Body Fluid Analysis:: Manual Hemacytometer Counts and Differential Slide Preparation

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Body Fluid Analysis:

Manual Hemacytometer Counts and Differential Slide Preparation

Learning Objectives

Using A Hemacytometer

Manual methods using a hemacytometer are often used to perform cell counts on body fluids such as cerebrospinal fluid (CSF), synovial fluid, pleural fluid, pericardial fluid, and peritoneal fluid, as well as peritoneal dialysates, bronchoalveolar lavages, and semen. In health, the numbers of red blood cells (RBCs) and white blood cells (WBCs) in these body fluids are low, and other cells or cellular debris can be present. As discussed in Chapter 16, automated cell counting analyzers can produce erroneous results when the cell count is low. It is the responsibility of each laboratory to define its lower limit for cell counts (RBCs and WBCs) and to have a protocol for performing manual cell counts using a hemacytometer when cell counts are below the laboratory-defined lower limit.1

Highly viscous body fluids (e.g., synovial fluid) and fluids that fail to appropriately liquefy (e.g., semen) require pretreatment before cell counting by manual or automated methods. Note that cell counts using a clotted body fluid are inaccurate. Because it may not be possible to obtain another body fluid specimen, every effort is made to work with the healthcare provider to provide valid, useful information. This may include performing a cell count and including on the report a statement such as, “Specimen clotted; cell counts must be interpreted with caution.”

Manual cell counts using a hemacytometer are time-consuming, require advanced technical skills, have poor precision (reproducibility), and are subject to numerous errors as a result of the multiple steps involved. Therefore it is imperative that well-trained and technically proficient laboratorians perform them and that appropriate materials are used to verify the achievement of quality goals.

Diluents and Dilutions

The visual appearance of the body fluid aids in determining whether a dilution should be made for cell counting and what dilution should be prepared. Body fluids that are clear do not require a dilution, and the fluid can be loaded directly onto a hemacytometer. Fluids that are visibly cloudy or bloody must be diluted to obtain accurate cell counts. Table 17.1 is provided as a guide to dilution selection based on visual appearance. When diluents that do not lyse RBCs are used, a higher dilution may be necessary. Body fluids that are visibly clear indicate a low cell count and are evaluated undiluted; sometimes cell counts in hazy or slightly cloudy fluids can also be performed on the undiluted fluid. To enhance visualization of WBCs in fluids other than synovial fluid, the fluid can be “exposed” to glacial acetic acid (Box 17.1). Blood-tinged or bloody fluids can be diluted using diluents that (1) lyse RBCs and (2) enhance visualization of nucleated cells.

Table 17.1

Body Fluid Dilution Guidelines for Cell Counts Based on Visual Appearance
Fluid Appearance WBC Count RBC Count
Clear Undiluted Undiluted
Hazy (slightly cloudy) 1:2a dilution Undiluted
Blood-tinged 1:2a dilution Undiluted
Cloudy 1:20 dilution Undiluted
Bloody 1:2a or 1:20 dilution 1:200 dilution

RBC, Red blood cell; WBC, white blood cell.

aUsing a diluent that lyses RBCs.

Often isotonic, particle-free commercial diluents used by the laboratory’s hematology analyzer can be used to dilute body fluids (e.g., Cellpack, Sysmex Corporation, Kobe, Japan). Laboratory-prepared isotonic solutions such as normal saline (0.85%) can also be used for RBC counts, whereas dilute acetic acid solutions are often used for the nucleated cell and WBC counts. Acetic acid in the diluent performs two functions: It lyses any RBCs present and it enhances the nuclei of WBCs. Table 17.2 summarizes diluents commonly used for body fluids when WBC and RBC counts are performed. Appendix D provides details on the preparation of these diluents.

