Hematological analyses
Red blood cell (RBC) counts were measured using an impedance counter with a floating threshold by a CellTac analyzer (model MEK 5108K, Nihon Kohden Corporation, Tokyo, Japan). Hemoglobin (Hb) was measured photometrically with the HemoCue analyzer (HemoCue, Prospect Diagnostics, Derbyshire, UK) with microcuvettes preloaded with reagent. Packed cell volume (PCV) was obtained with a hematocentrifuge by using plain capillary tubes sealed with Critoseal (Krackeler Scientific Inc., NY, USA). Samples were centrifuged at 10,000 g for 5 min and read manually with a hematocrit reader. White blood cell (WBC) counts were determined manually by mixing 0.38 ml of 1 % ammonium oxalate with 0.02 ml of EDTA blood and placing this on a rotor for approximately 5 min. The resultant mixture was used to fill the counting chamber of an improved Neubauer hemocytometer and allowed to stand for 5 min in a moist box to allow the cells to settle. Counts were performed with phase contrast microscopy with the 340/0.65 phase objective. A total of 4 large squares (64 small squares) was counted, and the total number was divided by 20 to obtain WBC X 109/L. Blood smears were stained with May-Grunwald-Giemsa. The differential count was based on 100 counted leucocytes. At the same time, thrombocytes were counted manually. For all samples, thrombocytes were estimated to be adequate and none were assessed as having a thrombocytopenia, and so were not considered further.