HE Body Fluid Analysis

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HE-32.02 Body Fluid Analysis

Document Number: Revision Number: 12.02

Document Type: Procedure Effective Date: 11/29/2019

Location: Clinical Biochemistry and Hematology\Hematology\General Hematology\32 CSF and Body Fluid Analysis

HE-32.02 Body Fluid Analysis 1. APPLICABILITY

This procedure applies to: DSC FMC RRL BMSH

2. PURPOSE

This procedure provides instructions for body fluid gross examination, cell count and WBC differential.

3. METHODOLOGY

Body fluid is examined visually for appearance and a WBC and RBC count is performed by automated or manual means. Cytospin preparations are made, stained with Wright Giemsa stain, and a WBC

differential, if ordered by physician, is performed. This procedure applies to body fluids including pleural, peritoneal, pericardial, and synovial fluids.

4. DEFINITIONS

Macrophages/

Monocytes

Monocytes are bone marrow derived cells that circulate in the blood.

Macrophages arise from bone marrow derived cells that migrate into tissues and evolve morphologically. Monocyte/macrophage morphology in fluids is quite variable, ranging from the typical blood monocyte of the peripheral blood to a vacuolated, activated stage with the morphology of a typical macrophage.

Monocytes are usually large (12-20 μm) with abundant blue-gray cytoplasm containing sparse azurophilic granules. The nucleus is round to oval and may show indentation, giving it a kidney bean or horseshoe shape. The chromatin is lacy and small nucleoli are apparent. Macrophages are larger cells (15-80 μm) with abundant cytoplasm showing evidence of active phagocytosis. This includes ingested material such as other blood cells or bacteria, hemosiderin, fungi, and remnants of digested materials as well as cytoplasmic vacuoles post- ingestion. One or more round to oval nuclei are present and occasionally prominent nucleoli. Macrophages can at times be difficult to differentiate from mesothelial cells. Mesothelial cells are usually larger than monocytes/

macrophages and usually show more biphasic staining cytoplasm and surface microvilli.⁸

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HE-32.02 Body Fluid Analysis Rev: 12.02

Mesothelial Cells Mesothelial cells (20-50 μm) normally line pleural, pericardial, and peritoneal surfaces. These cells can be shed individually or in clusters. When found in pairs or clusters, mesothelial cells have articulated or coupled cell borders with a discontinuous outer border (clear spaces or “windows”) between many of the cells. The nucleus is round to oval in shape with a definitive nuclear membrane and regular contour. Nuclear chromatin varies from dense to fine, but it is evenly distributed. Multiple nucleoli may occur and the nuclei may overlap; however, the nuclei remain of approximately equal size and shape. One or more nucleoli may be present. The nuclear-to-cytoplasmic ratio is low (less than 1:1) and the nucleus may be central or eccentrically placed. The cytoplasm is light to dark blue and may have a grainy texture, typically dense grainy basophilia or even a crystalline/ground glass appearance to the perinuclear area. With some staining techniques, the periphery and perinuclear cytoplasmic regions may appear as very lightly stained areas. With degeneration, additional small vacuoles may occur throughout the cell. Cytoplasmic budding or fragmentation may also occur. In chronic effusions or during inflammatory processes, mesothelial cells proliferate and become very large. Mitotic figures occasionally are seen within mesothelial cells. The nuclear chromatin is less condensed and nucleoli may be prominent; however, the nucleus still retains a definitive, smooth, nuclear membrane. Mesothelial cells can be phagocytic and resemble macrophages, resulting in forms that have a morphology intermediate between mesothelial cells and macrophages.⁸

Synovial Fluid Diarthrodial joints are lined at their margins by a synovial membrane with synovial cells lining the joint space. These cells have the capacity for protein synthesis and phagocytosis. Synovial fluid is basically an ultrafiltrate of plasma produced by the dialysis of plasma across the synovial membrane and

combined with a mucopolysaccharide hyaluronate that is synthesized by the cells of the synovial membrane. Synovial fluid lubricates the joint space and transports nutrients to the articular cartilage.

Analysis of synovial fluid plays a major role in the diagnosis of joint disease and determining whether there is an inflammatory or a non-inflammatory effusion.

Synovial fluids with a WBC count 25,000 x 10⁶/L are usually associated with septic arthritis or acute crystalline disease (e.g. Gout). Differentials performed on inflammatory fluids typically have 50% neutrophils while differentials performed on septic fluids typically have 75% neutrophils.

Mononuclear cells, including lymphocytes, monocytes, macrophages, and synovial tissue cells, are the primary cells seen in normal synovial fluid.

