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.