Comparative Clinical Pathology

https://doi.org/10.1007/s00580-018-2820-4

ORIGINAL ARTICLE

Equine blood reticulocytes: reference intervals, physiological

and pathological changes
M. Balan 1 & M. McCullough 1 & Peter J. O’Brien 1

Received: 2 May 2018 / Accepted: 7 September 2018

# Springer-Verlag London Ltd., part of Springer Nature 2018

Abstract
High-sensitivity haematology analysers detect equine blood reticulocytes, but their analysis and quantitation is largely unstudied.
We assessed whether equine reticulocytes are affected by pathological or physiological factors, determined reference intervals,
and examined reticulocyte and related, erythron parameters using 6-years’ data (Advia 2120) from our hospital archives. Data
was categorised according to horse age (foals < 12 months), breed (hot-, warm-, cold-blooded), presence of anaemia and various
diseases. In adults, reticulocytes (× 109/L) were 20% lower in cold-blooded horses compared to others (4.14 ± 2.3, 25 vs 5.15 ±
3.3, 157; data shown as mean ± SD, n; p < 0.05.) There was no gender effect. In thoroughbreds, foals had 50% higher reticu-
locytes than adults (7.5 ± 4.5, 18 vs 5.0 ± 3.5, 56). We determined the first reference interval for healthy adult horses as 5.0 ±
3.2,182, 1.16–13.1. Reticulocyte counts were 120% higher with marked anaemia (11.0 ± 9.4, 6) but 36% with other diseases (6.9
± 4.2, 74), versus reference mean. Erythrocyte size (MCV)/reticulocyte haemoglobin content (CHr) were 21/14% lower in foals
versus adults and 12/37% higher with marked anaemia. Immature reticulocytes (IRF-H) were 60% lower in foals versus adult
thoroughbreds, and 87% with marked anaemia. In conclusion, equine reticulocytes are only mildly increased by physiological
parameters (breed, age, but not gender), various non-blood diseases (colic, dysproteinaemia) and marked anaemia. Reticulocyte
counts are substantially lower in horses versus other species; their clinical use is limited to marked anaemias. This is consistent
with the greater erythrocyte storage and buffering capacity of the spleen of the horse. Finally, this study supports the theory of a
reticulocyte maturation role for equine spleen.

Keywords Equine . Reticulocyte . Physiological . Pathological . Reference interval

Introduction 2005). Also, before the introduction of modern haematology

Reticulocytes are an indicator of bone marrow activity. They
normally circulate in the blood of healthy dogs, cats, rodents,
rabbits, pigs and guinea pigs, whereas in other species (cows,
goats, sheep) are only detected during a regenerative response
to anaemia (Olver 2010). Equine reticulocytes are known to
be produced in the bone marrow, but measureable numbers are
not released during mild-to-moderate anaemia (Cooper et al.
A preliminary report of these findings has been made at the 17th Annual
conference of the Association for Comparative Clinical Pathology, Mar
12-13, 2016 (O’Brien 2017)
* Peter J. O’Brien
Peter.James.OBrien@ucd.ie
1 able, ranging from 0.5 to 85 × 109/L.
Clinical Pathology Laboratory, School of Veterinary Medicine,
Veterinary Sciences Centre, University College Dublin, Room 013,
Belfield D4, Ireland
analysers, reticulocytes were generally considered absent
from peripheral blood of horses (Walton 2013). However,
recent studies have demonstrated that reticulocytes can be
detected and measured in healthy and anaemic horses,
although at low numbers compared to other species. For
example, Cooper et al. (2005) reported basal reticulocytes
counts of ~ 3 × 109/L in mostly Standardbred horses, which
almost doubled with erythropoietin treatment (three doses of
73 U/kg, 48 h apart), but still remained 10- to 20-fold lower
than in dogs (Bauer et al. 2012). Weiss and Moritz (2003)
reported a 20-fold absolute reticulocyte increase in a horse
with severe haemolytic anaemia and a manual reticulocyte
count of 1.2%, as determined by light microscopy after new
methylene blue staining. Giordano et al. (2008) found reticu-
locyte measurements in 114 healthy horses to be highly vari-
The ADVIA analyser (Siemens Healthcare Diagnostics,
Inc., NY) is a widely used automated, laser-based