Table 17.2

Diluents for Body Fluid Blood Cell Countsa
Diluent Cell Counts Comments
Commercial isotonic diluents
Diluent used in hematology analyzers for cell counting
Isotonic saline (0.85%)
Also known as “normal” saline
Hypotonic saline (0.30%) WBC count •Lyses RBCs
Dilute acetic acid (3.0%) WBC count
Turk’s solution WBC count
Hyaluronidase (0.1 g/L) buffer solution



RBC, Red blood cell; WBC, white blood cell.

aSee Appendix D for diluent preparation.

The diluent used depends on the body fluid being evaluated. Note that synovial fluid cannot be diluted using weak acid diluents, such as acetic acid. Because of the high hyaluronic acid and protein content of synovial fluid, acetic acid will cause a mucin clot (i.e., the coprecipitation of hyaluronic acid and protein), which interferes with accurate cell counting. Instead, synovial fluid is diluted using a commercial isotonic diluent, normal saline (0.85%), hypotonic saline (0.30%), or a hyaluronidase buffer solution. A hyaluronidase buffer solution prevents mucin clots, and when toluidine blue stain is included, it aids in the visualization and identification of cellular elements. When WBC counts are performed on synovial fluid and hypotonic saline is used as the diluent, any RBCs present are lysed and mucin formation is not initiated.

To obtain accurate cell counts, dilutions of body fluids must be made using a quantitative technique. Calibrated automatic pipettes (e.g., Pipetman, Eppendorf, Drummond, MLA) are used to prepare these dilutions manually; commercial diluting systems (e.g., Unopettes) are not available in most locations. Note that when viscous fluids, such as synovial fluid and semen, are pipetted, a positive displacement pipette must be used because an air displacement pipette cannot accurately dispense these viscous fluids. In contrast, CSF, pleural, pericardial, and peritoneal fluids and pretreated synovial fluid can be diluted using either an air or a positive displacement pipette.

Pretreatment and Dilution of Synovial Fluid Specimens

When synovial fluid is evaluated without pretreatment, the viscosity of the fluid can cause an uneven distribution of cells in the hemacytometer. Also when preparing dilutions of untreated synovial fluid, a positive displacement pipette is required to accurately prepare dilutions of the fluid. An alternative is to pretreat synovial fluid using the enzyme hyaluronidase. This enzyme eliminates the fluids’ viscosity by depolymerizing hyaluronic acid, which will also prevent mucin clot formation. Two pretreatment approaches using hyaluronidase are provided in Appendix D. Basically, hyaluronidase is added to an aliquot of synovial fluid, which is mixed well. To enhance depolymerization the sample can be briefly incubated at 37°C. Note that pretreatment with hyaluronidase does not affect crystals that can be present in synovial fluid. Additionally, some excessively viscous synovial fluids may need to be pretreated with hyaluronidase despite the use of a hyaluronidase buffer solution as the diluent.

If pretreated synovial fluid is clear, it can be evaluated undiluted for cell counts. When a dilution is needed, a diluent that will not cause a mucin clot is required, such as a commercial or laboratory-made isotonic diluent, hypotonic saline, or a 0.1-g/L hyaluronidase buffer solution (see Appendix D for diluent preparation).

Semen Dilution and Pretreatment of Specimens

As with synovial fluid specimens, semen is viscous even after liquefaction, and positive displacement pipettes are required to accurately prepare dilutions.2 Often, the diluent used to dilute semen for sperm counts is a solution of sodium bicarbonate, formalin (a fixative), and, optionally, a stain—trypan blue or gentian violet (see Appendix D for diluent preparation). Including a stain enhances visualization, which assists in differentiating among sperm, immature sperm (spermatids, spermatocytes), and WBCs—primarily neutrophils, monocytes, and macrophages.

Semen specimens that fail to liquefy adequately after 60 minutes require treatment before sperm count, sperm motility assessment, and chemical testing can be performed. One treatment approach involves diluting the seminal fluid using an isotonic medium followed by mechanical mixing—repeated aspiration and dispensing of the mixture using a pipette. Equal parts of semen and a medium such as Dulbecco’s phosphate-buffered saline can be used.2 An alternate approach consists of digestion using the proteolytic enzyme bromelain. Semen is diluted 1:2 using this enzyme solution (i.e., 1 part semen+1 part bromelain solution). Note that any dilutions of the sample must be accounted for when the sperm concentration is calculated.