Neutrophils should account for less than 2% of the differential count. Increased neutrophils indicate a septic condition, whereas an elevated cell count with a predominance of lymphocytes suggests a non-septic inflammation. In both normal and abnormal specimens, cells may appear more vacuolated than they do on a blood smear. Besides increased numbers of these usually normal cells, other cell abnormalities include the presence of eosinophils, LE cells, Reiter cells (vacuolated macrophages with ingested neutrophils) and RA cells, or ragocytes (neutrophils with small, dark, cytoplasmic granules that consist of precipitated rheumatoid factor) ¹.

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Pleural, Peritoneal and Pericardial Fluid

These are each the result of a simple process of ultrafiltration and are found normally in small amounts (approximately 10 mL) in the pleural, peritoneal and pericardial cavities. The function of these fluids is to form a thin lubrication film between opposing surfaces. In pathological states several litres of fluid may be aspirated.

Peritoneal Dialysate/

Effluent²

Peritoneal dialysis cleans the blood without it being removed from the body.

Dialysate is injected into the peritoneal space in the abdomen through a two- way catheter. The membrane that lines the abdomen (peritoneum) allows waste and fluid to pass from the blood into the dialysate. Peritoneal dialysate, made up mostly of salts and sugar encourages ultrafiltration through the peritoneum.

About 2 weeks before dialysis begins a catheter is surgically inserted with one end in the peritoneal space and the other extending a few inches away from the skin. It remains permanently in place and is accessible any time. There are two methods of peritoneal dialysis, ambulatory and automated. Peritoneal dialysis must be performed every day and fluid must be in the abdomen at all times to clean the blood adequately.

Complications: Abdominal infection, amyloidosis (stiffening of kidney), diabetes, infected catheter, peritonitis, vitamin and mineral deficiencies. Some patients develop diabetes or obesity from the large sugar content of the dialysate.

BAL

(FMC only)

Broncoalveolar Lavage is a diagnostic procedure that involves sampling of the lower respiratory tract cells and acellular components by instillation and recovery of fluid through a fiberoptic bronchoscope. It provides important diagnostic information which can be interpreted in conjunction with radiologic and clinical findings, especially in the diagnosis of interstitial lung disease, malignancies and infections.

Paracentesis Procedure for aspiration of peritoneal fluid. Peritoneal fluid is also called abdominal or ascetic fluid.

Pericardiocentesis Procedure for aspiration of pericardial fluid.

Thoracentesis Procedure for aspiration of pleural fluid. Pleural fluid is also called thoracentesis fluid, chest fluid or empyema fluid.

5. MATERIALS

Equipment  Hematology analyzer for automated cell count

 Improved Neubauer Hemocytometer with glass coverslip

 INCYTO C-Chip Disposable Haemocytometer

 Light/phase microscope with 40X dry objective lens

 Automatic pipette and tips

 Automated slide stainer

 Cytospin and related equipment

 Specimen rocker

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Materials  Petri dish lined with moistened filter paper

 Wooden applicator sticks

 12 x 75 mm plastic test tubes plus caps

 Cytospin slides with pre-marked circle

 Frosted glass slides (FMC, ACH)

 Non-heparinized capillary tubes (blue tipped)

 Sterile transfer pipette

 Analyzer diluting fluid

 Disinfectant (e.g. Cavicide)

 95% Methanol

 Alcohol swabs Forms and

Records

 Peritoneal Dialysate Worksheet - ACH Only HE4031

 Manual Fluid Worksheet HE4032

 For retention guidelines refer to GL01-7.01_Attachment 1 Records Materials Retention 5.1. Reagents

Reagent Source Preparation Storage/Stability

Hyaluronidase Active Ingredient:

Hyaluronidase - a mucolytic agent used to reduce synovial fluid viscosity

Ready for use  Store at -20 ºC

 Date and initial when bottle is opened

10% Acetic Acid - ACH only

Prepared in lab  Add 5.0 mL glacial acetic acid to 45 mL distilled water

 Mix and add date prepared

 Stable indefinitely.

 Store in amber bottle on CSF bench

New Methylene Blue

- DSC only

Volu-Sol, Inc. Ready for use  Stable until manufacturer stated expiry date when stored at room temperature

 Store in a tightly sealed container away from direct sunlight and exposure to chemical fumes

6. SPECIMEN

Specimen

Type Pleural, peritoneal, pericardial, or synovial fluid, BAL.

Container 4 mL lavender top EDTA tube or 4 mL green top Sodium Heparin tube Maximum Maximum draw as indicated on tube

Minimum Optimum specimen volume is 3 - 4 mL, but testing can be performed on minimal quantities (see below).

Handling

Conditions SAFETY: Potential biohazard. Use Universal Precautions.

Process fluids for cell count and differential as soon as possible. Cellular elements may deteriorate upon standing.

Note: BAL for cell count and differential can only be ordered by Respirologists to assist in the diagnosis of ILD or Eosinophilic Pneumonia. Testing will be performed at FMC only.

