Research in Veterinary Science 1999, 67, 285293

Article No. rvsc.1999.0323, available online at http://www.idealibrary.com on

Bone marrow response to large volume blood collection

in the horse
N. MALIKIDES, A. KESSELL, J.L. HODGSON, R.J. ROSE, D.R. HODGSON
University Veterinary Centre, Camden, University of Sydney, PMB 4 Narellan Delivery Centre, Narellan,
NSW, Australia, 2567
SUMMARY
Evaluation of erythropoietic regeneration in horses is difficult unless serial bone marrow aspirates are performed. To investigate the
acute and chronic erythropoietic regenerative response of equine bone marrow following acute removal or loss of blood, sequential
bone marrow aspirates over 4 weeks were taken from the sternum of five horses from which 20 ml kg-1 of blood had been removed.
We found that the total number of erythroid cells counted (expressed as a percentage of the total number of erythroid and myeloid
cells counted) expanded initially by 137 per cent within 3 days after blood removal, the erythroid response peaking by 9 days with
a further 135 per cent increase. This peak coincided with the lowest M:E ratio. Concomitantly, a shift from proliferative phase cells
to maturing phase cells occurred, which appeared to persist beyond 31 days post collection. Thus, we found that the equine bone
marrow mounted a regenerative erythropoietic response more slowly than previously determined and, also, regeneration of the
erythroid compartment was incomplete 31 days after blood removal of this magnitude. 1999 Harcourt Publishers Limited

THE immediate effect of acute loss of up to 30 per cent of

total blood volume, is the activation of a variety of compensatory neuroendocrine homeostatic mechanisms, facilitating
rapid recovery of cardiovascular function, fluid volume and
plasma constituents (Runciman and Skowronski 1984,
Guyton 1986, Seeley 1987). Additionally, in the horse, resupply of up to one third of the total red cell volume rapidly
occurs as a result of splenic contraction (Persson et al 1973a,
1973b). Subsequently long term recovery of erythrocyte volume is controlled by the bone marrow. In normal horses, the
regenerative potential of bone marrow is proportional to the
volume of blood lost and the intensity of subsequent erythropoietin secretion (Giger 1992). Haemopoietic mechanisms in bone marrow induce, in a controlled manner, an
increase in the rate of erythropoiesis to meet the immediate
needs of the horse (Jain 1993).
Bone marrow examination is the most reliable technique
to evaluate erythroid regeneration in horses (Russell et al
1994). This is because equine erythrocytes remain in the
marrow until maturity (Morris 1989) and laboratory features
of regeneration, such as reticulocytosis, polychromasia,
macrocytosis, and anisocytosis, are not found after blood
loss. Significant erythropoiesis in bone marrow is not evident until 2 to 3 days after acute blood loss in dogs and cats
(Jain 1993) and up to 5 days in humans (Guyton 1986). In
contrast, maximal bone marrow response in the horse is
reported to occur 3 to 5 days after blood loss, reflected by a
decrease in the M:E ratio and an increase above 5 per cent in
the bone marrow reticulocyte count (Jain 1986).
Additional information about equine bone marrow regeneration following acute blood loss is restricted to one report
in which sequential bone marrow aspirates were taken over
25 days from three horses in which substantial volumes
(227 to 241 ml kg1) of blood were removed daily for three
0034-5288/99/000285 + 00 $18.00/0

days (Lumsden et al 1975). Little knowledge about marrow

regeneration was established though, because marked differences in the M:E ratio were found between animals. Other
reports have described the haemopoietic response to chronic
blood removal in the horse using sequential marrow aspirates over periods of up to 30 weeks (Franken et al 1982b,
Jain 1986, Tablin and Weiss 1985). However, scant information specifically is available about the acute and chronic
regenerative response of equine bone marrow following
acute blood loss. Furthermore, the time at which bone marrow completely recovers from acute blood loss has not been
accurately established.
The objective of this study was to determine the erythropoietic response of the bone marrow over time, in horses
from which 20 ml kg1 of blood (25 per cent of blood volume) had been collected.

MATERIALS AND METHODS

Horses
Five Standardbred geldings, ranging in age from 7 to 12
years and assessed as normal on physical examination, were
randomly selected from the population of blood donors at
the University of Sydneys Veterinary Centre, Camden. All
horses had undergone repeated blood collection in the past
and were well accustomed to the procedure. However, blood
collection had not been performed, or medications given for
six weeks prior to the commencement of experiments.
Preparation of donors and collection of blood
Twenty ml kg1 blood was collected from each of the
geldings on the same day. Two days prior to collection, all
1999 Harcourt Publishers Limited