haematology system (Clark et al. 2002; Giordano et al. 2008)
with specific software for multiple animal species, although
other systems have also been validated for veterinary use (e.g.
Sysmex XT-2000iV—Lilliehook and Tvedten 2009). The la-
ser technology allows for cell-by-cell measurement of cell size
and maturation, plotting the results on a cytogram. Compared
to impedance-based analysers and light microscopy, this is a
more sensitive method that identifies and quantifies reticulo-
cyte maturation by stained nucleic acid absorbance (Weiss and
Moritz 2003).
As some equine haematological parameters are influenced
by physiological, as well as pathological, factors, the reticulo-
cyte may be similarly affected. Age, breed, gender, reproduc-
tive status, physical activity and splenic contraction impact the
interpretation of the equine erythron (Jain 1993; Grondin and
Dewitt 2010). For example, hot-blooded horses have larger
spleens (Kline and Foreman 1991) with the capacity to store
between 6 and 12 l of blood (Satue et al. 2012), which may be
released with excitement or exercise and increase the
haematocrit by up to 0.25 L/L (McGowan 2008). Values re-
turn to normal approximately 1 h after exercise (Piccione et al.
2010).
The aim of this study was to determine the effect of phys-
iological and pathological factors on equine reticulocytes and
to determine reference intervals. We made multiple compari-
sons for reticulocyte parameters: population versus individual,
adult versus foals, healthy versus diseased, including anaemic.
Materials and methods
Data collection
In 2010, we began to routinely measure reticulocyte values in
all horses presented at University College Dublin Veterinary
Hospital (UCDVH), using the Advia 2120 haematology
analyser. Internal samples were processed rapidly after collec-
tion into EDTA(K3) anticoagulant. External samples were
mailed from nationwide clinics and arrived within 24 h, typ-
ically at ambient temperature, and comprised about one third
of all submissions. Samples were from horses of all ages,
breeds and genders of clinically sick or research study ani-
mals, and routine health screenings.
The following erythron parameters were retrieved from the
analyser’s database and transferred into GraphPad Prism v7
(La Jolla, CA 92037 USA), a statistics software program:
reticulocytes (× 109/L), red blood cell (RBC) counts (× 1012/
L), haematocrit (Hct; L/L), haemoglobin (Hb; g/L), mean cor-
puscular volume (MCV; fL) and mean corpuscular
haemoglobin concentration (MCHC; g/L) which is a calculat-
ed parameter based on measurements of Hb, MCV and RBC
(MCHC = (Hb ÷ [RBC × MCV]) × 100), mean reticulocyte
haemoglobin content (CHr; pg), immature reticulocyte
Comp Clin Pathol
fraction high (IRF-H; no units). Three identification numbers
were also retrieved from our internal laboratory information
system: sample, case and visit number.
Categorisation of horses according to age, breed
and health status
Only horses with complete information regarding breed, age
and health status, as defined below, were included in this
study.
Age Due to the limited numbers of horses available for study,
they were only separated into two broad age categories in
order to get sufficient animals in each category, adults or foals,
based on an age cut-off of 12 months. At our clinic, foals
typically had their age specified on their medical records.
However, age was occasionally not stated for adult horses.
When age was not precisely stated, cases were included in
the study only if they could be age-categorised (foal versus
adult) based upon other data that was age specific such as poor
performance, participation in a study of racing horses or hav-
ing a condition specific to adult horses.
Breed Horses were separated according to breed as stated on
the sample submission form. However, the only foals avail-
able for study were thoroughbreds. Adult breeds were identi-
fied as hot-blooded (Arabian, thoroughbreds), warm-blooded
(Connemara, Irish Draught, Holsteiner, hunter, sport horse)
and cold-blooded (drafts, Clydesdale, cob, Friesian,
Icelandic, ponies, donkey).
Data reduction for multiple blood collections from the same
horse Data was reduced for cases where there were multiple
blood collections for a horse to one single value per horse.
Data reduction was done similarly for all groups. The middle
value of the range, or median, was used when there were an
odd number of determinations. The average, or mean, was
used when there was an even number of blood samples.
Horses with multiple blood collections ranged from one to
eight repeat values/horse, and the time period of collection
of repeats ranged depending on whether the horses were
healthy or diseased. For healthy, adult horses, repeats were
obtained for a period of up to several years. For horses with
disease, repeats were obtained over the course of the disease,
which was typically a period of days up to several weeks..
Identification of horses with normal erythron Age- and breed-
specific reference intervals for normal Hct and Hb measure-
ments were determined from the mean values of six studies
(Lumsden et al. 1980; Clark et al. 2002; Giordano et al. 2008;
Lording 2008; Grondin and Dewitt 2010; Bauer et al. 2012)
and are reported in Table 1.
Comp Clin Pathol

Table 1 Literature and study

obtained equine erythron data. Published literaturea reference intervals
Breed-specific erythron values for
thoroughbred adults and foals Hot-bloods Warm-bloods Cold-bloods Foals
were obtained by calculating a RBC (× 1012/L) 7–12 6–11.3 5.5–9.5 7.7–13.3
mean of the minimum and Hct (L/L) 0.32–0.53 0.34–0.49 0.24–0.44 0.30–0.44
maximum values from several
published equine research papers Hb (g/L) 110–190 106–189 80–140 116–156
and textbooks (see text) compared MCV (fL) 36–50 38–49 40–48 30–45
to our own horse data MCHC (g/L) 310–390 370–400 320–380 328–376
Study obtained data
RBC (× 1012/L) 9 ± 1.2, 56 8.0 ± 0.96, 101 7.1 ± 1.2, 25* 10.1 ± 1.2, 18
(6.7–12.3) (4.8–12.5) (5.3–9.7) (8.1–12.3)
Hct (L/L) 0.40 ± 0.05, 56 0.38 ± 0.03, 101 0.33 ± 0.04, 25* 0.36 ± 0.03, 18
(0.32–0.52) (0.34–0.49) (0.27–0.42) (0.30–0.42)
Hb (g/L) 146 ± 16.9, 56 136 ± 12.1, 101 120 ± 15, 25* 132 ± 14.3, 18
(119–191) (113–172) (97–144) (108–158)
MCV (fL) 45.3 ± 4.8, 56 47.7 ± 4.6, 101 47.3 ± 5.8, 25 35.8 ± 5.1, 18*
(30–53) (33.8–59.4) (34–59.2) (30.3–44.8)
MCHC (g/L) 362 ± 14.3, 56 359 ± 14.4, 101 363 ± 12.2, 25 372 ± 17.2, 18*
(331–394) (334–390) (338–382) (331–398)

