The Staff Cytopathologists offer an onsite assessment of adequacy for clinicians at the University campus who use FNA. Onsite adequacy assessments are scheduled through the Cytology Office. Click here for more information.
|Benign soft tissue tumor of neck (schwannoma) specifically diagnosed with 25 gauge FNA needle. ThinPrep appearance (left), CellientTM cell block (middle), and S-100 confirmatory immunostain of cell block (right).|
Diffuse large cell lymphoma, follicular center B-cell type, diagnosed by FNA of inguinal lymph node. Direct smear (left), CellientTM cell block (second), CD10 immunostain on cell block (third) and BCL6 immunostain (right).
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Detailed Specimen Collection Instructions
General Labeling and submission instructions - All specimens must be accompanied by a Cytology Requisition that includes the patient name, medical record number, source of specimen and the submitting physician’s name. Any submitted microscope slides must be labeled with the patient’s name and medical record number using a pencil. Pertinent clinical history should be provided, and is essential for samples referred from outside the UMASS medical record system.
Turn around time - Turn-around time for Pap tests averaged 3.6 days in 2009. Routine Non-GYN cytologies have a turn-around time of about 90% in 2 days. Stat diagnostic requests can be requested, allowing diagnosis within 24 hours of receipt of the sample (same day for specimens received before 1PM) including cases with cell blocks. FNAs performed by Cytopathologists, or onsite evaluation by Cytopathology of clinician-performed FNAs (see FNA service) allows a definitive diagnosis in the majority of cases within minutes.
A. ThinPrep Pap tests: The ThinPrep vial must be labeled with the patient’s name and medical record number. Obtain cellular material as per Hologic’s instructions from the cervix using a spatula, endocervical brush or broom device. Avoid collecting a sample at time of menses, or in the presence of a severe purulent discharge. The goal is to capture exfoliated cells from the transformation zone, and to sample the endocervix. For Thin prep paps, immediately agitate the brush in the liquid “PreservCyt” fixative, against the inside of the container, to dislodge all mucous. It is essential to immediately dislodge the cells because the endocervical component becomes irreversibly fixed to the bristles after about 15 seconds.
B. HPV testing: Screening for high-risk types of HPV (“HR-HPV”) is available on material collected in Thin Prep Preservcyte vials with or without morphologic evaluation. Indicate on the cytology requisition whether HPV testing is needed, and whether testing should be only reflexed to patients with atypical squamous cells of uncertain significance. Reflex genotyping for HPV types 16/18 can also be requested (for patients with negative morphologic results by positive HR HPV screen results. Guidelines for ordering HPV high risk screening and HPV genotyping can be found at http://www.asccp.org/edu/hpv_testing.shtml.
C. Conventional Pap smears: Avoid collecting a sample at time of menses, or in the presence of an untreated infection. Transfer the material in one or two strokes (do not over-smear) and immediately spray fix the slide from a distance of about 12 inches with Cytology Spray Fixative, thoroughly saturating the cells. Allow the spray fixative to dry before sending the slide. HPV testing is not available on conventional pap tests.
D. Colposcopic Endocervical Brushing: Use an endocervical brush, preferably with a plastic straw covering the bristles. The purpose of the straw is to minimize collection from the portion of the cervix that can be directly visualized. Label a container of Cytorich red fixative (preferred) or a ThinPrep vial (available from Outreach Supplies or Cytology). Under Coloposcopic guidance, put the end of the straw at the endocervical os. Exend the brush 1-2 cm through the straw into the endocervical canal. Rotate the brush only 1 revolution. Withdraw the brush back into the straw. Extend the brush out of the straw into the fixative. Immediately dislodge sample from the bristles by rotating the brush against the inside of the specimen collection container. Endocervical curettage rather than colposcopic brushing is recommended as a follow-up for atypical glandular cells in a pap test.
E. Fine needle aspirates: The technical aspects of performing an FNA and triaging material for diagnosis are not trivial. Click here for a full description of the technique. Four options are available.
1. It is preferable for the staff Cytopathologists to perform FNAs of any palpable lesion at the bedside or in an outpatient setting at the University campus. The non-diagnostic rate for Cytopathologist-performed FNAs is significantly lower (about 15%) than FNAs performed by clinicians (about 30%) because we assess the adequacy of the sample while we do the procedure and triage material for needed ancillary studies. Cytopathology is available on a Stat basis between 8:30 and 4:30 Monday through Friday, or patients can be scheduled and registered to be seen by Cytopathology at the University outpatient Surgical Oncology clinic. Cytopathology can help answer patients’ questions, but we generally only give diagnoses to the referring clinician. We administer local anaesthetic (if needed), and can provide a definitive diagnosis within minutes for the majority of cases. Conversely, we can know within minutes if a larger core biopsy is needed and can refer patients to Surgery or radiology for the original physician.
2. For non-palpable lesions, or if a clinician prefers to perform the FNA, we recommend scheduling an immediate diagnostic evaluation of the FNA by calling Cytopathology 30 minutes in advance. This is available at the University and the Access Center. This is highly recommended if the procedure is performed under radiographic guidance or if lymphoma is suspected. We will prepare the smears, triage material for ancillary studies such as microbiology, flow cytometry, and cell block preparation and provide a rapid assessment of adequacy of the sample with a turn-around time of about 15 minutes. In most cases, a definitive diagnosis can be rendered within minutes.
3. For clinician-performed FNAs without on-site evaluation, please refer to the link providing detailed technical instructions. Rinse of the needle in CytoRich Red solution (available from Cytology) and shake the container to disperse the sample. Note that the Cytorich red solution lyses red cells after about 15 seconds. After the red cells are lysed, there should be visible particles if the FNA is adequate.