286

N. Malikides, A. Kessell, J.L. Hodgson, R.J. Rose, D.R. Hodgson

horses underwent a complete physical examination and

blood was drawn for haematological analysis [i.e. red blood
cell count (RCC), packed cell volume (PCV), hemoglobin
(HB), red cell distribution width (RDW), mean cell corpuscular volume (MCV), white cell count (WCC) and white cell differential analysis]. These haematological variables also
were determined from samples of blood taken on the days
bone marrow aspirates were collected.
Blood was collected from the right jugular vein using
either a 10-gauge 8-cm needle or a 10-gauge, 76-cm Teflon
catheter (Angiocath, Becton Dickinson, USA) introduced
against the flow of blood and secured in place using adhesive glue. Blood was collected sequentially into 3 L plastic
bags (Horizon Sterile Blood and Plasma Collection bags,
Horizon Animal Reproduction Pty Ltd, AUS) pre-filled with
150 ml of 4 per cent sodium citrate anticoagulant (pH range
48 to 50) until the required quantity of blood was removed.
The needle (or catheter) was then removed from the jugular
vein and a swab used to place pressure over the site until
haemorrhage ceased. After blood was collected, horses were
housed in yards for the next 31 days and fed lucerne hay and
a combination of lucerne chaff, white chaff and oats twice
daily. In addition, horses were clinically monitored twice
daily, after blood collection, during the 31-day period of
investigation.
Collection of bone marrow
Bone marrow samples were aspirated two days prior to
blood removal and on days 3, 5, 9, 14, 21 and 31 after blood
was removed. Prior to each bone marrow aspiration, a blood
sample was taken for haematological analysis. Blood and
bone marrow samples were taken from horses on the designated day at the same time and with minimal excitement.
The horses were placed in stocks and restrained using a
nose twitch. Sedation with xylazine (05 mg kg1 intravenously; Xylazil-100; Ilium; Troy Laboratories Pty Ltd.,
Smithfield NSW) was used when necessary. Bone marrow
aspirates were collected from the sternum using a SternumTemno bone marrow needle with stylet (Bauer; N. Stenning
and Co. Pty Ltd., Sydney, NSW). A standard technique was
used for marrow aspiration (Morris 1989, Russell et al
1994). Samples of stromal marrow particles, with as little
blood contamination as possible, were immediately transferred to a watch glass containing approximately 15 to 20 ml
of 3 per cent EDTA to prevent coagulation. While the quality
of marrow samples varied between horses for a number of
reasons, effort was made to ensure adequate marrow
spicules were obtained. Consequently, multiple aspirates
were sometimes necessary using the same needle in a redirected position along the sternum.

Laboratory preparation of bone marrow

Bone marrow samples were prepared for microscopic
evaluation within 10 minutes of collection. Using a Pasteur
pipette, marrow spicules were individually removed from
the watch glass and placed on pre-labelled glass slides.
Excess anticoagulant was subsequently sucked away from
the slide using the pipette. A squash preparation was made
using a second slide placed directly over the top of the

spicules and with application of light pressure the two slides

were slowly and evenly pulled apart. After air drying, the
slides were fixed in alcohol and stained using a
Romanovsky-stain (Diff-Quik; Labaids, Sydney, NSW).
Coverslips were glued to all slides after which the slides
were stored in containers until microscopic examination
could be performed.
From the two to three slides that were prepared from each
aspiration sample, only slides with good staining characteristics, normal haemopoietic cell distribution and minimally
damaged cells were selected for interpretation. Slides were
initially examined at low (10X) magnification. Staining
quality was assessed on the basis of stain evenness, uniformity of stain uptake, cell and organelle clarity and ease of
identification. Cellularity was determined by scanning for
the presence and number of marrow spicules or unit particles as well as the proportion of fat and haemopoietic cells
(Grindem 1989). A grade of hypocellular was given if less
than 25 per cent of the unit particle was cellular marrow,
normocellular if 50 per cent was cellular marrow and hypercellular if greater than 75 per cent of the unit particle was
cellular marrow. An estimate of megakaryocyte numbers per
low power field also was made. Using 20X and 40X magnification, a subjective assessment of the proportions of erythroid and myeloid cells, as well as the proportions of cells
that were within proliferating or maturing stages was made.
At high (100X) magnification, greater than 500 myeloid and
erythroid cells at various stages of differentiation were
counted and the M:E ratio accurately calculated. Cells that
were categorised as megakaryocytes or miscellaneous
(including basket or damaged cells), were also counted but
were not included in the 500 myeloid and erythroid cells
used for the M:E ratio. Interpretation of M:E ratios was
made in conjunction with the peripheral haematological
results. Reticulocytes were not counted as appropriate bone
marrow staining techniques (eg, new methylene blue) were
not used for identification. However, reticulocytes or polychromatic macrocytes were subjectively estimated in several
fields as either greater or less than 5 per high power field,
although accurate counts were not made at this time.
Classification of bone marrow cells and data generation
Bone marrow cells were identified and grouped according
to the classification described by Jain (1993) with minor
modifications. In the erythroid series, only pronormoblasts
(rubriblasts) and basophilic normoblasts (prorubricytes and
basophilic rubricytes) were classified as proliferative phase
cells. Early, intermediate and late normoblasts (polychromatophilic, normochromic rubricytes and meta-rubricytes
respectively) were classified as maturing phase cells.
Individual cells of the erythroid line were counted for each
horse on the selected day of investigation and then expressed
as percentages of the total number of erythroid cells
counted. The percentage [mean (SEM)] was subsequently
calculated. Similar percentages [mean (SEM)] were calculated for individual cells of the myeloid series (as percentages of the total number of myeloid cells counted) and
miscellaneous cell series (as percentages of the total number
of all cells counted). By adding the individual percentages
of cells that made up the proliferating and maturing pools, a
proliferating pool to maturing pool (P:M) ratio was calcu-

