PROCEDURE: CYTOLOGY SPECIMEN COLLECTION
PRINCIPLE:
Proper specimen collection for cytological interpretation is of primary importance and the recommended procedures must be correctly followed in order to obtain the best possible specimens for diagnostic purposes. The following are detailed protocols for the collection and fixation of various types of cytological specimens.
Handling Conditions:
Use Standard Precautions for handling all blood, body fluid and fresh tissue specimens. (All Springfield Hospital standard policies are available electronically in SMCS Policies and Procedures. Icon on computer desktop)
SPECIMEN:
Immediate and proper fixation cannot be stressed enough. As a general rule, CYTOLYT in equal volume to the specimen should be added to all fluid specimens.A spray or liquid fixative (eg. Shandon Cell-Fixx) should be applied to direct smears. The exception is fine needle aspirate smears that are air-dried and submitted for Diff-Quik stains such as breast aspirates. Sputum specimens must be fixed in Saccomanno fluid. Formalin should never be used as a cytology fixative for the collection of specimens. Water in 10% buffered formalin causes edema of the cells resulting in swollen amorphous nuclear content and loss of cytological detail. Materials required for obtaining and preserving cytology samples are available on request by contacting the Springfield Health Center Drawing Station (886-8915) or by leaving a message with the Pathology Secretary (885-7691). Basic supplies are no charge.
REQUISITION REQUIREMENTS:
All specimens must be identified with two identifiers, the patient's name, date of birth, and date of sample.Samples should also be identified with the source of each specimen. All specimens must be accompanied by a requisition which has all of the following information: Patient's name, date of birth, name of physician performing the procedure, names of other providers requiring a copy of the report, source of specimen, clinical history. and billing information (specimens from physician practices). Clinical indications for testing must be included. In-patient Cytology Requisitions (yellow "NON-GYNE") are available from the Pathology Department.
The pathologist will charge for the interpretation of all non-GYN cases and abnormal pap smears (Part "B" billing). Springfield Hospital will charge for the technical preparation of all cytology cases (Part "A" billing).
Specimens received without proper identification or information will be returned to the Physician or Nursing Unit. Specimens that are received unfixed, clotted, or not under refrigeration will be determined to be either satisfactory or unsatisfactory for processing. If they are deemed unsatisfactory they are rejected with a statement of reason for rejection and another specimen requested. Specimens which contain insufficient cellular material are reported unsatisfactory and another sample requested.
If a case is "urgent", it must be so indicated by marking RUSH on the requisition with prompt delivery to the laboratory. Every effort will be made to report the results as soon as possible and within 24 hours of receipt. All positive, unexpected findings and RUSH cases will be called directly to the physician by the pathologist or designee.
I. GYNECOLOGICAL CYTOLOGY – PAP TEST:
All pap testing, (liquid based ThinPrep and conventional pap smears) result reporting, and follow-up for Springfield Hospital are currently done at Rutland Regional Medical Center - Pathology Department.
Patient Preparation: The specimen should be taken from the cervix and/or vagina of a patient that is not menstruating and has not performed douching for 48 hours prior to sampling. If patient is using a topical vaginal medication, it should not be applied in the 24 hours prior to sampling. Complete specimen collection instructions are available from RRMC.
All pap supplies are available from RRMC Pathology Department: (802) 747-1787
II. FINE NEEDLE ASPIRATION
Advantages of Aspiration Biopsy Smears:
1. Aspiration is usually an office procedure or done on an out-patient basis in the Radiology
Department or One-Day surgery.
2. It may eliminate the need for more complicated diagnostic tests, eg. biopsy, frozen section.
3. The diagnosis may be made faster than with a biopsy.
4. Lymph nodes can be explored without removal.
5. Accurate needle aspiration of small peripheral lung tumors located radiologically can be of
great help in determining the nature of lesions that cannot or do not exfoliate in one of the
main bronchi.
6. For some diseases (fungal and viral infections and certain tumors) the smear can be easier to
interpret than histo-pathologic sections.
7. It can be repeated, as indicated, with minimal trauma.
PROCEDURE - STEPWISE:
There are several variants of the Fine Needle Aspiration Biopsy technique. The pathologist uses his own variation. The following technique is a suggested variation for FNA done in the physician's office.
1. Label 2-3 glass slides with the patient's name and date on the frosted end prior to starting
the procedure.
2. Attach a 23 or 25 gauge needle to a 10 ml syringe.
3. Insert syringe into a fine needle aspiration syringe holder (Cameco 2 holders) available at
Springfield Hospital.
4. Insert needle into lesion.
5. While applying negative pressure, move needle in short, stabbing motions, changing angle
of direction within the lesion.
6. Release negative pressure, and then remove needle and syringe together as a unit. Specimen
should not be drawn into barrel of the syringe. Pressure should be released as fluid
appears in the needle hub. The cells and tissue fragments obtained from a solid lesion should
remain in the barrel of the needle.
7. After withdrawing the needle, remove it from the syringe and fill the syringe with air.
8. Re-attach the needle and carefully eject one very small drop of specimen onto the slide.
9. Use another slide placed on top of the first in a pull-apart motion to spread specimen over
slide. More slides can be made as amount of specimen allows.
10. Fix or air dry smears immediately.
11. This procedure should be repeated 1-3 times with an attempt to sample different areas of
the mass each time.
12. If blood, fluid or cellular material in excess of one drop is obtained with a needle pass,
the excess should be expressed into a container of saline or CytoLyt (available from SH
Pathology Department. The needle and syringe should be rinsed with this same
preservative. Submit the liquid specimen with the fixed slides using one request form.
13. Please indicate on the request form the specific site, clinical diagnosis, whether the lesion
is solid or cystic, and the gross appearance of the aspirate if possible.