Data are expressed as mean ± SD, n (min–max)

*Significance level (p < 0.05) when comparing cold versus hot-blooded horses and hot-blooded adults versus hot-
blooded foals
a
Lumsden et al. (1980), Clark et al. (2002), Giordano et al. (2008), Lording (2008), Grondin and Dewitt (2010),
Bauer et al. (2012)

For reference interval determination, horses were screened
for a normal erythron, based on the above six published stud-
ies (summarised in Table 1) and categorised according to age
and breed. Data was only analysed for horses for which the
breed was stated on the sample submission form. Horses were
categorised by age into one of two groups: foals (less than
1 year of age) and adults. Adults were separated into hot-,
warm- and cold-blooded breeds. Only horses with normal,
age- and breed-specific Hct and Hb erythron measurements
(see Table 1) were included. RBC count reflects how Hb is
packaged according to number and size of RBCs, typically
used to screen for normalcy.
Identification of adult horses with anaemia Horses with
values lower than published reference interval were
categorised as anaemic. Adults were then screened for having
either clinical biochemistry abnormalities or a clinical history
of disease. Because there were frequently multiple measure-
ments for a single horse during the course of its illness, a
single value was determined by averaging multiple blood col-
lections after excluding statistical outliers..
Foals were separated into the above categories similarly,
except for the breed category. This reflected there only being
one breed with significant numbers. Anaemia and disease ef-
fects were not examined in foals because of the low numbers
and so as to avoid confounding age effect. For determination
of foals with normal erythron, we used the average Hct, RBCs
and Hb values reported in the literature (Grondin and Dewitt
2010; Harvey 1990).
Identification of adult horses with disease Most horses had
biochemistry profiles performed along with their haematology
profiles. Horses with biochemistry abnormalities or a clinical
history of disease were analysed separately. Dysproteinaemia
was defined as total protein (57–79 g/L), albumin (29–37 g/L)
and/or globulin (24–48 g/L) value below or above our
laboratory’s reference interval. Inflammation was defined as
a fibrinogen value > 4 g/L and/or a lipase value > 33 U/L.
Myopathy was defined as creatine kinase (CK) and/or aspar-
tate transaminase (AST) values > 1000 U/L. The category of
colic was based on clinical diagnosis made medically or
surgically.
Reference interval The reference interval was determined ex-
clusively for the adult horse, as sufficient data for foals was
only available for one breed, thoroughbreds. A reference in-
terval was determined as the 2.5 and 97.5 percentile values for
healthy horses of all breeds and both genders. Frequency his-
tograms were prepared from these data.
Statistical analysis
All data were tested for normality using the D’Agostino-
Pearson test. The breed effect was tested for by pooling
 Comp Clin Pathol

together the warm- and hot-blooded horses, referred to as non-
cold-bloods, and compared to the cold-bloods using a one-
tailed, unpaired t test with Welch’s correction. The gender
effect was tested for by using a two-tailed, unpaired t test. A
similar, non-parametric Mann-Whitney test was used in test-
ing for age effect. The anaemia and disease effects were tested
for by comparison to the previously established, reference
population by using a one-way ANOVA test. Statistical out-
liers were defined as being > 3 standard deviations away from
the mean of all the others and were excluded. All data are
reported as mean ± SD, n in the text and tables. p < 0.05 was
considered statistically significant. No microscopic examina-
tion of new methylene blue stained smears or bone marrow
glass slides was performed for this study.
Reticulocyte data was only normally distributed for healthy
cold-blooded horses and both gender groups (except the male
warm-bloods), but not for hot- or warm-bloods, nor the refer-
ence population, the anaemic and diseased groups and the
foals.
Physiological effects
Study obtained adult horse data had similar physiological ef-
fects as reported in the literature (Table 1). Adult cold-blooded
horses had lower values than hot-blooded by 22% for RBC
counts and by 18% for both Hct and Hb. Also, cold-blood
MCV values were 4% higher than for hot-bloods. Foals had
21% lower MCV values compared to the adults (Table 1).
Age, breed and gender effects on reticulocytes, MCV, CHr
and IRF-H are shown in Table 2.