SAFETY: For in vitro diagnostic use. Do not ingest. Avoid skin and eye contact.

Hyaluronidase - avoid contact and inhalation.

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Criteria for rejection

 Inadequate specimen identification

 Improper transportation/handling/storage of specimens

 Solidly clotted specimen

 Inadequate volume of specimen for analysis (cell count and differential) Minimum Volume Automated count/differential 1.0 mL Manual count/differential 0.5 - 1.0 mL

Test not performed

< 0.5 mL

Synovial Fluid: every attempt must be made to perform a count and/or differential prior to

cancelling the test.

 Use of incorrect collection tube (oxalate tube) or container:

o Specimens received in plain sterile containers or red top tubes are considered sub- optimal but may still be processed, provided NO other criteria for rejection are present.

o Hematology only – Specimens should NOT be accepted if they arrive in open syringes, transfusion bags, I.V. bottles or any container larger than 100 mL capacity.

o Cytopathology only – Requires a larger specimen volume to enable adequate cell recovery and will accept large volume containers.

Action taken

 For specimens outside of the listed rejection criteria, refer to Tech II/III before canceling the test.

 Contact Hematopathologist/Pathologist on call if required.

Specimen Retention

 5 days – refrigerated and then discard in biohazard container.

 Rack specimens with Chemistry for 7 days (PLC/SHC) and 5 days (RGH).

7. QUALITY CONTROL

Analyzer counts  Automated Body Fluid Controls – DxH (Coulter Body Fluid): Sysmex (XN BF Check).

 Analyzer background count is checked before performing a fluid cell count.

 DxH – Attach printout of instrument background counts whenever an automated count is reported.

 Sysmex – Do not need to record background count. Analyzer will not perform a cell count if background is above tolerance. If background is too high (Analysis too high error), Sysmex will ask for another background.

 Duplicate dilutions run on the analyzer must check within ± 10%.

 Prepare a second set of dilutions if results do not check.

Manual counts  A procedural control is performed on each specimen. All manual cell counts are done in duplicate and should check within:

Duplicate cell counts: 0-10 cells limit ±2 cells 11-50 cells limit ±5 cells

>50 cells limit 10%

 If the counts are not within acceptable limits, flood new counting chamber and repeat count until the duplicate count is within acceptable limits.

 Background count on diluting fluid is performed on hemocytometer before performing manual count on diluted sample. Hemocytometer tolerance up to 9 particles/9 squares in counting chamber.

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8. PROCEDURE

8.1. Preliminary Examination

Step Action Notes

1 Generate a fluid cell count worklist or select appropriate worksheet, if applicable.

2 Record fluid site and volume of hematology specimen.

3 Perform gross examination and record findings

Perform a gross examination of the fluid for appearance (volume, color and clarity, presence of clots/fibrin strands) and record findings on worksheet.

Refer to section 10.1 Reporting Format for report format and comments for volume, color, clarity, clots and/or fibrin strands present.

4 Prepare a wet mount to examine fluid microscopically for debris or cell clumps³

If: Then: Comments

Specimen is clear and wet mount does not show debris or cell clumps

Perform cell count on hematology analyzer.

BMSH – Manual cell counts only

Bloody fluids with no visible clumps or debris may also be run on the analyzer.

Clots, clumps, or debris are present on wet mount

Perform manual cell count, or at Technologist discretion if debris is not visible macroscopically, perform analyzer cell count. Refer to reporting format for comments to physician.

Clots may produce a decreased WBC count.² Debris may produce a falsely elevated WBC count.²

Clumps may compromise the accuracy of the WBC count.

Specimen contains a clot that is >1/2 of the total specimen volume

Perform only differential, if ordered.

Inform the physician or nursing unit immediately that specimen is clotted and the count will not be performed.

Specimen contains a clot(s) that is <1/2 of the total

specimen volume

Perform an automated or manual cell count. Refer to reporting format for comments to physician.

A differential will be performed only if ordered.

Specimen is solidly clotted

Do not process. Perform neither cell count nor differential.

Specimen is an extremely viscous synovial fluid or BAL

 Transfer approximately 1/2 the specimen volume to a 12 x 75mm test tube.

 Use a spatula to add a few grains of

hyaluronidase to the aliquot of fluid. Cap the tube and place on the mixer for 10 minutes.

 If not fluid in 10 min., place at 37C for 5 min.

Mix thoroughly by gentle inversion before counting or analyzing.

Hyaluronidase is a mucolytic agent used to reduce

synovial fluid viscosity.

Note: If used, both the cell count and the smears must be performed on the treated portion of the specimen⁵. Note: If fluid is still extremely viscous after hyaluronidase treatment, perform a manual cell count.

Note: If fluid is still too viscous for capillary tube/manual count, cancel test in Millennium as Lab-Unacceptable.