Bone marrow response to blood collection

lated for both erythroid and myeloid lines. The total number
of erythroid cells counted was expressed as a percentage of
the total number of erythroid and myeloid cells counted. A
similar calculation was made for the total number of
myeloid cells counted. In addition, the total numbers of proliferative phase cells and maturing phase cells of the erythroid series was expressed as a percentage of the total
number of erythroid and myeloid cells counted.

287

after blood removal. While increased numbers of proliferative erythroid cells were seen during this time, numbers of
maturing cells predominated 14 to 31 days post-collection,
which tended to maintain high overall erythroid cell numbers. Concomitantly, less myeloid cells were noted and
counted over the study period and subjectively the M:E ratio
remained low between 3 and 31 days post blood collection.
Objective microscopic evaluation

Statistical analysis
Statistical analysis was performed using a repeated measures, one-way analysis of variance (ANOVA; Statistica,
StatSoft Inc. Tulsa, Oklahoma, USA) to determine the effect
of removal of 20 ml kg1 of blood on different erythroid cell
types over time. Post hoc determination of least significant
difference was performed if the F value indicated a significant difference (P < 005) over time after blood collection.
All results are expressed as mean (SEM) unless otherwise
indicated. While statistical analysis was performed on the
other cell types described, emphasis was placed on the erythroid series only. However, general trends were noted, in
particular regarding descriptive analyses of the myeloid
series and miscellaneous cells.

RESULTS
Vital signs and results of haematology were consistently
within reference range (Jain 1986, Lumsden et al 1980) in
all horses 2 days prior to blood removal and before each
bone marrow collection. Body weight ranged from 455 to
580 kg [516 (51) kg] and volume of blood collected ranged
from 9 to 11 L [102 (10) L].
Smear quality and subjective microscopic evaluation
In general, the staining quality of all smears was good,
with clear cellular and organelle characteristics and easy
identification of cell types. Nevertheless, occasional smears
were moderately understained while others had uneven stain
distribution between different fields. Also, most smears had
moderate to high spicule density (>10 to 30 spicules) and
were classified as normocellular. However, while several
individual marrow aspirates were hypocellular, there was no
particular time during the period of bone marrow investigation when aspirates were more likely to have poor cellularity.
Megakaryocyte numbers for all smears were adequate,
ranging from 2 to 4 per low power field. Cells of the miscellaneous series that were identified included lymphocytes,
mitotic figures, monocytes/macrophages, unclassified cells
(unidentifiable cells) and basket cells (fragmented cells,
pyknotic nuclei or irregularly roundish, net-like, pinkish
structures). As a group, miscellaneous cells constituted less
than 12 per cent of the total number of erythroid, myeloid
and miscellaneous cells counted. The average number of
polychromatic macrocytes was 5 per field in smears before
blood collection although >5 per field in all smears up to 21
days after blood collection. By day 31, the average numbers
were only marginally >5 per field.
Subjective assessment of smears at low power showed
profound increases in cells of the erythroid series 3 to 9 days

Mean values and ranges for all individual erythroid and

myeloid cells as well as for the proliferating and maturing
pools of the erythroid and myeloid series, the total number
of erythroid and myeloid cells counted, the M:E ratio and
the P:M ratio of the erythroid and myeloid series, prior to
blood removal, are presented in Table 1. For comparison,
previously reported ranges and values also are shown
(Archer 1954, Calhoun 1954, Franken et al 1982a, Jain
1986, Tschudi et al 1975). In general, most of the means and
ranges for the erythroid series generated from the five normal horses in this investigation prior to blood removal, were
comparable to values reported elsewhere. A summary of all
data generated over the 31 days of investigation is shown in
Table 2. In addition, Figs 1 to 4 illustrate the regenerative
responses of cells of the erythroid series over 31 days and
the times at which results were significantly different from
time zero.
Erythroid series
After the removal of 20 ml kg1 of blood, the total erythroid cell compartment expanded initially by 137 per cent
in the first 3 days, peaking by 9 days with a further 135 per
cent increase. Total erythroid cell numbers were greater 3 to
31 days post-collection, than at time zero (P < 001). While
numbers declined after 9 days, values remained higher up to
31 days than at time zero and 3 days post-collection (P <
001).
Fig 1 shows the regenerative response of pronormoblasts
and basophilic normoblasts after blood removal. It is important to note that although pronormoblast numbers had
almost returned to time zero values by 14 days post-collection, numbers still tended to be higher than baseline after 31
days. In contrast, numbers of basophilic normoblasts were
still higher 14 days after collection than at time zero (P <
005), subsequently returning to time zero values between
days 21 and 31.
Fig 2 illustrates the response of proliferating phase cells
and maturing phase cells over 31 days. While the numbers
of proliferative phase cells began declining after peaking at
5 days post collection, values were still marginally (12 per
cent) higher than baseline percentages 31 days after collection [81 (06) at time zero vs. 93 (11) per cent at 31 days, P
> 005]. The maturing pool numbers were lower at 3, 5, 9
and 21 days following blood removal (P < 005) with values
returning toward baseline between 5 d and 31 days [919
(06) at time zero vs. 907 (11) per cent at 31 days, P >
005].
When cells of the proliferating and maturing pools were
considered as a percentage of the total number of erythroid
and myeloid cells counted, two specific details were added
to the above results. First, proliferative erythroid cells

