Author: Heidi Hanes Document Number: Pro64-E-01Effective (or Post) Date: 13 January 2009

Review History Date of last review: 7 Feb 2012Reviewed by: Penny Stevens

SOP should only be used as an example, change as needed. Items in red are suggestions.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.

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Laboratory NameAddress

Department

TITLE: Cell Count On Body Fluids – by Manual 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 Director’s designee should review all copies of this procedure at least once a year. Discontinued procedures must be retrievable in a reasonable timeframe.

Laboratory Name

Reviewed By: Date: Reviewed By: Date:

DEPARTMENT OF PATHOLOGYHEMATOLOGY

CELL COUNT ON BODY FLUIDS – by Manual Method

COMMON FLUID TYPE CODESJT JOINTPL PLEURALPERI PERICARDIALPERT PERITONEAL SYN SYNOVIALRKN RIGHT KNEELKN LEFT KNEEKN KNEEREL RIGHT ELBOW

PRINCIPLE:Special diluting fluid is used to stain cells present in fluids. The diluted sample is introduced into

a counting chamber and the number of erythrocytes and leukocytes are counted in a prescribed area. The number is then multiplied by an appropriate factor to obtain the number of erythrocytes and leukocytes per cm3 of body fluid.

CLINICAL UTILITY:CSF fluid is obtained to assist in diagnosing central nervous system infection, tumors or vascular

accidents. In Body fluids (pleural, peritoneal, synovial, etc.), results are used to determine whether a transudate due to increased hydrostatic or decreased plasma oncotic pressure; an exudate secondary to a wide variety of pleural diseases, e.g. increased capillary permeability, infection, infarction, rheumatoid disease, collagen disease; or secondary to malignancy.

SPECIMEN COLLECTION AND PREPARATION:

Good laboratory practice dictates that specimens should be processed as soon as possible.

The National Committee for Clinical Laboratory Standards (NCCLS) provides the following recommendations for handling, processing, and storing blood specimens:

Sterile preparation of spinal puncture site is necessary when collecting CSF. A non-traumatic tap using aseptic collection technique is required for all fluids. Sample should be placed in numbered glass or plastic tubes for CSF using order of draw for sequence. Body fluids may be collected in glass or plastic tubes using appropriate anticoagulant if necessary for analysis.

1. Storage requirements:Room temperature (25o C) 1 hourRefrigerator (2-8o C) 1 hour for CSF, 3 days for body fluids

After this time, distortion of morphology and cell lysis makes the specimen unsuitable for analysis

Freezer (-20o C) Not recommended for CSF. Supernatant from

centrifugation of other body fluids allowable for chemical analysis only, may be stored for up to 6 mos.

Some fluids may be delivered in a needle and syringe in order to maintain their integrity and sterility. Fluids received in such a way will be handled in the following manner:

a. If a culture is requested, Bacteriology will remove and discard the needle. The specimen will be transferred to a sterile screw cap tube and an EDTA vacutainer for further analysis. (Most chemistry analysis cannot be performed on EDTA samples).

b. If a culture is not requested, the first department receiving the specimen for analysis will perform the above step.

c. Specimens should be processed within one hour of collection. If there is a delay in analysis cells may start to deteriorate.

2. Rejection Criteria:A specimen will be rejected if:

It is improperly labeled or unlabeled. In the case of fluids, an exception report must be completed and signed by the physician and an incident report form filled out and forwarded to the lab Quality Assurance representative.

3. Cell Counts. Unless otherwise noted on the requisition, cell counts are performed on the last tubein the numbered series. Generally, tube 3 or 4 represents the least traumatic tube. If the highest numerical tube visually appears more cellular than other tubes, the physician is called to inquire about tubes being drawn out of numerical sequence. A decision is then made as to which tube to count.a. Perform count on last numbered (least traumatic) tube.b. Note appearance of all requested tubes. c. Testing is always performed on the tubes as follows unless stipulated other wise by

physician:Tube #1 – Chemistry ordersTube #2 – Microbiology ordersTube #3/4 – or last tube drawn – Cell Count

NOTE: Any fluid with flecks of material that may possibly be tissue should be referred to the Pathologist. He/she will determine whether to proceed with further testing on sample. (Histology may be required on this specimen). If after hours, preserve with formalin to maintain integrity. (See notes section.)