SAFETY: Potential biohazard. Use Universal Precautions. Use Biological Safety Cabinet when performing manual cell count.

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5 Whether or not a differential is ordered, prepare 2 Cytospin slides

 Refer to 32.04 Cytospin Preparation.

 FMC – 2 spots on one slide.

 ACH only – Prepare Cytospin slides only if differential is ordered.

 For slide retention refer to GL01-7.01_Attachment 1 Records Materials Retention

Note: For markedly bloody or cloudy/purulent fluids, make push smears as well as Cytospin preparations because even if diluted, the RBCs may obscure other cells present on the Cytospin preparations.

8.2. Automated Cell Count Procedure

Ensure body fluid controls have been run on the testing analyzer within the last 24 hours.

Note: WBC = TNC (DxH) or TC – BF# (Sysmex)

These mnemonics may be used interchangeably throughout the procedure.

8.2.1. DxH Cell Count Procedure

1 Invert the tube gently to mix thoroughly

2 Analyzer adjustment for body fluid

DxH: Run in single tube presentation.

 Specimen type: choose appropriate fluid

 Test: BFC 3 Perform and record

background count prior to analyzing fluid using analyzer Diluent

If above acceptable limit (WBC > 20 x 10⁶/L, RBC > 1000 x 10⁶/L), repeat background.

4 Mix fluid well. Run through analyzer in single tube presentation.

5 Report the cell count from the analyzer

 Report the TNC:

o If ‘P’ flag, (indicating partial aspiration), cellular interference, or

‘Bubbles’ system warning present, DO NOT report analyzer results. Perform manual cell count if insufficient specimen to repeat or if flags still present after rerun.

Note: May perform a dilution and run on analyzer if specimen is sufficiently cellular.

 Dilutions for instrumentation testing are to be performed in duplicate, sample volume permitting.

 Instrument dilution results must check within +/-10%.

 If instrument dilution results DO NOT check within +/-10% and the TNC from dilution is >100,000 x 10⁶/L with or without analyzer flags, do not report the number. Report the TNC with a “See Note” and attach Coded Phrase TCBF.

 ALL results need to be confirmed by a second technologist before verification.

6 Peritoneal dialysates/

effluents - ACH only

Automated counts less than 100 x 10⁶/L for WBC and less than 3000 x 10⁶/L for RBC need to be counted manually. Report the value counted.

Convert result to free text and enter a numerical result. Refer to section 9 Calculations.

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8.2.2. Sysmex Cell Count Procedure

1 Invert the tube gently to mix thoroughly.

2 Analyzer adjustment for body fluid

 Switch Mode to Manual Mode.

 Select Body Fluid in the Change analysis mode.

3 Background count  Automatically performs a background check after selecting Body Fluid mode.

 If background count is NOT within tolerance, (TC – BF# >1.0 x 10⁶/L or RBC >3000 x 10⁶/L), another background count will automatically be performed. Once the mode has initially been changed to Body Fluid, it is not necessary to run a background check before every Body Fluid

sample that succeeds it.

 There is no need to record/print a background count.

4 Mix fluid well. Run through analyzer in Manual Mode.

5 Report the cell count from the analyzer

 Report TC – BF# and RBC.

o TC – BF# <100 x 10⁶/L: Leave analyzer result in WAM. Millennium will convert result. If result = 0, select <100 x 10⁶/L in Millennium.

o RBC <3000 x 10⁶/L: Leave analyzer result in WAM. Millennium will convert result. If result = 0, select <3000 x 10⁶/L in Millennium.

o Perform a dilution if:

Non-Synovial fluid:

TC – BF# >10,000 x 10⁶/L or RBC >5,000,000 x 10⁶/L Synovial fluid:

TC – BF# >40,000 x 10⁶/L or RBC >5,000,000 x 10⁶/L or

TC – BF# <40,000 x 10⁶/L with flags

o Dilutions for instrumentation testing are to be performed in duplicate, sample volume permitting.

o Instrument dilution results must check within +/-10%.

o Prepare a second set of dilutions if results do not check. Refer to sections 9.2 Automated Cell Count and 9.4 Dilution Required for calculation instructions.

o If TC – BF# from dilution is >100,000 x 10⁶/L, with or without analyzer flags (and dilutions do not agree within 10%), do not report the

number. Report the TC – BF# with a “See Note” and attach Coded Phrase TCBF.

 ALL results need to be confirmed by a second technologist before verification.

8.2.2.1. FLHH (Available reportable parameters are Hgb, Hct and MCV)

Run body fluid as a CBC. Report Hgb and Hct. MCV is not a mandatory field.

