Diagnostic accuracy of using erythrocyte indices and polychromasia to identify regenerative anemia in dogs

Joanne Hodges Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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 DVM, DACVP
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Mary M. Christopher Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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 DVM, PhD, DACVP

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Abstract

Objective—To determine diagnostic accuracy of using erythrocyte indices and polychromasia to identify regenerative anemia in dogs.

Design—Retrospective and prospective cross-sectional study.

Animals—4,521 anemic dogs.

Procedures—CBC results obtained between July 2002 and July 2008 by use of an automated laser-based flow cytometric hematology analyzer from dogs with Hct values ≤ 35% were retrieved. Sensitivity, specificity, accuracy, and predictive values of using erythrocyte indices and polychromasia to identify regeneration were determined, with a reticulocyte count > 65,000 reticulocytes/μL considered the gold standard. Similarly, 134 blood samples from anemic dogs were analyzed prospectively with an in-house electrical impedance analyzer.

Results—Of 4,387 dogs with samples analyzed retrospectively, 1,426 (32.5%) had regenerative anemia. Of these, 168 (11.8%) had macrocytic hypochromic anemia. High mean cell volume and low mean cell hemoglobin concentration had low sensitivity (11%), high specificity (98%), and moderate accuracy (70%) when used to identify regenerative anemia. Use of polychromasia alone had an accuracy of 77%, and use of polychromasia combined with a high RBC distribution width (RDW) had an accuracy of 79%. Results obtained with the in-house analyzer were similar.

Conclusions and Clinical Relevance—Results suggested that most regenerative anemias in dogs were not macrocytic hypochromic. Polychromasia, with or without high RDW, was a more accurate indicator than other erythrocyte indices of regenerative anemia. To avoid a false diagnosis of nonregenerative anemia, a blood smear should be evaluated in anemic dogs when a reticulocyte count is not available.

Abstract

Objective—To determine diagnostic accuracy of using erythrocyte indices and polychromasia to identify regenerative anemia in dogs.

Design—Retrospective and prospective cross-sectional study.

Animals—4,521 anemic dogs.

Procedures—CBC results obtained between July 2002 and July 2008 by use of an automated laser-based flow cytometric hematology analyzer from dogs with Hct values ≤ 35% were retrieved. Sensitivity, specificity, accuracy, and predictive values of using erythrocyte indices and polychromasia to identify regeneration were determined, with a reticulocyte count > 65,000 reticulocytes/μL considered the gold standard. Similarly, 134 blood samples from anemic dogs were analyzed prospectively with an in-house electrical impedance analyzer.

Results—Of 4,387 dogs with samples analyzed retrospectively, 1,426 (32.5%) had regenerative anemia. Of these, 168 (11.8%) had macrocytic hypochromic anemia. High mean cell volume and low mean cell hemoglobin concentration had low sensitivity (11%), high specificity (98%), and moderate accuracy (70%) when used to identify regenerative anemia. Use of polychromasia alone had an accuracy of 77%, and use of polychromasia combined with a high RBC distribution width (RDW) had an accuracy of 79%. Results obtained with the in-house analyzer were similar.

Conclusions and Clinical Relevance—Results suggested that most regenerative anemias in dogs were not macrocytic hypochromic. Polychromasia, with or without high RDW, was a more accurate indicator than other erythrocyte indices of regenerative anemia. To avoid a false diagnosis of nonregenerative anemia, a blood smear should be evaluated in anemic dogs when a reticulocyte count is not available.

Early classification of anemia as regenerative versus nonregenerative is an imperative diagnostic step guiding further clinical evaluation and treatment of anemic dogs.1 Regenerative anemia typically results from erythrocyte destruction (hemolysis) or acute blood loss (hemorrhage), whereas nonregenerative anemia is observed with many types of chronic and inflammatory diseases, neoplasia, bone marrow disorders, and dietary iron deficiency. Iron deficiency anemia caused by chronic blood loss is variably regenerative.1–6 When the hypoxic stimulus is sufficiently severe, reticulocytosis develops in anemic patients with functional bone marrow, whereas patients with dysfunctional bone marrow may fail to respond appropriately to anemia.2,7,8 Diagnostic testing and treatment during the early clinical evaluation of anemic dogs is likely to vary on the basis of type of anemia.

A reticulocyte count is the gold standard test for assessing a regenerative response to anemia in most species, and reticulocytosis is the hallmark of intensified erythropoiesis in the bone marrow3,7–9 Low numbers of reticulocytes (1% of RBCs or < 60,000 to 65,000 reticulocytes/μL) are expected in healthy nonanemic dogs.1,8 In anemic dogs with regeneration, increased numbers of circulating reticulocytes are initially observed 2 to 4 days after bone marrow stimulation by erythropoietin.3,8 The reticulocyte count typically peaks between 4 and 7 days and declines after 2 to 3 weeks as anemia resolves.3 The degree of reticulocytosis and corresponding degree of polychromasia evident on blood smears should correlate inversely with the severity of the anemia. When regeneration is less than expected for the severity of anemia, assessment of the bone marrow may be indicated.10,11 Monitoring the reticulocyte count is also critical for monitoring response to treatment in anemic patients.1,12

A reticulocyte count can be determined manually or via automated methods. Manual counts are performed by means of light microscopic examination of supravitally stained blood smears. However, manual counts are labor-intensive, require high levels of technical precision, and are subject to inaccuracy.7,13,14 In our experience, most practitioners do not count reticulocytes manually and most diagnostic laboratories use automated methods. Reticulocytes can be enumerated automatically with large hematology analyzers that use flow cytometry, providing much more accurate, precise, and cost-effective results.7,13 Automated reticulocyte counts obtained by use of flow cytometry are also available with some, but not all, in-house hematology analyzers.

