ORIGINAL ARTICLE

Storage-associated changes in the bovine hemogramwith the ADVIA 120 hematology analyzer

Amy L. Warren & Tracy Stokol & Kent G. Hecker & Daryl V. Nydam

Received: 11 April 2012 /Accepted: 27 June 2012# Springer-Verlag London Limited 2012

Abstract Delayed hematological analysis occurs frequentlyin bovine practice. Interpretation of complete blood count(CBC) results may be affected by artifactual storage-associated changes. The objective of this study was to char-acterize changes in bovine CBC results that occur over 72 h ofstorage at room temperature (RT) and 4 °C. Blood samplesfrom 10 clinically healthy Holstein cows were analyzed withthe ADVIA 120 hematology analyzer at 0, 24, 48, and 72 h ofstorage at RT or 4 °C. A two-way repeated-measure ANOVAwas used to analyze time and temperature main effects. Time-associated changes in red blood cell (RBC) results were asignificant increase in mean corpuscular volume (MCV) (bothtemperatures) and hematocrit (RT) and a decrease in meancorpuscular hemoglobin concentration (RT). White blood cellcounts were relatively stable; however, automated absoluteconcentrations of monocytes and large unstained cells de-creased, whereas concentrations of lymphocytes and eosino-phils (RT) increased, over time. There was a statisticallysignificant increase in mean platelet volume (MPV) by 48 hof storage at both temperatures. Changes with storage weremore pronounced at RT. Mean CBC results, with the excep-tion of MPV, did not exceed reference intervals. With storage,bovine RBCs become macrocytic and hypochromic, variablechanges occur in the automated differential cell count, and the

MPV increases. However, most changes, with the exceptionof MCV, are of little diagnostic relevance. For the mostaccurate CBC results, bovine blood should be stored at 4 °Cand analyzed within 24 h of collection.

Keywords Artifact . Bovine . Hematology . Storage .

Temperature

Introduction

A complete blood count (CBC) is a routine diagnostic testused for identifying a broad range of disease states in cattle.Many bovine practitioners submit blood for CBC analysis tolocal or remote diagnostic laboratories. These laboratoriesoften use automated analyzers that yield a substantialamount of CBC data, including information on red bloodcell (RBC) and platelet (PLT) number and size, and auto-mated total and differential white blood cell (WBC) counts.The laser-based hematology system, the ADVIA 120, withmultispecies software is one such analyzer that is commonlyused in veterinary diagnostic laboratories.

The ADVIA 120 hematology analyzer uses light scatter,differential WBC lysis, and myeloperoxidase staining toyield automated differential WBC counts and light scatter-ing and colorimetric measurements to provide informationon RBC numbers, size, and hemoglobin (Hgb) content.Platelet counts and size are also determined by light scatter-ing (Harris et al. 2005). By analyzing the refractive index ofcells, the ADVIA 120 can differentiate RBC from PLT andflags for the presence of RBC fragments and ghosts andcellular debris.

Due to the often-remote location of cattle farms fromveterinary clinics, the large number of animals examined inone visit, and the trend for many veterinarians to submit bloodto specialized diagnostic laboratories rather than performing

This study was performed at the College of Veterinary Medicine,Cornell University, Ithaca, NY, USA.

A. L. Warren (*) :K. G. HeckerDepartment of Veterinary Clinical and Diagnostic Sciences,Faculty of Veterinary Medicine, University of Calgary,3330 Hospital Dr. NW,Calgary, AB T2N 4N1, Canadae-mail: alwarren@ucalgary.ca

T. Stokol :D. V. NydamDepartment of Population Medicine and Diagnostic Sciences,College of Veterinary Medicine, Cornell University,Ithaca, NY, USA