8.2.2.2. Intrauterine Transfusion Samples - FMC only

1. Samples are send from Maternal Fetal Medicine in the pneumatic tube system to the Transfusion Medicine station.

2. TM will pre-order 3 accession numbers as FLHH in Millennium: #1, #2, #3.

3. TM delivers sample(s) to Hematology along with manual requisition and Millenium labels. If more than one sample is received, label based on time of collection of #1, #2, #3.

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4. Run sample STAT, print out result from analyzer. Write on printout #1 or

#2 or #3. Fax instrument printout (attach Millennium label) to Maternal Fetal Medicine department. Phone number and contact information is listed in the Entry Bench binder.

5. Reporting:

a. Modify Collection date and/or time b. ARE – Accession mode

c. Convert fluid source result field to free text. Type “fetal blood”

d. Comment in Result Comment tab – sample # 1 (or #2 or #3) e. Enter Hgb, Hct and MCV result and Platelet count if requested (in

“See Note”) f. Perform results g. Print Screen

h. Check result entries and Verify 8.3. Manual Cell Count Procedure

1 Invert the tube gently to mix thoroughly 2 Perform manual WBC and RBC cell

count based on clarity of specimen

 Refer to section 8.4 Manual Cell Count Procedure - Based on Appearance

 Refer to section 8.5 Microscopic Cell Count Procedure

 Count RBCs and WBCs.

 Note: Manual RBC count not usually done on Synovial fluids unless ordered.

 All cell counts are performed on light/phase microscope under 40X dry objective. Count both sides of chamber.

 If unable to perform cell count due to presence of debris, report “See Note” in Result field. Enter CCNP in Result Comment attached to “See Note”.

3 Flood both sides of counting chamber using a capillary pipette and allow to sit for approximately 2 minutes

Clean the hemocytometer and glass coverslip using lens paper and 95% methanol.

4 Perform appropriate required calculation Refer to section 9 Calculations.

5 Perform differential if ordered by physician

6 After cell count is completed, disinfect counting chamber

Note: If using disposable chamber, discard in a biohazard container.

 Remove coverslip and immerse counting chamber and coverslip in disinfectant using one of the following methods:

o 10% Bleach - soak for 10 minutes o Accel TB - soak for 5 minutes o Cavicide - soak for 3 minutes

 After applicable time, remove the counting chamber and rinse with distilled water, then alcohol.

 Blot chamber and coverslip dry with scratch-free paper and store until next use.

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8.4. Manual Cell Count Procedure - Based on Appearance

Refer to section 8.5 Microscopic Cell Count Procedure.

If Fluid is: Then: Count in Chamber

Visibly Clear RBC: All nine squares

WBC: All nine squares Visibly Cloudy

and/or Bloody (Dilution NOT required)

<200 RBCs or <200 WBCs present in ALL 9 squares (Calculation in section 9.3.1. All Nine Squares Counted)

Count RBCs and/or WBCs in all 9 squares

>200 RBCs or >200 WBCs present in ALL 9 squares (Calculation in section 9.3.2. 5 “R” Squares Counted)

Count RBCs and/or WBCs in 4 corner squares labeled “W”

>200 RBCs or >200 WBCs present in ONE of 9 squares (Calculation in section 9.3.3. 5 “R” Squares Counted)

Count RBCs and/or WBCs in 5 small squares labeled “R”

Visibly Cloudy and/or Bloody (Dilution required)

1. Make appropriate duplicate dilutions of fluid in analyzer diluting fluid. Ensure background count on diluting fluid is performed on hemocytometer before performing manual count.

 DSC – Add 2 drops of New Methylene Blue (retic stain) to 5 mL analyzer diluting fluid

 Suggested dilutions: X50 (100 L fluid in 4.9 mL diluent), X100 (20 L Synovial Fluid in 1.98 mL diluent), X200 (50 L fluid in 9.95 mL diluent).

2. Flood both sides of the counting chamber with diluted specimen using a capillary pipette and allow it to sit for approximately 2 minutes.

3. Count RBCs and WBCs.

4. Perform calculation in section 9.4 Dilution Required using appropriate dilution factor.

Notes:

 If a specimen is grossly bloody, prepare dilutions in dilute 10% acetic acid.

 Check acetic acid solution for contamination (microscopically) before use and document on fluid worksheet (can use urine DeciSlides).

 DO NOT use 10% acetic acid for synovial fluid.

Refer to section 5.1 Reagents.

RBC: All nine squares WBC: All nine squares

8.5. Microscopic Cell Count Procedure

 Refer to Figure 1: Improved Neubauer Hemocytometer

 All cells touching any one of the triple lines at the top or left of the square being evaluated are counted.

 All cells touching any one of the triple lines at the bottom or to the right are excluded from the count.

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Figure 1: Improved Neubauer Hemocytometer

8.6. Fluid Differential Procedure (Perform Only When Ordered)

1 If WBC is ≤200 x 10⁶^/L, report the cell count only

 Report the numerical value of all WBC/TNC ≥ 100.

