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CYTOSPIN METHOD
Author: Heidi HanesDocument Number:Pro64-E-03Effective (or Post) Date:13 January 2009Review HistoryDate of last review:NAReviewed by:Penny StevensSOP should only be used as an example, change as needed. Anything in red should be adjusted per laboratory policy.SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your labs specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering their use in other applications. If you have any questions contact SMILE.
Laboratory Name
Address
Department
TITLE: CYTOMORPHOLOGY SMEARS FOR BODY FLUIDS-CYTOSPIN METHOD
Prepared By _________________________________ Date: ____________________
Approved By _________________________________ Date: ____________________
_________________________________ Date: ____________________
Effective Date Discontinued Date:____________________ (retain this procedure for at least 2 years)
Supersedes an Earlier Procedure : (Y or N) Earlier Procedure Discontinuance Date: ________ The Medical Laboratory Director or the Directors designee should review all copies of this procedure at least once a year. Discontinued procedures must be retrievable in a reasonable timeframe.
Reviewed By:Date:Reviewed By:Date:Laboratory Name
CYTOMORPHOLOGY SMEARS FOR BODY FLUIDS
CYTOSPIN METHOD
PRINCIPLE:
Cytocentrifugation can be regarded as membrane filtration performed horizontally. The cells are directly settled onto the slide. The blotter is the filter's pores through which the liquid component of the specimen passes. Centrifugal force acts as the negative pressure that drives the separation of cells from their suspension medium. Cells are flattened by cytocentrifugation allowing better enhancement of cytomorphology.
ORDERING:
This section should include any special ordering steps.
Cytospins are automatically order through the workshheet, FLD1, if more than nine nucleated cells are seen. To order manually use function RE, test code CYTO.
REQUIRED MATERIALS:
Cytospin Instrument
Cytofunnelsdisposable or reusable
Filter paper--brown and white
Microscope slides cleaned in 95% alcohol or Superfrosted Plus slides (See comment in procedural notes)
10 x 75 test tubes
REAGENTS:
Bovine Albumin, 30%: Store stock bottle at 2-8(C. Acceptable within expiration date if there is no evidence of contamination (i.e.: turbidity).
Hyaluronidase Acid: Store at 0-5(C. Use a few crystals, as needed, to diminish viscosity of certain fluids (joint, synovial, etc). Briefly warm to 37(C after addition of Hyaluronidase to an aliquot of fluid.
Balanced Electrolyte or Salt Solution: As a diluent and to add to sample chamber if total volume is less than 5 drops. Acceptable in absence of turbidity.
GENERAL CYTOSPIN 2 NOTES:
1. Speed range: 200-2000 RPM (enter as 20-200)
2. Acceleration rate: Hi/Lo-Use Lo accel for CSF
3. Time range: 1-99 minutes.
4. 0.5 ml is the maximum capacity of sample chamber (approximately 12 drops with disposable Pasteur pipets).
5. Drop liquid in cytofunnel without draining down funnel walls.
PROCEDURE:
Sample preparation:
Using dilution chart below to decide what dilution to use.
Label test tube with specimen identification.
Label slide.
For samples requiring the addition of Hyaluronidase acid add a few crystals. Allow to sit 1-2 minutes to allow crystals to dissolve. (If a heating block is available place tube in block for 30 seconds to speed up dissolving.)
Assemble cytofunnel.
Load cytofunnel with a drop of albumin first if Superfrosted Plus slides are not used followed by 5 drops of appropriate dilution or undiluted fluid. (See note below)
Spin at 600 RPM for 100 minutes or 700 RPM for 5 minutes. Use Lo Accel for CSF and Hi Accel for all other fluids.
When done spinning carefully remove the slide from the cytofunnel.
Air dry for several minutes.
Stain slide and perform differential.
NOTE: Cell count should be performed first to know what type of dilution will be needed. The specimen is generally diluted to yield WBC ( 100/mm3 and RBC( 4000/mm3 to assure a monolayer of cells.
If RBC is < 4000/mm3, and WBC falls in dilution chart, proceed with volumes on chart. If RBC is > 4000/mm3, dilute as necessary to reduce it to < 4000/mm3, then proceed to use dilution chart.