Most hematology analyzers report erythrocyte indices, including MCV, MCH, MCHC, and RDW. One automated hematology analyzera used in many university and diagnostic laboratories operates on the basis of laser-based flow cytometry to measure erythrocyte indices and other RBC parameters, including reticulocyte count. Another hematology analyzerb commonly used in veterinary practices uses electrical impedance technology. This analyzer, like many in-house analyzers, reports erythrocyte indices without measuring reticulocytes, with the result that erythrocyte indices alone may be used by practitioners to classify anemia as regenerative, when macrocytosis and hypochromasia are identified.

Many reference texts and case examples emphasize the correlation between macrocytosis and hypochromasia and a regenerative response to anemia,4,15,16 and a few studies17–20 have evaluated the use of erythrocyte indices for the classification of anemia in dogs and people. Reticulocytes are larger (macrocytic) than mature erythrocytes and contain less Hgb (hypochromic). Therefore, a regenerative response might result in macrocytosis and hypochromasia when sufficient reticulocytes are present to affect the mean values.3,4,9 However, a regenerative response of lower magnitude would not be expected to affect erythrocyte indices and might go undetected if other methods are not used to evaluate regeneration. The number of polychromatophilic RBCs in a routinely stained blood smear generally parallels the number of aggregate reticulocytes in a new methylene blue–stained smear, and although more subjective, estimation of polychromasia can be used to identify a regenerative response.7–9 To our knowledge, however, most veterinary practices performing in-house hematology analysis do not routinely review blood smears, regardless of the analyzer used.

We hypothesized that the sole use of erythrocyte indices for identification of regenerative anemia would result in substantial underdetection of regeneration in dogs and inappropriate classification of many regenerative anemias as nonregenerative. We also hypothesized that evaluation of polychromasia in a blood smear would be more accurate than use of erythrocyte indices to correctly identify regenerative anemia in dogs. The specific aims of the study reported here were to retrospectively determine the prevalence of macrocytosis and hypochromasia, as determined with a contemporary hematology analyzer, in dogs with regenerative anemia examined at a large referral hospital; determine the diagnostic accuracy and predictive values of using erythrocyte indices (MCV, MCH, MCHC, and RDW) to identify regenerative anemia, with reticulocyte count measured via automated methods as the gold standard; determine the diagnostic accuracy and predictive values of using polychromasia, as assessed semiquantitatively through examination of a blood smear, to identify regenerative anemia; and compare diagnostic accuracy of erythrocyte indices with that of polychromasia. To further assess the applicability of these findings to clinical practices, we also measured these parameters prospectively with an in-house analyzer. It is our hope that the results of this study will better define the prevalence of macrocytosis and hypochromasia in dogs with regenerative anemia and provide guidance regarding the best laboratory methods for identifying regenerative anemia in veterinary practice.

Materials and Methods

Retrospective study—A computerized search of the electronic medical records system of the William R. Pritchard Veterinary Medical Teaching Hospital at the University of California-Davis was conducted to identify CBC results for dogs examined between July 2002 and July 2008 in which Hct was ≤ 35%. All blood samples submitted during the study period had been analyzed with a single automated hematology analyzer.a Results for Hgb concentration, RBC count, Hct, MCV, MCHC, MCH, RDW, reticulocyte count, and an estimate of degree of polychromasia were downloaded into a spreadsheet program,c and data were verified for accuracy. Information on patient age, sex, and breed was also downloaded. Degree of polychromasia was determined by licensed medical technologists at the time of sample analysis through examination of a blood smear. Degree of polychromasia was reported as 0 (none), 1+ (rare [ie, ≤ 1 polychromatophil/hpf {100× magnification}]), 2+ (slight [ie, 2 to 7 polychromatophus/hpf]), 3+ (moderate [ie, 8 to 15 polychromatophils/hpf]), or 4+ (marked [ie, 15 polychromatophils/hpf]).21 The reticulocyte count measured with the automated hematology analyzera was used as the gold standard for classifying anemia as nonregenerative (≤ 65,000 reticulocytes/μL [ie, the laboratory's upper reference limit for reticulocyte count in dogs]), mildly regenerative (65,001 to 150,000 reticulocytes/μL), moderately regenerative (150,001 to 300,000 reticulocytes/μL), or markedly regenerative (> 300,000 reticulocytes/μL).4,9

Complete blood count results were included in the study if the Hct was ≤ 35% because this was the cutoff used by the laboratory during the study period to trigger an automatic reticulocyte count via an automated method. For dogs with multiple CBCs that met the inclusion criteria during the study period, all but the first CBC were excluded from analysis. In addition, samples were excluded if ≥ 1 of the CBC results was not reported. Samples from breeds with hereditary macrocytosis (Greyhounds and Italian Greyhounds) or microcytosis (Akitas and Shiba Inus) were excluded from analysis.22–24 Samples from dogs < 6 months of age were also excluded because of potential age-related differences in erythrocyte indices and reticulocyte counts and lack of age-specific reference intervals.

