Disodium EDTA used as anticoagulant causes hemolysis in common carp blood

Malgorzata WITESKA, Wioleta WARGOCKA Department of Animal Physiology, University of Podlasie, Prusa 12, 08-110 Siedlce – POLAND

Nur Wahidah


Blood for diagnostic analyses is usually treated with anticoagulants to prevent clotting. Heparin is a natural endogenous anticoagulant agent acting both in blood vessels in vivo, and ex vivo when added to a blood sample. Heparin inhibits conversion of prothrombin into active thrombin, and thus prevents conversion of fibrinogen into fibrin. The mechanism of EDTA anticoagulant action is based on inhibition of thrombocyte aggregation and various reactions of hemostatic cascade due to chelation of free Ca2+ ions.

Materials and methods

Blood of 10 healthy 18-month-old common carp (Cyprinus carpio L.) of body mass 80-100 g was used in the experiment. The fish were obtained from the rearing pond of the Inland Fisheries Institute in Żabieniec, and kept for 12 months under laboratory conditions in a flow-through aerated tank, at 20-22°C, and fed once a day carp starter Aller Aqua Classic 4 mm. Blood (about 500µL from each of 10 fish) was collected by heart puncture, using chilled needles, into chilled plastic Eppendorf tubes containing no anticoagulant. Then blood of each fish was

immediately transferred to 4 tubes containing dried anticoagulants: sodium heparin or Na2EDTA, to obtain fi nal concentrations of anticoagulants in blood: for heparin 50 IU/mL, and for Na2EDTA 0.1, 0.5, and 1.0 mg/mL of blood, similar to the method applied in human hematology. Blood was stored for 2 h at 4°C (standard blood storage procedure), and gently mixed every 15 min. After this time, hematocrit (Ht) was measured in microhematocrit capillaries, after centrifugation for 5 min at 12,000 rpm. Percentage of hemolysis was evaluated using a hemolysis test: 20 µL of blood from each of 10 fish was added to 2 cm3 of physiological NaCl solution (0.6%), and incubated for 30 minutes at room temperature. After this time the samples were centrifuged for 5 min at 1000 rpm, supernatant was carefully transferred into new tubes, and extinction was read spectrophotometrically at 540 nm wavelength. Percentage of hemolysis was calculated according to the formula:

hemolysis [%] = (A × 100%) / B, where

A – extinction of sample with physiological solution,

B – extinction of sample with distilled water (100% hemolysis).

Blood smears were also made from samples of 8 fish treated with each anticoagulant, and stained with May-Grunwald and Giemsa solutions. A detailed

erythrocyte morphology analysis was done: 300 cells were viewed in each smear, and number of normal, abnormal, and hemolyzed erythrocytes was noted. Percentage of erythrocytes showing various anomalies was calculated. Photographs of anomalies were taken using a Nikon Coolpix digital camera connected to a Nikon-Eclipse E 600 microscope, and computer image analysis system CoolView. The significance of differences was evaluated using the non-parametric Mann-Whitney U test, at P ≤ 0.05.

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