0511
1744
110328
165345
189327
335421
47172
647835
0205
393478
065092

42119
28104

08 (01)
32 (05)
205 (35)
244 (30)
263 (27)
386 (15)
105 (20)
754 (31)
03 (006)
452 (15)
08 (005)

88 (13)
64 (13)

28148
04124

0412
1054
45365
110378
143383
320452
15195
614894
006054
386520
0610

057
2585
59103
130262
297449
322388
897941
005013
486610

Reference range%

174

637
243

83316

11102

102

263

191417

25226

024
05
194

0066
014099
118309

2616

176

634

22130a
42392

034
188

Mean%

0087
016435

Range%*

Archer (1954)
(12 ponies)

32112

0938

2224

05
26656

5242

832a

0434

67

5674
164

145

18
381

3466

137

209

16b

Mean%

Calhoun (1954)
(7 horses)
Range%

i. myelocytes include neutrophilic, basophilic and eosinophilic forms

ii. metamyelocytes include neutrophilic, basophilic and eosinophilic forms
iii. bands include neutrophilic, basophilic and eosinophilic forms
iv. segmenters include neutrophilic, basophilic and eosinophilic forms
v. miscellaneous cells include monocytes/macrophages, mitotic figures, plasma cells, lymphocytes and unclassified cells
a. range for early and itermediate normoblasts (classified as rubricytes by Jain, 1986).
b. defined by Calhoun as stem cells and not included in total erythroid count.
c. classified as prorubricytes and basophilic rubricytes by Jain, 1986.
* mean (2 sd).

0754
4079
6394
145247
322431
319371
906938
00601
522607

Min-Max range%

26 (09)
55 (07)
81 (06)
199 (16)
373 (19)
350 (09)
919 (06)
009 (001)
548 (15)

Mean (sem)%

Current Study
(n = 5 horses)

Bone marrow differential cell counts (%) from normal horses in the current study and reported in literature

Erythroid Series
Pronormoblasts
Basoph. normobl.
Proliferating Pool
Early normoblasts
Interm. normoblasts
Late normoblasts
Maturing Pool
P:M ratio
Total erythoid cells
Myeloid Series
Myeloblasts
Promyelocytes
Myelocytesi
Proliferating Pool
Metamyelocytesii
Bandsiii
Segmentersiv
Maturing Pool
P:M ratio
Total myeloid cells
M:E ratio
Miscellaneous
Series
Miscellaneous cellsv
Basket cells

Cell Type

TABLE 1:

1010

0308

11311

5116

0320
0030
1053

2545

1023a

0604
2090

Range%

62

3453
06

2074

81

119
127
3 33

6048

349

162

218
578

Mean%

Tschudi et al (1975)
(15 horses)

1515

0509

1515
6265
3 225

0050
0535
1075

1436

14544a

0 020
1095

Range%

55

379
071

56
157
105

10
1 7
32

559

232

282

07
36

Mean%

Franken et al (1982a)
(24 horses)

22114
2390

281484
052145

2391
74159
114254

0315
1019
2141

332562

14726
0743
107154

0611
55131c

Range%

Jain (1993)
(4 horses)

288
N. Malikides, A. Kessell, J.L. Hodgson, R.J. Rose, D.R. Hodgson

289

Bone marrow response to blood collection

TABLE 2: Results (mean [sem]) of bone marrow aspirate percentage cell counts taken from five horses on selected days over 4 weeks and cell
counts from two horses after 6 weeks.

Erythroid Series1
Pronormoblast
Basoph. normoblast
Proliferating pool (P)
Early normoblast
Interm-normoblast
Late normoblast
Maturing pool (M)
P:M ratio (erythroid)
Total erythroid cells2
Myeloid Series1
Myeloblasts
Promyelocytes
Myelocytes
Proliferating pool
Metamyelocytes
Bands
Segmenters
Maturing pool
P:M ratio (myeloid)
Total myeloid cells2
M:E ratio
Miscellaneous
Series3
Miscellaneous cells
Baskets cells
Comparative
Haematology4
PCV
RCC
HB
MCV
RDW
WCC

Day 0

Day 3

Day 5

Day 9

Day 14

Day 21

Day 31

26 (01)
55 (07)
81 (06)
196 (16)
373 (19)
350 (09)
919 (06)
01 (00):1
548 (15)

87 (18)
73 (15)
160 (14)
136 (19)
330 (17)
374 (35)
840 (15)
02 (00):1
685 (35)

