Changes in canine hematology measurements may occur when analyses are delayed due to shipment of specimens to a laboratory.
Changes in canine hematology measurements may occur when analyses are delayed due to shipment of specimens to a laboratory.
The purpose of this study was to report changes in hematologic variables in healthy and diseased canine blood measured with a Sysmex XT-2000iV during storage at room temperature for 24 and 48 hours.
EDTA-K3 blood samples from 42 healthy and diseased dogs were measured on a Sysmex XT-2000iV analyzer within one hour of sampling, and after storage for 24 and 48 hours at room temperature in the dark.
Storage caused little or no change in RBC count, HGB concentration and MCH, while there was a moderate increase in HCT, MCV and reticulocytes count, and a moderate decrease in MCHC. Decreased platelet counts by impedance (PLT-I) and optical (PLT-O) measurements were associated with increased mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet distribution width (PDW), including a right shift in the platelet histogram and a dispersion of the platelet dot plot on the scattergram. The total and differential WBC count remained stable except for decreased monocyte counts. In the scatterplots, monocytes shifted into the lymphocyte population after 24 hours, and neutrophil population shifted to the right appearing in the eosinophil gate at 48 hours of storage. The disease status had only a small effect on storage-induced changes, and observed changes had no consequences for clinical decisions.
Blood storage at room temperature was accompanied by moderate variations in some hematologic variables, awareness of which helps in avoiding misinterpretations.
In veterinary practices and clinical pathology laboratories, hematology analyses are usually performed rapidly or, as recommended, within 24 hours[1, 2] on specimens kept at 4°C, as in human medicine.[3, 4] However, in many cases, specimens cannot be processed immediately, and delays of up to 48 hours can occur due to shipment to a referral laboratory or due to lack of service on weekends and holidays. This raises questions about the stability of the measured variables and about the validity of data interpretation, especially for blood specimens kept at room temperature (RT).
The main quantitative changes reported for canine blood samples stored for 24 and 48 hours at 4°C or RT using impedance, flow cytometry (ADVIA 120) or combined impedance-flow cytometry (XT-2000iV) hematology analyzers are summarized in Table 1. Moreover, a recent study reported the percentage deviation kinetics of some analytes obtained from 10 healthy dogs after blood storage at 4°C or 22°C using the XT-2000iV analyzer. Whatever the analyzer technology, variables such as HGB concentration and RBC count appear to be stable, while HCT and MCV were increased, and MCHC was decreased. WBC count was stable if measured with the XT-2000iV regardless of storage temperature, while it was increased with impedance technology or decreased in samples stored at RT and measured by the ADVIA 120. Finally, the platelet (PLT) count was stable in specimens stored at 4°C and determined by the ADVIA 120, whereas it was decreased with XT-2000iV and impedance analyzers. If stored at RT, PLT count was decreased with all analyzers tested, changes were always more pronounced at RT than at 4°C except for PLT.