Comp Clin PatholDOI 10.1007/s00580-012-1556-9

analysis in-house, there is often a substantial time delay be-tween sample collection and analysis. Such delays can resultin artifactual changes in CBC results, which can adverselyimpact interpretation. Studies done in dogs, horses, monkeys,rabbits, rats, and mice with the ADVIA 120 have shown thatRBC and PLT results are themost affected by storage (Clark etal. 2002; Furlanello et al. 2006; Ameri et al. 2011). Erythro-cytes progressively become macrocytic and hypochromic (at-tributable to RBC swelling from water uptake), resulting inincreased hematocrit (HCT) (which is a calculated value withthe ADVIA which is dependent on the mean corpuscularvolume (MCV) and RBC count). Platelet numbers decrease(likely due to clumping) with increased storage time (Clark etal. 2002; Furlanello et al. 2006; Ameri et al. 2011). In contrast,WBC counts tend to be resistant to age-changes, althoughthere are species differences. Furthermore, the temperature atwhich blood samples are stored and transported will affect therate and degree to which these changes occur. Storage atrefrigerated temperatures helps reduce time-related artifacts(Wood et al. 1999). Since storage-related changes in CBCresults cannot necessarily be extrapolated from one speciesto another, we sought to document the artifactual changes thatmay occur in bovine CBC results following storage at bothroom (RT) or refrigerator temperature (4 °C) with the ADVIA120 hematology analyzer.

Materials and methods

Ten milliliters of blood was collected from the coccygealvein of 10 clinically healthy adult female Holstein dairycows into vacutainers containing K2 EDTA (BD Bioscien-ces, Franklin Lakes, NJ) on the same day. The samples wereanalyzed immediately to provide baseline values. The sampleswere then equally divided into two aliquots, one of which wasstored at 4 °C and the other kept at RT (average temperature of24 °C). The stored samples were then reanalyzed at 24, 48,and 72 h after collection.

For each time point, blood samples were thoroughlymixed, warmed to RT, and hematological analysis was per-formed following standard laboratory procedures using theADVIA 120 hematology analyzer and multispecies software(version 3.1.8.0-MS, Bayer Corporation, Tarrytown NJ.).The analyzer yielded the following RBC, WBC, and PLTresults: (1) RBC: HCT, RBC count, and Hgb concentration,and the RBC indices of MCV, mean corpuscular hemoglo-bin (MCH), mean corpuscular hemoglobin concentration(MCHC), and RBC distribution width (RDW); (2) WBC:count, differential percentages, and absolute concentrationsof neutrophils, lymphocytes, monocytes, eosinophils, baso-phils, and large unstained cells (LUC); and (3) PLT: count andmean platelet volume (MPV). ADVIA-generated cytograms

(peroxidase and basophil WBC, RBC, and PLT) were visuallyassessed at each time point.

Statistical analysis was performed using PAWS Statistics17 software. Kolmogorov–Smirnov (K–S) tests were usedto assess the normalcy of the data for each result at bothtemperatures. A total of 136 K–S were performed, 12 ofwhich (9 %) were significantly non-normal. Because therewere relatively few non-normal cases and they were ran-domly distributed among the results, parametric analyseswere used. A two-way repeated-measure ANOVAwas usedto assess two main effects, time and temperature, and oneinteraction effect for time×temperature, for 17 dependentmeasures. Post hoc tests were performed using a one-wayrepeated-measure ANOVA to determine time effects and apaired t test to determine the between temperature andbetween-time point differences if present. Because of themultiple tests, a Bonferroni-adjusted p value of 0.003 wasused. For some results, the percentage change compared tobaseline values was calculated.

Results

Time-dependent changes in RBC, WBC, and PLT resultsoccurred with the ADVIA 120 in bovine blood at bothstorage temperatures. However, with the exception ofMPV, mean results stayed within established reference inter-vals. In general, changes were more pronounced in samplesstored at RT and the majority of changes were noted after48 h of storage (Table 1).

There were time- and temperature-dependent changes inRBC results and indices. The mean HCT and MCHC sig-nificantly increased and decreased, respectively, with time atRT but not at 4 °C. The mean MCV increased significantlyover time at both temperatures, although slightly largerchanges occurred at RT. A temperature-, but not time-,dependent effect was observed for RDW (Table 1).

Changes in mean WBC count and absolute differentialWBC concentrations were time-dependent, with no signifi-cant differences observed due to storage temperature. Themean WBC count significantly increased in samples storedat 4 °C, but not at RT. Mean lymphocyte and eosinophilabsolute concentrations increased significantly (by 15–20 %and 25 % within 72 h, respectively), whereas mean mono-cyte and LUC absolute concentrations decreased significant-ly (by 30–62 and 56–55 % within 72 h, respectively) withstorage at 4 °C (the change was not significant for eosino-phils) and RT. These changes were more pronounced at RT,except for the absolute concentration of lymphocytes, wherethe degree of change was greater at 4 °C (Table 1). Asignificant time and temperature interaction effect was notedfor basophil and monocyte absolute concentrations.