73 (09)
94 (21)
167 (23)
126 (15)
373 (26)
334 (26)
833 (23)
02 (00):1
730 (26)

69 (18)
65 (05)
134 (18)
200 (28)
304 (15)
362 (33)
866 (18)
02 (00):1
820 (16)

38 (08)
85 (15)
123 (20)
173 (26)
358 (30)
346 (34)
877 (20)
01 (00):1
795 (22)

53 (12)
84 (09)
137 (17)
112 (15)
286 (18)
465 (27)
863 (17)
02 (00):1
783 (36)

39 (03)
53 (13)
93 (11)
118 (09)
390 (29)
399 (22)
907 (11)
01 (00):1
784 (38)

08 (01)
32 (05)
205 (35)
244 (30)
263 (27)
386 (15)
105 (20)
754 (31)
03 (01):1
451 (15)
08 (00):1

30 (02)
52 (16)
108 (16)
190 (31)
147 (37)
471 (32)
191 (56)
818 (31)
02 (00):1
315 (35)
05 (01):1

23 (08)
46 (14)
105 (17)
174 (19)
127 (19)
448 (40)
251 (35)
826 (19)
02 (00):1
270 (26)
04 (01):1

18 (05)
21 (10)
86 (16)
125 (25)
135 (41)
412 (71)
331 (75)
877 (26)
01 (00):1
180 (16)
02 (01):1

27 (11)
34 (14)
99 (33)
159 (47)
188 (21)
402 (31)
250 (66)
841 (47)
02 (01):1
205 (22)
03 (01):1

16 (09)
35 (11)
65 (32)
115 (37)
168 (27)
417 (60)
300 (83)
885 (37)
01 (01):1
217 (36)
03 (01):1

11 (06)
43 (13)
78 (12)
13 3 (28)
135 (38)
375 (62)
357 (87)
867 (28)
02 (00):1
216 (38)
03 (01):1

88 (13)
64 (14)

83 (20)
65 (18)

38 (13)
20 (08)

85 (34)
57 (29)

54 (08)
39 (07)

101 (14)
72 (09)

116 (52)
55 (26)

033 (00)
71 (03)
119 (38)
465 (21)
180 (02)
82 (07)

032 (00)
69 (03)
116 (49)
463 (19)
190 (04)
89 (09)

031 (00)
66 (03)
111 (42)
467 (13)
182 (02)
97 (07)

033 (00)
72 (04)
120 (64)
462 (16)
181 (02)
97 (07)

030 (00)
65 (03)
109 (55)
457 (14)
182 (02)
81 (06)

032 (00)
70 (04)
119 (72)
455 (12)
183 (01)
80 (05)

031 (00)
69 (04)
118 (69)
448 (19)
180 (03)
82 (05)

1. Results [mean (sem)] of individual cells of the erythroid and myeloid series are expressed as a percentage of the total erythroid and myeloid cells counted
respectively.
2. Results [mean (sem)] of total erythroid or total myeloid cells are expressed as a percentage of the sum of the erythroid and myeloid cells counted.
3. Results [mean (sem)] of the total miscellaneous (all miscellaneous cells including basket cells) and basket cells are expressed as a percentage of the total
number of all cells counted.
4. All results [mean (sem)] of selected haematological variables were calculated from blood samples collected prior to bone marrow aspiration.

increased from values prior to blood collection of 44 (02)

per cent to 122 (17) per cent, (P < 001) 5 days after collection. Numbers subsequently declined, reaching values not
significantly different from time zero 31 days post collection
[44 (02) at time zero vs. 73 (10) per cent at 31 days, P >
005]. Second, while cells entered the proliferating phase
after blood collection, maturing phase cells also concomitantly increased from time zero percentages of 505 (17) to
710 (35) per cent, (P < 001) 9 days after collection, maintaining this level up to 31 days.
Fig 3 shows the variable changes of the early, intermediate and late normoblast cells of the maturing pool during the
post-collection period.
The mean M:E ratio prior to blood removal was 082
(012) to 1 with a lower and upper range of 065 to 092 and
a reference range [mean (2 sd)] of 06 to 10 (Table 1).
Although numbers of horses in this study were few, these
results were similar to published values (Franken et al
1982a, Jain 1993, Latimer and Andreasen 1992). The M:E
ratio was lower at all days after blood collection than at time
zero (P < 001). The lowest M:E ratio occurred after 9 days,
beyond which the M:E ratio remained significantly lower up
to day 31, than at time zero and day 3 (P < 001) (Fig 4). In
comparison, the P:M ratio increased sharply after blood
removal with values higher after 3 days (P < 005), 5 days (P

< 001) and 9 days (P < 005), than at time zero.