The effects that these treatments have on sperm function, morphology, or the biochemistry of the seminal plasma are not known.2 Therefore when a semen specimen is specially treated for testing, this must be documented on the report. Note that any laboratory analyzing semen for any purpose other than postvasectomy analysis should have available the WHO Laboratory Manual for the Examination and Processing of Human Semen.2 This comprehensive and indispensable text is a vital resource for all aspects of testing when semen analysis is performed.

Hemacytometer Cell Counts

The total nucleated cell or WBC count is an important count that is requested on almost all body fluids. In contrast, little clinical value is derived from an RBC count other than to identify a traumatic puncture procedure, so it may not be performed, particularly when counts are done manually. Box 17.2 summarizes a protocol for a manual cell count using an “improved” Neubauer hemacytometer (Fig. 17.1).

Box 17.2

Manual Cell Count Using a Neubauer Hemacytometer

  1. 1. Using a disposable pipette, fill both sides of a standard or disposable “improved” Neubauer hemacytometer (Fig. 17.1) with well-mixed undiluted or appropriately diluted body fluid.
  2. 2. Allow the chamber to remain undisturbed for 3 to 5  minutes for the cells to settle (and red blood cells to lyse, depending on the diluent used).
  3. 3. Examine the hemacytometer chambers for an even distribution of cells without overlap or clumping. If overlapping or clumping is present, the specimen needs to be recharged. Mix the specimen well or possibly prepare a dilution of the fluid. Clean the hemacytometer or use a new disposable hemacytometer and fill the chambers; examine for even distribution.
  4. 4. Cerebrospinal fluid, synovial, pleural, pericardial, and peritoneal fluids

    1. a. If less than an estimated 200 cells are present in all 9 squares1:

      1. i. Count cells in all 9 large squares in both chambers of the hemacytometer.
      2. ii. Area counted: 9 × 1 mm2 = 9 mm2 on each side = 18 mm2.

    2. b. If more than an estimated 200 cells are present in all 9 squares1:

      1. i. Count cells in the 4 large corner squares (the “W” squares) in both chambers of the hemacytometer. See the “W” squares in Fig. 17.1.
      2. ii. Area counted: 4 × 1 mm2 = 4 mm2 on each side = 8 mm2.

    3. c. If more than an estimated 200 cells are present in 1 large square1:

      1. i. Count cells in five red blood cell squares (i.e., the 4 corner squares and the center square within the central large square on both sides of the hemacytometer). See the “R” squares in Fig. 17.1.
      2. ii. Area counted: 5 × 0.04 mm2 = 0.20 mm2 on each side = 0.40 mm2.

  5. 5. Semen

    1. a. Spermatozoa concentration

      1. i. Count sperm present in five red blood cell squares (i.e., the four corner squares and the center square within the central large square on both sides of the hemacytometer). See the “R” squares in Fig. 17.1. (An alternate approach is to count two large “W” squares.)
      2. ii. Area counted: 5 × 0.04 mm2 = 0.20 mm2 on each side = 0.40 mm2

    2. b. Round cell count

      1. i. For the “round cell” (germ cells and white blood cells) count, count the round cells in the four large corner squares and the center large square (the “W” squares) in both chambers of the hemacytometer (see Fig. 17.1).
      2. ii. Area counted: 5 × 1 mm2 = 5 mm2 on each side = 10 mm2.

  6. 6. The number of cells counted in each chamber of the hemacytometer must agree within a percentage or absolute cell number. If counts from both sides do not agree, the cell count procedure must be repeated. Note that each laboratory establishes the acceptable precision criteria required between counts on each side of the hemacytometer (e.g., the number of cells counted in each chamber must agree within 20% or ±8 cells, whichever is greater).

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Oct 18, 2022 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Body Fluid Analysis:: Manual Hemacytometer Counts and Differential Slide Preparation

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