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Platelet counts and MPV were affected by storage andtemperature, with mean PLT count and MPV decreasing andincreasing, respectively, with storage at both temperatures.These changes were larger for samples stored at RT versus4 °C; however, only the changes in mean MPV (time- andtemperature-dependent) were significant (MPV increasingby 42 and 63 %, respectively). The mean MPV wasoutside the established reference interval by 48 h at bothtemperatures (Table 1).

Consistent qualitative changes were observed in theADVIA 120 cytograms over time at both storage temper-atures. By 72 h, the entire RBC cluster shifted slightly up(macrocytic) and to the left (hypochromic) in the samples

stored at RT. Similar changes were not observed in thesamples stored at 4 °C (Fig. 1a). Within 24 h, there wereincreased numbers of events in the noise/platelet gate of theperoxidase cytogram at both temperatures in most samples,which worsened over time. The individual cell clusters(particularly neutrophils and eosinophils) became more dif-fuse over time. These changes were more apparent in sam-ples stored at RT (Fig. 1b). At 48 and 72 h, platelet cytogramevents were more diffusely spread, particularly in samplesstored at RT (Fig. 1c). Mild RBC hemolysis was evident inthe platelet cytograms of most (70 %, 7/10) samples at 48and 72 h of storage at 4 °C (Fig. 1c), whereas this changewas only evident in 10 % (1/10) of samples stored at RT.

Table 1 Mean±SD CBC results from bovine blood (n010) obtained with the ADVIA 120 hematology analyzer immediately after collection and24, 48, and 72 h of storage at 4 °C or room temperature (RT)

CBC result Storage temperature Storage time (h) Reference intervala

0 24 48 72

RBC HCT (%) 4 °C 29±3 29±3 29±3 29±3 23–35RT* 30±3 30±3 30±3

Count (x 1012/L) 4 °C 6.4±0.6 6.3±0.5 6.3±0.6 6.2±0.5 5.4–8.2RT 6.2±0.6 6.3±0.6 6.3±0.5

Hemoglobin (g/dL) 4 °C 10.8±1.0 10.7±1.0 10.7±1.0 10.8±1.0 8.6–13.2RT 10.7±1.0 10.8±1.0 10.8±1.0

MCV (fL) 4 °C* 45±2 45±2 46±2 46±2 36–49RT* 46±2 47±2 47±2

MCH (pg) 4 °C 17±1 17±1 17±1 17±1 14–19RT 17±1 17±1 17±1

MCHC (g/dL) 4 °C 37±1 37±1 37±1 37±1 36–40RT* 38±1 36±1 36±1

RDW (%) 4 °C** 17.4±0.8 17.3±1.0 17.2±1.0 17.1±0.9 16.2–19.7RT 17.4±1.0 17.4±1.0 17.1±0.9

WBC Count (x109/L) 4 °C * 9.3±2.3 9.5±2.3 9.3±2.3 9.7±2.5 5.6–13.2RT 9.4±2.3 9.4±2.3 9.4±2.3

Neutrophil (x109/L) 4 °C 3.8±1.0 3.7±0.90 3.6±0.98 3.4±1.0 1.7–6.0RT 3.8±1.0 3.8±0.94 3.7±0.81

Monocyte (x109/L) 4 °C* 0.5±0.2 0.4±0.1 0.4±0.1 0.3±0.1 0.2–1.0RT* 0.4±0.2 0.2±0.1 0.2±0.1

Lymphocyte (x109/L) 4 °C* 4.4±1.7 4.8±1.7 4.8±1.6 5.4±1.7 2.3–7.4RT* 4.5±1.6 4.8±1.6 4.9±1.7

Eosinophil (x109/L) 4 °C 0.4±0.2 0.4±0.3 0.5±0.3 0.4±0.2 0.1–1.2RT* 0.5±0.3 0.5±0.3 0.5±0.3

Basophil (x109/L) 4 °C 0.1±0.02 0.1±0.02 0.1±0.02 0.1±0.02 0–0.2RT 0.1±0.02 0.1±0.03 0.1±0.04

LUC (x109/L) 4 °C* 0.08±0.04 0.05±0.02 0.02 ±0.02 0.03±0.02 0–0.2RT* 0.04±0.02 0.03±0.01 0.03±0.02

PLT Count (x 109/L) 4 °C 352±94 315±85 304±75 320±51 232–596RT 302±60 311±77 295±75

