In vitro-induced Heinz bodies showed no impact on feline reticulocyte haemoglobin content measurement using the Advia 2120i analyser

Abstract

Objectives

Reticulocyte haemoglobin content (CHr) is a promising tool for evaluating iron status in dogs; however, its usefulness in cats varies. Heinz bodies interfere with haematological analyses on the Advia 120/2120i analyser (Siemens Healthineers), but the effect of Heinz bodies on CHr measurement in feline blood samples is unknown.

Methods

In an analytical intervention study, CHr concentration was measured pairwise in 30 feline blood samples with and without Heinz bodies chemically induced in vitro using 1-acetyl-2-phenylhydrazine (APH).

Results

The percentage of Heinz bodies and the amount of Heinz body material were assessed by microscopy after staining with New Methylene Blue, and both increased significantly after in vitro APH incubation. After incubation, CHr levels decreased significantly in both samples without APH and in APH-treated samples, but no significant difference was detected between the samples with and without APH.

Conclusions and relevance

In vitro-induced Heinz bodies did not impact CHr levels. Explaining the lesser diagnostic utility of CHr measurements in cats compared with dogs as an effect of Heinz bodies is therefore not substantiated by this in vitro study. Accordingly, variations in feline patient populations and varying definitions of iron deficiency in previous studies are likely the main explanations for the lesser diagnostic utility of CHr measurements in cats.

Introduction

Iron is essential to haemoglobin and involved in several cellular energy-generating oxidative processes. ¹ Commonly employed tests to diagnose iron deficiency in veterinary medicine include haematocrit, red blood cell count (RBC), haemoglobin, mean cell haemoglobin concentration, mean cell volume, serum iron, total iron-binding capacity, transferrin saturation, serum ferritin and iron content of bone marrow.^{2 –5} Currently, no single optimal test for diagnosing iron deficiency in cats exists. Bone marrow iron staining is invasive and subjective, ⁶ a lack of stainable iron is not always predictive of iron deficiency ⁷ and, in cats, iron is not commonly stored in the bone marrow. ³ Serum iron, transferrin and ferritin are affected by acute-phase responses,^5,8 and although serum ferritin is believed to reflect body iron stores best, it has only a weak correlation to liver iron content in cats (r = 0.365) ⁹ and is not widely available in veterinary practice for routine first-line testing. ³ Because of the long lifespan of erythrocytes, ¹⁰ the microcytic, hypochromic anaemia characteristic of iron deficiency anaemia takes weeks to months to develop and is seen only in the very late stages of iron depletion.^3,5

Reticulocyte haemoglobin content (CHr on Advia 120/2120i haematology analysers or RET-He on other analysers) has been applied to evaluate iron status in dogs and cats.^11–17 CHr provides a measure of iron available for erythropoiesis over the previous 1–4 days.^16,18,19

In dogs, CHr is considered a suitable screening tool for detecting iron deficiency or iron-deficient erythropoiesis,^13,14,20 with diagnostic sensitivity and specificity in the range of 85–95.2% and 90.5–99%, respectively.^11,14 In cats, however, studies using the Advia 120/2120i analyser have reported diagnostic sensitivity in the range of 35–93.8% and specificity in the range of 76.9–87%.^{14 –16} Variations in the feline patient populations included and different definitions used to define iron deficiency likely explain part of the variation in sensitivity and specificity.

Heinz bodies are clumps of denatured, precipitated haemoglobin within erythrocytes near or protruding from the surface. ²¹ They form readily in cats,^22,23 most likely because of the unique characteristics of feline haemoglobin, with eight reactive sulfhydryl groups on the haemoglobin molecule and easy dissociation to a dimer form,^21,22 and the unique non-sinusoidal spleen ²⁴ believed to be ineffective in removing Heinz body-containing erythrocytes. Heinz bodies interfere in automated haematology assays in cats,^{25 –27} including interference with haemoglobin measurement. ²⁸ In humans, it has been reported that Heinz bodies can occur within reticulocytes, ²⁹ and a study in rats has shown similar findings when treating rats with phenylhydrazine. ³⁰ Because of this, we hypothesised that Heinz bodies interfere with CHr measurements in feline blood samples using the Advia 120/2120i analyser, thereby contributing to explaining the large variation in the diagnostic utility of CHr in diagnosing iron deficiency in cats.