Results
Reticulocyte determinations had been made for 458 horses for
a total 688 determinations. Eighty-eight of horses had multiple
determinations for which mean or median values were used as
described above.
One hundred eighteen (~ 25%) horses were missing signal-
ment information; approximately half were missing breed in-
formation and the other half missing age information. These
were excluded from the study. Consequently, only 322 adults
were studied, 182 healthy and 140 with anaemia or clinical
disease. There were 42 foals for which breed and age infor-
mation was available. However, there was only one breed with
sufficient numbers for analysis: the thoroughbred. There were
insufficient numbers to study the effect of disease.
Consequently, 18 healthy foals were studied.
Age effect Of the 322 adults, age was specifically stated in
their medical records for 297 but had to be deduced, as de-
scribed above, for 25. Of the 297 adults with precisely stated
age, this ranged from 1 to 30 years with a mean of 8.3 (SD =
5.1). The 18 thoroughbred foals ranged from 1 day to
11 months, with a mean of 4.4 months (SD = 0.3).
Erythron measurements showed that foal Hb and Hct
values were 10% lower than in adults, while RBC counts were
12% higher. MCV was 21% lower in foals than adults and
MCHC was 3% higher (Table 1). Reticulocyte measurements
demonstrated that foals had 50% higher reticulocyte counts
when compared to adults. Foal CHr and IRF-H values were 14
and 60% lower, respectively (Table 2).
Breed effect Adult cold-blooded horses had 20% lower retic-
ulocyte counts than all other adult horses.

Table 2 Physiological effects on equine reticulocytes. The effects of breed, age and gender on reticulocyte counts and reticulocyte-related parameters
(MCV, CHr, IRF-H)

Breed reticulocyte (× 109/L) Gender MCV (fL) MCHC (g/L) CHr (pg) IRF-H
Female
Male
(× 109/L)

Cold-blooded 4.14 ± 2.3, 25* 3.83 ± 1.69, 10 47.3 ± 5.8, 25 363 ± 12.2, 25 ND ND
4.50 ± 2.68, 12
Warm-blooded 5.22 ± 3.3, 101 5.94 ± 3.17, 38 47.8 ± 4.6, 101 359 ± 14.4, 101 ND ND
4.89 ± 3.35, 58
Hot-blooded Adults 5.04 ± 3.5, 56 5.76 ± 3.15, 19 45.3 ± 4.8, 56 362 ± 14.3, 56 19.4 ± 2.06, 46 12.8 ± 9.8, 46
5.37 ± 4.1, 26
Foals 7.5 ± 4.5, 18* 6.18 ± 1.4, 4 7.57 ± 1.3, 13 35.8 ± 5.1, 18* 372 ± 17.2, 18* 16.7 ± 2.6, 16* 5.2 ± 6.6, 16*
Non-cold breedsa 5.15 ± 3.33, 157 5.88 ± 0.4, 57 5.03 ± 0.4, 84 46.9 ± 4.77, 157 360 ± 14.4, 157 ND ND

Data expressed as mean ± SD, n

ND not determined
*Significance level (p < 0.05) when comparing cold versus non-cold breed reticulocytes; hot-blooded adult versus hot-blooded foal reticulocytes, MCV,
CHr and IRF-H. Note, foals were only available for the hot-blooded horses
a
Obtained by pooling together the warm- and hot-blooded horses
Comp Clin Pathol

Gender effect There was no gender effect on reticulocyte
counts when comparing within the same breed. Thirty-three
horses did not have gender information.
Pathological effects
Anaemia and disease effects on reticulocytes, MCV, MCHC,
CHr and IRF-H are shown in Table 3 and Fig. 1.
Anaemia effect
Compared to the reference interval for all adults, reticulocytes
for anaemic horses were 36% increased, with minimum and
maximum values of 1 and 39.8 × 109/L, respectively.
Anaemic adults (n = 66) were mostly warm-blooded horses
(n = 51) and fewer hot- (n = 11) and cold-bloods (n = 4), with a
mean Hct value of 0.28 ± 0.05, 66, range of 0.12 to 0.33 L/L.
Marked anaemia was defined as Hct values < 0.20 L/L and
had a mean of 0.15 ± 0.03, 6, range of 0.09 to 0.19 L/L with a
120% increase compared to the reference population.
Moderate anaemia was defined as Hct values ≥ 0.20 and <
0.30 L/L with a mean of 0.25 ± 0.03, 20, range of 0.2 to
0.29 L/L. Mild anaemia was defined as Hct values ≥ 0.30
but < 0.34 L/L with a mean of 0.31 ± 0.01, 40, range of 0.30
to 0.33 L/L. Mild and moderate anaemia did not have effects
on reticulocyte values (Fig. 1 top).
Of eight anaemic foals, not included in the analyses, only a
2-month-old thoroughbred diagnosed with marked intravas-
cular immune-mediated haemolytic anaemia (IMHA) had
significant reticulocyte increases. The highest reported value
for this foal was 91.3 × 109/L and did not correlate with the
expected regeneration peak. For anaemic adults, the highest
values were observed in a few warm- and cold-blooded horses
with moderate and marked anaemia, lacking clinical history.
There were two adult horses, excluded from analysis due to
lack of breed information or absence of anaemia or disease,
which had higher reticulocyte values (106 and 97 × 109/L), the
basis for which the increase in reticulocytes was unknown.
One adult horse also had IMHA for which there was a tran-
sient, 4-fold over basal values increase in reticulocytes; how-
ever, this rise was not sustained during the course of the
anaemia.
CHr data was available for 55 adult, anaemic horses. It was
increased by up to 37% with marked anaemia when compared
to the mild and moderate groups and the healthy thorough-
breds. Furthermore, with marked anaemia, CHr increases
showed both mean and individual values to exceed a previ-
ously published reference interval of 14 to 23 pg from clini-
cally healthy horses (Giordano et al. 2008).
IRF-H data was inversely proportionate to the degree
of anaemia, as progressively lower values were observed
with moderate and marked anaemia. Mean values de-
creased by 87% with marked anaemia compared to the
mild group.
MCV mean values had a 12% increase with marked anae-
mia compared to the reference interval. No correlation was
observed between the reticulocyte counts and MCV (r =
0.22; p = 0.087).

