Diagnostic performances of manual and automated reticulocyte parameters in anaemic cats

Abstract

Objectives

The objective of this study was to evaluate the diagnostic performances of manual and instrumental measurement of reticulocyte percentage (Ret%), reticulocyte number (Ret#) and reticulocyte production index (RPI) to differentiate regenerative anaemia (RA) from non-regenerative anaemia (NRA) in cats.

Methods

Data from 106 blood samples from anaemic cats with manual counts (n = 74; 68 NRA, six RA) or instrumental counts of reticulocytes (n = 32; 25 NRA, seven RA) collected between 1995 and 2013 were retrospectively analysed. Sensitivity, specificity and positive likelihood ratio (LR+) were calculated using either cut-offs reported in the literature or cut-offs determined from receiver operating characteristic (ROC) curves.

Results

All the reticulocyte parameters were significantly higher in cats with RA than in cats with NRA. All the ROC curves were significantly different (P <0.001) from the line of no discrimination, without significant differences between the three parameters. Using the cut-offs published in literature, the Ret% (cut-off: 0.5%) was sensitive (100%) but not specific (<75%), the RPI (cut-off: 1.0) was specific (>92%) but not sensitive (<15%), and the Ret# (cut-off: 50 × 10³/µl) had a sensitivity and specificity >80% and the highest LR+ (manual count: 14; instrumental count: 6). For all the parameters, sensitivity and specificity approached 100% using the cut-offs determined by the ROC curves. These cut-offs were higher than those reported in the literature for Ret% (manual: 1.70%; instrumental: 3.06%), lower for RPI (manual: 0.39; instrumental: 0.59) and variably different, depending on the method (manual: 41 × 10³/µl; instrumental: 57 × 10³/µl), for Ret#. Using these cut-offs, the RPI had the highest LR+ (manual: 22.7; instrumental: 12.5).

Conclusions and relevance

This study indicated that all the reticulocyte parameters may confirm regeneration when the pretest probability is high, while when this probability is moderate, RA should be identified using the RPI providing that cut-offs <1.0 are used.

Introduction

The differentiation between regenerative anaemia (RA) and non-regenerative anaemia (NRA) may drive further diagnostic or therapeutic procedures. ¹ The identification of RA relies on the quantification of reticulocyte responses. The current literature therefore recommends the use of the absolute number of reticulocytes (Ret#) rather than the reticulocyte percentage (Ret%) or the reticulocyte production index (RPI).^2,3 However, the cut-offs reported in the literature for feline Ret# are variable (eg, 40–60 × 10³/µl) and have been determined using different methods, including laser-based counters, which have a higher analytical sensitivity and provide higher Ret# than manual counts.^2,4–8 Moreover, the Ret# and the Ret% are higher in some feline breeds than in others (the Ret% may be as high as 0.8% in Norwegian Forest cats, 1.2% in Holy Birman cats, 1.9% in Siberian cats and 3.3% in Maine Coon cats; and the Ret# may be as high as 250.00 in Maine Coon cats).^9,10 Additionally, the magnitude of reticulocytosis should inversely correlate with the severity of anaemia.^2,11 In human medicine the RPI has been proposed as a tool to correct the Ret% for the severity of anaemia. ³ The calculation of RPI is based on the maturation time of human circulating reticulocytes, which is higher in RA, when reticulocytes released in blood are younger than in healthy individuals. ³ The maturation time of feline reticulocytes is unknown but it is likely different from other species, as feline erythroid cells have some peculiarities such as a shorter erythrocyte lifespan, a prolonged maturation time of punctate reticulocytes and a weaker maximal response of aggregate reticulocytes. ¹² Independent of the correctness of the formula, we recently demonstrated that the Ret% and the RPI may be used to diagnose canine RA. ¹³ However, no studies on the utility of the three reticulocyte parameters in cats are available.

Hence, this study aimed to assess the diagnostic performances of Ret%, Ret# and RPI counted manually or by a laser-based analyser, for the diagnosis of RA in cats.