Prospective study—From September 2008 through October 2008, blood samples from dogs that were submitted to the Veterinary Medical Teaching Hospital Hematology Laboratory for a CBC were first analyzed by use of an automated hematology analyzera and, if the Hct was ≤ 35%, were subsequently analyzed by use of an in-house analyzer.b Samples were stored at 4°C and gently remixed prior to analysis with the in-house analyzer; all samples were analyzed with the in-house analyzer within 8 hours after analysis with the automated hematology analyzer. Additional blood samples from dogs that were submitted from January 2009 through March 2009 and had reticulocyte counts > 150,000 re-ticulocytes/μL were selectively included in the study. Samples with insufficient volume for both analyses were excluded. As for the retrospective study, results for only the first CBC were included for dogs that had multiple CBCs performed during the study period. The in-house analyzer was calibrated daily, and known blanksd were analyzed weekly for quality control purposes, in accordance with the manufacturer's recommendations. Complete blood count data were downloaded into a spreadsheet program.c

Statistical analysis—Retrospective data were analyzed by use of the Shapiro-Wilk test for normality and the box plot outlier test.e Outliers were identified as values > 3 times the interquartile range. Because not all data sets were normally distributed, nonparametric statistical tests were used, and results were reported as median, interquartile range (ie, 25th and 75th percentiles), and 90% confidence interval. The Mann-Whitney test was used to compare RBC parameters (Hct, RBC count, Hgb concentration, MCV, MCH, MCHC, and RDW) between dogs with nonregenerative versus regenerative anemia. The Kruskal-Wallis test followed by a Bonferroni post hoc adjustment was used to compare values among dogs with nonregenerative anemia and dogs with mild, moderate, or marked reticulocytosis. Values of P < 0.05 were considered significant.

Sensitivity, specificity, accuracy, PPV, and NPV of using MCV, MCH, MCHC, RDW, combined high MCV and low MCHC, combined high MCV and high RDW, polychromasia, and combined polychromasia and high RDW to detect regenerative anemia were calculated, with the reticulocyte count measured with the automated hematology analyzer as the gold standard. Whether results obtained with the automated hematology analyzer were normal or abnormal was determined by comparison with the lower and upper reference limits established by the laboratory. Results obtained with the in-house analyzer were analyzed on the basis of analyzer-specific canine reference intervals developed in our laboratory. When evaluating diagnostic performance of combined high MCV and low MCHC, MCHC, rather than MCH, was used because MCHC takes into account variations in erythrocyte volume and thus is considered to be more accurate.4 When evaluating diagnostic performance of polychromasia, slight to marked (+2 to +4) polychromasia was considered indicative of regenerative anemia and none or rare (0 to +1) polychromasia was considered indicative of nonregenerative anemia.

Results

Retrospective population characteristics—Results of 45,246 CBCs performed with the automated hematology analyzer were retrieved with the retrospective electronic medical records search. Of these, 40,030 were excluded because Hct was > 35% (n = 32,164), they represented multiple CBCs performed on individual dogs (6,155), or data were missing (1,711; most often, this involved samples with an Hct of 35% without a concurrent reticulocyte count). Results of an additional 820 CBCs were excluded because they had been obtained from Greyhounds (n = 124), Italian Greyhounds (40), Akitas (189), Shiba Inus (76), or dogs < 6 months of age (391). Results of 9 CBCs with outliers were also excluded. Six of these samples had markedly high MCHC or MCH secondary to severe hemolysis or oxyglobin treatment; 2 had markedly high MCV because of moderate to marked agglutination, and 1 had a low MCV of undetermined cause. Results of the remaining 4,387 CBCs were included in the study.

Dogs ranged in age from 0.6 months to 24 years (mean ± SD, 8.5 ± 4.0 years); age was not reported for 86 dogs. There were 372 sexually intact females, 1,835 spayed females, 527 sexually intact males, and 1,651 castrated males; sex was not reported for 2 dogs. Overall, 1,025 (23%) of the dogs were of mixed breeding. The most common breeds were Labrador Retriever (433 [10%]), Golden Retriever (295 [6.7%]), and Cocker Spaniel (128 [3%]). Other breeds represented in the study included Rottweiler (n = 162), German Shepherd Dog (106), American Pit Bull Terrier (86), Australian Shepherd (84), Yorkshire Terrier (71), Miniature Schnauzer (68), Shih Tzu (63), Boxer (66), Shetland Sheepdog (53), Pomeranian (53), Border Collie (52), Jack Russell Terrier (50), Chihuahua (50), Beagle (49), Australian Cattle Dog (46), Pug (45), Dachshund (45), Doberman Pinscher (44), Bouvier des Flanders (44), English Springer Spaniel (43), Maltese (43), Bernese Mountain Dog (39), Dalmatian (39), Standard Poodle (37), West Highland White Terrier (36), Pembroke Welsh Corgi (34), English Bulldog (32), Mastiff (31), Siberian Husky (30), Cavalier King Charles Spaniel (29), Brittany (28), Rhodesian Ridgeback (28), Bichon Frise (27), Boston Terrier (25), Toy Poodle (24), German Shorthaired Pointer (23), Miniature Poodle (23), Bassett Hound (22), Chinese Shar-Pei (22), Lhasa Apso (22), Malamute (22), and Chow Chow (22). Other breeds were represented by < 20 dogs each. Breed was unspecified for 3 dogs.