Alternative Specimen Preparation Method (For bloody or fluid specimens.)
1. Perform steps 1-7 above.
2. Eject specimen directly into container of CytoLyt. Flush syringe
with same fixative.
3. Submit separate body sites or lesions in separate, appropriately labeled containers.
4. Provide all pertinent clinical information. Indicate if lesion is solid or cystic.
III. BLADDER BARBOTAGE WASHINGS:
Bladder and kidney washings are generally quite successful in producing good specimens. The repeated irrigation of the urinary bladder, ureter, and kidney pelvis with 50ml of isotonic saline solution after voiding usually yields an excellent specimen rich in well preserved cells.
Fix bladder, ureter, or kidney washing specimens immediately with an equal volume of CytoLyt. Do not refrigerate.
IV. URINE
Preliminary hydration of the patient is strongly recommended.
Hydration of the patient is accomplished by administration of one glass of water every 15 minutes for a 2 hour period. Diuretic drugs or intravenous fluid can also be given. Voided urine can be accepted from the male in certain cases, but catheterization is always preferred in the female in order to avoid contamination from vaginal secretions. Urine collected in the morning, but not first morning specimen, is the richest in cells. Urine specimens should be fixed immediately with an equal volume of CytoLyt. The only exception to this is if a Urine sample needs to be split between Urine analysis and Cytology. Do not refrigerate. CytoLyt fixative in bulk and "CytoLyt added" stickers are available from the Pathology Office (885-7691) to all Physician practices.Samples should also be identified with the source of specimen
V. BRONCHIAL SECRETIONS - BRONCH BRUSH AND BRONCH WASH
The use of the flexible broncho-fibroscope permits the scraping of an increased number of bronchial lesions under direct observation. The few cells obtained are often highly diagnostic and well-preserved. The smears should be fixed immediately.
The brush tip should be submitted to the laboratory in a container of Saccomanno fixative.
Bronch washing from the instilling of normal saline into the bronchus usually yields a larger number of cells than the brush technique. The fluid recovered from this procedure should be added to an equal volume of Saccomanno fixative.
An instruction sheet for preservation of bronch wash/brush specimens is included with the bronch kit. See sample at end of the specimen collection section.
VI. CEREBRO-SPINAL FLUID
The first few drops of the spinal fluid lumbar tap, if very bloody, may be discarded. Then as much spinal fluid as clinical judgment allows should be collected. If a spinal fluid cannot be processed immediately, it should be mixed with the same amount of CytoLyt fixative. Refrigeration is not necessary.
VII. EFFUSIONS and Other BODY FLUIDS
Includes: pleural/thoracentesis, pericardial, ascites/paracentesis fluids, pelvic washings, etc.May be collected in tubes or syringes which may be either plain or heparinized to prevent coagulation. Three (3) units of heparin per ml. of fluid may be placed in the collecting vessel.
After collection, a minimum of 100 ml. should be should be sent to the laboratory in a Urine cup. Upon arrival Laboratory personnel will further aliquot the sample into multiple containers to permit the addition of an equal volume of CytoLyt fixative. Refrigeration is not necessary.
VIII. ESOPHAGEAL BRUSHING
Brushing of the esophageal lesion under direct vision may be done with excellent results by using the gastro-esophageal fibroscope and a small brush. This method is gentler and safer than biopsy and reduces the risk of perforation of the thin wall of the esophagus. Barium contamination of the smears may be avoided if the lesion is washed with saline solution before brushing. Smears should be immediately spray fixed and then air dried.
IX. SPUTUM
A deep cough expectorated sputum sample is examined for the presence of malignant cells. The first, early morning specimen yields the best diagnostic results. All sputum specimens are processed using the Saccomanno-Carbowax technique. This helps to prevent cell shrinkage and to retain nuclear detail.
1. Plastic cups (100 ml) half filled with Saccomanno fixative, (50% ethyl alcohol containing
1 ml of 50% polyethylene glyco-Carbowax 540) are prepared by the Pathology department.
These are labeled SACCOMANNO FIXATIVE.
2. Nursing service or Physician offices may obtain specimen containers from the laboratory.
Saccomanno may also be added to specimens by Laboratory personnel for in-patient specimens
or if additional microbiology tests are also required. .
3. Patients are told to deeply cough the material directly into these cups, thus fixing the
material immediately. It is best to shake the cup gently for a few seconds after the lid
has been tightly closed. These containers do not need to be refrigerated for out-patients
bringing in specimens later in the same day of collection. But if laboratory delivery is
delayed, they should be refrigerated.
X. SKIN SCRAPING
In patients with a skin lesion where biopsy is not possible, e.g. patient refusal, inaccessible location, etc., or where biopsy is not indicated as in a benign appearing lesion, cytology of the surface scraping may be of great assistance and can render accurate and specific diagnosis of the nature of the lesion. Some fungal and viral infections are easier to recognize in a smear than in a biopsy.
Since the surface of the skin is physiologically dry, most of the superficial lesions should be moistened with a dripping wet compress before scraping. A compress left for a least 30 minutes or longer not only makes it softer for easier scraping but also removes most of the loose, degenerated cellular debris and serum crust that otherwise would clutter the smear.
After removal of the crust, the small ulceration produced should be energetically scraped with a sharp curette. With a vesicle or bulla, the dome should be removed and the margins of the ulceration scraped. Some of the non-ulcerated subepidermal lesions may need to be aspirated with a needle.
The material collected by scrapings or aspiration should be spread on glass slides and may be air-dried or immediately spray fixed. Please label "air-dried" or "fixed" on the slides.
REFERENCE:
Koss, Leopold G.; Diagnostic Cytology and Its Histopathologic Bases; J.B. Lippincott Co., Philadelphia, PA; 4th edition 1992, Volume 2.