CYTOLOGY PROTOCOLS

Table of Contents
SPUTUM PREPARATION
SPUTUM SMEARS
SPUUTUM (Blender method)
BRONCHIAL/GI BRUSHINGS
BRONCHIAL/GI WASHINGS

 

 

SPUTUM PREPARATION

A sputum series is the least invasive method for detecting pulmonary cancer. Patients with a history of smoking, chronic obstructive pulmonary disease or airflow obstruction, long term exposure to uranium or asbestos, or a history of metastatic carcinoma are at a higher risk for the development of tumor. A sputum series consists of one fresh specimen collected the FIRST THING IN THE MORNING for three consecutive mornings. The rate of detection for pulmonary carcinoma improves when more specimens are submitted. One specimen may be a post bronchoscopy sputum specimen which is produced immediately after the bronchoscopy Procedure. This sputum should never be discarded as it may contain valuable diagnostic material treed during the procedure. The minimum volume of specimen accepted is 1.0 ml, however the quality of sputum is Judged by its contents rather than quantity. If a series of consecutive early morning sputum specimens is collected and submitted to the laboratory together as a complete group, they may be pooled during processing resulting in one final cytologic report.

 

SUPPLIES NEEDED

COPLIN JAR
WOODEN STICKS OR MICROSCOPE SLIDES
95% ETHANOL
TONGUE DEPRESSOR
BIOHAZARD HOOD

METHOD

 

  1. Verify the patient name on the requisition and specimen container.
  2. Complete a NONGYN form describing the specimen appearance in detail, number of slides prepared, and stains performed.
  3. Label 3 slides with the pathology accession number and last name of patient using lead pencil.
  4. Using wooden sticks or tongue depressor, select areas that appear opaque or bloody and spread the material thinly and evenly over / of the slide surface.
  5. Immediately immerse 2 of the slides in coplin jar containing 95% alcohol and set aside until ready for staining process (see Staining Schedules).
  6. Use a color coded slide and make a third smear, allow it to dry, then stain with DIFF QUIT Give to cytotechnologist for preliminary check. (See Staining Schedules>).
  7. Record the date of preparation on the specimen container and refrigerate remaining specimen. Specimen is stored for one week before disposal.

 

 

PROCEDURE: CYTOPREPARATION METHODS

 

 

SUPPLIES NEEDED FOR BLENDER METHOD

COPLIN JAR
MICROSCOPE SLIDES
WARING BLENDER
SACCOMANNO FIXATIVE
BIOHAZARD HOOD
BLENDER CUPS
50 ML CENTRIFUGE TUBES

BLENDER METHOD

  1. Verify the patient name on the requisition and specimen container.
  2. Complete a NONGYN form describing the specimen appearance in detail, number of slides prepared, and stains performed.
  3. Label 2 slides with the pathology accession number and last name of patient using lead pencil.
  4. Add 50 ml of Saccomanno fixative to specimen if none was added previously.
  5. Shake thoroughly to mix.
  6. Pour into blender cup and homogenize for 20 to 30 seconds.
  7. Pour homogenate into prelabelled 50 ml centrifuge tube.
  8. Centrifuge for 5 minutes at 1500 RPMs.
  9. Decant all supernatant and mix on vortex for 15 seconds at High setting.
  10. Place 2 or 3 drops of mixed concentrate on one slide.
  11. Make smears by laying second slide over first allowing material to flow evenly until of the slide surface is covered. Gently slide apart and lay on a flat surface to dry.
  12. When completely dried, immerse in 95% ethanol for 15 minutes before staining. (See Staining Schedules)
  13. Record the date of preparation on the specimen container and refrigerate remaining specimen. Specimen is stored for one week before disposal.

 

PROCEDURE NOTES

Ancillary Studies: If specimens are collected for culture and cytology, the sample must go to Microbiology first. Detection of Pneumocystis carini may be done on sputum; however, bronchoscopic lavage is more successful in detecting the organism. If Pneumocystis is requested, make an additional smear and submit to Histology for Grocott's Methenamine Silver staining (GMS).

Sputum from the same patient which is collected on consecutive days may be poured together or pooled.