Retrospective hematologic results—Given the definition of regenerative anemia as a reticulocyte count > 65,000 reticulocytes/μL, 2,961 of the 4,387 (67.5%) dogs had nonregenerative anemia and 1,426 (32.5%) had regenerative anemia. Dogs with regenerative anemia had mild (911/1,426 [64%]), moderate (365/1,426 [25.5%]), and marked (151/1,426 [10.5%]) reticulocytosis. Significant differences were found between samples from dogs with nonregenerative anemia and samples from dogs with regenerative anemia for all variables except MCV (Table 1). Severity of anemia and hypochromasia (MCHC) were inversely correlated with degree of reticulocytosis, and severity of macrocytosis (MCV) and anisocytosis (RDW) were directly correlated with degree of reticulocytosis.

Table 1—

Median (range) erythrogram results obtained with an automated hematology analyzer for 4,387 dogs with anemia.

Anemia classificationNo. of dogsHct(%)Hgb(g/dL)RBC count (× 10a cells/μL)MCV (fL)MCH (pg)MCHC (g/dL)RDW(%)Reticulocyte count (cells/μL)
Nonregenerative2,96131.0a (5.0–35.0)10.9a (1.1–16.6)4.6a (0.8–8.0)67.1a (39.5–94.3)23.5a (10.8–39.5)34.9a (23.4–63.0)13.6a (10.5–42.1)26,600 (0–65,000)
Mildly regenerative91130.0b (4.0–35.0)10.2b (0.6–14.1)4.4b (0.4–6.6)67.8b (42.5–103.4)23.2b (4.6–97.6)34.2b (8.3–100.0)15.2b (11.2–39.4)95,700 (65,100–150,000)
Moderately regenerative36427.0c (6.0–35.0)8.9c (2.0–13.1)3.8c (0.7–7.2)70.4c (47.8–104.0)23.7c (15.2–57.0)33.3c (26.0–58.8)173c (12.0–36.9)196,800 (150,010–300,000)
Markedly regenerative15124.0d (7.0–35.0)77d (1.8–12.0)3.3d (0.8–6.0)74.7d (40.8–114.8)23.7d (9.0–29.6)31.6d (22.1–37.4)197d (13.3–38.5)394,000 (300,100–1,080,000)
Regenerative (all)1,42629.0* (4.0–35.0)9.6* (0.6–14.1)4.1* (0.4–7.2)69.0 (40.8–114.8)23.5* (4.6–97.6)33.8* (8.3–100.0)16.0* (11.2–39.4)122,100 (65,100–1,080,000)

Significantly (P < 0.001) different from value for dogs with nonregenerative anemia.

In each column, values with different superscript letters were significantly (P < 0.05) different.

Of the 1,426 dogs with regenerative anemia, 240 (16.8%) had macrocytic anemia, 450 (31.5%) had hypochromic anemia (as determined on the basis of MCHC), and 168 (11.8%) had macrocytic hypochromic anemia. Diagnostic sensitivity, specificity, accuracy, and predictive values of using erythrocyte indices and polychromasia to identify regenerative anemia were tabulated (Table 2).

Table 2—

Sensitivity, specificity, diagnostic accuracy, PPV, and NPV of using erythrocyte indices (measured with an automated hematology analyzer) and polychromasia (determined by evaluation of a blood smear) to identify anemia as regenerative versus nonregenerative in 4,387 doqs with Hct ≤ 35%.

VariableCutoff valueTPSensitivity (%)TNSpecificity (%)TP + TNDiagnostic accuracy (%)PPV (%)NPV (%)
MCV> 75 fL240172,831963,071706571
MCHC< 33 g/dL450322,691913,141666273
MCH< 22 pg317222,545862,862654370
RDW> 14%1,194841,830623,024695189
MCV and RDW> 75 fL and > 14%225162,905983,130718071
MCV and MCHC> 75 fL and <33 g/dL163112,916983,079707870
Polychromasia≥ 2+1,262892,102713,364775993
RDW and polychromasia> 14% and ≥ 2+1,094772,363803,457796588

Of the 4,387 dogs, 1,426 were classified as having regenerative anemia on the basis of reticulocyte count > 65,000 reticulocytes/μL (ie, the gold standard) and 2,961 were classified as having nonregenerative anemia. Sensitivity was calculated as (TP/1,426) × 100; specificity was calculated as (TN/2,961) × 100; diagnostic accuracy was calculated as ([TP + TN]/4,387) × 100; PPV was calculated as (TP/[TP + false positives]) × 100; and NPV was calculated as (TN/[TN + false negatives]) × 100.