|Temperature||Time(hour)||N (animal status)||RBC||HGB||HCT||MCV||MCHC||WBC||PLT||Reticulocyte||Instrument||Reference|
|4°C||24||10 (H, D)||−3%||+2%||=||+4%||=||=||−50%||NR||Impedance||F-800|||
|5 (H)||=||=||+2%||+1%||−2%||+7%||NR||NR||Impedance||Counter ZBI|||
|5 (H)||+1%||+1%||NR||+2%||NR||+18%||−20%||NR||Impedance||Several instrumentsb|||
|8 (H)||+0.9%a||=||NR||+3.2%a||NR||−1.5%a||−37%a||+4.6%a||Impedance and FCM||XT-2000iV|||
|16 (H)||NR||NR||NR||NR||NR||NR|| |
|NR||Impedance and FCM||XT-2000iV|||
|5 (H)||−1.1%||+0.9%||+6.7%||+1.1%||−5.4%||+3.6%||−0.9%||NR||FCM||ADVIA 120|||
|48||10 (H, D)||−4%||=||+2%||+6%||−2%||−3%||−50%||NR||Impedance||F-800|||
|5 (H)||=||=||+3%||+3%||−3%||+14%||NR||NR||Impedance||Counter ZBI|||
|8 (H)||+0.9%a||+0.6%a||NR||+5.6%a||NR||−0.4%a||−20%a||+4.6%a||Impedance and FCM||XT-2000iV|||
|14 (H)||NR||NR||NR||NR||NR||NR|| |
|NR||Impedance and FCM||XT-2000iV)|||
|5 (H)||−1.4%||=||+8.9%||+10.4%||−8.4%||+1.9%||−0.45%||NR||FCM||ADVIA 120|||
|24–25°C or RT||24||152 (H, D)||−3.5%||+0.8%||NR||+4.2%||NR||+6.7%||−16.2%||NR||Impedance||T54|||
|10 (H, D)||−5%||=||+7%||+12%||−5%||+5%||−20%||NR||Impedance||F-800|||
|40 (H, D)||−4%||+3%||+2%||+5%||−1.5%||+32%||−24%||NR||Impedance||VetABC|||
|5 (H)||+1%||=||+4%||+2%||−3%||+6%||NR||NR||Impedance||Counter ZBI|||
|NR||Impedance and FCM||XT-2000iV|||
|5 (H)||−1.1%||+0.9%||+13.4%||+14.4%||−10.7%||−2.25%||−10.3%||NR||FCM||ADVIA 120|||
|9 (H)||+1.4%||+2.2%||+10.3%||+5.5%||−4.5%||−5.2%||−0.1%||NR||FCM||ADVIA 120|||
|48||152 (H, D)||−2.5%||+1.5%||NR||+8.3%||NR||+7.4%||−17.4%||NR||Impedance||T540|||
|10 (H, D)||−6%||−1%||+20%||+25%||−10%||+5%||−35%||NR||Impedance||F-800|||
|5 (H)||+3%||=||+7%||+4%||−7%||+14%||NR||NR||Impedance||Counter ZBI|||
|NR||Impedance and FCM||XT-2000iV|||
|5 (H)||−0.8%||+0.4%||+20%||+20.6%||−16.5%||−8.9%||−19.7%||NR||FCM||ADVIA 120|||
|9 (H)||+0.9%||+1.7%||+17.5%||+13%||−11%||−13.7%||+3.6%||NR||FCM||ADVIA 120|||
|?||384 (H, D)||=||NR||+8%||+9%||+8%||NR||NR||NR||Impedance||NR|||
So far, no investigations of modifications due to storage of canine blood cells in histograms (impedance measurement) and scattergrams (flow cytometry measure) have been reported in diseased animals. Little information is available on changes in the WBC differential count, and particularly WBC scattergrams. Increased eosinophil counts and decreased monocyte counts have been reported in canine[5, 6] and human blood[7, 8] stored at RT for 24–48 hours. A significant decrease in canine lymphocyte counts was observed at 22°C using flow cytometry analyzers.[5, 9] Moreover, an overlap of neutrophil and eosinophil cell clusters was seen in scattergrams of XT2000iV of blood specimens stored for 72 hours at 22°C.
Most of the analytes measured in hematology are not chemically defined molecules but cells, which may undergo autolysis during storage. In addition, the reagents used to dilute or stain these already fragile cells may cause further morphologic change. This implies that the stability of a variable may be affected not only by preanalytical but also by analytical conditions, thus that the changes observed may differ according to the analyzer used.
The objective of this study was therefore to report changes in hematologic variables in blood collected from healthy and diseased dogs, and measured by the Sysmex XT-2000iV during storage in EDTA-K3 plastic tubes at RT for 24 and 48 hours. The results were interpreted from statistical, analytical, and clinical points of view. Special emphasis was placed on variables previously not addressed, namely cell histograms and scattergrams, and on possible effects of disease on blood cell stability.