WBC/TNC <100 will be reported as < 100 x 10⁶/L.

 A differential will not be performed when the WBC count is

≤200 x 10⁶/L.

 In the Neut% field, enter “See Note” and append chartable comment BFDIF.

2 ACH only – If peritoneal dialysate WBC <100 x 10⁶/L do not perform the differential

In the Neut% field, enter “See Note” and append Result Comment PERDIF.

3 Air-dry prepared Cytospin slides then stain using Wright/

Wright-Giemsa

4 Perform a WBC differential on the stained Cytospin

 Assess Cytospin for adequate cell yield, uniform cell

distribution, proper staining, and ready recognition of cell types.

 Count all nucleated cells including mesothelial cells and macrophages if present.

 If number of cells counted is <100, do not report differential as

%. Report only the actual number of cells counted.

 In Total Cells Counted text box, add chartable comment CCD.

Differential Report Notes

Normal cells - normal body fluids with inflammatory cells the Technologist is confident in reporting

 Verify the differential.

 Do not refer the Cytospin to the Hematopathologist.

FMC RRL – Refer Cytospin to Tech II/III

Unable to identify cells due to excessive debris

 Report the differential as “See Note”.

 Append the Result Comment CCNP.

Unable to identify cells due to degenerative changes

 Report the differential as “See Note”.

 Append Result Comment FLUIDEG.

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Differential Report Notes

Unidentified cells or suspicious morphology

 Count the differential; unidentified cells/suspicious morphology are counted as OTHER.

 Append Result Comment (chartable) FLDPRELIM.

 If an urgent result is required, contact LIC at 403-770-3600 for the On Call Hematopathologist/Pathologist contact number.

FMC RRL – Refer Cytospin to Tech II/III

Synovial fluid - DSC BSMH only

Count cells as neutrophils, eosinophils and mononuclear cells.

 Mononuclear cells: include all cells seen that are not neutrophils, except eosinophils, which should be

counted and reported separately.

 If a dilution was used for the cell count, use a dilution for the Cytospin preparations as well. Dilution must be in saline only.

BAL

- FMC only

 Report the differential, if able.

 Refer Cytospin to Tech II/III.

 Cytospin is forwarded on to Hematopathologist.

 Any cells you cannot identify (i.e.

non-hematopoietic), do not include in differential.

 Do not count as OTHER.

9. CALCULATIONS

9.1. Basic Equation for Cell Count on Improved Neubauer Hemocytometer WBC x 10⁹/L = n x Depth x Dilution Factor

÷ Unit conversion factor Area

n = average number of cells counted Depth (mm) = 10

Area (mm²) = 9

Unit conversion factor = 1000

To report the WBC value to a whole number, round up if the number in the first decimal place is 5 or greater; round down if the number in the first decimal place is 4 or lower.

Example: 88.6 is reported as 89; 88.4 is reported as 88.

9.2. Automated Cell Count

WBC and RBC Reporting units: x 10⁶/L

If a dilution is required, instrument counts must be multiplied by the appropriate dilution factor before reporting.

NB: Prepare 2 dilutions of the sample and run each through the analyzer. Take the average prior to multiplying by the dilution factor. See example in section 9.4 Dilution Required.

9.3. Manual Cell Count

 The following equations are used to calculate both RBC counts and WBC counts.

 Count RBCs and WBCs on both sides of chamber.

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9.3.1. All Nine Squares Counted (Entire Chamber)

Equation Example

WBC/RBC x 10⁶/L = Side 1 + Side 2

= Side 1 + Side 2

2(0.9) 1.8

Applies to WBC or RBC

# Cells counted Side 1 = 144

# Cells counted Side 2 = 148 Cell count x 10⁶/L = 144 + 148

2 (0.9)

= 162 x 10⁶/L 9.3.2. 4 “W” Squares Counted

 The cell count is obtained by counting the number of WBCs or RBCs in the 4 large corner squares of the counting chamber (labeled W in Figure 1: Improved

Neubauer Hemocytometer).

 Area counted = 0.4 mm³= total area of 4 large squares ([4 x (1 x 1 x 0.1)]).

= Side 1 + Side 2

2(0.4) 0.8

# Cells counted Side 1 = 44

# Cells Counted Side 2 = 46 Cell count x 10⁶/L = 44 + 46

2(0.4)

= 113 x 10⁶/L 9.3.3. 5 “R” Squares Counted

 The RBC or WBC count is obtained by counting the number of cells in 5 small squares in the center square of the counting chamber (labeled R in Figure 1:

Improved Neubauer Hemocytometer).

