Artifactual Changes in Equine Blood Following Storage, Detected Using the Advia 120 Hematology Analyzer
Abstract
Background — Delayed analysis of blood samples may be caused by restricted access to laboratories. Artifactual changes may occur in the measured analytes as a consequence of delayed analysis and may complicate interpretation of the data.
Objective — The purpose of this study was to characterize artifactual changes in equine blood, due to storage, using the Advia 120 hematology analyzer.
Methods — Samples of blood from 5 horses were analyzed using the Advia 120 soon after collection and again after 24 and 48 hours of storage at either 4°C or ambient laboratory temperature (∼24°C).
Results — Delayed analysis of equine blood samples resulted in increased numbers of normocytic hypochromic RBCs, increased numbers of macrocytic hypochromic RBCs, misclassification of granulocytes as mononuclear cells using the basophil reagent method, and pseudothrombocytosis, due to misclassification of ghost RBCs as platelets. The latter artifact was corrected by an amended version of the software. Many of the artifactual changes were identified by morphology flags.
Conclusion — Characteristic changes in cytograms produced by the Advia 120 allowed recognition of artifactual changes in stored equine blood samples. These changes were less pronounced in samples stored at 24°C than at 4°C.
Delayed analysis of blood samples may be caused by restricted access to laboratories due to either geographical remoteness or the operating hours of the laboratory. In many places, samples collected by practicing veterinarians may take more than 24 hours from the time of collection until analysis at the laboratory. The Advia 120 (Bayer Corporation, Tarrytown, NY, USA) is a hematology analyzer that employs laser light-scattering flow cytometry based on methods developed for the Technicon H1 hematology analyzer (Bayer Corp). The characteristics of horse blood, when assessed using the Technicon H1, have been published.1 However, the stability of equine samples and changes in numerical data and cytograms have not previously been reported. We undertook this study to determine whether changes occurred in measured hematological characteristics following storage of equine blood samples.
Materials and Methods
Samples of blood were collected, by jugular venipuncture, from 5 horses (2 Thoroughbred mares, 2 Standardbred mares, and 1 Thoroughbred gelding) maintained at Massey University in accordance with institutional animal ethics guidelines. Samples were mixed with EDTA in commercial tubes (Becton Dickinson, Rutherford, NJ, USA) and were initially analyzed within 1 hour of collection using the Advia 120 hematology analyzer and multispecies software (Version 1.01.07). The samples were then divided: one aliquot was stored at 4°C and the other aliquot was stored at ambient laboratory temperature (about 22–25°C; hereafter referred to as 24°C). Samples stored at 4°C were warmed to room temperature prior to analysis. Each sample was analyzed again at 24 and 48 hours postcollection.
The following analytes were measured: WBC concentration, RBC concentration, hemoglobin concentration, HCT, MCV, MCH, MCHC, red cell distribution width (RDW), hemoglobin distribution width (HDW), platelet concentration, mean platelet volume (MPV), platelet distribution width (PDW), and automated WBC differential count, including neutrophils, lymphocytes, monocytes, eosinophils, basophils, and large unstained cells (LUC). Automated WBC differential counts and cytograms were obtained from the peroxidase channel. Neutrophil (BASO%PMN) and mononuclear cell (BASO%MN) counts and cytograms also were obtained from the basophil channel. In addition, results for the following morphology flags were recorded: macrocytosis, microcytosis, anisocytosis, hypochromasia, hyperchromasia, hemoglobin content (HC) variation, large platelets, and platelet clumps. Cytograms were also obtained for RBC volume/hemoglobin concentration (V/HC) and platelet scatter.
Numerical data were expressed as mean± SD or minimum and maximum values. At the completion of all blood analyses, the manufacturer made a modification to the analysis software. Stored, raw data from the initial analyses were reanalyzed using the updated software (V2.206). Because of small sample size, statistical analyses were not done.
Results
There were no apparent changes in most hematologic values (Table 1), however changes were observed in the RBC V/HC cytograms from all 5 horses after storage (Figure 1). Increased numbers of normocytic hypochromic RBCs and macrocytic hypochromic RBCs were observed (Figure 1, Table 2). Changes in RBC subpopulations were most pronounced after 48 hours of storage at 4°C, with 4.2%-15.7% normocytic hypochromic RBCs and 0.4%-2.7% macrocytic hypochromic RBCs. No changes were observed in MCV or MCHC values.
