<p> Manual Preparation for Fluid Differential SOP (Insert Laboratory Name)</p><p>Heidi Hanes Document Effective Author(s), Name & Number Date Title International QA/QC Coordinator Pro64-E-09- 2 April 2009 SOP SMILE Comment: This document is provided as an example only. It must be revised to accurately reflect your lab’s specific processes and/or specific protocol requirements. Any comments in red should be deleted and replaced as needed. SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s 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.</p><p>Name, Title Signature Date Approved By</p><p>Name, Title Signature Date SOP Annual Review</p><p>Version # [0.0] Revision Date Description (notes) [dd/mm/yy] Revision History</p><p>Name (or location) # of copies Name (or location) # of copies Distributed Copies to</p><p>0929da12208a72a847d56b8107af09cd.doc 1 of 9 SMILE Document Manual Preparation for Fluid Differential SOP (Insert Laboratory Name) Associated Forms:</p><p>0929da12208a72a847d56b8107af09cd.doc 2 of 9 SMILE Document Manual Preparation for Fluid Differential SOP (Insert Laboratory Name)</p><p>I acknowledge that I have read, understand and agree to follow this SOP. Name (print) Signature Date</p><p>0929da12208a72a847d56b8107af09cd.doc 3 of 9 SMILE Document Manual Preparation for Fluid Differential SOP (Insert Laboratory Name)</p><p>PRINCIPLE: </p><p>This procedure can be use in place of a cytocentrifuge slide preparation. The procedure produces a monolayer smear that is required for accurate analysis of fluid differentials. It involves the concentrating of a fluid and preparation of a smear using the cover slip technique.</p><p>ORDERING: </p><p>This section should include any special ordering steps as needed.</p><p>REQUIRED MATERIALS: </p><p>1. Centrifuge (Need a speed of 600-700 RPMs) 2. Centrifuge tubes, such as conical tubes 3. Cover slips 4. Frosted Microscope slides 5. Mounting material 6. Transfer Pipettes 7. Stain(Same used for blood smear staining)</p><p>REAGENTS: </p><p>1. Hyaluronidase: Store at 0-5C. Needed for fluids other than CSF such as joint fluid.Use a few crystals, as needed, to diminish viscosity of certain fluids (joint, synovial, etc). Should be labeled with open date and expiration date as needed. Briefly warm to 37C after addition of Hyaluronidase to an aliquot of fluid. </p><p>2. Balanced Electrolyte or Salt Solution: As a diluent if a dilution is required for cellular fluids. Should be labeled with open date and expiration date as needed. If turbidity is noticed discard, may be contaminated.</p><p>PROCEDURE: Slide preparation:</p><p>Step Action 1 Label test tube with specimen identification.</p><p>0929da12208a72a847d56b8107af09cd.doc 4 of 9 SMILE Document Manual Preparation for Fluid Differential SOP (Insert Laboratory Name) 2 Label slide. 3 It there is more than 1 ml of fluid mix well and place 1 ml into labeled tube. If less than 1 ml place entire sample into tube. 4 If sample is cellular use Dilution Chart below to dilute sample to required cellularity. 5 For samples requiring the addition of Hyaluronidase 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.) 6 Centrifuge sample for 5 minutes at either 600 or 700 RPMs. 7 When done spinning carefully remove tube from centrifuge without disturbing pellet at bottom of tube. 8 Pipette most of supernate off of pellet and reserve for any other testing as needed, such as chemistry. 9 Gently re-suspend pellet at bottom of tube. 10 Take two clean cover slips. 11 Hold one cover glass by its two adjacent corners, with the thumb and index finger of one hand. 12 Place a small drop of well-mixed sediment on this cover slip. 13 With the other hand, hold a second cover slip in the same manner as the first. 14 Gently place the second cover slip over the cover slip containing the drop of fluid (with the drop of blood in between the two cover slips), so that the two cover slips, one on top of the other form a 16-sided figure (Figure 1). As soon as the two cover slips come together, the blood begins to spread.</p><p>Figure 1</p><p>15 Just before the spreading of the blood is complete, separate the two cover slips by a rapid, even, horizontal lateral pull. Care should be taken to avoid any squeezing together of the cover slips. 