* CSF cell counts may be requested on more than 1 tube from a single lumbar puncture from the Emergency Department. If count is requested on more than one tube, on receipt the second tube requested should be placed on rotator for 2-4 min. Retain and refrigerate approximately 1 ml in original tube for further testing. Forward a well-mixed aliquot of tube #1 to next appropriate section (usually chemistry) for centrifugation and processing. (Fluid cannot sit for long periods before glucose, protein, etc. are performed). If original order for

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CSF includes two counts, call physician if RBC count from tube #3 or #4 is less than 20 RBC’s to see if second count is necessary.

** For CSF samples: if amount of fluid in tube #1 is less than 1 ml, chemistry tests can be performed on supernatant from tube #2 and tube #1 retained for additional count. If quantity of specimen is not enough for all test requested, physician should be called to decide which test should be performed.

Any request for additional cell count should be performed for Red Cells only.

SAFETY PRECAUTIONS:ALL SAMPLES ARE POTENTIALLY INFECTIOUS AND MUST BE HANDLED WITH CARE AT ALL TIMES

REAGENTS:Reagents are checked for contamination (fibrin or cells) before each use and re-filtered monthly.All reagent containers are to be properly labeled with preparation, expiration date, storage temperature and tested date.

1. Crystal violet solution: Combine 0.2 gm crystal violet, 2 ml glacial acetic acid and 100 ml Type II deionized water in a brown bottle. (Only use this solution for 9:10 dilutions). Reagent is good for 1 year.

Warning: Crystal Violet is carcinogenic and an irritant to eyes, skin and mucous membrane. May alter genetic material. Wear impervious lab gown, protective gloves and eye/face protection. Do not breathe dust. See MSDS sheets for further information.

2. Methylene blue/saline solution Isotonic: Place 0.1 gm methylene blue in 100 ml volumetric flask and dilute with 0.85% NaCl. Reagent is good for 1 year.

Warning: Methylene blue is a severe eye irritant. Avoid contact with eyes and skin. Do not ingest. Avoid inhalation. Wear impervious lab gown, protective gloves and eye/face protection. See MSDS sheet for further information.

3. Stock solution Buffered Sodium Chloride (10%) solution: Using a volumetric flask, dissolve 90 gm NaCl, 13. 66 gm Na2HP04, and 2.43 gms NaH2PO4..H20 in 1000 ml Type II deionized water. This solution is osmotically equivalent to 10% NaCl. It is stable in stoppered bottle for one year.

a. 1% Sodium Chloride Solution: Dilute the stock solution 1:10 by pipetting 10 ml stock solution into a 100 ml volumetric flask and dilute to the mark with Type II deionized water. Good for 1 year.

b. 0.30% Sodium Chloride Solution: Pipette 30.0 ml of 1 % Sodium Chloride solution into a 100 ml volumetric flask and dilute to the mark with Type II deionized water. Good for 1 year.

4. Hypotonic: Place 0.1 gm methylene blue in 100 ml volumetric flask and dilute with 0.30% NaCl. Reagent good for 1 year.

5. Turk's Solution: Combine 30 ml glacial acetic acid and 10 ml 1% aqueous gentian violet in a volumetric flask and dilute to the 1000 ml mark with Type II deionized water. Filter before use.

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Label bottle correctly with preparation date, expiration date and tested date. Turk' solution is good for 5 years.

NOTE: Glacial acetic acid is corrosive. Avoid contact with eyes and skin. Wear safety goggles and face shield, impervious lab gown and protective gloves. See MSDS sheet for further information.

Crystal Violet is carcinogenic and an irritant to eyes, skin and mucous membranes. May alter genetic material. Wear impervious lab gown, protective gloves and eye/face protection. Do not breathe dust. See MSDS sheets for further information.

6. Gower's Solution: Using a volumetric flask, dissolve 62.5 gm sodium sulfate and 166.5 ml glacial acetic acid in 1000 ml Type II deionized water. Label properly with preparation date, expiration date and tested date. Gower's is good for 5 years.