Subsequently, the P:M ratio gradually declined to values
comparable to time zero in a manner similar to the response
of the proliferating pool (Fig 4).
Myeloid and miscellaneous series
In general, the proliferative phase cells (myeloblasts,
promyelocytes and neutrophilic, eosinophilic and basophilic
myelocytes) of the myeloid series tended to decline over the
31 days after blood removal despite early increases in
myeloblasts and promyelocytes. In contrast, the cells of the
maturing myeloid pool (neutrophilic, eosinophilic and
basophilic metamyelocytes, bands and segmenters) tended
to increase over the 31 days, due mostly to increases in neutrophilic, eosinophilic and basophilic segmenters. The
myeloid P:M ratio, therefore, tended to decrease over the 31
days after collection.
The numbers of individual cells classified as miscellaneous (including lymphocytes, monocytes/macrophages,
mitotic figures, unclassified and basket cells) fell within
similar ranges to those reported (Jain 1993). The total number of miscellaneous cells (not including basket cells)
increased as time progressed beyond 14 days and as more
bone marrow aspirations were taken. However, average

290

N. Malikides, A. Kessell, J.L. Hodgson, R.J. Rose, D.R. Hodgson

Proliferating pool (percentage)

10
*
8

6
4
2
12
10

Maturing pool (percentage)

Basophilic normoblasts (percentage)

Pronormoblasts (percentage)

12

8
6
4
2

20.0
17.5

*
15.0

12.5
10.0
7.5
5.0
92.5
90.0
87.5

85.0

*
*

82.5
80.0

3 5

14

21

31

3 5

Time (days)

14

21

31

Time (days)

FIG 1: Bone marrow regenerative response over 31 days after 20 ml kg1

blood removal: Values [mean (sem)] for pronormoblasts and basophilic normoblasts expressed as a percentage of the total number of erythroid cells
counted.
*significantly different than time zero (P < 005).

FIG 2: Bone marrow regenerative response over 31 days after 20 ml kg1

blood removal: Values [mean (sem)] for the erythroid proliferative pool
(pronormoblasts and basophilic normoblasts) and maturing pool (early, intermediate and late normoblasts) expressed as a percentage of the total number
of erythroid cells counted.
*significantly different than time zero (P < 005).

.9

16

12

.7
.6
.5

**

.4

**

.3

8
44
40
36
32
28
24
48
44
40
36
32
28

.2

**

**

**

**

.1

#
d

3 5

14

21

31

Time (days)
FIG 3: Bone marrow regenerative response over 31 days after 20 ml kg1
blood removal: Values [mean (sem)] for early, intermediate and late normoblasts expressed as a percentage of the total number of erythroid cells
counted.
*significantly different than time zero and 9 days after blood collection (P <
005).
#significantly different than time zero and 31 days after blood collection (P <
005).
dsignificantly different than all previous times (P < 005).

P:M ratio (erythroid series)

Early (%)
Intermediate (%)
Late (%)

20

M:E ratio

.8
24

.24
**

.20

.16

.12
0.8
2

3 5

14

21

31

Time (days)
FIG 4: Bone marrow regenerative response over 31 days after 20 ml kg1
blood removal: Values [mean (sem)] for 1) the myeloid to erythroid ratio (M:E)
ratio between the total numbers of myeloid and erythroid cells counted; and
2) the proliferative pool to maturing pool ratio (P:M) ratio between the values
[mean (sem)] for the erythroid proliferative pool and maturing pool expressed
as a percentage of the total number of erythroid cells counted.
*significantly different than time zero (P < 005)
**significantly different than time zero (P < 001).

Bone marrow response to blood collection

numbers of basket or smudge cells remained relatively constant in most smears over the study period.

DISCUSSION
The use of horses previously used for blood donation may
have influenced the findings of this investigation.
Unfortunately, restrictions on economics and horse availability precluded the use of a more ideal or normal population of experimental horses. However, with the exception
one inconclusive report (Lumsden et al 1975), no previous
information describing similar work in horses or humans is
available. In addition, other sources of information about
equine bone marrow response are restricted to reports of single aspirates for the purposes of determining response to
anaemia caused by certain toxicological, neoplastic,
haemolytic and inflammatory diseases (Jain 1986, Schalm
1980). Despite the paucity of information, it probably is reasonable to assume that bone marrow obtained from normal
horses following acute blood loss of the magnitude produced in the current study, would follow similar trends to
marrow responses observed in this study. However, it is
likely that bone marrow from blood donor horses would be
more responsive and may recover more rapidly, following
acute blood removal or loss, than normal horses.
Interestingly, this point may place greater importance to the
finding that regeneration of the erythroid compartment was
not complete 31 days after blood removal and that normal
horses, under similar circumstances, might take even longer
to recover.
This study demonstrated that there was an intense erythropoietic regenerative response of bone marrow over time,
after acute removal of 20 ml kg1 of blood from donor
horses. The marked expansion of the erythroid compartment
in the first 3 days was due to a doubling in numbers of proliferative phase cells. Numbers of proliferative phase cells
began to decline 5 days after blood removal, coinciding with
more cells entering the maturing phase, so that total numbers of erythroid cells counted reached highest values after 9
days. In contrast, previous reports have suggested that maximal bone marrow response occurs 3 to 5 days after blood
loss in the horse (Jain 1986). The peak erythroid response 9
days after collection found in this study also corresponded
with the lowest M:E ratio. Thereafter, recovery of the erythroid compartment to control values commenced with proliferative phase cells continuing to decrease while maturing
phase cells increased in numbers.
Results of this study also showed that the erythroid compartment was not completely recovered 31 days after blood
removal. Although the proportions of proliferative and
maturing phase cells had returned to numbers not significantly different from pre-blood removal levels 31 days after
blood removal, total erythroid cell numbers were still
increased and M:E ratios remained significantly below
reference range. No studies have determined how long bone
marrow takes to fully recover to the pre-collection state. The
findings of the present study imply that equine bone marrow
requires at least 1 month to recover from blood loss of this
magnitude. Although close to completion at this time, bone
marrow maturing phase cells were still required to meet the
immediate needs of the horse (Jain 1993).