Materials and methods

Case selection criteria

The laboratory information system was analysed to retrieve data recorded between January 1995 and January 2013 from anaemic cats. The database included data generated using an impedance counter (SEAC Hemat 8) followed by reticulocyte counts on brilliant cresyl blue-stained smears, or using a laser counter validated in cats (Sysmex XT2000-iV).^14,15 Only aggregate reticulocytes were counted manually, as punctate reticulocytes do not indicate active or recent regeneration in cats. ¹² Moreover, a moderate-to-high correlation between aggregate reticulocyte counts and automated reticulocyte counts with Sysmex has previously been reported.^14,15

The inclusion criteria were the following: presence of anaemia based on the comparison with the reference intervals (RIs) in use at our institution for cats (red blood cell [RBC] count <5.0 × 10³/µl; haematocrit [Hct] <27%; haemoglobin <10 g/dl); availability of manual or instrumental reticulocyte counts; availability of stored glass slides to review the original classification; diagnosis of RA or NRA based on history, diagnostic tests (necropsy, cytology, serum biochemistry, bone marrow cytology, serology/PCR for infectious diseases); or follow-up (recovery within 1 month for RA; no improvement during 1 year follow-up for NRA). Post-haemorrhagic acute anaemia was classified in the RA group when sampling was undertaken at least 5 days after the haemorrhage, or within the NRA group when sampling was carried out 1–2 days after the haemorrhage (preregenerative phase of acute anaemia).

Samples from cats treated with drugs that influence bone marrow activity, or belonging to breeds known to have high reticulocyte counts,^9,10 were excluded from the study.

Calculation of reticulocyte parameters

Based on RBC numbers and on the Ret% generated by manual or instrumental counts, the following parameters were calculated using the formula reported in literature: ³ (i) Ret# = number of erythrocytes × (Ret%/100); (ii) corrected Ret% = Ret% × (Hct/37); (iii) RPI = corrected Ret%/[(–0.05) × Hct + 3.25].

Given that the maturation time of feline reticulocytes is unknown, the RPI was calculated using the formula used in humans, ³ assuming that in cats the maturation time is also longer if younger reticulocytes are released in blood. Moreover, as further support to this assumption, the application of this formula in dogs provided useful information for patient management. ¹³

Statistical analysis

Data from manual and instrumental counts were analysed separately. The Ret%, Ret# and RPI for each group (RA, NRA) were compared with the Friedman test with the Bonferroni correction using a commercial software (Analyse-it).

For each parameter, the number of true positives and false negative (samples with RA with values higher and lower, respectively, than each operating point), true negative and false positive (samples with NRA with values lower and higher, respectively, than each operating point) were calculated. ¹⁶ Sensitivity, specificity and positive likelihood ratio (LR+) were calculated using standard formulae, ¹⁷ either at the cut-offs determined with receiver operating characteristic (ROC) curves, or using the upper RIs reported in the literature (Ret% = 0.5%; Ret# = 50 × 10³/µl; RPI = 1.0). ² ROC curves were designed to determine the ability of each parameter to identify cats with RA and to identify which cut-off best differentiates RA from NRA. ¹⁸ The level of significance was set at P <0.05

Results

Composition of groups

The retrospective search in the database according to the selection criteria allowed us to include 106 cases in the study (Figure 1; Table 1). The RA group included cats with haemoplasma infection or with acute haemorrhage that had occurred at least 5 days before sampling (Table 1). The NRA group included either recent haemorrhage or infectious, metabolic or neoplastic conditions that depress the bone marrow activity.

Figure 1

Flow diagram illustrating the selection procedure employed in this study. Ret = reticulocyte

Table 1

Final diagnosis in the 106 cats with regenerative anaemia (RA) or non-regenerative anaemia (NRA) included in this study

		n	Breed	n	Condition	n
Manual counts	RA	6	Domestic shorthair	3	Haemorrhage	3
			Persian	2	Mycoplasma haemofelis	3
			Exotic Shorthair	1
	NRA	68	Domestic shorthair	56	Tumours	20
			Persian	8	FIP	19
			Abyssinian	2	CKD	14
			Siamese	2	FIV/FeLV	9
					Inflammation	4
					Recent haemorrhage	2
Instrumental counts	RA	7	Domestic shorthair	4	Haemorrhage	3
			Persian	2	Mycoplasma haemofelis	4
			Scottish Fold	1
	NRA	25	Domestic shorthair	14	Tumours	9
			Abyssinian	3	FIP	6
			Persian	3	CKD	5
			Siamese	2	FIV/FeLV	3
			Chartreux	1	Inflammation	1
			Devon Rex	1	Recent haemorrhage	1
			Exotic Shorthair	1