MPV (fL) 4 °C*,** 7.3±1.2 7.3±0.9 8.9±1.1 10.3±1.2 5.6–8.0RT* 8.0±1.1 10.8±1.4 12.0±1.6

*p<0.003, significant time-dependent differences at each indicated storage temperature (4 °C or RT); **p<0.003, significant temperature-dependent differences (4 °C versus RT) over timea Reference intervals established with the ADVIA 120 hematology analyzer at Cornell University

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Discussion

This study documents the time- and temperature-inducedchanges in RBC, WBC, and PLT results from bovineEDTA-anticoagulated blood obtained with the ADVIA120. The predominant changes were time-associated andwere generally more pronounced in samples stored atRT. Our findings in cattle are generally similar to thosedocumented in other veterinary species (Clark et al. 2002;

Furlanello et al. 2006; Ameri et al. 2011), reflecting RBCand PLT swelling and WBC degradation.

Time- and temperature-dependent changes noted in RBCresults were similar to previous reports in dogs (Furlanelloet al. 2006; Prins et al. 2009), horses (Clark et al. 2002;Prins et al. 2009), monkeys, rabbits, rats, and mice (Ameri etal. 2011) using the ADVIA 120. Similar changes have alsobeen observed in bovine, caprine, and porcine blood, usingmanual techniques (packed cell volume, manual RBC count,

Fig. 1 Representative ADVIA 120 cytograms at baseline and 72 h ofstorage at 4 °C or RT. a RBC cytogram: scatterplot of hemoglobinconcentration (X-axis) and cell volume (Y-axis) of events falling withinthe RBC gate. The central square represents normocytic normochro-mic RBCs. By 72 h of storage, the entire RBC population had shiftedslightly upwards (macrocytic) and towards the left (hypochromic) insamples stored at RT (arrow); however, most events are still within thecentral normocytic normochromic box. b Peroxidase WBC cytogram:the ADVIA separates different WBCs into gates based on peroxidasestaining (X-axis) and size (Y-axis): A noise, B noise or platelet clumps,C lymphocytes, D monocytes (small box with arrowhead), E large

unstained cells, F neutrophils, and G eosinophils. With storage, in-creased numbers of events were observed in the noise/platelet clumpgate (arrow) at both storage temperatures, but more were seen insamples stored at RT. The gated cell clusters also become less compactover time, particularly at RT. c Platelet cytogram: the ADVIA distin-guishes PLT by internal complexity or granularity (X-axis) and size (Y-axis). A more diffuse scatter (with decreases in cell complexity) oc-curred with storage, particularly those samples stored at RT. In mostsamples stored at 4 °C, a linear vertical stream of events, correspondingto lysed RBCs (Tvedten 2010) (arrow), is observed on the left handside of the cytogram

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hemoglobin concentration) (Ihedioha and Onwubuche 2007).The most substantial change occurred with the MCV, whichincreased over time at both temperatures. The change inMCVwas more pronounced at RT (both mean results and cytogramplots) and was accompanied by a mild but significant decreasein MCHC and increase in HCT, both of which are calculatedresults that are dependent on the MCV. The changes in MCVcan be attributed to osmotic swelling of RBC (Furlanello et al.2006). Although the mean HCT, MCV, and MCHC remainedwithin reference intervals, results for these RBC tests couldbecome abnormal with storage, particularly at RT, in individ-ual animals. Changes in the mean RDW were minor atboth storage temperatures and the significant temperature-associated difference in this test result is not considered clin-ically relevant. Since changes in these RBC tests can behelpful in identifying and classifying anemias in ruminants(Jones and Allison 2007), it is possible that storage-relatedchanges may result in incorrect interpretation of CBC resultsand potential misdiagnosis.