Table 3 Pathological effects on equine reticulocytes. The effects of mild, moderate and marked anaemia and of other diseases (dysproteinaemia,
myopathy, colic, inflammation) on reticulocytes, and reticulocyte-related parameters are shown

Reference Anaemia Disease

interval§

Retics 5.0 ± 3.2, 182 6.8 ± 6.8, 66* 6.9 ± 4.2, 74*
× 109/L Mild Moderate Marked Dysproteinaemia Myopathy Colic Inflammation
6.0 ± 4.8, 40 7.3 ± 9, 20 11.0 ± 9.4, 6* 7.1 ± 5, 30* 6.6 ± 3.6, 20 6.9 ± 2.8, 6.9 ± 4.7, 9
15*
MCV (fL) 47 ± 4.9, 182 46 ± 5.8, 66 45.8 ± 4.8, 74
45.3 ± 5, 40 45.6 ± 5.8, 20 52.4 ± 7.5, 6* 44 ± 5.4, 30 47.3 ± 4.7, 46.1 ± 3.6, 47.7 ± 3.6, 9
20 15
MCHC (g/L) 360 ± 14.1, 182 365 ± 28, 66 366 ± 16.6, 74 *
368 ± 12.3, 373 ± 19.1, 355 ± 20.5, 6 ND
40* 20*
CHr (pg) 14–23§ 20.1 ± 3, 55
19.3 ± 2.1, 33 20 ± 2.9, 18 26.4 ± 3.6, 4*
IRF-H ND 8.5 ± 7.9, 55
9.3 ± 8.5, 33 8.7 ± 7.1, 18 1.1 ± 0.4, 4*

All data are expressed as mean ± SD, n

Retics reticulocytes, ND not determined
*Significance level (p < 0.05) when comparing reference interval versus anaemia and disease reticulocytes, MCV, CHr and IRF-H
a
All reference interval data are derived from the current study, except for the CHr for which the reference interval is taken from Giordano et al. (2008)
 Comp Clin Pathol

97.5 percentile reference interval was 1.16 to 13.1 × 109/L and

M a rk e d
A n a e m ia * the range was 0.5 to 15.0 × 109/L (Fig. 2). In determining this
interval, up to 5% values were excluded based on the 3-SD
M o d e ra te
statistical rule described above: 3 of 59 hot-bloods, 4 of 105
M ild
warm-blood and 1 of 26 cold-bloods. Data were not normally
distributed and had long tails to the right (skewness of 0.96)
R I and was truncated at 0.5, approximately the lower limit of
measurement.
0

0
1

4
D y s p r o te in e m ia
*
Discussion
M y o p a th y
As equine reticulocytes are little studied, we investigated
C o lic whether they are affected by pathological or physiological
*
changes, analysing horses with anaemia and various other
In fla m m a tio n
diseases. The physiological effects of age, breed and gender
R I on reticulocytes were also assessed, as such effects on mature
erythron parameters (RBC, Hct and Hb) are well documented
(Grondin and Dewitt 2010; Satue et al. 2012; Walton 2013).
0

0
1

Uniquely, we validated our selection of healthy horses using

9
R e t ic u lo c y t e s ( x 1 0
Fig. 1 Pathological effects on equine reticulocytes. Line indicates mean
with SEM. *Significance level (p < 0.05). Top—The effects of mild (Hct
≥ 0.30 but < 0.34 L/L), moderate (Hct ≥ 0.20 and < 0.30 L/L) and marked
(Hct < 0.20 L/L) anaemia on equine reticulocytes compared to the
reference interval (RI). Bottom—Effect of common equine diseases on
reticulocytes compared to the RI
MCHC was ~ 3% higher with mild and moderate anaemia,
respectively, when compared to the reference interval.
However, the reticulocytosis of marked anaemia was associated
with mildly decreased MCHC compared to all other horses.
Non-anaemic disease effect
Compared to the reference interval values for all adults, retic-
ulocytes from the diseased horses were 36% increased, with
minimum and maximum reticulocyte counts of 0.6 and 18.4 ×
/L ) consensus, literature-derived reference intervals for standard
erythron parameters of healthy horses of different breed and
age. We demonstrated the pathological effects of anaemia on
reticulocytes by analysing and comparing erythron and retic-
ulocyte parameters (MCV, MCHC, CHr and IRF-H). Also,
individual clinical history and biochemical abnormalities were
used to identify cases with disease, the effects of which were
analysed for the first time. We found that reticulocyte counts
were only mildly affected by physiological parameters, mild-
to-moderate anaemia and examined disorders. And they were
of limited diagnostic or prognostic value when compared to
reference intervals. Only in horses with marked anaemia, es-
pecially immune-mediated, was there significant change in
reticulocyte counts compared to normal horses. We
hypothesise that this lack of reticulocyte count difference in
the horse reflects the large buffering effect of this species
N u m b e r o f h o rs e s