FIP = feline infectious peritonitis; CKD = chronic kidney disease; FIV = feline immunodeficiency virus; FeLV = feline leukaemia virus

Reticulocyte parameters

With both methods, results for all the reticulocyte parameters were significantly higher in cats with RA than in cats with NRA (Figure 2). In cats with RA, the Ret%, Ret# and RPI were higher than the upper RI reported in the literature, ² in 6/6, 4/6 and 0/6 cases, respectively, after manual counts, and in 6/6, 0/7 and 6/7 cases, respectively, after instrumental count. In the NRA group, the Ret%, Ret# and RPI were abnormal in 13/68, 4/68 and 0/68 cats, respectively, after manual counts, and in 7/25, 4/25 cats and 2/68 cats, respectively, after instrumental counts.

Figure 2

Distribution of reticulocyte percentage (Ret%), absolute number of reticulocytes (Ret#) and reticulocyte production index (RPI) in cats with regenerative anaemia (RA) and non-regenerative anaemia (NRA), after (a) manual or (b) automated counts. The boxes indicate the I–III interquartile range (IQR), the horizontal line the median value, and the whiskers extend to further observation within quartile 1 minus 1.5 × IQR or to further observation within quartile 3 plus 1.5 × IQR. The grey area represents the reference interval of the laboratory. For each parameter and method, results of the RA group were significantly higher (P <0.001) than results of the NRA group

Hence, using both methods, the Ret% was more sensitive than specific, the RPI was very specific but not sensitive, and the Ret# had sensitivity and specificity >80.0% and the highest LR+ (Table 2). However, for all the parameters, sensitivity and specificity approached 100.0% if cut-offs determined by the ROC curves were used. These cut-offs were higher than those reported in the literature for Ret%, lower for RPI and variably different, depending on the method used to enumerate the reticulocytes, for Ret#. Using these cut-offs, the RPI had the highest LR+ using both methods. However, the discriminating power of all the parameters, as defined by the ROC curves (Figure 3), was always close to 100% and significantly higher (P <0.001) than the line of no discrimination, without significant differences between the three parameters.

Table 2

Sensitivity (Sens), specificity (Spec) and positive likelihood ratio (LR+) of reticulocyte percentage (Ret%), absolute number of reticulocytes (Ret#) and reticulocyte production index (RPI) using predetermined cut-offs (ie, the cut-off corresponding to the upper reference interval reported in the literature for humans ² ) or using the cut-offs determined by the receiver operating characteristic (ROC) curve

		Manual counting (n = 74)				Automated counting (n = 32)
		Cut-off	Sens %	Spec %	LR+	Cut-off	Sens %	Spec %	LR+
Predefined cut-off	Ret%	0.5	100.0 (54.1–100.0)	73.5 (61.4–83.5)	3.78	0.5	100.0 (59.0–100.0)	72.0 (50.6–87.9)	3.57
	Ret#	50.0	83.3 (36.9–99.6)	94.1 (85.6–98.4)	14.17	50.0	100.0 (59.0–100.0)	84.0 (63.9–85.5)	6.25
	RPI	1.00	0.0 (0.0–45.9)	98.5 (92.1–100.0)	0.00	1.00	14.3 (0.04–57.9)	92.0 (74.0–99.0)	1.79
Cut-off determined by the ROC curve	Ret%	1.7	100.0 (54.1–100.0)	94.1 (85.6–98.4)	17.00	3.06	100.0 (59.0–100.0)	88.8 (68.8–97.5)	8.33
	Ret#	41.4	100.0 (54.1–100.0)	92.6 (83.7–97.6)	13.60	57.7	100.0 (59.0–100.0)	88.8 (68.8–97.5)	8.33
	RPI	0.35	100.0 (54.1–100.0)	95.6 (87.6–99.1)	22.67	0.59	100.0 (59.0–100.0)	92.0 (74.0–99.0)	12.50

Values in parentheses are 95% confidence intervals

Figure 3

Comparison of receiver operating characteristic curves of the reticulocyte percentage (grey circle), absolute number of reticulocytes (black circle) and reticulocyte production index (open circle) obtained after (a) manual count or (b) instrumental count. The grey line indicates the line of no discrimination