Prospective population characteristics—For the prospective study, which involved samples analyzed with the in-house analyzer, 210 samples were submitted during the initial study period and 19 samples were selected for analysis during the second study period. After samples with incomplete data (n = 14) and multiple samples from the same patient (81) were excluded, 134 samples were included in the study. No samples were excluded because of breed or age of the dog.

Dogs ranged in age from 6 months to 17 years (mean ± SD, 8.2 ± 4.3 years) and included 14 sexually intact females, 51 spayed females, 15 sexually intact males, and 54 castrated males. There were 26 mixed-breed dogs, 13 Labrador Retrievers, 11 Golden Retrievers, and 81 dogs of other breeds. Breed was unspecified for 3 dogs.

Prospective hematologic results—Given the definition of regenerative anemia as a reticulocyte count determined with the automated hematology analyzer of > 65,000/μL, 86 of the 134 (64%) dogs had nonregenerative anemia and 48 (36%) had regenerative anemia. Dogs with regenerative anemia had mild (27/48 [56%]), moderate (14/48 [29%]), or marked (7/48 [15%]) reticulocytosis. Of the 48 dogs with regenerative anemia, 7 (14.6%) had macrocytic anemia, 2 (4.2%) had hypochromic anemia (as determined on the basis of MCHC), and none had macrocytic hypochromic anemia, as determined on the basis of erythrocyte indices obtained with the in-house analyzer. Analysis of the same 48 samples with the automated hematology analyzer revealed that 9 (18.8%) dogs with regenerative anemia had macrocytic anemia, 18 (37.5%) had hypochromic anemia (as determined on the basis of MCHC), and 8 (16.7%) had macrocytic hypochromic anemia. Diagnostic sensitivity, specificity, accuracy, and predictive values of erythrocyte indices obtained with the in-house analyzer and polychromasia evaluated by examination of blood smears were tabulated (Table 3).

Table 3—

Sensitivity, specificity, diagnostic accuracy, PPV, and NPV of using erythrocyte indices (measured with an in-house analyzer) and polychromasia (determined by evaluation of a blood smear) to identify anemia as regenerative versus nonregenerative in 134 dogs with Hct ≤35%.

VariableCutoff valueTPSensitivity (%)TNSpecificity (%)TP + TNDiagnostic accuracy (%)PPV (%)NPV (%)
MCV> 71 fL715849891687888
MCHC< 33 g/dL2486100886610065
MCH< 22 pg817738581603865
RDW> 17%3471606994705781
MCV and RDW> 71 fL and > 17%4886100906710066
MCV and MCHC> 71 fL and < 33 g/dL00000000
Polychromasia≥2+45946473109816595
RDW and polychromasia> 17% and ≥ 2+32678194113848784

Of the 134 dogs, 48 were classified as having regenerative anemia on the basis of reticulocyte count (determined with an automated hematology analyzer) > 65,000 reticulocytes/μL (ie, the gold standard) and 86 were classified as having nonregenerative anemia. Sensitivity was calculated as (TP/48) × 100; specificity was calculated as (TN/86) × 100; diagnostic accuracy was calculated as ([TP + TN]/134) × 100; PPV was calculated as (TP/[TP + false positives]) × 100; and NPV was calculated as (TN/[TN + false negatives]) × 100.

Discussion

Results of the present study suggested that in dogs with Hct ≤ 35%, the use of MCV and MCHC alone to classify the anemia as regenerative or nonregenerative resulted in significant underdetection of a regenerative response, supporting our initial hypothesis. Only about 1 in 10 dogs in the present study with regenerative anemia had macrocytic hypochromic anemia. Although the PPV of using macrocytosis and hypochromasia to identify regenerative anemia was high, the sensitivity was low, such that between 30% and 33% of dogs with regenerative anemia would have been misclassified as having nonregenerative anemia on the basis of this combination of erythrocyte indices. For both analyzers in the present study, use of high RDW to identify regenerative anemia had higher diagnostic sensitivity than did MCV and MCHC but lower specificity. However, combining high RDW with detection of polychromasia yielded the highest diagnostic accuracy. Polychromasia, alone or together with RDW, was a more accurate indicator of a regenerative response than were other erythrocyte indices, which supported our second hypothesis. On the basis of our findings, blood smear examination is indicated for anemic dogs when a reticulocyte count is not available. Blood smear examination has previously been shown to be more sensitive than erythrocyte indices for detecting low numbers of macrocytic, polychromatophilic, or hypochromic RBCs.3

Our results were obtained from a population of anemic dogs examined at a tertiary referral hospital, which may have been more likely to have certain types of diseases that could bias the results of this study. However, the prevalence of macrocytic hypochromic regenerative anemia and the sensitivity and specificity of these indices were similar to those reported in a preliminary studyf of canine samples submitted by veterinary practices to a large regional diagnostic laboratory. Therefore, the diagnostic accuracy and predictive values of the erythrocyte indices in our study population are likely applicable to other canine populations and in fact may represent the best-case scenario for detection of a regenerative response to anemia, further emphasizing the need for blood smear review or reticulocyte counts when assessing anemia.