Whole blood samples collected in 5-mL EDTA-K3 tubes (VenoJect EDTA (K3) K3E, Terumo Europe- Belgium) and submitted to the laboratory of the hospital of the veterinary school of Toulouse were selected from 42 dogs, independently of breed, age, sex, or health status, the sole criterion being a correctly filled tube with no macroscopically visible clot.
The tubes were placed on an agitator (Specie Mix Drew, Oxford, CT, USA) for approximately 15 minutes, followed by final mixing by 30 gentle inversions. Duplicate measurements were immediately obtained using a Sysmex XT-2000iV analyzer (Sysmex, Kobe, Japan) with settings for canine blood (software version 00-09). Measurements included RBC count by optical (RBC-O) and impedance (RBC-I) methods, HGB concentration, HCT, MCV, MCH, MCHC, total and relative reticulocyte (RET) counts, low-, medium- and high-fluorescence ratios (LFR, MFR, and HFR, respectively) for stages of reticulocyte maturation, immature reticulocyte fraction (IRF) as the sum of MFR and HFR, RDW expressed as RDW-SD (standard deviation) and RDW-CV (coefficient of variation), WBC count, neutrophil, lymphocyte, monocyte, and eosinophil counts, platelet count by optical (PLT-O) and impedance (PLT-I) methods, mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR) as an index of platelet activity, and plateletcrit (PCT). The different measurement methods used by this analyzer have been fully described in previous articles[10, 11] and are summarized in Table 2. Before each series of analyses, 3 manufacturer's control solutions (Sysmex e-check L1, L2, and L3 levels) were run. Imprecision was as previously reported.
|Spectrophotometry||Impedance||Fluorescence Flow Cytometry||Flow Cytometry|
|Measured||HGB||RBC-I, HCT, PLT-I, PCT||RBC-O, Reticulocytes, PLT-O, WBC-DIFF(Neutrophils, Eosinophils, Lymphocytes,Monocytes)|| |
|Calculated||MCV, MCH, MCHC, RDW, MPV, PDW, P-LCR||Reticulocyte ratios (LFR, MFR, HFR, IRF)|
All measurements were performed within one hour of sampling (T0). After storage for 24 hours at RT in the dark, the tubes were gently homogenized by 30 inversions, and re-analyzed (T24). The same procedure was repeated at 48 hours (T48). A blood film was prepared at T0 and stained with May-Grünwald Giemsa (Aerospray Hematology Slide Stainer Cytocentrifuge 7150, Wescor, Logan, UT, USA) to check blood cell morphology and to detect possible platelet aggregates. Platelet aggregation was determined at low magnification (×100 to ×200) at the feathered edge.
The means of duplicate results obtained at T0, T24, and T48 were tested for effects of storage deviation and a dog's health status by a general model with interaction between the 2 parameters. When an effect of storage deviation was observed, comparison between T0, T24, and T48 was done by Dunnett's test. When an effect of health status on stability was observed, comparisons were performed in the healthy and diseased subgroups. The differences between the results for each time point were compared with the maximum difference, which could be due to analytical variability (2.77*CV of between series imprecision at a median concentration). The results of each variable were then classified according to the corresponding reference interval to see if the differences would have affected the medical interpretation. Calculations were performed with an Excel spreadsheet (Microsoft, Redmond, WA, USA), Analyse-It (Analyse-It, Leeds, UK), and Systat 13 (Chicago, IL, USA).
In total, 42 canine blood specimens were analyzed, 30 (71.5%) of which were obtained from sick animals. Twenty-three of the latter (54.5%) showed hematologic abnormalities. Of the 23 cases, no results were available for PCT, MPV, PDW and P-LCR at T0 in 13 cases due to the error message “PLT abnormal distribution” due to PLT anisocytosis with macroplatelets, as verified by blood smear evaluation and histogram analysis in 12 cases, very low platelet counts (PLT-I < 100 109/L) in 8 cases, and/or PLT aggregates in 7 cases. Overall, PLT aggregates were observed microscopically in 13 of the 42 specimens, and all originated from diseased animals. Consequently, no results were obtained for PCT, MPV, PDW and P-LCR in 7 of these 13 cases. Differential WBC counts were unavailable at T0 with the message “WBC abnormal scattergram” for 3 dogs exhibiting severe leukopenia (1.4 109/L) associated with lymphoma, marked leukocytosis (30.4 109/L) associated with hypereosinophilia syndrome, and WBC aggregates in a case of babesiosis. The final numbers of available variables for each time point, and observed changes over time, are listed in Table 3.