109/L, respectively. 3 0

Of 74 adult horses with biochemistry abnormalities, 30 had

dysproteinaemia, mostly due to hypoproteinaemia; 20 had
2 0
myopathy; 15 had colic and 9 had inflammation.
Reticulocyte counts of dysproteinaemic and colic cases were
2-fold higher than the mean for the adult reference interval 1 0
(Fig. 1 bottom). Similarly as for mild and moderate anaemia,
MCHC was 2% higher in the diseased group when compared
to the reference interval. 0
0 5 1 0 1 5

Reference interval R e t ic u lo c y t e s ( x 1 0
9
/L )
Fig. 2 Reticulocyte histogram of all breeds and genders of adult, healthy
For absolute, reticulocyte counts of all breeds and genders of horses with a normal erythron that were used to establish the reference
adult horses, the mean was 5.0 ± 3.2 to 182 × 109/L, the 2.5 to interval
Comp Clin Pathol
spleen masking peripheral signs of regeneration (Lording
2008; Satue et al. 2012; Walton 2013).
Physiological effects
We used published reference intervals to confirm that horses
used for physiological comparisons were all healthy, as well as
screened for absence of clinical signs and abnormalities of
clinical pathology profiles. After Hct and Hb screening of
horses, RBC values for healthy horses in our study were typ-
ically within literature reference interval. Only occasional,
mild, clinically insignificant differences from the published
reference interval were observed within the warm-blood
population.
Our study confirmed physiological differences for the
erythron and extended these to include reticulocytes.
Analysis of our categorised data confirmed the well-known,
haematological breed differences of smaller erythrocyte vol-
ume and compensatory-increased erythrocyte number in hot-
blooded compared to cold-blooded horses (Grondin and
Dewitt 2010). In health, reticulocytes also reflect this breed-
specific physiological difference, with higher values observed
in non-cold-blooded breeds. In contrast, gender-related differ-
ences of the erythron are subtle in horses (Satue et al. 2012)
and have no influence on reticulocytes.
Foals are reported to have erythron values at the lower end
of adult reference intervals which are typically attained at
1 year of age (Axon and Palmer 2008; Grondin and Dewitt
2010; Satue et al. 2012; Walton 2013). The Hct, Hb, RBC and
MCV data for foals in our study were the same as for these
reported references.
Young animals have age-dependent higher reticulocyte
counts than adults, reflecting the growth and lack of full matu-
ration of the erythron (Rizzi et al. 2010). We had similar find-
ings, as foal reticulocyte numbers were 50% higher relative to
adults. It is noteworthy that some of the increase in reticulocyte
numbers of foals compared to adults may be attributable to the
increase in erythrocyte numbers. However, erythrocyte counts
were only increased 10% whereas reticulocyte counts were
increased an order of magnitude more than this.
This study provides new data to support the proposal that
foal microcytosis, which peaks at 3 to 5 months of age, is
attributable to iron deficiency (Axon and Palmer 2008;
Grondin and Dewitt 2010; Satue et al. 2012). This has been
speculated on the basis of low body iron of neonates and low
iron in mare’s milk (Walton 2013). We demonstrated that re-
ticulocyte haemoglobin content (CHr), which is the most sen-
sitive biomarker of iron status (Brugnara et al. 1994; Steinberg
and Olver 2005), was 14% decreased in the foal compared to
adults of the same breed. This supports iron deficiency as a
basis of foal microcytosis. The alternative proposal that
microcytosis could be caused by removal of the larger foetal
erythrocytes is unlikely since this process occurs earlier in the
neonate (Pearson 1967; Brace et al. 2000).
This study is the first to examine reticulocyte maturity in
the horse. Reticulocyte numbers were higher in foals than in
adults, as found in other species (Rizzi et al. 2010), but their
maturity based on reticulocyte ribonucleic acid (RNA) content
was higher, unlike in other species. Immature reticulocyte
(IRF-H) numbers are correlated with the degree of erythropoi-
etin stimulation of bone marrow (Buttarello and Plebani 2008)
and are reported to be increased in neonates compared to
adults (Christensen et al. 2016). However, we found immature
reticulocytes to be 60% lower in foals than in adults. The
physiological basis for this is unknown but may relate to pref-
erential, splenic sequestration of more immature reticulocyte
or preferential splenic release of more mature reticulocytes
(Rhodes et al. 2016; Song and Groom 1972). The greater
maturity in the horse may reflect a greater splenic role in
reticulocyte storage or maturation.
Pathological effects
Our study confirmed the findings of three previous reports on
reticulocyte increases in anaemic horses (Weiss and Moritz
2003; Cooper et al. 2005; Rout et al. 2015) and further qual-
ified this finding to indicate that mild-to-moderate increases
only occur with marked anaemia. The reticulocytosis was ac-
companied by mild increases in reticulocyte haemoglobin
content (CHr), as found for other species (Brugnara et al.
1994), but with decreases in reticulocyte immaturity (IRF-
H), in contrast to what is found for other species (Chang and
Kass 1997). In other species, the response to anaemia may be
evaluated by a reticulocyte production index that incorporates
a gradual increasing factor due to the progressively increased
immaturity of reticulocytes with more severe anaemias
(Cowgill et al. 2003). Also, we determined that mild reticulo-
cyte increases occur with various, non-haematological dis-
eases. We assessed regeneration in our study, using both
MCV and reticulocyte counts alongside CHr and IRF-H data.
Anaemia
In our data, the pathological effect of anaemia was observed
with most of the studied haematological parameters and was
primarily attributable to a small proportion of cases with
higher reticulocytes in moderate-to-marked anaemia. The
most notable change was observed in a 2-month-old thor-
oughbred foal diagnosed with severe IMHA, with reticulocyte
values ranging from 27.9 to 91.3 × 109/L (reference interval of
1.16 to 13.1). Such variability, and up to 6.5-fold increases,
has previously been reported with severe IMHA (Weiss and
Moritz 2003) and severe haemorrhage (Rout et al. 2015) and
is considered to be of clinical significance. Also, the few