WARNING Contains glacial acetic acid. Corrosive. Avoid contact with eyes and skin. Wear safety goggles and face shield, impervious lab gown and protective gloves. See MSDS sheets for further information.

7. Body Fluid Cell Count Controls: (Include description of the control, how many levels, stability information, such as storage and expiration date upon opening).

EQUIPMENT:

1. MLA Pipettes 2. Pipette tips3. 10 x 75 test tubes4. 16 x 100 mm test tubes5. Gauze.6. Hemocytometers.7. Petri dishes.8. Coverslips.9. Mixer

QUALITY CONTROLTo monitor system performance and chart trends, at least one level of control must be run within any 8 hour period prior to running patient samples. If no fluid is performed, no QC is required. .Include commercially available control sera that cover at least two levels of analyte. Follow manufacturer’s instructions for reconstitution and storage. Quality control samples should also be assayed after maintenance or replacement of MLA pipettes. Treat all quality control samples the same as patient samples.

1. Refer to the quality control product insert or the suggested Expected Values. If the quality control results do not fall within the suggested Expected Values or within the laboratory’s established range, then perform the following:a. Review the instructions to ensure that the assay was performed according to the

procedures recommended by vendor.

b. Verify that the materials are not expired.

c. If necessary, rerun the quality control samples or contact control manufacturer for more assistance.

2. First fluid of the day:a. Check log sheet in fluid area to see what level of control to be run. Alternate the level of QC per

shifts run.

b. Remove control from refrigerator, it is not necessary to allow to warm to room temperature.

c. Mix control thoroughly by rapid inversion of the vial to ensure mixing to obtain reproducible results.

Make a 9/10 dilution of the control using Crystal Violet reagent as follows:i. In a glass tube, pipette 450 ul of mixed control.

ii. In the same tube, pipette 50 ul of Crystal Violet.iii. Allow to sit 1-5 minutes.iv. Thoroughly mix and plate both sides of hemacytometer.v. Allow to settle, count all 9 squares on both sides, enumerating nucleated and RBC.

vi. Calculate total cells and record results. Follow basic QC rules.

NOTE: Fluid dilutions for QC do not need to be made in duplicate. For patient fluids, duplicate dilutions should be made when fluid volume allows for it. Plate one of the dilutions on each side of the hemacytometer, results should match within 10%. If only one dilution can be performed, both sides of the hemacytometer should be counted and match within 10%.

PROCEDURE: (For all dilutions stated in procedure, see dilution chart following directions.)1. Colorless Clear Fluid:

a. For Colorless clear fluids, a 9/10 dilution is used.b. CSF - Crystal Violet is the diluting fluid of choice.c. Other Body Fluids – 0.85% Methylene Blue dilution fluid is usedd. Allow dilution to sit for 2-3 minutes; mix before plating.e. Fill both sides of counting chamber. Allow cells to settle out, see notes on settling.f. Using the high power objective (43x dry):g. Count all nucleated cells in entire ruled area (9 sq mm). For CSF differentiate nucleated

cells if possible.h. If 9 or less nucleated cells seen, a differential can be calculated from the chamber for CSF

Fluids.i. If more than 9 nucleated cells seen, perform a cytospin for differential count, see procedure

on cytospin.j. Count all erythrocytes in the entire ruled area (9sq mm)..k. Use formula to calculate the number of nucleated cells and erythrocytes.l. Report nucleated cells and erythrocytes in /MM3 and differential in percentage.

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2. Hazy or slightly bloody fluids:a. Diluting fluid of choice is 0.85% methylene blue.b. Use a 1/10 dilution c. Allow dilution to sit for 2-3 minutes; mix before plating.d. Fill both sides of counting chamber. Allow cells to settle out, see notes on settling.e. Using the high power objective (43x dry):f. Enumerate both nucleated cells and erythrocytes on chamber. Counting area will depend

on cell distribution. The more cells seen, the less area counted. (e.g. 4 large corner squares, entire middle squares or 5 small middle squares)

g. Make a cytospin slide to perform differential count. h. Use formula to calculate the number of nucleated cells and erythrocytes.i. Report nucleated cells and erythrocytes in /MM3 and differential in percentage.

