Artifactual Prolongation of the Activated Partial Thromboplastin Time Associated With Hemoconcentration in Dogs

Abstract

An inappropriate blood-to-anticoagulant ratio can cause an artifactual prolongation of the activated partial thromboplastin time (APTT) and prothrombin time (PT). In a drug safety study in dogs, w e observed a 4-to 5-second increase in the APTT from baseline coincident with increased hematocrit values (56% to 65%) secondary to drug-induced vomiting and diarrhea. The PT and platelet counts were unchanged, and there was no clinical evidence of bleeding associated with venipuncture. Although we were unable to sample the same dogs to investigate the possible effect of hemoconcentration on the prolonged APTT, the question was addressed by an in vitro study. The hematocrit value for citrated blood samples collected from t is well documented in human patients that a marked I increase in the hematocrit can artifactually prolong the APTT and PT because of an inappropriate blood-to-anticoagulant ratio.' When the hematocrit value is above 55%, there is a relative decrease in plasma volume. As a result, when the blood is centrifuged, the plasma fraction will contain an increased concentration of sodium citrate, causing excessive anticoagulation. The ideal concentration in the standard blood/anticoagulant (9: 1) mixture must be maintained between 0.109 mol/L (3.2%) and 0.129 mol/L (3.8%) of sodium citrate for accurate interpretation of the APTT and PT results.2In a drug toxicity study conducted in beagle dogs, we observed a 4-to 5-second prolongation for the APTT in dogs with high hematocrits (ie, 57% to 65%) ( Table I). The hemoconcentration was attributed to dehydration associated with drug-induced vomiting and diarrhea. The observation prompted an in vitro assessment of the effects of increased hematocrit on the APTT values in dogs.A 4.5-mL whole blood specimen anticoagulated with 0.5 mL of 0.129 mol/L (3.8%) buffered citrate solution (9: 1) was obtained from 3 healthy beagle dogs. A spun hematocrit (Drummond Microhematocrit, Broomall, PA) was determined for each specimen. The blood tubes were centrifuged at 2200 rpm for 15 minutes at 4°C and aliquots of plasma and red blood cell layer were retained to prepare blood specimens with high hematocnts.The 3 dogs were bled again as described above. Appropriately measured aliquots of this whole blood were dispensed into each of two 2.7 mL tubes each containing 0.3 mL of 0.129 mol/L buffered citrate. One tube was filled completely with whole blood, the other tube was filled with whole blood and an aliquot of hemoconcentrated citrated blood. The proper fill level was maintained in all tubes. A spun microhematocrit was determined on all tubes before centrifugation at 2200 rpm for 15 minutes at 4°C to harvest test plasma for determination of the PT and APTT by a photometric clot detection method using the Electra 700 (Medical Laboratory Automation Inc, Mt. Vernon, NY). Dade Actin Activated Cephaloplastin Reagent (liquid rabbit brain) and Dade Calcium Chloride, 0.02 mol/L (Baxter Diagnostics, Deerfield, IL) were used for APTT determinations and Dade Thromboplastin C (dried rabbit br...

Quantification of Surgical Blood Loss

“…Factors such as hemoconcentration, hemolysis, handling of blood samples, etc. are important to consider when evaluating the precision of coagulation analysis in dogs 15,16 . Several authors conclude that APTT is prone to variations because of sensitivity of sampling and handling of the citrate stabilized blood as well as instability in relation to freezing 17–20 .…”

Blood Coagulation Tests in Toxicological Studies-Review of Methods and Their Significance for Drug Safety Assessment-

“…The excess of citrate is known to induce prolongation of PT and thromboplastin time (PTT) in human plasma (Koepke et al, 1975;Ingram and Hills, 1976). Similar effects have been observed on these PT and APTT in plasma from rats and dogs (O'Brien et al, 1995;Kurata et al, 1998). Quick et al (1935) introduced prothrombin time (PT) as a screening test for factors VII, X, V and II of the extrinsic pathway.…”

Canine Reference Intervals for Coagulation Markers Using the STA Satellite® and the STA-R Evolution® Analyzers

“…1,13 These conditions are routinely used in veterinary clinical pathology; although, to the authors' knowledge, they have never been validated except for absence of a significant effect of citrate concentration between 3.2% and 3.8%, 8 lack of effect of sampling by direct venipuncture or through a catheter, 7 significant effect of abnormal colors of plasmas, 9 and of dehydration. 12 Prothrombin time, APTT, and fibrinogen are almost unchanged in canine plasma stored for up to 2 days at room temperature or at 4uC, 3 which is longer than the delays recommended for human specimens. 1 The durations of storage tested in the current study were chosen according to the actual conditions of the experiment where blood specimens collected at the individual breeders' locations usually arrived at the laboratory after less than 4 hr and, in a very few cases, after up to 7.5 hr.…”

“…The pre-analytical conditions were chosen according to recommendations for coagulation testing in human clinical pathology 1,13 . These conditions are routinely used in veterinary clinical pathology; although, to the authors' knowledge, they have never been validated except for absence of a significant effect of citrate concentration between 3.2% and 3.8%, 8 lack of effect of sampling by direct venipuncture or through a catheter, 7 significant effect of abnormal colors of plasmas, 9 and of dehydration 12 …”