If specimen is received in alcohol or Saccomanno fixative, make crush smears or use blender method. Place a small quantity of hardened material between two slides and crush the material until it begins to coat the slide uniformly. Immediately immerse the slides in 95% alcohol.

If the specimen is watery, it probably represents saliva. Make direct smears if a cell button is obtained after centrifugation. If not, use cytocentrifugation to prepare the smears. Follow procedure for preparing brushing specimen. URINARY TRACT SPECIMENS: (Voided Urine, Catheterized Urine, Ureteral, Renal Pelvis, Bladder Washing, Urethral Specimens)

 

 

 

PROCEDURE: CYTOPREPARATION METHODS

 

 

BRUSHING SPECIMENS (Bronchoscopy and Gl tract)

Brushing specimens may be submitted from suspicious areas seen during bronchoscopy or endoscopy.

 

SUPPLIES NEEDED

COPLIN JAR
SLIDES as needed
95% ETHANOL
3 SAMPLE CHAMBER ASSEMBLIES
PIPETS CYTOCENTRIFUGE
VORTEX MIXER
MICROSCOPE
BRUSH WASH VIALS
BIOHAZARD HOOD
SACCOMANNO FIXATIVE
BALANCED SALT SOLUTION

METHOD

  1. Verify the patient name on the requisition and specimen container.
  2. Complete a NONGYN form describing the specimen appearance in detail, number of slides prepared, and stains performed.
  3. Label 3 slides with the pathology accession number and last name of patient using hard lead pencil.
  4. Load 3 slides and sample chamber assemblies into the cytocentrifuge.
  5. Brushings are usually collected in a Saccomanno Brush Wash vial that contains Saccomanno fixative. Because the quantity is small, precentrifugation is usually not needed. If the brush is submitted, tease any remaining material from the brush by pushing it back and forth through special insert in the Brush Wash vial. If the brush is separate, add a few milliliters of Saccomanno fixative to the container and vortex it on high speed for 15 seconds.
  6. Saccomanno fixative will change the structure of cells, so one must estimate the cellularity visually. The solution should appear slightly hazy. For specimens appearing clear, the sample may be equally divided among three chambers. Saccomanno fixative may also be added to dilute specimens appearing cellular.
  7. Load three sample chamber assemblies and spin for 5 minutes at 1000 RPMs.
  8. Carefully remove sample chambers and clips as described earlier. Drying is permissible due to the addition of Saccomanno fixative and should occur in a few seconds. Immerse slides in coplin jar containing 95% alcohol and set aside until ready for staining process. One slide may be stained with DlFF-QUIK for preliminary check. (See Staining Schedules).
  9. Record the date of preparation on the specimen container and refrigerate any remaining specimen. Specimen is stored for one week before disposal.

 

PROCEDURE NOTES

Ancillary Studies: Brush specimens may be submitted in saline, although saline is a poor

transport medium for maintaining cell morphology. Add an equal volume of Saccomanno fixative

upon receipt in the Lab. This specimen must be centrifuged by pouring into a clean prelabeled

centrifuge tube and spinning for 5 minutes at 1500 RPMs. Decant supernatant down to the conical

portion of the centrifuge tube and follow instructions for visual estimation of cellularity.

 

 

 

PROCEDURE: CYTOPREPARATION METHODS

 

If Microbiology studies are requested on the same specimen, it must go to Microbiology first. Requests may be for fungal identification or ova and parasites. Ask Microbiology to return remaining specimen to cytology for processing or indicate this information directly on the requisition.

If Brush Wash vial is submitted with only a drop or two of fluid, use additional Saccomanno fixative to thoroughly rinse the container and brush. Use vortex mixer to remove as much material as possible.

 

 

PROCEDURE: CYTOPREPARATION METHODS

WASHING SPECIMENS (Bronchoscopy and Gl tract)

Cytology specimens may be collected during bronchoscopy or endoscopy to augment the biopsy sample. The surface of suspicious areas should be aspirated, brushed and then biopsied. If insufficient secretions are present, or if a lesion is not visualized, the area in question should be ravaged. Sometimes, rinsing the biopsy forceps in the saline used for the washing will provide additional material for examination. For full diagnostic value, all material should be identified as to site of origin. The minimum acceptable volume for washing specimens is 1 ml.