IMHA cases observed in our data had higher reticulocyte
values than the small number of haemorrhagic anaemic cases.
We confirmed and extended the well-known finding that in
the horse, and in other species, increased MCV values reflect
effective erythrocyte regeneration (Lumsden et al. 1975;
Weiser et al. 1983). Additionally, we found that both MCV
and reticulocytes increased with marked anaemia, but not with
mild or moderate anaemia. Previous papers have found serial
evaluations of individual MCV values to be more sensitive in
detecting macrocytosis than comparison with a population-
based reference interval and changes greater than 2.0 fL
interpreted as a true increase (Radin et al. 1986). However,
in our retrospective study, we did not have serial measurement
of reticulocyte and MCV parameters, covering the time period
over which the anaemia developed and resolved.
With marked anaemia, there was a mild increase in MCV
and a mild decrease in MCHC. The basis for why horses with
mild-to-moderate anaemia and disease had slightly higher
MCHC than for the reference interval is unknown; however,
it may be related to a mild effect on RBC hydration or it may
be an artefact of sample numbers or of pooling different
breeds for determination of the reference interval.
Findings of this study are consistent with the well-accepted
buffering effect of the equine spleen on circulating erythrocyte
numbers, and on erythrocyte maturity. With anaemia in hors-
es, erythrocyte replacement is mediated largely by the spleen
at least acutely, whereas in other species, the bone marrow
mediates this with the reticulocytes. This likely explains
why reticulocytes are not increased in equine anaemia. The
increased CHr and the decreased IRF-H indicate that more
mature reticulocytes circulate with marked anaemia. The find-
ing that peripheral blood reticulocytes are predominantly ma-
ture in marked anaemia was also observed by examination of
reticulocyte scattergrams in one study of an adult horse with
clostridium-associated IMHA (Weiss and Moritz 2003). As
suggested above, regarding maturation of the erythron in
foals, this may be explained by the preferential splenic seques-
tration of immature reticulocytes and preferential splenic re-
lease of mature reticulocytes.
Our study suggests that the spleen is responsible for
RBC mass repletion, in lieu of bone marrow release of
immature reticulocytes. In contrast to findings in other spe-
cies, in which marked anaemia is associated with release of
immature reticulocytes from the bone marrow, anaemia
was associated with release of mature reticulocytes in hors-
es. This suggests that the erythropoietic stimulation of the
bone marrow was less than occurs in other species, possi-
bly because of the buffering capacity of the spleen and its
sequestration of immature reticulocytes and releases ma-
ture reticulocytes. Similar findings were observed in dogs,
as splenectomised canines had mild reticulocytosis with
increase immaturity (Lorber 1958), implying that the
spleen has a role in reticulocyte maturation.
Comp Clin Pathol
Disease
The reticulocyte effect of non-blood diseases in horses with
normal erythron values is unknown. This study demonstrates
mild reticulocytosis in horses with colic or dysproteinaemia,
the latter typically attributed to hypoproteinaemia of gastroin-
testinal disease. Other reticulocyte increases in non-anaemic
horses and humans have previously been associated with an-
oxia such as with recent anaemia, cardiac or respiratory dis-
ease or high altitude (Jelkmann 2007; Satue et al. 2014). Also,
reticulocytosis may occur with decreased erythrocyte lifespan
due a genetic or acquired RBC defect causing increased fra-
gility (O’Brien et al. 1995), or due to low-level haemorrhagic
loss (Cowgill et al. 2003). Consequently, there is a compen-
satory increase in RBC turnover and therefore in reticulocytes.
Reticulocytosis may also be associated with marrow trophic
effects of certain drugs such as anti-inflammatory and chemo-
therapeutic medications and certain nutraceuticals (Pattullo
et al. 2015).
Reference interval
We have defined the first reticulocyte reference interval for
adult horses based on a large number of individuals (182)
and screened for pathological and age effects and for statistical
outliers. We are aware of only one other study where the
equine reference interval was reported (Giordano et al.
2008). Their 2.5 to 97.5 percentile interval was 6-fold wider
than in our study, 0.5 to 85 versus 1.2 to 13 × 109/L, mostly
because of the skewed distribution towards high values. This
skewness as characterised by the ratio of their mean to median
values was 12-fold greater than in our study: their mean was 3-
fold their median value whereas our mean was only 25%
larger than our median value. Our distribution was much less
skewed due to the rigorous screening of values for a wide
interval of diseases other than anaemia that we have demon-
strated cause reticulocytosis. Furthermore, we use a validated,
outlier exclusion policy that removed up to 5% of outliers.
Limitations of the study
There are a few potential limitations to this study. The first is