Thus, the aim of the present study was to investigate whether Heinz bodies interfere with CHr measurements in feline blood samples using the Advia 2120i analyser.

Materials and methods

Study

The study was designed as an analytic intervention study ³¹ comparing CHr in blood samples before and after chemical in vitro preparation of Heinz bodies. The study was approved by the local Ethical and Administrative Committee (2024-10). All samples were anonymised relative to individual patients as the aim was to assess methodological characteristics and not the relation to other patient characteristics. The study was performed between January 2024 and June 2024.

Blood samples

All samples originated from blood samples in K₃-EDTA-coated blood collection tubes (Beckton-Dickinson Vacutainer; BD) submitted for haematological analysis to the Veterinary Diagnostic Laboratory, University of Copenhagen. Samples were deemed eligible for use in the study until 48 h after blood collection, as CHr values are reportedly stable for this period.^14,32,33 Blood samples were included irrespective of the final diagnoses, since the present study was an in vitro methodological study.

In vitro production of Heinz bodies by 1-acetyl-2-phenylhydrazine

For the in vitro induction of Heinz body formation, 1.49 g of 1-acetyl-2-phenylhydrazine (APH; molecular weight 150.18 g/mol; Fisher Scientific) was dissolved in 100 ml phosphate-buffered saline (PBS).^34–37 Each EDTA-stabilised blood sample was divided into two separate portions (400 µl each). One was mixed with 200 µl APH (case samples), the other with 200 µl PBS (control samples) and both samples were then incubated at 37°C for 3 h.

Heinz body percentage and the amount of Heinz body material

To confirm that Heinz bodies had been produced in the APH-incubated samples, 20 µl of each sample before and after incubation was mixed with 20 µl 1% (w/v) New Methylene Blue for 15 mins. ³⁸ The percentage of Heinz bodies was estimated using microscopy by the same observer and based on the number of Heinz body-containing erythrocytes out of 250 erythrocytes. The amount of Heinz body material was assessed by evaluating 250 erythrocytes using the index suggested by Magos and Sziza. ³⁹ As the index was originally formulated for use on rats with lower numbers and intensities of Heinz body presence, the index definitions were slightly modified as follows:

$$Heinzbodyindex=\frac{\left(1.n1\right)+\left(2.n2\right)+(4.n4)}{n0+n1+n2+n4}$$

where n0 is erythrocytes with no Heinz bodies, n1 is erythrocytes with one to five small Heinz bodies (modified from ‘one small Heinz body’), n2 is erythrocytes with more than five small or one moderate-sized Heinz body (modified from ‘two small Heinz bodies or one of moderate size’) and n4 is erythrocytes with two or more moderate-sized Heinz bodies or one large Heinz body.

Measurement of CHr

After incubation, CHr was measured in both samples using the Advia 2120i analyser (Siemens Healthcare Diagnostics), using the protocol and feline setting in the multispecies software provided by the manufacturer. The analyser was subjected to daily internal quality control and quarterly external quality control.

Statistical analysis

Statistical analyses were performed using MedCalc. ⁴⁰ The variables were non-normally distributed as assessed by the d’Agostino-Pearson test and thus values are presented as median and range. The Wilcoxon matched-pairs signed-rank test was used to compare Heinz body percentage and index before and after incubation with PBS and APH. The Friedman test was employed to calculate CHr values of the paired case and control samples. The significance level was set at P <0.05.

Results

Incubation with APH caused a variable degree of poikilocytosis that did not affect the assessment of Heinz bodies. The percentage of Heinz bodies as well as the amount of Heinz body material increased significantly (P <0.0001) after incubation with APH at 37°C for 3 h while incubation with PBS slightly, but significantly, decreased the Heinz body index (P = 0.0085) (Table 1).