We used slight (2+) instead of moderate (3+) polychromasia as a cutoff for a regenerative response to anemia to optimize both sensitivity and specificity. Although low numbers of polychromatophilic RBCs (≤ 1 polychromatophil/hpf) may be seen in nonanemic dogs and in dogs with nonregenerative anemia,1,9,21 we do not expect slight polychromasia (2 to 7 polychromatophils/hpf) without a physiologic or pathological demand for increased erythropoiesis in dogs with functioning bone marrow. In our study, for example, 2,110 of the 2,961 (71%) dogs with nonregenerative anemia (as determined on the basis of reticulocyte count) had ≤ 1 polychromatophil/hpf. If the cutoff for a regenerative response to anemia was increased to moderate polychromasia (8 to 15 polychromatophils/hpf), specificity would be increased (from 71% to 99.2%), but sensitivity would be decreased (from 89% to 29%). Therefore, in practice, ≥ 2 polychromatophils/hpf is a more diagnostically useful cutoff for identifying a regenerative response to anemia.

Although grading of polychromasia is semiquantitative and may be affected by operator variability, it is not altered by many of the preanalytic, analytic, and disease-related factors that can influence MCV, MCHC, MCH, and RDW, such as agglutination and hemoglobinemia. All blood smears in our hospital were reviewed by licensed medical technologists, with the result that diagnostic accuracy of polychromasia may have been higher in this study than it is in clinical practice, where blood smears may be reviewed by less qualified or less experienced staff. However, evaluation of polychromasia can be standardized, and grading guidelines are available.21

The low sensitivity of MCV, MCH, and MCHC for detecting regenerative anemia likely reflects the inability of these indices to detect low numbers of macrocytic or hypochromic RBCs. If dogs with marked reticulocytosis (> 300,000 reticulocytes/μL) were considered separately, 41% (62/151) had macrocytic hypochromic anemia, consistent with the larger number of immature RBCs. False increases in MCH and MCHC resulting from in vivo or in vitro hemolysis; spectral interference resulting from lipemia, severe icterus, or Heinz bodies; or previous oxyglobin treatment could have contributed to the low sensitivity of these indices. The 6 excluded samples with markedly high MCHC or MCH were from dogs that had intravascular hemolysis or had received oxyglobin, and given the wide range of results for these indices, especially among dogs with mild regeneration, it is likely that other samples contained extracellular Hgb, even if they did not qualify as outliers.

To minimize false-negative results for MCV, known Asian breeds with microcytosis were excluded from the study; however, anecdotal reports3,23,24 suggest that Chow Chows, Chinese Shar-Peis, and Japanese mongrel dogs also may have microcytic erythrocytes. Although we did not exclude these breeds, any potential effect on the sensitivity of MCV was likely small because of the low number of dogs of these breeds (n = 44).

Red blood cell distribution width may be more sensitive than MCV for detection of regenerative anemia because it increases with mixed populations of macrocytic and microcytic erythrocytes, whereas MCV may be normal. High RDW and normal MCV may occur when a regenerative response to anemia occurs concomitantly with microcytosis, as with chronic iron deficiency5,18,26 and portosystemic shunting.27 Of the 1,194 dogs with regenerative anemia and high RDW, 277 (23%) had a low MCV (< 65 fL) and 707 (60%) had an MCV within reference limits (65 to 75 fL), suggestive of concurrent macrocytosis and microcytosis. However, because RDW also increases with mixed populations of normocytic and microcytic erythrocytes,17,20,26 it was less specific for detection of a regenerative response, compared with MCV, MCH, and MCHC. The combined use of RDW and MCV could help distinguish the size of erythrocytes in mixed populations. In a study20 that used manual reticulocyte counts as the gold standard, 72% (156/217) of dogs with anemia were accurately categorized as having regenerative or nonregenerative anemia on the basis of high RDW and high MCV, similar to results in the present study (71%). However, the combined use of RDW and polychromasia resulted in even higher diagnostic accuracy (77%). Therefore, examination of a blood smear is indicated to verify whether polychromasia is present and whether high RDW is a result of microcytosis, macrocytosis, or both.

The specificity of macrocytosis for regenerative anemia was high in this study because, other than reticulocytosis, only a few uncommon conditions in dogs cause high MCV and could have caused false-positive results. These conditions include dyserythropoiesis28; hereditary stomatocytosis in Alaskan Malamutes, Drentse Patrijshonds, and Schnauzers29; and hereditary marrow dyscrasia in Miniature and Toy Poodles.30 Because the latter 2 disorders are rare,29,30 we did not exclude dogs of breeds in which these conditions have been reported. A false increase in MCV also can occur with erythrocyte agglutination,31 as was seen in 2 of the 9 samples excluded because of outliers in the retrospective portion of the present study. In the prospective portion of the present study, 1 of 4 samples with agglutination had macrocytosis but also had marked reticulocytosis. Red blood cell agglutination is typical of immune-mediated anemia, which can be highly regenerative.1,32 A significant difference in MCV was found between dogs with regenerative anemia and dogs with nonregenerative anemia in a previous study32 involving 151 dogs with immune-mediated hemolysis, 118 of which had agglutination. We did not exclude all samples in which agglutination may have been present; however, the number of false-positive results was low, suggesting a minimal effect of agglutination.