291

In the current study, prior to blood removal, the proliferative phase cells made up 81 (06) per cent, with maturing
phase cells accounting for 919 (06) per cent, of the total
number of erythroid cells counted. In comparison, Jain
(1993) and Latimer and Andreasen (1992) report that about
5 per cent of the erythroid population consist of proliferating
cells, the remaining 95 per cent consisting of maturing cells.
Additionally, while it is reported that less than 15 per cent of
myeloid cells are proliferative and greater than 85 per cent
are maturing phase cells (Latimer and Andreasen 1992), we
found higher proportions of myeloid proliferative phase
cells and lower percentages of myeloid maturing phase cells
[244 (30) vs. 754 (31) per cent] prior to blood removal.
This difference in results may be a reflection of the type of
horses used in this investigation, all being frequent blood
donors. However, it is important to note that all horses had
M:E ratios within published reference ranges prior to blood
removal, suggesting that there was little ongoing erythropoietic demand because of previous use.
Response of bone marrow to acute blood removal or loss
in this study was characterised by expansion of the erythroid
proliferating pool initially but with the majority of cells still
within the maturing pool and no, or few, morphologic abnormalities (Grindem 1989). When the proliferative phase and
maturing phase erythroid cells were assessed as percentages
of the total number of erythroid and myeloid cells counted,
it was found that the initial expansion of the proliferative
pool of cells over the first 5 days after blood removal was
accompanied by a 205 per cent increase in maturing cells
over the first 9 days as well. This phenomenon is simply
explained by the progressive differentiation and maturation
of proliferative cells into maturing cells. It is important to
note that proportions of proliferative phase cells and maturing phase cells as percentages of the total number of erythroid cells counted and the total number of erythroid and
myeloid cells counted, as well as the M:E ratio, conveyed
the most information in this study. Interpretation of changes
in individual cells (erythroid or myeloid) was difficult
because of the limited number of horses used. In addition,
variation occurred in numbers of individual cells in smears
from different horses, as well as in ratios of cells between
certain fields on the same smear, especially near marrow
particles. While every effort was made to minimise variation
by counting cells from a variety of fields and repeating
counts if necessary, this remains an important limitation to
this type of study.
The M:E ratio reflects activity of the erythroid and
myeloid cell lines and usually is interpreted in conjunction
with results of a complete blood cell count (Grindem 1989).
In the present study, results of haematology including PCV,
RCC, HB, MCV, RDW, and WCC were within reference ranges
(Jain 1986, Lumsden et al 1980) at the time of bone marrow
aspiration, indicating that decreased M:E ratios were due to
erythroid hyperplasia (Latimer and Andreasen 1992). When
calculating the M:E ratio, Franken et al (1982a) state there is
little difference between differentiating 200 cells compared
to 500 cells. Also, Grindem (1989) reports that subjective
estimation of changes in the M:E ratio may be more informative, easier and less time consuming than performing and
interpreting a count of 500 or more cells. In this study, subjective evaluation of cells was made and at least 500 erythroid and myeloid cells were differentiated to define the