Discussion

This study indicated that all the reticulocyte parameters may identify cats with RA. Moreover, the different counting methods provided similar results, contrary to what has been demonstrated in dogs. ¹³ This was likely owing to the fact that instrumental counts mostly detect aggregate reticulocytes (ie, the only cells that are counted manually in cats) ¹⁵ and not punctate reticulocytes, which are included in manual counts in dogs. However, all the reticulocyte parameters had better performances if cut-offs different from those reported in the literature are used. This is particularly true for the RPI, which corrects the magnitude of reticulocytosis for the severity of anaemia.^2,3 The RPI is based on the maturation time of human reticulocytes, which is likely different than in cats, owing to the peculiarities of feline erythroid cells. ¹² However, it is very likely that in cats the maturation times of circulating reticulocytes increases if these cells are released earlier than in normal conditions. Hence, we assessed whether RPI, as well as the other reticulocyte parameters, may provide useful diagnostic information at the cut-off reported in the literature or at a different cut-off, identified through a ROC curve analysis. This approach revealed that the cut-off reported in the literature for humans is very specific but not sensitive. This is not surprisingly, based on the peculiarities of feline reticulocytes described in the introduction. ¹² Hence, further studies on the maturation time of feline reticulocytes are needed in order to increase the accuracy of the formula used to calculate the RPI in cats. However, using lower cut-offs, the RPI had the highest LR+. A similar finding was recorded in dogs, ¹³ which have different maturation times than humans. Although the ROC curves did not detect significant differences between the discriminating power of the three parameters, the RPI, at cut-offs lower than those of humans, is preferred if the pretest probability of regeneration is unknown, as in this case a test with high LR+ increases the post-test probability of disease.^16,18 Conversely, if the pretest probability of regeneration is high (evident blood loss or haemolysis), a test with high specificity, which avoids false-positive results, may be appropriate as a confirmatory test. Based on our results, any reticulocyte parameter, except the Ret% at the cut-off reported in literature, may play this confirmatory role.

The main limitation of this study is the low number of cats, especially in the RA group. However, the performance of each parameter was expressed in terms of LR+, which, differently from predictive values, is not affected the prevalence of the diseases, ¹⁸ thus minimising the effect of the low number of cats with RA. The prevalence of RA in our caseload ranged from 8% (manual counts) to approximately 20% (instrumental counts). This is in agreement with the opinion that haemolytic anaemia is uncommon in cats, ¹⁹ although recent reports suggest that haematological patterns consistent with RA or pathogenic mechanisms responsible for RA are present in more than 40% of anaemic cats.^8,20 However, the latter studies were based on cut-offs that, according to the current study, are poorly specific and may have overestimated RA. The low prevalence of RA in our study, and the lack of cases of immune-mediated haemolytic anaemia, were likely owing to the application of strict exclusion criteria, which excluded cases of unknown aetiology, or biased by multiple pathogenic mechanisms or by inaccurate classification of RA. This is important as the bone marrow cytology, which is considered the most accurate marker with which to assess the presence of regeneration, is not recommended in routine practice when blood loss or haemolysis are suspected based on clinical or haematological findings. ² Hence, cats were classified based on the final diagnosis and on the restoration of the RBC mass during the follow-up, as in a similar study in dogs. ¹³ With this aim, we established a long follow-up time to assess whether restoration of the RBC mass occurred or not (1 year), to prevent misclassification of cases of RA in the NRA group. Independent of the time of recovery, acute post-haemorragic anaemia was included in the NRA group when samples were obtained in the first days after the haemorrhage, as during preregenerative anaemia reticulocytosis is not fully detectable in peripheral blood. Conversely, in a similar study in dogs, ¹³ the ability of reticulocyte parameters to detect regeneration was assessed either including preregenerative anaemia in the NRA group, to detect full regeneration, or in the RA group, to detect early regeneration. However, in the current study this approach was hampered by the low number of cases with preregenerative anaemia. Future studies including higher number of cases, and especially a higher number of cats with preregenerative anaemia, are needed to confirm that Ret#, Ret% or RPI may be early indicators of regeneration in cats.

Conclusions

This study indicated that all the reticulocyte parameters confirm regeneration when the pretest probability is high, while when this probability is moderate, RA should be identified using the RPI at cut-offs <1.0.