Table 1

Heinz body percentage and Heinz body index before and after incubation of 30 feline blood samples with either PBS or APH

Variable	Before incubation	After PBSincubation	After APH incubation
Heinz body percentage (%)	0.80 (0.40–1.13)	0.40 (0.00–3.60)	98.60* (84.80–100.00)
Heinz body index	0.016 (0.000–0.024)	0.008 † (0.000–0.056)	2.926* (1.080–4.000)

Data are median (range)

*

P <0.0001 (before incubation compared with after APH incubation)

†

P = 0.0085 (before incubation compared with after PBS incubation)

APH = 1-acetyl-2-phenylhydrazine; PBS = phosphate-buffered saline

Compared with CHr levels before incubation (PBS median 1.04 fmol; range 0.75–1.17; APH median 1.03 fmol, range 0.75–1.22), CHr levels decreased significantly after incubation in both PBS-treated samples (PBS median 0.95 fmol, range 0.78–1.08) and APH-treated samples (APH median 0.96 fmol, range 0.77–1.11). However, no significant difference in CHr levels was detected between PBS-treated samples and APH-treated samples (Figure 1).

Figure 1

CHr before and after incubation with either PBS (T0 PBS and T3 PBS) or APH (T0 APH and T3 APH). Median CHr concentrations (T0 PBS 1.04 fmol; T3 PBS 0.95 fmol; T0 APH 1.03 fmol; T3 APH 0.96 fmol) are indicated by black horizontal lines. T0 PBS and T0 APH differed significantly (P <0.00001) from T3 PBS and T3 APH. No statistically significant difference was found between T3 PBS and T3 APH. APH = 1-acetyl-2-phenylhydrazine; CHr = reticulocyte haemoglobin content; PBS = phosphate-buffered saline

Discussion

The presence of Heinz bodies interferes with many haematological analyses in cats, for example, mean cell haemoglobin and mean cell haemoglobin concentration.^{25 –28,41} In the Advia 2120i analyser and its earlier versions, the RBC cluster of cats with a significant occurrence of Heinz bodies extends to the lower right of the scatterplot, indicating a hyperchromic RBC population.^25,28

The Advia2120i analyser measures the haemoglobin concentration in individual erythrocytes following their isovolumetric sphering with sodium dodecyl sulphate and glutaraldehyde. ⁴² The haemoglobin concentration in individual erythrocytes is then calculated using low-angle (2°–3°) and high-angle (5°–15°) light-scatter analysis, with the resulting scatter measurements converted into volume and refractive index values using the Mie theory of light scattering for homogeneous spheres. ⁴³ Reticulocytes are differentiated from erythrocytes by the absorption of Oxazine 750, which binds to cellular RNA. In feline blood, the Advia 2120i analyser measures aggregate reticulocytes. ⁴⁴ Haemoglobin content is calculated on a cell-by-cell basis by multiplying erythrocyte or reticulocyte volume by haemoglobin concentration, with separate values reported for the mean haemoglobin content of reticulocytes (CHr) and of mature erythrocytes (CH). In humans, Heinz bodies occur in erythrocytes as well as in reticulocytes. Assuming that Heinz bodies also occur in feline reticulocytes after incubation with APH, Heinz bodies could potentially interfere with intracellular haemoglobin measurement in reticulocytes in the Advia 2120i analyser by disturbing light scattering and subsequent conversion into refractive index values. Counting Heinz bodies in aggregated reticulocytes was, however, not attempted in this study, yielding a limitation.

Conclusions

Incubating EDTA-stabilized blood with APH significantly induced the development of Heinz bodies; however, the impact of this on CHr levels was not different to incubating with PBS. The reason why CHr levels decreased after incubation with PBS is unknown. Incubation with PBS resulted in a lower Heinz body index, and it is possible that incubation with PBS at 37°C for 3 h increased surface remodelling or permeability with subsequent loss or dilution of reticulocyte haemoglobin. In cats, Heinz body formation is associated with diabetes mellitus, hyperthyroidism and lymphoma. ²² If Heinz bodies, as seen in this study, in fact interfere by lowering CHr values, then the number of false positives, that is, sick cats with low CHr values but without iron deficiency, would increase. This would increase the diagnostic sensitivity but reduce the specificity of CHr measurements, which conflicts with earlier studies reporting diagnostic sensitivities in the range of 35–93.8% in cats compared with 85–95.2% in dogs. Explaining the lesser diagnostic utility of CHr measurements in cats compared with dogs as an effect of Heinz bodies is, therefore, not substantiated by this study.