 

SUPPLIES NEEDED

COPLIN JAR
SLIDES as needed
95% ETHANOL
LABELED TISSUE CASSETTE
PIPETS CONTAINER with FORMALIN
VORTEX MIXER 3 SAMPLE CHAMBER ASSEMBLIES
50 ML CENTRIFUGE TUBE
CYTOCENTRIFUGE
BALANCED SALT SOLUTION
MICROSCOPE
SACCOMANNO FIXATIVE
BIOHAZARD HOOD

MIXING VIAL

M ETHOD

  1. Verify the patient name on the requisition and specimen container.
  2. Complete a NONGYN form describing the specimen appearance in detail, number of slides prepared, and stains performed.
  3. Label 3 slides with the pathology accession number and last name of patient using hard lead pencil.
  4. Pour specimen into 50 ml centrifuge and spin for 5 minutes at 1500 RPMs.
  5. If specimen quantity is sufficient, make a cell block. Carefully remove the cell button without breaking it apart, wrap it in tissue, and enclose it in a tissue cassette. Place the cassette in formalin. Send cassette to grossing room for processing and paraffin embedding.
  6. If material Is insufficient for cell block make 2 smears by placing 1 or 2 drops of specimen on a slide. Lay the second slide on top, allow the material to spread uniformly until it covers of the slide surfaces, and gently slide them apart. Immediately immerse both slides in 95% alcohol.
  7. Use vortex mixer or a pipes to thoroughly mix remaining sample concentrate.
  8. If sample concentrate appears thick, dilute with several drops of balanced salt solution or reserved supernatant until the sample concentrate appears hazy.
  9. Prepare 3 sample chamber assemblies and load in Cytospin. Use a color coded slide for DIFF-QUIK staining. Add 6 drops of concentrate to each sample chamber. For slides that will be stained with Papanicolaou and Hematoxylin/Eosin, add 4 to 5 drops of Saccomanno fixative.
  10. Spin for 5 minutes at 1000 RPMS.
  11. Remove the alcohol fixed slides first. Remove the sample chamber assemblies from cytocentrifuge and carefully unlock slide clip. Peel off blotter being careful not to disturb cell button.
  12. For Papanicolaou and H&E staining immediately immerse the slides in a coplin jar containing
  13. 95% ethanol and set aside until ready for the staining process (See Staining Schedules). Remove the color coded slide last. When dry, stain with DIFF-QUIK (See DIFF-QUIK Staining Schedule)
  14. Give to cytotechnologist for preliminary exam/ check for cross contamination potential. If the cytotechnologist makes any recommendations, adjust the number of drops as indicated and make new cytospins. Discard the first set of slides.
  15. Record the date of preparation on the specimen container and refrigerate remaining specimen. Store specimen for one week before disposal.

 

 

PROCEDURE NOTES

Ancillary Studies: If specimens are collected for culture and cytology, the sample must go to Microbiology first.

Detection of Pneumocystis carinii is more successful on bronchoscopic ravage. If requested, make additional smear for Grocott's Methenamine Silver (GMS) staining as described for alcohol-fixed slides. Or if a cell block is obtained, cut an additional section for GMS staining. GMS staining is performed in Histology.

Specimens that are received in alcohol cannot be used for Microbiology.

Specimens received in formalin will be prepared for cell block only. No additional cytologic smears will be made.

If specimen is received in alcohol or Saccomanno fixative, make crush smears. Place a small quantity of hardened material between two slides and crush the material until it begins to coat the slide uniformly. Immediately immerse the slides in 95% alcohol. Alternatively, use blender method for 15 seconds to homogenize specimen. (See Sputum).

To estimate cellularity of fresh unfixed specimens, place 1 drop of specimen on a slide and coverslip. Rack the condenser knob down and examine the slide. Select a representative area of cells and switch to the high power objective. To achieve the optimal dilution, there should be 10 cells per high power field. If there are too many cells, add more balanced salt solution or reserved supematant. If there are too few, recentrifuge the specimen and remove more of the supernatnant.

Specimens containing mostly red blood cells may be diluted to 20 cells per high power field.