F. Pleural, peritoneal and pericardial fluids: Do not add any fixative. If the sample is grossly bloody, it is preferable to collect the sample directly into a container containing at least 2 units per ml heparin.
G. Urine: A clean-catch, mid-stream, fresh voided urine is optimal as a screen for bladder cancer. 50 ml is adequate. Add one volume of Saccomanno’s fixative (which contains mixed alcohols and 2% polyethylene glycol) to the sample at the time of collection. If microbiology and urinalysis is needed, split off part of the sample for these studies before adding fixative. Avoid sampling during a clinical urinary infection, or during a bout of gross hematuria, otherwise the urothelium can be too diluted with inflammatory cells or blood cells. A 24-hour urine is not satisfactory for diagnosis because the cells will be degenerated. Two or three samples on different days have been shown to boost sensitivity of the test.
H. Fluorescence in situ hybridization (FISH): UroVysion FISH testing for bladder cancer is available on urine samples submitted in Saccomanno fixative, with or without morphological evaluation. Indicate whether UroVysion FISH testing is desired directly on the cytology requisition. The FISH testing is performed by the Diagnostic Molecular Oncology Laboratory and is reported as an addendum to Cytology reports.
I. CSF: At least 1 ml CSF is needed for cytopathology. Send the sample promptly to the cytology laboratory so that the cells can be prepared before they degenerate. If the sample is collected on Friday, add one volume of Saccomanno’s fixative to prevent degeneration in case the sample is delayed over the weekend. If flow cytometry is needed, a separate aliquot of at least 3 mL of fresh, unfixed CSF is required. CSF samples for flow cytometry should be separately submitted to Hematopathology who will issue the reports. Separate samples also must be sent for cell count studies, handled by the Hematology lab.
J. Pelvic and Peritoneal Washing fluid specimens: The washing/lavage fluid should be a physiologic solution (E.g. Lactated Ringer’s, Normosol, or Hank’s) rather than normal saline. Write “refrigerate” and deliver promptly.
K. Other Lavage fluid specimens: Other useful specimens for diagnosis include lavages of the pulmonary tree or various GI sites. The lavage fluid should be a physiologic solution (E.g. Lactated Ringer’s, Normosol, or Hank’s) rather than normal saline. Write “Refrigerate” and deliver promptly.
L. Sputum samples: An induced sputum is optimal. Have patient rinse mouth with water thoroughly before collecting sample to decrease the number of oral squamous cells, which otherwise obscure the pulmonary sample. In order to be adequate for diagnosis, the specimen must consist of a high proportion of pulmonary material since the pulmonary tree sheds so few cells compared to the oropharynx. Write “Refrigerate” and deliver promptly. If collected on Friday add one volume of Saccomanno’s fixative (available from Cytology) to preserve cells in case they cannot be processed over the weekend.
M. Brush samples (e.g. GI tract, biliary tract, and bronchial tree): If only one brushing per site is performed, use Cytorich red fixative (provided by Outreach Supplies or Cytopathology). Immediately swirl the brush against the side of the cytorich red container, or immediately retract and extend the brush within the plastic sleeve (for some brush designs) in order to dislodge the cells. It is essential to dislodge cells from the brush within about 30 seconds because the cells will become permanently fixed to the bristles of the brush. It is not necessary to cut off the brush. If more than one brushing per site is performed, dislodge the sample into 2 ml of normal saline as described above, then repeat the brushing as many times as needed to sample the area, and finally transfer the 2 ml of saline (containing the sample) into at least 10 ml of cytorich red fixative. If scheduled in advance, an immediate intra-procedural diagnostic evaluation by cytopathologist may be able to be performed. (see Contact Us).
N. Anal pap tests: Screening for anal squamous cell carcinoma in high risk individuals involves use of Dacron-type swab with a sturdy plastic handle (not provided by Cytology). The swab is pre-moistened with water, inserted about 1-2 inches, and rotated one to two revolutions. Rinse the sample into a ThinPrep vial. HPV testing is not recommended.
O. Tzanck prep (Skin scrape cytology to rule out herpes infection): Use Cytorich red fixative (provided by Cytopathology) Moisten the skin for a few minutes by placing a paper towel saturated with water over the area. Scrape the blister with the edge of a clean microscope slide. This microscope slide may be pre-cleaned/sterilized before use with an alcohol-soaked pad. The blunt edge of a scalpel blade or a pap test brush may also work. Put the edge of the microscope slide into the Cytorich red fixative (or brush or scalpel blade) and immediately agitate to dislodge the (often sparse-appearing) sample.
P. Amyloid detection in abdominal fat aspirates: Cytopathology can perform the FNA to get an optimal preparation sample. If clinicians perform the FNA, sample the subcutaneous fat about 10 cm to the left and to the right (two samples) of the umbilicus. Express the entire sample on one slide. Smear the material between two slides, trying to flatten the particles of fat to make them as thin as possible. There should be 5-10 particles per side. Send the four air dried slides unfixed to Cytology with a note “CONGO RED STAIN” on the requisition. For patients with known monoclonal gammopathy, express an additional pass into Cytorich Red solution (available from Cytology.
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Hours of operation are 8:00 a.m. – 4:30 p.m. Monday – Friday
Main Phone Number: 508-793-6120
Director: Andrew Fischer, M.D.
508-579-9342 (cell phone pager)
Supervisor: Marypatricia Scott
Use main phone number for requests for Cytopathologist-performed FNAs, request for immediate adequacy assessment of FNAs, inquiries about supplies, or general questions.
Immediate adequacy assessments should be requested with 1 hour, and then again 5 minutes, advanced notice.
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