3. Moderately bloody or cloudy fluid:a. Diluting fluid of choice is 0.85% methylene blueb. Use a 1/20 dilutionc. Follow steps c-i in step 2 above.

3. Very Bloody fluid:a. Due to the high erythrocyte count two dilutions are necessary to achieve an accurate count

for both cell lines.b. For the erythrocyte dilution, the diluting fluid of choice is 0.85% methylene blue, using

either a 1/100 or 1/200 dilution.c. For nucleated cell the diluting fluid of choice in the 0.30% methylene blue (hypotonic

solution) using either a 1/10 or 1/20 dilution.d. Allow dilutions to sit for 2-3 minutes.e. Fill 2 separate chambers with mixed diluted fluid, allow settling.f. Use high power objective (43x dry).g. For erythrocyte dilution count either the entire middle square or 5 little squares, depending

on cell distribution.h. For nucleated dilution count either all 9 squares or 4 corner squares, depending on cell

distribution.i. Make a cytospin slide to perform differential.j. Use formula to calculate both the nucleated cells and erythrocyte counts.k. Report nucleated cells and erythrocytes in /MM3 and differential in percentage.

NOTE: Gowers or Turk's solutions can be used for the erythrocyte and nucleated cell counts, but due the proteinous nature of fluids the acetic acid will cause precipitation, making counting difficult to perform.

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Dilution Procedure:

Dilution Required Amount of Sample Amount of diluting fluid1:10 100 ul 0.9 ml1:20 100 ul 1.9 ml1:100 100 ul 9.9 ml1:200 50 ul 9.95ml9:10 0.9 ml (450 ul) 100 ul (50 ul)

Mix dilution well, allow to sit 1-5 minutes, fill hemacytometer and follow directions based on fluid appearance for counting and calculations:

Alternate dilutions:1. For clear to slightly cloudy fluids with a final dilution of ½:

a. Dilute specimen 1:2 with appropriate diluting fluid. (0.2ml specimen to 0.2 ml diluent).b. Mix in a glass tube and allow to sit 1-5 minutes.c. Fill hemacytometer and count the appropriate area.d. Calculate as follows multiplying cells counted:

18 squares - multiply by 1.1 9 squares - multiply by 2.2 4 squares - multiply by 5

1 square - multiply by 20 5 small squares multiply by 100

2. For moderately cloudy fluids with a final dilution 1/20:a. Dilute specimen 1: 10 with normal saline or Coulter Isoton (0.1 ml of specimen to 0.9 ml

diluent).b. From the above dilution, make an additional dilution 1:2 with appropriate fluid reagent

(0.2ml of dilution to 0.2 ml of diluent).c. Mix in a glass tube and allow to sit 1-5 minutes. Fill hemacytometer and count appropriate

area.d. Calculate as follows:

18 squares - multiply by 119 squares - multiply by 22 4 squares - multiply by 50 1 square - multiply by 200 5 small squares - multiply by 1000

3. For very cloudy or bloody fluids requiring a final dilution of 1/200a. Dilute specimen 1:100 with saline or Isoton (0.1 ml of specimen to 9.9 diluent).b. From the above dilution, make an additional dilution 1:2 using the appropriate diluting

reagent (0.2 ml to 0.2 diluting fluid).c. Mix in glass tube and allow to sit 1-5 minutes. Fill hemacytometer and count appropriate

area.

d. To calculate:18squares multiply by 110 9 squares multiply by 220 4 squares multiply by 500 1 square multiply by 2000

5 small squares multiply by 10,000

CALCULATIONS:

1. Erythrocyte and leukocyte counts should be performed within 30-60 minutes after the specimen is received because cells lyse on prolonged standing yielding inaccurate counts.

2. Alternate dilution procedures serve as guideline. However, due to variability in cell number, it may sometimes be necessary to increase the dilution and/or decrease the area counted in order to

facilitate counting. Common sense judgements should be the rule and a factor calculated for the revised dilution and/or area counted using the following formula:

1 x Reciprocal of dilution = FactorArea counted X Depth (0.1 mm)

Multiply cells counted by factor for final concentration.