|Variable||Unit||n at T0a||T0b||T24c||Percentage Change||T48c||Percentage Change|
|RBC-I||1012/L||42 (14-24-4)||< 0.0001||5.95 (1.56/9.19)|| |
6.01 (1.54/9.29) **
|NS||5.61 (1.38/8.56)|| |
|< 0.0001||0.39 (0.14/0.57)|| |
0.48 (0.15/0.71) **
|< 0.0001||135.3 (35/202)|| |
135.8 (36/206) **
135.3 (36/206) **
|< 0.0001||66.8 (58.1/90.4)|| |
75.1 (65.5/94.8) **
81.4 (69.8/104.7) **
|NS||23.0 (19.1/26.1)|| |
23.1 (19.3/26.1) **
23.0 (19.3/26.4) **
|< 0.0001||345 (249/363)|| |
301 (247/330) **
278 (239/312) **
|< 0.0001||35.9 (30.5/70.5)|| |
41.2 (36.1/69.6) **
44.1 (30.6/86.6) **
|< 0.0001||16.5 (13.6/25.7)|| |
15.7 (13.0/21.6) **
15.8 (13.3/25.0) **
|0.0015||111.7 (9.8/380.4)|| |
116.4 (14.35/457.9) **
121.4 (13.2/646.0) **
|0.0478||1.79 (0.19/15.27)|| |
1.76 (0.28/21.1) **
1.88 (0.25/35.0) **
|< 0.0001||76.2 (46.3/93.2)|| |
70.5 (43.4/87.0) **
69.9 (43.0/85.5) **
19.5 (3.3/33.1) **
|< 0.0001||6.9 (1.3/27.2)|| |
12.4 (2.4/35.1) **
13.6 (3.2/35.3) **
|< 0.0001||23.8 (6.8/53.8)|| |
29.5 (13.0/56.6) **
30.2 (14.5/57.0) **
|0.0030||13.1 (1.4/62.6)|| |
12.6 (1.5/61.4) **
12.6 (1.7/58.7) **
|0.0379||9.06 (1.03/51.22)|| |
9.59 (1.42/52.15) **
9.52 (1.59/52.14) **
|0.0019||2.08 (0.13/6.81)|| |
|< 0.0001||0.98 (0.28/4.19)|| |
0.71 (0.20/3.42) **
0.52 (0.08/2.80) **
|NS||2.35 (0.10/13.6)|| |
|< 0.0001||267 (5/817)|| |
233 (7/725) **
191 (6/596) **
|0.0012||274 (23/984)|| |
|< 0.0001||9.8 (7.8/12.5)|| |
11.3 (7.4/14.4) **
11.2 (8.4/13.4) **
|< 0.0001||23.8 (9.6/40.6)|| |
34.0 (20.8/54.8) **
33.6 (20.3/49.8) **
|< 0.0001||3.4 (1.5/6.4)|| |
2.8 (1.2/5.9) **
|< 0.0001||10.6 (8.0/16.1)|| |
13.6 (10.4/21.8) **
14.3 (10.6/19.8) **
Effects on storage-induced changes were only observed for HCT, MCV, MCHC, HFR, and PLT-I, but the statistically significant effects observed were mild and had no effect on the clinical interpretation of the results.
No statistical difference was observed for RBC-O and MCH (Table 3). HGB was statistically increased at T24, but this had no effect on the clinical classification. Analysis of the RBC population on scatter- and histograms showed no or only minor changes over time (Figure 1D–O), with the exception of 2 very ill dogs suffering idiopathic regenerative immune-mediated hemolytic anemia (IMHA, Figure 2D–O). The reticulocyte and PLT-O scattergrams of these 2 dogs revealed an abnormally enlarged RBC cluster at T0 that merged with the platelet cluster from T24 onward. In these 2 cases, the general message “RBC abnormal distribution” was displayed.
