Effect of time and storage on toxic or pseudo‐toxic change in canine neutrophils

Abstract

| INTRODUC TI ONThe CBC is an essential component of patient health evaluations.Automated cell counters have been used since the mid-20th century 1 to rapidly obtain certain hematology data, including several red blood cell (RBC), white blood cell (WBC), and platelet (PLT) indices. However, blood smear evaluation remains a requisite part of the CBC in veterinary species, as automated analyzers do not reliably detect many clinically relevant morphologic abnormalities, such as immature granulocytes, toxic change, infectious agents, and neoplastic cells. Evaluation and, therefore, interpretation of CBC findings can be limited by certain preanalytical factors. For example, prolonged storage could cause artifactual increases in hematocrit, MCV, and MPV, and decreases in the MCHC and PLT variables. 2-6 Additionally, in 1984, Gossett and Carakostas 7 showed that Abstract Background: The presence of toxic change in neutrophils is frequently used as a biomarker of inflammation in dogs. Objective: We aimed to evaluate the effect of time and storage on toxic change in canine neutrophils. Methods: One hundred and fifty microliters of EDTA blood were obtained from eight dogs with no toxic neutrophil changes observed on fresh blood smears (T0). Blood was stored at room temperature (RT), in a box with an icepack (ICE), and at 4°C. For each storage condition, smears were prepared 2 (T2), 4 (T4), 8 (T8), and 24 (T24) hours post blood draw. Smears were randomized, and each smear was evaluated for the presence of toxic neutrophil change. Results: A statistically significant effect of time and storage on the presence of toxic neutrophil change was observed. Compared with T0, the number of neutrophils containing Döhle bodies was significantly higher at T8 and T24 for the RT (P < 0.0001) and ICE (P < 0.0001) samples and at T24 for 4°C samples (P < 0.0001). Additionally, smears were falsely classified as having 1+ toxic change in 0/8 (T2), 1/8 (T4), 3/8 (T8), and 8/8 (T24) for RT samples; 0/8 (T2 and T4), 2/8 (T8), and 5/7 (T24) smears for ICE samples; and 0/8 (T2, T4, and T8) and 2/8 (T24) for 4°C samples. Conclusions: Smears can be falsely classified as having neutrophils with toxic change as early as 4 hours post blood draw in samples stored at RT, 8 hours when stored with icepacks, and 24 hours when stored at 4°C. Canine blood smears should be prepared and evaluated for toxic neutrophil change as early as possible. K E Y W O R D S artifact, delay, Döhle bodies, icepack, preanalytic, refrigerator

Blood Storage Conditions Affect Hematological Analysis in Samples From Healthy Donkeys and Donkeys With Experimentally-Induced Endotoxemia

“…These discrepancies between donkeys and horses in the effect of temperature on blood parameters could be due to different viability of neutrophils, although variations amongst analyzers used in each study should also be considered ( 12 , 15 ). The pseudotoxic changes observed on blood smears kept at 24°C also has been reported in dogs ( 20 ). Lysed WBCs significantly increased (compared to basal) at 12 h in samples kept at 4 and 24°C.…”

“…Samples stored at 4°C showed no increase in toxic indicators for at least 48 h, which is markedly longer than reported in dogs, where pseudo-Döhle bodies and other toxic changes were apparent at 24 h ( 20 ).…”

“…In addition, some of the artifactual changes caused by deficient storage in humans and dogs, such as pseudoleukocytopenia or pseudotoxic changes, can mimic typical features of the systemic inflammatory response syndrome (SIRS) or endotoxemia, leading to erroneous diagnosis (19,20). Endotoxemia is assumed to be as common in donkeys as in horses (21), but little is known about the hematological response to this disturbance in this species (21,22).…”

Assay variability and storage stability of the myeloperoxidase index of the ADVIA 2120i hematology analyzer in canine and equine whole blood samples

“…Analyte stability is important given that many blood samples submitted to veterinary diagnostic laboratories are stored for at least 24 hours before analysis. Considering that neutrophils undergo morphologic changes with storage, 15 we had expected that MPXI would become unstable with storage. We found that MPXI results largely decreased with storage, resulting in significant changes in median MPXI at some time points, but results were not consistent between or within individual animals on the same instrument.…”

“…It is also possible that the reference intervals were not optimal for evaluating MPXI, and reference change values might be preferable, 16 however, there is no guarantee the latter would not differ between analyzers. We did not evaluate other storage temperatures, and it is possible that more consistent changes in MPXI would be seen at room temperature, based on reported morphologic changes in neutrophils 15 . We did not make blood smears to evaluate for these changes in the stored samples.…”