Hemocytometer

A hemocytometer is a transparent glass chamber that holds a cell suspension for microscopic cell counting. The hemocytometer is 0.1 mm deep and is divided into subunits by a grid with a precise 3-mm by 3-mm surface area (Figure 2-2). The surface area of the grid used in a procedure thus determines volume of the hemocytometer containing the cells counted. One can mathematically determine the number of cells in a cubic millimeter (i.e., mm³; see Figure 2-2) based on this volume. One should understand these calculations so that the hemocytometer can be used to count cells in other fluids such as cerebrospinal and synovial fluid. In the United States, cell numbers are reported in cells/µl, which equals cells/mm³. This was likely because originally cells were counted in a hemocytometer designed to represent portions as mm³. Most other countries report hematologic cell concentrations in cells × 10⁹/L. A liter contains 1 million (10⁶) µl; therefore a liter would have a million times more cells than 1 µl (see Chapter 1).

FIGURE 2-2 Dimensions of a hemocytometer. The hemocytometer grid consists of 9 mm² and it is 0.1 mm deep. The central 1 mm² is additionally divided into 25 squares. The areas usually used for erythrocyte, leukocyte, and platelet counts are indicated by R, WBC, and PLT, respectively. To determine the cells in 1 mm³ (which is equal to 1 µl), conversion factors for RBCs, WBCs, and platelets are listed in Table 2-2.

The blood (or other fluid) must be diluted so that the number of cells in the chamber is easy to count. A dilution system with plastic containers is easiest. Becton Dickinson (BD) discontinued the Unopette System and now recommends, as an alternative, similar products made by Bioanalytic GmbH (www.bioanalytic.de). The amount of dilution needed to provide a reasonable concentration of cells in the chamber varies with expected concentration of cells in blood (or other fluids such as abdominal fluid). Erythrocytes in blood are very numerous, so blood is greatly diluted (e.g., 1 : 200). Note that manual RBC counts are so inaccurate and time consuming that a manual RBC count is not recommended. A microhematocrit is more practical if one does not have an automated hematology instrument. A microhematocrit is even useful for bloody fluids such as abdominal fluid to estimate the amount of hemorrhage. WBC dilution is usually 1 : 20 and platelets are diluted 1 : 100. The reciprocal of the dilution (i.e., 1/dilution) is used in calculation of the final conversion factor (Table 2-2).

TABLE 2-2 HEMOCYTOMETER CONVERSION FACTORS

The portion of grid in which cells are counted varies for each cell type and even type of counting chamber. For WBC counts in the United States, the outer four squares are counted (see Figure 2-2). For platelets, two squares (2 mm²) are counted. The reciprocal of the area is used in calculating the final conversion factor. Because the hemocytometer is only 0.1 mm deep, counting 1 mm² represents one tenth of 1 mm³.

To convert cell counts to number/mm³, cell count must be adjusted for the portion of 1 mm³ counted and the dilution of the blood sample (see Table 2-2). The WBC factor of 50 is based on adjustments for depth of the hemocytometer (0.1 mm), area counted (4 mm²), and dilution factor (1 : 20). A different factor can be determined if one chooses to vary from the standard approach.

Hemocytometers have two counting chambers (one on each side). Both chambers should be filled and counted. The number of cells counted from each side should vary by less than 10% for WBC counts. If the cell suspension was not evenly distributed, as indicated by greater variation, the test should be repeated.

Manual counts with a hemocytometer have significant error (e.g., 20% error may occur with WBC counts). This magnitude of error should be considered when interpreting day-to-day changes in WBC counts in animals. For example, a count of 2100 WBCs/µl varies too little from a count of 1900 WBCs/µl the previous day to be considered an improvement. Despite imprecision, manual counts are adequate for most clinical diagnoses for clinicians who do not have need of or access to an automated hematology instrument. Manual counts may be performed when errors in automated counts are suspected.

Impedance Counters

The impedance principle was the standard method of cell counting in hematologic instruments and remains a common method in many current instruments. Using the impedance counting principle (i.e., Coulter principle), cells are diluted in an electrolyte solution and drawn through an aperture (hole in an electrode). The electrical resistance across the aperture changes as a pulse with each cell because cells are poor conductors of electricity. The frequency of the change in resistance indicates cell number, and the magnitude of the change in resistance indicates cell size. Impedance counters must be electronically adjusted to count only cells within an appropriate size interval. This size interval is set by adjusting electronic thresholds for each species. Impedance counters directly count cell numbers, measure cell size, and then mathematically calculate the Hct, MCHC, and MCH.

Platelet-erythrocyte histograms should be inspected for adequate separation between the platelet and erythrocyte peaks to avoid errors. The histogram should show two peaks and indicate which cell types were counted as platelets or RBCs by the impedance instrument (Figure 2-3). There must be a clear separation “valley” between the RBC and platelet peaks. This valley and a dashed line correctly placed between RBC and platelet peaks assures that the counts were accurate. A frequent error is that there is overlap in the size of platelets and RBCs so that the instrument cannot correctly determine the division between them.