0929da12208a72a847d56b8107af09cd.doc 5 of 9 SMILE Document Manual Preparation for Fluid Differential SOP (Insert Laboratory Name) 16 Allow the smears to air-dry completely. 17 The cover slips are now ready for staining with the blood cell stain in use. 18 After staining, place a drop of mounting material on cell side of the cover slip. 19 Carefully flip the cover slip onto the labeled frosted slide and allow the mounting fluid to spread. Gently press any air bubbles out. 20 Smear is ready for viewing.</p><p>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.</p><p>DILUTION CHART</p><p>WBC DILUTION DROPS DROPS DROPS IN* COUNT FACTOR FLUID SALINE SAMPLE CHAMBER </p><p>0-101 --- 5 0 5</p><p>101-300 1:2 6 6 5</p><p>301-700 1:4 3 9 5</p><p>701-1500 1:10 1 9 5</p><p>1501-3000 1:20 1 19 5</p><p>Differential:</p><p>Step Action 1 The slide should be scanned to insure that a monolayer of cells were created and to detect any unusual cell formation. 2 If Then</p><p>0929da12208a72a847d56b8107af09cd.doc 6 of 9 SMILE Document Manual Preparation for Fluid Differential SOP (Insert Laboratory Name) A monolayer was not Make a larger dilution and achieved. spin again. 3 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 when to perform differential on low counts. 4 Follow the policy of your laboratory for cell identity.</p><p>EXPECTED RESULTS:</p><p>Differential in Normal Body Fluid CSF: Adults Neonates Lymphocytes 40-80% 5-35% Monocytes 15-45% 50-90% Polynucleated 0-6% 0-8%</p><p>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.</p><p>All other fluids: PMN - Less than 25% Macrophages and mesothelial cells may be present.</p><p>PROCEDURE NOTES: </p><p>1. 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.</p><p>2. 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.) (Note – Insert the policy for your lab) </p><p>3. Fragile cells may require a lower RPM setting, lower </p><p>0929da12208a72a847d56b8107af09cd.doc 7 of 9 SMILE Document Manual Preparation for Fluid Differential SOP (Insert Laboratory Name) acceleration and increased spin time. Try this if cells are disintegrated on initial smear.</p><p>4. Smears with blasts or abnormal cells for the first time need to be referred for Pathologist review. </p><p>5. For fluids that are thick due to the protein content of the specimen, (sticky), try using the hyaluonidase acid. Usually the problem occurs with joint fluids. </p><p>6. Fluid slides are filed in the slide cabinet and stored for at least one year. Follow policy of your laboratory for retention of slides.</p><p>7. For macrophages in CSF with possible siderophages result as follows: Add comment on to the macrophages -; occ/mod/num possible siderophages seen. If iron stain is available another slide can be made and stained with the iron stain to confirm siderophages.</p><p>8. For morphology consistency, all technologists participate in each quarterly survey. Only one technologist's result is submitted for EQA; all other results are compiled, reviewed and kept for competency training. (Follow the policy of your laboratory. Can be used for competency testing of technologist.)</p><p>References: </p><p>1. 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 2. Fascicle VI, Patient Preparation & Specimen Handling, Chemistry/Clinical Microscopy, Peritoneal Fluid Analysis, College of American Pathologists. CAP.5M293 3. Modified from Davidson, Israel and Henry, John B. : Todd- Sanford Clinical Diagnosis by Laboratory Methods, 15th edition, Philadelphia, 1974, W.B. Saunders Company. 4. Medical Laboratory Observer, Tips on Technology - "Cell Counts on Joint Fluids", August, 1983. 5. KJeldsberg, Carl R., Knight, Joseph A. Body Fluids - Laboratory Examination of Amniotic, Cerebrospinal, Seminal, Serous & Synovial Fluids, Second edition, American Society of Clinical Pathologists , Chicago, 1986</p><p>0929da12208a72a847d56b8107af09cd.doc 8 of 9 SMILE Document Manual Preparation for Fluid Differential SOP (Insert Laboratory Name) 6. 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. Brown,Barbara A., Hematology: Principles and Procedures, Third Edition, Lea and Febiger, Philadelphia, 1980: pages 83- 84.</p><p>0929da12208a72a847d56b8107af09cd.doc 9 of 9 SMILE Document</p>