For significantly altered RBC variables, the highest frequency of analytical and clinical differences over time was observed in descending order for MCV, RDW-SD, MCHC, HCT, and RDW-CV. According to the same criteria, the highest frequencies of differences in PLT variables were observed for MPV, P-LCR, PLT-I, and PCT.
Except for the 2 severely diseased dogs with IMHA, in which the PLT-O counts were increased, the PLT counts decreased as storage time increased from T24 onwards, based on impedance and optical measurements (Figure 3). In addition, there was no statistically significant difference between impedance and flow cytometry (paired t-test, P = .10 at T24 and T48).
The lower PLT counts were associated with a right shift of the curve on the PLT-I histogram, and with a diffusion of the PLT dot plot on the PLT-O scattergram from T24 onwards (Figure 1G–I, M–O). All variables derived from the PLT population calculated from impedance measurement were consequently modified, with increased MPV, P-LCR and PDW, and decreased PCT at T48.
The absolute RET count increased significantly with storage duration, but with very little effect on the clinical classification (Table 3). Differences exceeded the analytical variability in 15/42 and 23/41 cases at T24 and T48, respectively. A marked increase in RET count at T24 was observed in 3 specimens (Figure 3C). The RET maturation index MFR remained stable, LFR decreased, while HFR and IRF were increased.
Total and differential WBC counts were available even at T48, except for one dog at T24 and 2 dogs at T48, where differential counts were not obtained. The overall counts decreased significantly from T24, but this had no effect on the clinical classification (Table 3). All differential WBC counts were statistically different from T0 except for eosinophils. The most important difference was observed for monocytes, as 5/38 and 9/37 cases would have been misclassified relative to the reference intervals at T24 and T48, respectively. The differential WBC scattergrams showed that many monocytes, appeared to shift into the lymphocyte cluster from T24 onward, and the neutrophils shifted to the right, mixing with eosinophils on T48 (Figure 1A–C).
In this study, we report changes of the XT-2000iV analytes from healthy and diseased dog blood stored at RT. We emphasized the analysis of histo- and scattergrams and compared the impact of storage according to health status. Most of the changes observed in this study were predictable as they had been previously reported. Storage times had a significant effect on almost all variables, and in many cases, the differences were higher than could be explained by analytical variability. Although moderate variations occurring over time were both statistically and analytically significant, they would probably not be considered clinically relevant by most users. The definition of in vitro stability of a variable in a specimen is ambiguous, and to our knowledge, there is no internationally accepted definition. One proposition is that “a constituent is stable under specified conditions when measurements demonstrate, with prespecified risks of decision-error, that its mean concentration in the tested specimens has changed by less than an amount δ, where δ is a function of the precision of the method.” This implies that the same variable may be considered as stable or not if the method has high or low imprecision, which is unacceptable but occurs frequently. Other options, discussed within the larger framework of desirable test performance, involve the use of paired statistics, arbitrary thresholds or percents of variation, clinical expert criteria, maximum acceptable variability based on intra-individual and analytical variability, or classification of results according to reference intervals or decision limits.[16-18] From a practical point of view, any change in the test results must not be so great that it modifies the medical interpretation, which may also differ according to the person actually interpreting the results.
It was recently shown in human clinical pathology that stability depended on the analyzer used and that “the stability of hematologic analytes is shorter than assumed in the past,” probably a consequence of the improved analytical sensitivity of new technologies. These observations may have different causes. Preanalytical parameters may influence the stability of some analytes. For instance, plastic tubes have been reported to provide superior platelet stability over glass tubes, or using K3-EDTA instead of K2-EDTA supposedly less affects the MCV. The complex mixtures of diluents and stains used in the latest cytometers may cause more damage to cells slightly autolyzed by storage than the normal saline used in older systems.