 Area counted = 0.02 = 2 x (total area of 5 small squares on one side of counting chamber) 0.02 mm = [5 x (0.2 x 0.2 x 0.1)]

= Side 1 + Side 2

2(0.02) 0.04

# Cells counted Side 1 = 44

# Cells counted Side 2 = 46 Cell count x 10⁶/L = 44 + 46

2(0.02)

= 2250 x 10⁶/L 9.4. Dilution Required

WBC = TNC = TC – BF#

Analyze the dilution. Multiply the result of the WBC/RBC by the dilution factor to obtain the final result.

Example: WBC result = 6000 x 10⁶/L (Average of the 2 dilutions) Dilution of x 2

WBC final result = 6000 x 2 = 12,000 x 10⁶/L

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10. REPORTING RESULTS

Reference Ranges Body fluid: No ranges given

Synovial Fluid: WBC 0-200 x 10⁶/L Abnormal Results Criteria for referral to Pathologist:

 Increase in any non-hematological cell population (e.g. mesothelial cells, atypical mononuclear cells, tumor cells, macrophages, etc.)

 Increase in any immature hematological cell population.

 Unable to identify cells.

 Presence of crystals, either intracellular or extracellular.

 Refer to section 10.2 Referral of Body Fluids.

Reportable Results  Upper reportable value (automated count):

o Refer to specific analyzer method for upper linear limit RBC and WBC

 Lower reportable value (automated):

o RBC <3000 x 10⁶/L o WBC <100 x 10⁶/L

 Manual Count: Report the value counted. Covert result to free text and enter a numerical result.

Downtime Reporting Refer to:

 GL04-7.13 Miscellaneous Hematology Downtime Workflow and Reporting DSC FMC RRL

 Temporary Report - Coagulation and Miscellaneous Hematology HE4050

Coded Phrase Expandable Phrase

BFDIF Differential not performed on WBC less than or equal to 200 x 10⁶/L.

CCD Only __ cells counted. The reported % may not be represented.

CCNP Cell count not performed due to the presence of debris.

CNTR Specimen received in a container other than EDTA/Na Heparin. The results may be compromised and should be interpreted in the context of the patient’s clinical condition.

CPWBCC Clumps present. WBC count may have been compromised.

DPWBCC Debris present. WBC count may have been compromised.

FLUIDEG Unable to perform fluid differential due to degenerative changes in the cellular elements.

FLMICRO Micro-organisms present. Correlate to Microbiology report for definitive diagnosis.

FLDPRELIM

Preliminary report. Differential referred to the Hematopathologist/Pathologist for

morphological assessment. Final report to follow. If an urgent result is required contact LIC at 403-770-3600 for the on call Hematopathologist/Pathologist contact #.

PERDIF Differential not performed on peritoneal dialysate when WBC less than 100 x 10⁶/L.

TCBF Fluid WBC >100,000 x 10⁶/L. WBC differential count is precluded by degenerative changes. Recommend follow-up of gram stain and bacterial culture with Microbiology.

UNIDFLD Unidentified cells present in fluid. Classification and differential to follow after review by Hematopathologist/Pathologist.

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10.1. Reporting Format

Volume Report approximate volume of fluid in mL.

Appearance Report appearance as:

 Color: colorless, yellow, slightly bloody, moderately bloody, or markedly bloody.

 Clarity: clear, slightly cloudy, moderately cloudy, or markedly cloudy.

 Small clots (less than 1/2 of specimen volume) and/or fibrin strands present: “Small clots present”, or “Fibrin strands present”.

 Large clot (greater than 1/2 of specimen volume) present: “Fluid clotted.

Unable to do cell count”.

 Solid clot present: “Fluid clotted. Unable to do cell count”. Differential is reported as “Clotted”.

WBC Count  WBC count is reported up to 10 digits, no decimal place; reporting unit is x10⁶/L.

 If WBC count is less than 100, report as <100 x 10⁶/L.

 IF WBC clumps and/or debris are present in the wet mount, report WBC count with the following comments:

o CPWBCC for clumps present o DPWBCC for debris present

RBC Count  DSC BMSH: Report Synovial fluid cell count as N/A.

 RBC count is reported up to 10 digits, no decimal place; reporting unit is x10⁶/L.

 If RBC count is less than 3000, report as 3000 x 10⁶/L.

WBC Differential  WBC differential is reported as a percentage (%).

 DSC BMSH: Report only neutrophils, eosinophils, and mononuclear cells.

 If 100 cells counted for WBC differential, do not report as a percentage. Enter the actual number of cells counted (refer to section 7.6.1 Fluid Differential, step #4).

 In the Total Cell Counted text box, add the Result Comment (chartable) CCD.

 If WBC is <200 x 10⁶^/L, report the cell count only, with the exception of BAL.

Report the numerical value of all WBC/TNC ≥100.

 In the Neut% field, enter “See Note” and append the chartable comment BFDIF.