DILUTION CHART
WBC
COUNT DILUTION
FACTOR
DROPS
FLUID DROPS
SALINE
DROPS IN*
SAMPLE CHAMBER 0-101 --- 5 0 5
101-300
1:2
6
6
5
301-700
1:4
3
9
5
701-1500
1:10
1
9
5
1501-3000 1:20 1 19 5
Differential:
The slide should be scanned to insure that a monolayer of cells were created and to detect any unusual cell formation.
If a monolayer was not achieved make a larger dilution and spin again.
A hundred cell differential should be performed if cellularity of the slide allows it. If cell count is low perform differential on as many cells possible and calculate the percentage.
Follow the policy of your laboratory for cell identity.
EXPECTED RESULTS:
Differential in Normal Body Fluid
CSF: Adults Neonates
Lymphocytes 40-80% 5-35%
Monocytes 15-45% 50-90%
Polynucleated 0-6% 0-8%
Macrophages, Ependymal or choroid plexus cells may be present.
Synovial:
PMN - Less than 25%
Mononuclear cells, including lymphocytes, monocytes, macrophages and synovial lining tissue cells are the primary cells seen in normal synovial fluid.
All other fluids:
PMN - Less than 25%
Macrophages and mesothelial cells may be present.
PROCEDURE NOTES:
Use Cytospin 2 on CSF with > 9 WBC/mm3 and all other fluids with nucleated cells present.
Superfrosted Plus slides are slides that have been given an electrically positive charge. This allows the cells to adhere to the surface more readily.
Identify: Lymphs, Polys, Large Mononuclear Cells and any other cell that can be identified (i.e. eos, etc.). When possible, specify type of Large Mononuclear Cells through keyboard (i.e., monocytes, macrophages, mesothelial cells, etc.). Follow laboratory policy.
If identification is in question and Supervisor or Pathologist are not available, report out count and note differential to follow. Show slide to Supervisor or Pathologist as soon as possible. (See Glossary for cell description.)
If cells fail to stick to slides, pre-clean slide in 95% alcohol.
Fragile cells may require a lower RPM setting, low acceleration and increased spin time. Try this if cells are disintegrated on initial smear (i.e. 3,000 RPM and 15 minutes).
Smears with blasts or abnormal cells for the first time need to be referred for Pathologist review.
For fluids that you can not get a nice cytospin off of due to the protein content of the specimen, (sticky), try using the hyaluonidase acid. Usually the problem occurs with joint fluids. Place either 6-8 drops of the fluid, straight, or diluted if nucleated count is high in a glass tube. With a pipette, transfer a few crystals of the acid into the tube and mix. Heat 10-30 seconds in the heating block to dissolve crystals. Place 5-6 drops into the cytospin funnel and spin as directed.
Fluid slides are filed in the slide cabinet and stored for at least one year.
For macrophages in CSF with possible siderophages result as follows: Add comment on to the macrophages -; occ/mod/num possible siderophages seen.
For morphology consistency, all technologists participate in each quarterly survey. Only one technologist's result is submitted; all other results are compiled, reviewed and kept for further reference.
Forms:
Fluid Count Worksheet
References:
Fascicle VI, Patient Preparation & Specimen Handling, Chemistry/Clinical Microscopy, Cerebrospinal Fluid, analysis, routine color and appearance, cell count and differential, College of American Pathologists. CAP.5M293
Fascicle VI, Patient Preparation & Specimen Handling, Chemistry/Clinical Microscopy, Peritoneal Fluid Analysis, College of American Pathologists. CAP.5M293
Modified from Davidson, Israel and Henry, John B. : Todd-Sanford Clinical Diagnosis by Laboratory Methods, 15th edition, Philadel phia, 1974, W.B. Saunders Company.
Medical Laboratory Observer, Tips on Technology - "Cell Counts on Joint Fluids", August, 1983.
KJeldsberg, Carl R., Knight, Joseph A. Body Fluids - Laboratory Examination of Amniotic, Cerebrospinal, Seminal, Serous & Synovial Fluids, econd edition, American Society of Clinical Pathologists , Chicago, 1986
Kreig AF, Kjeldsberg CR. Cerebrospinal fluid and other body fluids. In: Henry JB, ed. Clinical Diagnosis and Management by Laboratory Methods, Philadelphia PA: W.B. Saunders Co.;1991:445-473
7. Operators Manual for Cytospin 2 by Shandon
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