The principle time-dependent change in WBC results atboth storage temperatures was an altered automated WBCdifferential count characterized by reduced absolute concen-trations of monocytes and LUC, with a concomitant increasein the absolute concentration of lymphocytes and eosino-phils. The degree of change in the automated WBC differ-ential counts was generally greater in samples stored at RT.Similar artifactual changes have been described for mono-nuclear cells in human, dog, horse, rabbits, rats, and miceblood with storage (Wood et al. 1999; Clark et al. 2002;Furlanello et al. 2006), but not for eosinophils. The changesin the WBC differential count can mostly be attributed totime-associated degradation (Ameri et al. 2011), nuclearswelling, or apoptosis of cells resulting in shrinkage andpotential misclassification of monocytes and LUC as lym-phocytes by the ADVIA. An artifactual increase in absolutelymphocyte concentrations may affect neutrophil/lympho-cyte ratios, a figure sometimes used by clinicians to detectearly inflammation in ruminants. Pathologic lymphocytosisin cattle is uncommon, but can be seen in chronic bacterialand viral infections, particularly bovine leukemia virus in-fection (Jones and Allison 2007). However, the small arti-factual increases in lymphocytes noted in our study areunlikely to affect the clinical interpretation of the leuko-gram. The reason for the mild increase in absolute eosino-phil concentrations is unclear but may be due to inclusion ofdebris or platelet clumps in the eosinophil gate combinedwith less-defined cell clusters with time. The mean WBCcount also increased mildly with storage at 4 °C for un-known reasons. Despite the changes in total and automatedWBC differential counts, mean counts fell within estab-lished reference intervals, and overall, the changes weresmall and unlikely to impact interpretation of the CBC.Our results suggest that an automated differential count will

be reasonably reproducible in samples stored at RT or 4 °Cfor up to 72 h after collection.

The mean PLT count decreased over time at both storagetemperatures. This change can be attributed to platelet acti-vation and clumping with increasing duration of storage(Handagama et al. 1986; Stokol and Erb 2007). Plateletclumping and cell fragments would also contribute to theincreased number of events observed in the noise/PLT gateof the peroxidase scattergram. We did not examine Wrights-stained blood smears for the presence of platelet clumps;however, storage-associated PLT clumping causing de-creased PLT counts has been reported previously for otherspecies (Zelmanovic and Hetherington 1998). Although thechange in mean PLT count was not statistically significant(likely due to the overlap in results in individual animals),the decrease in PLT count with storage could result inerroneous interpretation of thrombocytopenia in individualanimals, particularly if smears are not reviewed for thepresence of PLT clumps. The mean MPV increased signif-icantly at both storage temperatures and was higher than thereference interval at both temperatures by 48 h of storage.This is similar to findings in monkeys, rabbits, rats, andmice (Ameri et al. 2011). The increased MPV is attributedto a combination of osmotic cell swelling and activation(Handagama et al. 1986; Stokol and Erb 2007). The broaderscatter of events within the PLT cytogram, which corre-sponds to decreased internal complexity or mean plateletcomponent concentration, is additional support for dilutionor loss of cytoplasmic granularity (Stokol and Erb 2007). Itis also possible that ghost erythrocytes (which appearedover time, especially at 4 °C) may have contributed to theincreased MPV (Tvedten 2010). The changes in MPV weremore pronounced at RT versus 4 °C, which contrasts with aprevious published report in dogs (Handagama et al. 1986),indicating that there are species differences in PLT responsesto storage temperature. An increased MPV is associatedwith active thrombopoiesis (Dircks et al. 2009) and, in somestudies in human patients, may be predictive of thromboticrisk (since large platelets are considered to be hyperfunc-tional) (Martin et al. 1983; Braekkan et al. 2010). However,similar associations have not been made in cattle and thesubstantial significant changes in mean MPVare unlikely tobe of diagnostic relevance in this species.

In conclusion, we have documented that, similar to otherspecies, artifactual changes do occur in bovine blood withstorage over 72 h at 4 °C or RTwhen tested with the ADVIA120 hematology analyzer. Changes of diagnostic relevanceoccurred for MCV and potentially HCT and PLT countswithin 48 h of storage, whereas the total and automateddifferential WBC count was reasonably accurate up to72 h of storage. However, it should be noted that we onlyexamined healthy cows in our study. It is possible that theartifactual changes could be accentuated in diseased cattle

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and may impact test interpretation. Refrigeration of samplesmitigated some of the time-associated changes. We thus rec-ommend that blood should be analyzed within 24 h of collec-tion and samples stored at 4 °C for the most accurate CBCresults in cattle. Our results will be a useful guide to cliniciansand clinical pathologists in interpreting changes that occur inCBC results derived from stored bovine blood.

Acknowledgments The authors wish to thank the clinical pathologystaff at Cornell University for analyzing the blood with the ADVIA 120and the clinical pathology laboratory manager, Richard DeFrancisco, fordata input.

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