292

N. Malikides, A. Kessell, J.L. Hodgson, R.J. Rose, D.R. Hodgson

relative distribution of cells in proliferative and maturing

phases and for the calculation of M:E ratios (Grindem 1989,
Jain 1993, Tyler and Cowell 1989). However, some limitations in accurately quantifying the M:E ratio were still
encountered. For example, apart from limitations already
discussed, some cells were difficult to classify, ratios from
the same site often differed due to uneven distribution of
myeloid and erythroid cells within the marrow and on
smears (Grindem 1989), while reported M:E ratios encountered in normal horses varied widely (Jain 1986, Tyler and
Cowell 1989). In view of these limitations, counting at least
500 cells probably increased the accuracy of classification
and quantification of myeloid and erythroid cells in this
investigation.
Aspirates for cytology are adequate to characterise marrow erythropoiesis (Morris 1989), so marrow biopsies were
considered unnecessary in this study. The sternum was used
for aspiration of marrow samples because this site was reliable and diagnostic specimens could frequently be obtained
(Russell et al 1994). Also, the sternum is not covered by a
large muscle mass and the marrow cavity is covered by a
thin layer of bone only (Morris 1989, Russell et al 1994).
However, the major disadvantage of this approach was the
awkward position the investigator was forced to assume
when obtaining the sample. Another drawback is the relative
proximity of vital organs, including the heart (Russell et al
1994). Inadvertent thoracic puncture (Berggren 1981) and
cardiac laceration (Jacobs et al 1983) was avoided in this
study by taking care during needle placement, needle guards
and close monitoring of needle depth in bone.
No complications of bone marrow aspiration were noted
over the study period. There was only minimal haemorrhage
after marrow sampling, little evidence of swelling or inflammation at the sites of aspiration, and the procedure appeared
to have little effect on horses. Although by the final (8th)
aspirate on day 31 samples became difficult to collect, this
generally did not result in a decrease in smear cellularity.
The reason for collection difficulty of the last aspirate probably was related to repeated needle trauma and fibrosis in
adjacent sites. In general, there was enough space along the
sternebrae to repeat aspirates, although this made the procedure more time consuming and extra needles were often
necessary.
While smears of bone marrow aspirates can be made
directly without anticoagulant and before the sample clots
(Grindem 1989, Morris 1989, Russell et al 1994), bone marrow samples in this study were first placed in anticoagulant
and smears made subsequently. This allowed one person to
perform the aspirate and process the sample. In addition, it
provided the opportunity to collect repeat aspirates if necessary and more time to produce multiple smears of good
quality. Routinely, several slides are stained with any
Romanovsky- or Wright-type stain for morphological evaluation, while Prussian blue stain is required for semi-quantitation of iron stores. However, iron store evaluation was
beyond the scope of this investigation. Also, because of concerns of minor contamination of most marrow aspirates with
peripheral blood (Franken et al 1982a, Schalm 1980) as well
as constraints on time, reticulocyte staining [using new
methylene blue stain (Morris, 1989)] and counts were not
performed. Nevertheless, reticulocytes or polychromatic
macrocytes were subjectively assessed while performing the

differential erythroid and myeloid count in Diff-Quik

stained smears. In limited data presented by Schalm (1980),
counts of greater than five polychromatic macrocytes per oil
immersion field were found to be more indicative of effective erythropoiesis compared to the M:E ratio alone. In the
present study, average polychromatic macrocyte numbers on
all measurement days after blood collection, varied from
well above 5 to marginally above 5 by day 31. However, it
should be noted that there was wide variation in numbers of
polychromatic macrocytes between fields due to the oftenuneven distribution of these cells. Consequently, interpretation of subjective estimates of polychromatic macrocyte
numbers was difficult. Accurate counting of cells in 20 or
more fields, as described by Schalm (1980), may have
placed further weight to the results found in the current
study. Interestingly, in this study, polychromatic macrocytes
before blood removal numbered just less than 5. While M:E
ratios before blood collection fell within reference range, the
presence of borderline numbers of macrocytes may be interpreted to suggest some residual, ongoing erythropoietic
activity. This most likely was a reflection of the function of
horses used in this study, all being blood donors though no
blood collection had been performed for 6 weeks prior to the
study.
Finding any megakaryocytes in bone marrow aspirates in
horses is evidence of an adequate number (Jain 1993). These
cells were easily visualised in smears in this study, most
commonly near marrow particles. Also, while numbers of
miscellaneous cells designated as either unclassified or basket cells were within acceptable ranges, occasional smears
had disproportionately increased numbers of basket cells.
This was probably the result of poor aspiration, sample handling and smearing technique and may have caused an
increase in the total miscellaneous cell count toward the end
of the study period. Basket cells are the swollen free nuclear
material of damaged older cells or granulocytes, which are
more susceptible to rupture during preparation of smears
(Jain 1986). According to Jain (1986), they should be
included in a bone marrow differential count. However,
because of the difficulty in definitively identifying the origin
of these cells as either erythroid, myeloid or miscellaneous,
baskets cells were not included in the count for calculation
of the M:E ratio in this study. For this reason also, speculation about the effect of adding basket cell numbers to the
myeloid count, resulting in higher M:E ratios, would
unlikely be useful. The mean numbers of basket cells over
the study period were never greater than 72 per cent of the
total number of erythroid, myeloid and miscellaneous cells
counted. Therefore, deleting low numbers of basket cells
from the myeloid count, would probably have had minimal
overall effect on the M:E ratio.
In conclusion, this study demonstrated that the intense
erythropoietic response of equine bone marrow following
acute removal of 25 per cent of blood volume was characterised by marked expansion of the erythroid compartment
in the first 3 days and a peak erythroid response 9 days after
blood removal. This peak response coincided with the lowest M:E ratio and a shift from erythroid proliferative phase
cells to maturing phase cells over approximately 31 days.
Results of this study also showed that equine bone marrow
not only takes more time to mount a regenerative erythropoietic response than previously reported, but also regenera-

Bone marrow response to blood collection

tion of the erythroid compartment probably was not complete 31 days after blood removal of this magnitude. This
information may add to the body of knowledge about bone
marrow responses in the horse, be useful for equine clinicians when monitoring horses post-haemorrhage and enable
clinicians to better predict the expected response times of
equine bone marrow.

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Accepted June 27, 1999