our method for determination of reticulocyte reference inter-
vals. Although this did not follow published guidelines
(Friedrichs et al. 2012), which are prospective, our unique
retrospective approach was valid, effective and necessary for
the identification of healthy horses. We used clinical exami-
nation, clinical pathology data and screening against horses
with abnormal erythron to identify healthy horses.
Secondly, we had only small numbers of anaemic cases and
did not have measurements that tracked with the development
and recovery of the anaemia. However, the study was suffi-
cient powered to resolve differences associated with marked
Comp Clin Pathol
anaemia and demonstrate that mild-to-moderate anaemia did
not produce differences. Data was not available in this study to
demonstrate that monitoring reticulocytes was of prognostic
or therapeutic value for individual cases in which comparisons
are made to the individual’s baseline rather to the reference
range. This approach has been shown to be effective in at least
one study of marked anaemia (Weiss and Moritz 2003). This
would need to be addressed in future studies.
Thirdly, CHr and IRF-H data were not available for all
horses and categories of horses. However, there was sufficient
data to conclude that there are mild-to-moderate changes with
marked anaemia and mild changes with age. Further studies
are needed to assess the diagnostic and prognostic value of
these novel reticulocyte parameters.
Fourthly, there is need to consider pre-analytical variation
as a confounder of our interpretation of changes. Up to one
third of our samples were posted to us at ambient temperature
from external practices arriving within 24 h of collection. Our
subjective experience is that effects of up to 24 h at our (Irish)
ambient temperatures are minimal, although one of two stud-
ies of storage stability of haematologic parameters have found
minor changes within 24 (Clark et al. 2002; Bauer et al. 2012)
at ambient temperature. These changes are attributable to loss
of cell energy and loss of cell water balance with erythrocytes
swelling from water uptake which results in mild increases in
MCV and Hct and decrease in MCHC. Other haematological
parameters, including Hb, are unaffected. However, we can
exclude this source of pre-analytic variation for two reasons.
Such variation would equally well affect all groups, as the
frequency of external samples was approximately the same
across groups. Secondly, those groups with higher MCV or
Hct would have unaltered Hb if the changes were due to this
artefact. In this study, the Hct and Hb were affected similarly,
ruling out storage artefact as the cause.
It is noteworthy that we defined foals as aged 0 to
12 months, and that haemogram changes peak at 3–5 months
for microcytosis, with adult values around 1 year, and in the
neonate for foetal macrocytosis (Axon and Palmer 2008;
Walton 2013). However, we had insufficient numbers of foals
to resolve changes occurring within different age groups. Our
finding confirmed that of the literature for the same age group
of 0 to 12 months, namely that there was mild microcytosis
and erythrocytosis relative to the adult. Additionally, we found
mild relative reticulocytosis and that these reticulocytes were
more mature.
Finally, we did not validate reticulocyte counts and param-
eters for the horse. However, reticulocyte counts in the horse
have been validated for the ADVIA by comparison to direct
microscopic counts in three other studies which considered
that ADVIA counts were more sensitive than the manual
method and were specific (Giordano et al. 2008; Weiss and
Moritz 2003; Cooper et al. 2005). Furthermore, there is no
reason to expect that they would be. With regard to equine
reticulocyte parameters, the CHr was studied and found to
respond similarly as in humans (Giordano et al. 2008;
Cooper et al. 2005).
Conclusions
Equine reticulocytes are only slightly increased by physiolog-
ical parameters, such as breed and age, or by various non-
blood diseases (colic, dysproteinaemia) and only mildly in-
creased by marked anaemia. Compared to other species, retic-
ulocyte counts are substantially lower in horses and their clin-
ical use is limited to marked anaemias. The first reference
interval was established for adult, healthy horses. Our data
support that in the horse, the spleen has a major role in buff-
ering erythron mass and in reticulocyte maturation. The spleen
restored lost blood, preferentially retained immature reticulo-
cytes and released mature reticulocytes and may have released
reticulocytes with various non-blood diseases.
Acknowledgements The authors wish to thank the staff of the clinical
pathology laboratory. We also thank Prof Kurt Zimmerman, Virginia-
Maryland College of Veterinary Medicine, for his thorough review of
the manuscript and helpful comments.
Compliance with ethical standards
Ethical approval For this retrospective type of study, formal consent is
not required.
Conflict of interest The authors declare that they have no conflict of
interest.
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