In the present study, criteria incorporating intra-individual variability were not considered relevant as all analyses were performed on the same specimen; moreover, such information was not available for most variables. The option of considering possible changes in medical interpretation, according to classification within or outside the reference interval, has already been adopted. The efficiency of this procedure in the present study was limited by the relatively few cases available, and therefore by the number of cases located close to the reference limits. However, differences in medical interpretation were clearly apparent when major changes occurred during storage, such as for HCT, MCV, or MCHC.
As in previous studies, RBC, HGB, and MCH were quite stable in canine blood stored at RT.[5, 9, 21-24] Changes due to erythrocyte swelling during storage were similar to those previously reported for HCT, MCV, and MCHC. PLT-I and PLT-O decreased with storage at RT as in previous studies, which showed a PLT decrease independent of analyzer technology. The decrease was reported to be more pronounced with impedance[5, 21, 23, 25] or XT-2000iV optical measurement than with the ADVIA 120 flow cytometer.[9, 22, 24] In the present study, the decrease in PLT count was comparable between impedance and optical measurements.
The decreases in PLT-I and PLT-O were associated with increases in MPV, P-LCR and PDW, a right shift on the PLT-I histogram, dispersion of the platelet dot plot on the PLT-O scattergram and decrease in PCT at T48. The right shift and the increase of P-LCR could be explained by the increase in MPV previously reported.[9, 26, 27] In addition, the upper limit of MPV measurement by XT-2000iV is 30 fL. This explains why no results were obtained for PCT, MPV, PDW, and P-LCR in the 13 dogs with macroplatelets. This increase in PLT volume was associated with an overlap of the RBC-I and PLT-I histograms, and could also be observed on the PLT-I histograms, which did not return to the baseline value after T24. It can also be hypothesized that PLT aggregation or PLT ghosts occurred during storage. It would have been interesting to verify these hypotheses on blood films at T24 and T48. In conclusion, PLT and PLT- derived variables obtained by impedance or optical technology in specimens from healthy and diseased dogs were difficult to interpret after 24 and 48 hours of storage at RT. The results obtained from dogs with significant hematologic abnormalities were even less reliable.
The stability of RET has been little studied in veterinary clinical pathology. The RET percentages measured in healthy dogs with the XT-2000iV were mildly to markedly increased at 4°C or 22°C at T24 and T48.[5, 22] In people,[19, 28, 29] RET were shown to be stable at 4°C up to 72 hours and unstable at RT, with decreasing counts from T24 on. In the current study, a slight increase in absolute RET count was observed, except in 3 cases which had a high RET count at T0 and showed an marked increase with time. No explanation could be found for these changes.
As previously reported with different storage conditions and analyzers, WBC counts were stable at RT from T24 onward and the differential WBC counts were slightly modified.[5, 7, 9, 24, 30] As previously described in dogs[5, 6] and people[7, 8], monocyte counts decreased during storage. It could be seen on the scattergrams that the samples lacked a clear separation of lymphocytes and monocytes. In one human study, the alterations of Sysmex NE-8000 differential WBC scattergrams with storage concluded in a poor correlation of the monocyte counts because of poor separation between monocytes and granulocytes. Special attention should therefore be paid to WBC differential dot plots location, which differed according to the analyzer and animal species. This could account for the observed discrepancy and would warrant a study of blood storage effects with each new analyzer and for each species.
In conclusion, delayed analysis of canine blood specimens stored at RT by the XT-2000iV analyzer led to increases in MCV and HCT, and to decreases in MCHC, PLT, and monocyte counts. RBC, MCH, HGB, RET counts, WBC, neutrophil, lymphocyte and eosinophil counts remained stable. The impact of storage according to health status had no effect on the clinical interpretation of the results. Most discrepancies were observed in dogs with severe clinical illness and when abnormalities could be observed on the blood smear or scattergrams, emphasizing that in hematology it is not sufficient to only consider numerical results.
The Sysmex analyzer used in this study was a free loan by Sysmex Europe GmbH.
Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.