| Analyte* | Initial Analysis | 4°C | 24°C | ||
|---|---|---|---|---|---|
| 24 Hr | 48 Hr | 24 Hr | 48 Hr | ||
| WBC(×109/L) | 8.8 ± 1.2 | 9.0 ± 1.3 | 9.0 ± 1.5 | 8.9 ± 1.6 | 8.9 ± 1.2 |
| RBC(×1012/L) | 7.7 ± 0.7 | 7.5 ± 0.6 | 7.4 ± 0.5 | 7.7 ± 0.6 | 7.6 ± 0.5 |
| Hemoglobin (g/L) | 129 ± 10 | 128 ± 10 | 128 ± 10 | 129 ± 10 | 129 ± 9 |
| HCT (L/L) | 0.34 ± 0.04 | 0.34 ± 0.03 | 0.34 ± 0.03 | 0.35 ± 0.03 | 0.35 ± 0.04 |
| MCV (fL) | 45.0 ± 2.2 | 45.5 ± 2.5 | 46.2 ± 2.8 | 45.3 ± 2.4 | 46.4 ± 2.7 |
| MCH (pg) | 16.9 ± 1.0 | 17.2 ± 0.9 | 17.4 ± 0.9 | 16.8 ± 1.0 | 17.0 ± 0.8 |
| MCHC (g/L) | 380 ± 10 | 378 ± 10 | 378 ± 11 | 370 ± 11 | 367 ± 14 |
| RDW (%) | 19.3 ± 0.9 | 20.4 ± 1.0 | 21.8 ± 1.3 | 19.4 ± 0.8 | 19.5 ± 0.7 |
| HDW (g/L) | 18.1 ± 2.0 | 27.4 ± 5.3 | 34.3 ± 6.5 | 19.5 ± 2.6 | 20.6 ± 3.2 |
| Platelet (×109/L) | 140 ± 23 | 165 ± 25 | 274 ± 26 | 145 ± 25 | 139 ± 26 |
| MPV (fL) | 6.9 ± 1.1 | 10.8 ± 3.7 | 22.0 ± 3.5 | 7.9 ± 0.8 | 10.4 ± 3.0 |
| PDW (%) | 64.4 ± 8.3 | 107.0 ± 10.9 | 80.0 ± 13.8 | 84.0 ± 16.0 | 96.0 ± 19.7 |
| Neutrophils(×109/L) | 4.3 ± 1.0 | 4.6 ± 1.1 | 4.9 ± 1.4 | 4.4 ± 1.0 | 4.4 ± 1.2 |
| Lymphocytes (×109/L) | 3.5 ± 0.9 | 3.4 ± 0.9 | 3.2 ± 0.9 | 3.3 ± 0.8 | 3.4 ± 0.9 |
| Monocytes (×109/L) | 0.5 ± 0.1 | 0.5 ± 0.04 | 0.5 ± 0.1 | 0.7 ± 0.4 | 0.5 ± 0.1 |
| Eosinophils(×109/L) | 0.3 ± 0.1 | 0.3 ± 0.2 | 0.2 ± 0.1 | 0.2 ± 0.1 | 0.2 ± 0.2 |
| Basophils(×109/L) | 0.06 ± 0.05 | 0.10 ± 0.05 | 0.10 ± 0.04 | 0.40 ± 0.20 | 0.30 ± 0.20 |
| LUC(×109/L) | 0.08 ± 0.04 | 0.10 ± 0 | 0.10 ± 0.04 | 0.10 ± 0 | 0.10 ± 0 |
| BASO%PMN | 46.8 ± 5.9 | 24.8 ± 3.9 | 14.3 ± 7.0 | 24.1 ± 6.9 | 36.7 ± 9.6 |
| BASO%MN | 51.8 ± 5.9 | 74.2 ± 4.0 | 84.5 ± 7.5 | 71.3 ± 5.9 | 59.7 ± 8.7 |
- *See text for abbreviations.
Representative cytograms from the Advia 120 illustrating changes in equine blood stored for up to 48 hours at 4°C or 24°C. (A) Initial analysis (within 1 hour of collection). (B) Analysis after 24 hours at 4°C. Note the subpopulation of ghost cells, which appear as nor-mocytic hypochromic RBC (left panel, arrow) and in the platelet cytogram (right panel, arrow). (C) Analysis after 48 hours at 4°C. Changes are more pronounced than those at 24 hours. (D) Analysis after 24 hours at 24°C. Note the smaller subpopulation of normocytic hypochromic RBC compared with that in the sample stored 24 hours at 4°C. (E) Analysis after 48 hours at 24°C.
| RBC Subpopulation | Initial Analysis | 4°C | 24°C | ||
|---|---|---|---|---|---|
| 24 Hr | 48 Hr | 24 Hr | 48 Hr | ||
| Normocytic normochromic (%RBCs) | 99.2–99.5 | 88.6–96.8 | 81.1–95.1 | 98.2–99.2 | 97.0–99.2 |
| Macrocytic normochromic (%RBCs) | 0–0.4 | 0.1–0.7 | 0–0.6 | 0.1–0.7 | 0–1.2 |
| Macrocytic hypochromic (%RBCs) | 0 | 0–0.9 | 0.4–2.7 | 0–0.1 | 0–0.3 |
| Normocytic hypochromic (%RBCs) | 0 | 2.2–9.8 | 4.2–15.7 | 0.2–1 | 0.2–1.10 |
| Ghost RBCS(×1012/L) | 0–0.01 | 0–0.02 | 0–0.01 | 0–0.02 | 0–0.01 |
There were no changes in differential WBC counts obtained by the peroxidase channel after storage at either temperature (Table 1). The total WBC concentration measured by the basophil reagent method also did not change with sample storage, however, more granu-locytes were classified as mononuclear cells after storage of the sample (at both temperatures).