FIGURE 2-3 Two examples of red blood cell (RBC) platelet and platelet (PLT) histograms from an impedance platelet and RBC count from a Cell-Dyn instrument. The upper histograms are from a normal dog. In the upper right histogram, two distinct peaks are seen, a smaller orange platelet peak and a large red RBC peak. The orange platelet peak is repeated on the left as only a PLT histogram, which shows distribution of platelets by size. When the platelets and RBCs are distinctly different in size, an impedance instrument can differentiate and count them accurately. The lower histograms are from a dog with iron deficiency anemia. There are three populations (peaks) of cells in the right histogram, and the abnormal middle peak represents microcytic RBCs. Because there was no clear separation of platelets from RBCs (no valley between two peaks), the instrument could not correctly distinguish between two cell types and drew the border right through the middle of the microcytic RBCs. Part of the microcytic RBCs were counted as platelets (orange) and part as RBCs (red). Thus the platelet count was in error. Usually the relative error with an RBC count is minor because there are a great many more RBCs than platelets, but in a case of severe anemia and strong thrombocytosis, even the RBC count can be prominently in error.

Impedance counters count the number of RBCs and determine the size of each RBC in that sample and then calculate a hematocrit (Hct) essentially by multiplying the number of RBCs by their size (e.g., RBCs × MCV ÷ 10) or by a summation of individual RBC sizes. This is unlike the classic method of centrifugation of blood to pack RBCs and determine a PCV. Therefore instrument counts are better called a hematocrit (Hct) instead of packed cell volume (PCV). For simplicity, PCV is usually used in this book to indicate Hct or PCV. PCV by the microhematocrit method is a good quality control addition to performing a CBC with a hematology instrument. If the variation between the microhematocrit’s PCV and the instrument’s Hct is greater than 3% to 5%, this suggests a technical error, which should be clarified.

Newer impedance instruments provide additional information, such as three to four cell differential leukocyte counts, mean platelet volume (MPV), platelet distribution width (PDW), and reticulocyte count, and they may identify the presence of NRBCs.⁴

Laser Light Scatter Cell Counters

Newer automated hematology analyzers often use a laser detection system in a flow cytometer to measure size and internal complexity of cells based on light scatter at different angles.⁷ Stains are added to help differentiate types of leukocytes or reticulocytes. Two veterinary instruments designed for large referral laboratories are the Advia 120/2120 (previously the H-1; Siemens Medical Solutions) and the Sysmex XT-2000iV (Sysmex Corporation, Kobe, Japan). The Sysmex reports both laser (optical; PLT-O) and impedance (PLT-I) platelet counts. These newer instruments provide abundant information about each cell type. The RDW numerically describes the variability in RBC size (anisocytosis), and the hemoglobin distribution width (HDW) describes variability in Hgb concentration (see Chapter 3). Tvedten prefers interpreting the morphology of the graphics rather than many numerical results such as RDW, HDW, and several others.

Graphic displays of cells aid in diagnosis and detection of laboratory errors.¹³ The Advia measures and reports the volume and Hgb content of each erythrocyte analyzed (Figures 2-4 and 2-5). The Advia system is the most sensitive and specific way to correctly classify anemia as normocytic normochromic, macrocytic hypochromic, or microcytic hypochromic. As described in RBC indices, the use of MCV and MCHC more often gives an incorrect classification than the true type of anemia. RBC cytograms illustrate normal and abnormal RBC populations based on cell size and Hgb concentration. The Advia 120/2120 automated reticulocyte analysis provides not only the absolute and relative reticulocyte counts but even mean reticulocyte volume, which may aid in judging the response of a dog with iron deficiency to treatment. The color-coded reticulocyte graphics in Figure 2-5 show at a glance that the anemia in the dog was clearly regenerative. Advia 120/2120 and Sysmex XT-2000iV reticulocyte analysis works well for canine reticulocytes. The Advia and Sysmex instruments detect mainly the aggregate-type feline reticulocyte but do not detect even great changes in punctate reticulocytes.

FIGURE 2-4 The Advia 2120 instrument graphics best illustrate the three basic types of anemia, which are normocytic normochromic anemia (A), macrocytic hypochromic anemia (B), and microcytic hypochromic anemia (C). The graphics include a red blood cell (RBC) cytogram, which is a nine-box grid, in the top row. The RBC cytogram displays each individual erythrocyte analyzed based on that cell’s volume (V) on the vertical axis and that cell’s Hgb concentration (HC) on the horizontal axis. There are two histograms under this nine-box grid. The RBC volume histogram is equivalent to the vertical axis of the cytogram and shows the number of RBCs based on volume. The two vertical lines reflect upper and lower reference limits. The Hgb histogram is equivalent to the horizontal axis of the cytogram and shows the number of RBCs based on cell hemoglobin concentration. A, The dog with normocytic normochromic anemia had all RBCs within the central box or vertical lines on the histograms, as would a normal nonanemic dog. B, The dog with macrocytic hypochromic anemia was an IMHA case with many immature (macrocytic hypochromic) RBCs that extended up and to the left from the central box on the cytogram. Extension of cells to the right on the RBC volume histogram reflect that they were macrocytic. These RBCs also extended to the left on the cytogram and Hgb histogram, indicating they were hypochromic. Unique to the Advia 2120 is the display of hypochromic RBCs. The Hgb histogram shows two peaks, and the area “under the curve” reflects the relative number (%) of RBCs in the left peak, which were hypochromic. In the IMHA case, there were more hypochromic (left peak) than normochromic (right peak) RBCs. Thus at a glance one can determine the magnitude of a regenerative response of immature RBCs (usually reticulocytes). Additionally, a cluster of RBCs in the upper central box of the cytogram were RBCs in autoagglutination. C, This dog had a microcytic hypochromic anemia due to iron deficiency that was so extreme that there were too few normal normocytic normochromic RBCs remaining to show where normal cells should be found. The straight lines at the bottom and left of the cells in the cytogram and a line on the left of the two histograms indicate that the Advia 2120 did not believe canine RBCs could be small or hypochromic and excluded them from analysis (truncated).

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