 Always perform and verify a differential on BAL sample regardless of WBC count.

 ACH only – Peritoneal dialysate - If WBC less than 100 x 10⁶/L, report the cell count only. In the Neut% field, enter “See Note” and append Result Comment PERDIF.

Specimen received in a container other than EDTA/

Na Heparin

Report Results: Process according to the procedure and append the chartable Result Comment CNTR.

10.2. Referral of Body Fluids

Cytospin slides are referred to senior technologist as per individual site protocol. All abnormal slides are then referred to a pathologist.

Abnormalities present in fluid

 When performing the fluid differential and abnormalities are noted (refer to section 10.1 Reporting Format), select Yes from the Fluid Referral drop down and Verify the result.

 Leave the smear for the senior technologist review.

Create a Fluid Referral Worklist

 Performed by senior technologist after reviewing the smear and abnormalities are confirmed.

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Send referred fluids for

Pathologist review

 Pack slides in a slide box in the same order as the Body Fluid Referral worklist.

 Send worklist, a chart print for each patient on the list, and slides to the Pathologist performing PBS duties according to the Rota.

 Once the Pathologist verifies the fluid interpretation in ARE, the .FLD REF will autocomplete.

 When the slides are returned to your site, ensure all slides on the worklist are returned and retain according to GL01-7.01_Attachment 1 Records Materials Retention.

11. ADDITIONAL INFORMATION

 Synovial fluids:

o Normal synovial fluid is light yellow and clear; it does not clot. The color of synovial fluid becomes a deeper yellow in the presence of inflammation and may have a greenish tinge with bacterial infection. Turbidity occurs when the cell count is elevated and is usually proportional to the number of cells present. However, a milky fluid may also indicate the presence of crystals³. o Abnormal synovial fluid does clot and must be collected with heparin in the syringe or must be put

directly in an EDTA or heparin tube immediately after collection.

o Hyaluronidase is a powerful protein-digesting agent. When adding to synovial fluid, use minimally (only a few crystals).

o Rice bodies are whitish flakes suspended in synovial fluid. These flakes are derived from degenerating proliferating synovial lining cells and may be mistaken for small clots¹. Primary Cells Seen in Normal Synovial Fluid³

Cell/Inclusion Description Significance

Neutrophil Polymorphonuclear leukocyte  Bacterial sepsis

 Crystal induced inflammation Lymphocyte Mononuclear leukocyte  Non-septic inflammation Macrophage

(monocyte)

Large mononuclear leukocyte, may be vacuolated

 Normal

 Viral infections Synovial lining cell Similar to macrophage but may be

multinucleated, resembling a mesothelial cell

 Normal

Neutrophils may contain vacuoles, fat droplets, bacteria, or crystals. It is common for the nuclei to show pyknosis and karyorrhexis.¹

12. REFERENCES

1. Kjeldsberg CR, Knight JA. Body Fluids. Laboratory Examination of Cerebrospinal, Seminal, Serous

& Synovial Fluids: A Textbook Atlas. 3rd Edition. ASCP; 1993. p. 129-136, 167-169.

2. Steine-Martin EA, Lotspeich-Steininger CA, Koepke JA. Clinical Hematology Principles, Procedures, Correlations. Philadelphia: JB Lippincott Company; 1998. p. 400-414.

3. Strasinger SK. Urinalysis and Body Fluids. 3^rd ed. Philadelphia: FA Davis Company; 1994. p. 165- 171.

4. MTS University of Washington, Department of Laboratory Medicine. CD-ROM Lab Training Library.

Body Fluids.

5. Gatter RA, Schumacher. A Practical Handbook of Joint Fluid Analysis. Philadelphia: Lea & Febinger;

1991. p. 32.

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6. Ringsrud KM, Urinalysis and body fluids: A Color Text and Atlas. 1st Ed. St Louis: Mosby; 1995. p.

182-191.

7. CLSI. Body fluid analysis for cellular composition; Approved Guideline. CLSI document H-56A [ISBN1-56238-614-X]. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute; 2008.

8. College of American Pathologists. 2017 Hematology and Clinical Microscopy Glossary.

9. DxH Coulter Body Fluid Control Package Insert.

10. Sysmex XN BF Check Package Insert and Assay Values Sheet.

11. CAP Standard – HEM.35340 Body Fluid Cell Counting.

13. RELATED DOCUMENTS

GL01-7.01 Retention Schedule Attachment 1 Records Materials Retention

GL04-7.13 Miscellaneous Hematology Downtime Workflow and Reporting DSC FMC RRL HE-32.04 Cytospin Preparation

Manual Fluid Worksheet HE4032

Peritoneal Dialysate Worksheet ACH HE4031

Temporary Report - Coagulation and Miscellaneous Hematology HE4050