Color Atlas of Hematology - Practical Microscopic and Clinical ...

14Physiology and Pathophysiology of Blood CellsLeukocyte CountLeukocytes, unlike erythrocytes, are completely colorless in their nativestate. Another important physical difference is the stability of leukocytesin 3% acetic acid or saponins; these media hemolyze erythrocytes (thoughnot their nucleated precursors). Türk’s solution, used in most countingmethods, employs glacial acetic acid for hemolysis and crystal violet (gentianviolet) to lightly stain the leukocytes. A 50-µl EDTA blood sample ismixed with 500 µl Türk’s solution in an Eppendorf tube and incubated atroom temperature for 10 minutes. The suspension is again mixed andcarefully transferred to the well of a prepared counting chamber using apipette or capillary tube. The chamber is allowed to fill from a dropletplaced at one edge of the well and placed in a moisture-saturated incubatorfor 10 minutes. With the condenser lowered (or using phase contrastmicroscopy), the leukocytes are then counted in a total of four of the largesquares opposite to each other (1 mm 2 each). The result is multiplied by27.5 (dilution: 1 + 10, volume: 0.4 mm 3 ) to yield the leukocyte count permicroliter. Parallel (control) counts show variation of up to 15%. The normal(reference) ranges are given in Table 2.In an automated blood cell counter, the erythrocytes are lysed and cellswith a volume that exceeds about 30 fl (threshold values vary for differentinstruments) are counted as leukocytes. Any remaining erythroblasts,hard-to-lyse erythrocytes such as target cells, giant thrombocytes, or agglutinatedthrombocytes are counted along with the leukocytes, and thiswill lead to an overestimate of the leukocyte count. Modern analyzers canrecognize such interference factors and apply interference algorithms toobtain a corrected leukocyte count.Visual leukocyte counts using a counting chamber show a variance ofabout 10%; they can be used as a control reference for automatic cellcounts. Rough estimates can also be made by visual assessment of bloodsmears: a 40 objective will show an average of two to three cells per fieldof view if the leukocyte count is normal.