When counting Body Fluids, scan entire GRID (low power) to confirm even distribution. Alter the area based on cellularity of fluid.

All 9 squares = 9 mm2

Complete Center Square =1 mm2

Center square has 25 smaller squares- One small square in center square = 0.04 mm2

Five small squares in center square = 0.2 mm2

3. When counting RBC and WBC on chamber, identifying WBC may be useful as guide to what to expect on smear.

EXPECTED RESULTS: CSF should be clear and watery. Other body fluids can be clear or a straw to yellow color. In all fluids, turbidity may be caused by leukocytes, erythrocytes, micro-organisms, or by contrast media. A pink to orange color represents xanthrochromia from the breakdown of RBC.

Cell Counts in Normal Fluids:CSF:

RBC 0-2/cmm3

WBC - Adults 0-5/cmm3

Neonates 0-30/cmm3

Synovial: RBC 0-2000/cmm3

WBC <200/cmm3

Pleural: RBC 0-10,000/cmm3

WBC <1000/cmm3

Peritoneal/ RBC 0-10,000/cmm3

Pericardial: WBC <500/cmm3

Differential in Normal Body FluidCSF: 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.

COMPUTER ENTRY:For laboratory that has a computer system this section should include fluid codes, ordering of fluids and resulting of fluid.A. Order Entry:

**ALWAYS ENTER TYPE – CODE FOR FLUID BEING TESTED FIRST OR RESULTS WILL NOT BE APPENDED TO THE CORRECT FLUID

At TEST 1 prompt enter: TYPE - (enter code for type of fluid being tested)TEST 1 will again display - the following test s may then be entered:

Chemistry tests: Hematology tests:TEST 1: SFAPTEST 2: SFTPTEST 3: SFG

TEST 4: FAPPTEST 5: FDF

TEST 6: FCT

B. Result Entry:Enter the following results under Function MEM, Worksheet FLD1:

1. Fluid Type if not previously entered2. Fluid appearance3. Cell Count

Enter differential results through Function DF, keyboard DF2 using either "O" (online) or M (manual) entry.

LIMITATIONS AND NOTES:

1. The following times are noted and will eliminate cell lysis due to excessive standing.Maximum time – Sitting after dilution - 5 minutes

Sitting after plating - 10 minutes

2. In cases where small clumps/clusters of cells are seen on the hemacytometer (but not visible in the undiluted specimen) a comment should be attached that the cell count may be inaccurate.

3. When performing a cell count on bloody fluids only intact red cells should be enumerated. If there are ghost red cells present these should be accounted for in a comment attached to the red cell count using few, moderate or numerous as a modifier as appropriate. This is particularly

important in CSF indicating an intra-cranial bleed. If the cells are not intact they are probably not from a current bleed. Usually CSF will be yellow and fairly clear in this case.

4. A fluid is considered clotted if one or more bloody or gelatinous clots can be seen in the tube. If tissue fragments appears in the tube it needs to be viewed by a pathologist who will decide if histology needs to section it. If clumps of cells are seen on the hemacytometer, but not visible in the undiluted specimen the specimen is not considered to be clotted. If the specimen is clotted, no cell count shall be reported.

5.

A. If physician insists on having the cell count, ask the physician to call the Pathologist. The Pathologist will then call the lab if there is clinical indication to process the clot as a surgical specimen (to section it in histology). To preserve the specimen for histology place in formalin and mix well into the clot(s) with applicator sticks. (Formalin can be found in Hematology in the draw next to the stainer in coulter cups.)

B. The cell count and fluid differential should be credited in the computer with "sample clotted, count not possible, notified:" and name of person notified. The appearance and color may be reported. In cases where clumps of cells are seen on the hemacytometer, but not visible in the undiluted specimen, a comment should be hyphenated to the cell count indication the cell count may be inaccurate.

FORM:

Fluid Count Worksheet

APPENDIX:A: Guideline for Counting Cells in Body FluidB. Hemacytometer Grid

RELATED DOCUMENTS:

Cytospin SOP

REFERENCES:

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, econd edition, American Society of Clinical Pathologists , Chicago, 1986

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. Cell-Chex Body Fluid Cell Control insert.(Fluid Control Insert)