Analytic methodology may have an effect on erythrocyte indices and, therefore, on their diagnostic accuracy. Past emphasis on macrocytosis and hypochromasia as evidence of a regenerative response was based on early work that used centrifuged microhematocrit tubes to determine PCV and subsequently used PCV to calculate MCV (PCV/RBC) and MCHC (Hgb/PCV).3,33 This contrasts with the methodologies incorporated in the hematology analyzers used in the present study. The automated hematology analyzer that was used incorporates laser-based flow cytometry and the in-house analyzer uses electrical impedance technology to directly measure both RBC count and MCV, which are used to calculate Hct. The Hct is then used to calculate MCHC (Hgb/Hct). Differences between PCV measured on centrifuged samples and calculated Hct may affect the MCHC, altering the sensitivity and specificity of erythrocyte indices determined by use of current technologies. In the present study, hypochromasia was less sensitive for detection of reticulocytosis, as determined with the in-house analyzer rather than the automated hematology analyzer, resulting in more false-negative results for regenerative anemia. Thus, none of the dogs in the prospective portion of the present study were considered to have macrocytic hypochromic anemia on the basis of results obtained with the in-house analyzer, whereas 8 of 48 dogs for which samples were analyzed with the automated hematology analyzer had macrocytic hypochromic anemia. Although both analyzers measure Hgb concentration via the cyanomethemoglobin method, impedance analyzers may underestimate the RBC count when small RBC aggregates are present or when the axis of the RBC is not aligned properly.34 If this occurs, MCH and MCHC may be falsely increased. This might explain the lower sensitivity of these indices when results of the in-house analyzer were examined. However, MCV measured with the in-house analyzer was more, rather than less, specific than MCV measured with the automated hematology analyzer.

In the present study, we used a laboratory-specific reference interval for determining the diagnostic accuracy of erythrocyte indices obtained with the in-house analyzer. For 11 of 36 samples, however, the classification of regenerative anemia would have been different if reference intervals provided by the manufacturer had been used. The in-house analyzer used in the present study does not report a reference interval for RDW, and given the usefulness of this parameter in the present study, development of a reference interval is strongly recommended to optimize interpretation. Regardless, the diagnostic accuracy of RDW was highest when this parameter was used in conjunction with polychromasia. Thus, blood smear review is recommended together with automated analysis for the most accurate classification of anemia when a reticulocyte count is not available.

ABBREVIATIONS

Hgb

Hemoglobin

MCH

Mean cell hemoglobin

MCHC

Mean cell hemoglobin concentration

MCV

Mean cell volume

NPV

Negative predictive value

PPV

Positive predictive value

RDW

RBC distribution width

TN

True negative

TP

True positive

a.

ADVIA 120 hematology analyzer, Siemens Healthcare Diagnostics, Deerfield, Ill.

b.

Abaxis VetScan HM5 hematology analyzer, Abaxis, Union City, Calif.

c.

Excel, Microsoft Corp, Redmond, Wash.

d.

Abaxis VetScan HM5 Control Package, Abaxis, Union City, Calif.

e.

Analyse-it, Analyse-it Software Ltd, Leeds, West Yorkshire, England.

f.

DeNicola DB, Matthews JA, Fernandes PJ, et al. Comparison of reticulocyte counts to mean corpuscular volume and mean corpuscular hemoglobin concentration in anemic dogs (abstr), in Proceedings. 12th Cong Int Soc Anim Clin Biochem 2006;51–52.

References

  • 1.

    Cowgill ESNeel JAGrindem CB. Clinical application of reticulocyte counts in dogs and cats. Vet Clin North Am Small Anim Pract 2003; 33: 12231244.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Lucroy MDChristopher MMKraegel SA, et al. Anaemia associated with canine lymphoma. Comp Haematol Int 1998; 8: 16.

  • 3.

    Jain NC. Hematologic characteristics of anemia, part II: interpretive aspects. Calif Vet 1979; 33: 1518.

  • 4.

    Stockham SLScott MA. Fundamentals of veterinary clinical pathology. 2nd ed. Ames, Iowa: Iowa State University Press, 2008; 107220.

  • 5.

    Weiser GO'Grady M. Erythrocyte volume distribution analysis and hematologic changes in dogs with iron deficiency anemia. Vet Pathol 1983; 20: 112.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Burkhard MJBrown DEMcGrath JP, et al. Evaluation of the erythroid regenerative response in two different models of experimentally induced iron deficiency. Vet Clin Pathol 2001; 30: 7685.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Riley RSBen-Ezra JMGoel R, et al. Reticulocytes and reticulocyte enumeration. J Clin Lab Anal 2001; 15; 267294.

  • 8.

    Laber JPerman VStevens JB. Polychromasia or reticulocytes—an assessment of the dog. J Am Anim Hosp Assoc 1974; 10: 399406.

  • 9.