Platelet concentrations obtained initially and after 24 hours of storage at either 4° or 24°C were not noticeably different (Table 1). However, after 48 hours of storage at 4°C, platelet concentration was higher than the initial value. The MPV increased from 6.9 fL to 10.4 fL after 48 hours of storage at 24°C and to 22.0 fL after 48 hours of storage at 4°C.
A number of morphology flags were noted in the results from stored samples. No morphology flags were observed when samples were initially analyzed. After 24 hours of storage at 24°C, 1+ platelet clumps (2/5 samples) and 1+ anisocytosis (1/5) flags were evident. After 24 hours of storage at 4°C, 2+ hypochromasia (1/5), 1+ anisocytosis (3/5), 1+ HC variation (5/5), and 2–3+ large platelets (2/5) flags were evident. After 48 hours of storage at 24°C, 3+ large platelets (2/5), 1+ platelet clumps (5/5), and 2+ HC variation (1/5) flags were evident. After 48 hours of storage at 4°C, 1+ macrocytosis (2/5), 1–3+ hypochromasia (5/5), 2–3+ anisocytosis (4/5), 3+ large platelets (5/5), 1+ platelet clumps (2/5), and 3+ HC variation (5/5) flags were seen.
Platelet values obtained following the software update were different than those obtained initially (Table 3). The artifactual increase in platelet concentration observed after 48 hours of storage at 4°C (274± 26 ×109/L) was no longer evident in the re-analyzed data (160± 44 ×109/L). There was no change in distribution of RBC subpopulations in RBC V/HC cytograms following re-analysis of the data.
| Analyte* | Initial Analysis | 4°C | 24°C | ||
|---|---|---|---|---|---|
| 24 Hr | 48 Hr | 24 Hr | 48 Hr | ||
| Platelets (×109/L) | 142 ± 2713 | 148 ± 34 | 160 ± 44 | 142 ± 27 | 130 ± 30 |
| MPV (fL) | 6.6 ± 1.2 | 5.8 ± 1.0 | 5.4 ± 0.4 | 6.3 ± 0.5 | 7.2 ± 2.4 |
| PDW (%) | 51.3 ± 14.8 | 43.0 ± 28.6 | 31.5 ± 4.0 | 32.6 ± 13.6 | 43.3 ± 38.5 |
- *See text for abbreviations.
Discussion
The Advia 120 uses laser light-scattering flow cytometry to assess RBCs and platelets by both size and refractive index. The characterization of RBCs by both cell volume and hemoglobin concentration allows subpopulations of RBCs to be recognized. Detection of small RBC subpopulations may help identify a disease process that is not detected by changes in RBC indices.2 In the present study, artifactual changes in a subpopulation of RBCs were detected following sample storage. These normocytic hypochromic cells likely had partial loss of hemoglobin following altered membrane permeability and may be considered ghost cells, which need not be totally lysed or have lost their entire hemoglobin content. The formation of ghost RBCs in horse blood was temperature dependent and more pronounced at 4°C. This was in contrast with results of studies showing that human blood stored at 4°C had fewer artifactual changes than blood stored at room temperature.3–4
Individuals using the Advia 120 need to recognize the pattern of ghost cell formation as an artifact rather than as a regenerative response to anemia. Horses with regenerative anemia typically have increased numbers of macrocytic normochromic RBCs when assessed using the Advia 120.5
The automated WBC differential counts from the peroxidase channel did not notably change during the 48–hr storage period. The differential WBC results generated by the basophil method, however, did change. The observed increase in the proportion of mononu-clear cells probably resulted from decreased chromatin density in deteriorating granulocytes and subsequent misclassification as mononuclear cells. The Advia 120 analyzer reports the differential WBC count results from the former method, and flags any discrepancy between the 2 methods.
The Advia 120 uses size and refractive index to distinguish platelets from RBCs.6 Platelets up to 60 fL can be distinguished from RBCs. This new capacity is a distinct advantage in assessing platelets but also makes possible the false identification of larger particles as platelets. The increase in platelet concentration after 48 hours at 4°C was due to misclassification of ghost RBCs as platelets, resulting in artifactual thrombocytosis. The updated software largely corrected this problem. The MPV increase in stored samples prior to software modification was probably due to a combined effect of the classification of ghost cells as platelets, and platelet clumping during storage.
Delayed analysis of equine blood samples produced artifactual changes that were detected by the Advia 120 analyzer and which were easily recognized from the graphical display. When sample analysis is delayed, care must be taken to not misinterpret these changes as a pathologic change. If delayed submission or analysis is likely to occur, samples of equine blood should be stored at room temperature.