    Christopher MMHarvey JW. Specialized hematology tests. Semin Vet Med Surg 1992; 7: 301310.

  • 10.

    Weiss DJ. Bone marrow pathology in dogs and cats with non-regenerative immune-mediated haemolytic anaemia and pure red cell aplasia. J Comp Pathol 2008; 138: 4653.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Weiss DJ. A retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital (1996–2004). J Vet Intern Med 2006; 20: 955961.

    • Search Google Scholar
    • Export Citation
  • 12.

    Piek CJJunius GDekker A, et al. Idiopathic immune-mediated hemolytic anemia: treatment outcome and prognostic factors in 149 dogs. J Vet Intern Med 2008; 22: 366373.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Abbott DLMcGrath JP. Evaluation of flow cytometric counting procedure for canine reticulocytes by use of thiazole orange. Am J Vet Res 1991; 52: 723727.

    • Search Google Scholar
    • Export Citation
  • 14.

    Savage RASkoog DPRabinovitch A. Analytic inaccuracy and imprecision in reticulocyte counting: a preliminary report from the College of American Pathologists Reticulocyte Project. Blood Cells 1985; 11: 97112.

    • Search Google Scholar
    • Export Citation
  • 15.

    Willard MDTvedten H. Small animal clinical diagnosis by laboratory methods. 4th ed. Philadelphia: WB Saunders, 2003.

  • 16.

    Fernandez FBGrindem CB. Reticulocyte response. In: Feldman BEZinkl JGJain NC, eds. Schalm's veterinary hematology. 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2000; 110116.

    • Search Google Scholar
    • Export Citation
  • 17.

    Bessman JDGilmer PRGardner FH. Improved classification of anemias by MCV and RDW. Am J Clin Pathol 1983; 80: 322326.

  • 18.

    Sirdah MTarazi IAl Najjar E, et al. Evaluation of the diagnostic reliability of different RBC indices and formulas in the differentiation of the B-thalassaemia minor from iron deficiency in Palestinian population. Int J Lab Hematol 2008; 30: 324330.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Irwin JJKirchner JT. Anemia in children. Am Lam Physician 2001; 64: 13791386.

  • 20.

    Neiger RHadley DPfeiffer DU. Differentiation of dogs with regenerative and non-regenerative anemia on the basis of their red cell distribution width and MCV. Vet Rec 2002; 150: 431434.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Weiss DJ. Uniform evaluation and semiquantitative reporting of hematologic data in veterinary laboratories. Vet Clin Pathol 1984; 13: 2731.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Shiel REBrennan SFO'Rourke LG, et al. Hematologic values in young pretraining healthy Greyhounds. Vet Clin Pathol 2007; 36: 274277.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Gookin JLBunch SERush LJ, et al. Evaluation of microcytosis in 18 Shibas. J Am Vet Med Assoc 1998; 212: 12581259.

  • 24.

    Fujise HHiga KNakayama T, et al. Incidence of dogs possessing red blood cells with high K in Japan and East Asia. J Vet Med Sci 1997; 59: 496497.

    • Search Google Scholar
    • Export Citation
  • 25.

    Meinkoth JHClinkenbeard KD. Normal hematology of the dog. In: Feldman BFZinkl JGJain NC, eds. Schalm's veterinary hematology. 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2000; 10571063.

    • Search Google Scholar
    • Export Citation
  • 26.

    Weiser MGKociba GJ. Sequential changes in erythrocyte volume distribution and microcytosis associated with iron deficiency in kittens. Vet Pathol 1983; 20: 112.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Center SAMagne ML. Historical, physical examination, and clinicopathologic features of portosystemic vascular anomalies in the dog and cat. Semin Vet Med Surg (Small Anim) 1990; 5: 8393.

    • Search Google Scholar
    • Export Citation
  • 28.

    Weiss DJ. Evaluation of dysmyelopoiesis in cats 34 cases (1996–2005). J Am Vet Med Assoc 2006; 228: 893897.

  • 29.

    Brown DEWeiser MGThrall MA, et al. Erythrocyte indices and volume distribution in a dog with stomatocytosis. Vet Pathol 1994; 31: 247250.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Canfield PJWatson ADJ. Investigations of bone marrow dyscrasia in a poodle with macrocytosis. J Comp Pathol 1989; 101: 269278.

  • 31.

    Porter REWeiser MG. Effect of immune-mediated erythrocyte agglutination on analysis of canine blood using a multichannel blood cell counting system. Vet Clin Pathol 1990; 19: 4550.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Weinkle TKCenter SARandolph JF, et al. Evaluation of prognostic factors, survival rates, and treatment protocols for immune-mediated hemolytic anemia in dogs: 151 cases (1993–2002). J Am Vet Med Assoc 2005; 226: 18691880.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    Schalm OWKaneko JJ. Laboratory notes. Calif Vet 1961; 3: 1618.

  • 34.

    Mohandas NKim YRTycko DH, et al. Accurate and independent measurement of volume and hemoglobin concentration of individual red blood cells by laser light scattering. Blood 1986; 68: 506513.

    • Crossref
    • Search Google Scholar
    • Export Citation
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