r

Sra

eb o

ssftesnlova

Research in Veterinary Science 1999, 67, 285293nTHE immediate effect of acute loss of up to 30 per cent oftotal blood volume, is the activation of a variety of compen-satory neuroendocrine homeostatic mechanisms, facilitatingrapid recovery of cardiovascular function, fluid volume andplasma constituents (Runciman and Skowronski 1984,

Bone marrow response to lain the

N. MALIKIDES, A. KESSELL, J.L. HO

University Veterinary Centre, Camden, University ofNSW, Aust

SUMM

Evaluation of erythropoietic regeneration in horses is difficult unlacute and chronic erythropoietic regenerative response of equine bone marrow aspirates over 4 weeks were taken from the sternumWe found that the total number of erythroid cells counted (exprecells counted) expanded initially by 137 per cent within 3 days aa further 135 per cent increase. This peak coincided with the lowto maturing phase cells occurred, which appeared to persist beyomarrow mounted a regenerative erythropoietic response more serythroid compartment was incomplete 31 days after blood remo

Article No. rvsc.1999.0323, available online at http://www.idealibrary.com oGuyton 1986, Seeley 1987). Additionally, in the horse, re-supply of up to one third of the total red cell volume rapidlyoccurs as a result of splenic contraction (Persson et al 1973a,1973b). Subsequently long term recovery of erythrocyte vol-ume is controlled by the bone marrow. In normal horses, theregenerative potential of bone marrow is proportional to thevolume of blood lost and the intensity of subsequent ery-thropoietin secretion (Giger 1992). Haemopoietic mecha-nisms in bone marrow induce, in a controlled manner, anincrease in the rate of erythropoiesis to meet the immediateneeds of the horse (Jain 1993).

Bone marrow examination is the most reliable techniqueto evaluate erythroid regeneration in horses (Russell et al1994). This is because equine erythrocytes remain in themarrow until maturity (Morris 1989) and laboratory featuresof regeneration, such as reticulocytosis, polychromasia,macrocytosis, and anisocytosis, are not found after bloodloss. Significant erythropoiesis in bone marrow is not evi-dent until 2 to 3 days after acute blood loss in dogs and cats(Jain 1993) and up to 5 days in humans (Guyton 1986). Incontrast, maximal bone marrow response in the horse isreported to occur 3 to 5 days after blood loss, reflected by adecrease in the M:E ratio and an increase above 5 per cent inthe bone marrow reticulocyte count (Jain 1986).

Additional information about equine bone marrow regen-eration following acute blood loss is restricted to one reportin which sequential bone marrow aspirates were taken over25 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 $18.00/0days (Lumsden et al 1975). Little knowledge about marrowregeneration was established though, because marked differ-ences in the M:E ratio were found between animals. Otherreports have described the haemopoietic response to chronicblood removal in the horse using sequential marrow aspi-

ge volume blood collection horse

DGSON, R.J. ROSE, D.R. HODGSON

ydney, PMB 4 Narellan Delivery Centre, Narellan,lia, 2567

ARY

ss serial bone marrow aspirates are performed. To investigate theone marrow following acute removal or loss of blood, sequentialf five horses from which 20 ml kg-1 of blood had been removed.ed as a percentage of the total number of erythroid and myeloider blood removal, the erythroid response peaking by 9 days witht M:E ratio. Concomitantly, a shift from proliferative phase cellsd 31 days post collection. Thus, we found that the equine bonewly than previously determined and, also, regeneration of the l of this magnitude. 1999 Harcourt Publishers Limited

rates over periods of up to 30 weeks (Franken et al 1982b,Jain 1986, Tablin and Weiss 1985). However, scant informa-tion specifically is available about the acute and chronicregenerative response of equine bone marrow followingacute blood loss. Furthermore, the time at which bone mar-row completely recovers from acute blood loss has not beenaccurately established.

The objective of this study was to determine the erythro-poietic response of the bone marrow over time, in horsesfrom which 20 ml kg1 of blood (25 per cent of blood vol-ume) had been collected.

MATERIALS AND METHODSHorses

Five Standardbred geldings, ranging in age from 7 to 12years and assessed as normal on physical examination, wererandomly selected from the population of blood donors atthe University of Sydneys Veterinary Centre, Camden. Allhorses had undergone repeated blood collection in the pastand were well accustomed to the procedure. However, bloodcollection had not been performed, or medications given forsix weeks prior to the commencement of experiments.

Preparation of donors and collection of bloodTwenty ml kg1 blood was collected from each of the

geldings on the same day. Two days prior to collection, all

1999 Harcourt Publishers Limited

N. Malikides, A. Kessell, J.L. Hodgson, R.J. Rose, D.R. Hodgson286horses underwent a complete physical examination andblood was drawn for haematological analysis [i.e. red bloodcell count (RCC), packed cell volume (PCV), hemoglobin(HB), red cell distribution width (RDW), mean cell corpuscu-lar volume (MCV), white cell count (WCC) and white cell dif-ferential analysis]. These haematological variables alsowere determined from samples of blood taken on the daysbone marrow aspirates were collected.

Blood was collected from the right jugular vein usingeither a 10-gauge 8-cm needle or a 10-gauge, 76-cm Tefloncatheter (Angiocath, Becton Dickinson, USA) introducedagainst the flow of blood and secured in place using adhe-sive glue. Blood was collected sequentially into 3 L plasticbags (Horizon Sterile Blood and Plasma Collection bags,Horizon Animal Reproduction Pty Ltd, AUS) pre-filled with150 ml of 4 per cent sodium citrate anticoagulant (pH range48 to 50) until the required quantity of blood was removed.The needle (or catheter) was then removed from the jugularvein and a swab used to place pressure over the site untilhaemorrhage ceased. After blood was collected, horses werehoused in yards for the next 31 days and fed lucerne hay anda combination of lucerne chaff, white chaff and oats twicedaily. In addition, horses were clinically monitored twicedaily, after blood collection, during the 31-day period ofinvestigation.

Collection of bone marrowBone marrow samples were aspirated two days prior to

blood removal and on days 3, 5, 9, 14, 21 and 31 after bloodwas removed. Prior to each bone marrow aspiration, a bloodsample was taken for haematological analysis. Blood andbone marrow samples were taken from horses on the desig-nated day at the same time and with minimal excitement.

The horses were placed in stocks and restrained using anose twitch. Sedation with xylazine (05 mg kg1 intra-venously; Xylazil-100; Ilium; Troy Laboratories Pty Ltd.,Smithfield NSW) was used when necessary. Bone marrowaspirates were collected from the sternum using a Sternum-Temno bone marrow needle with stylet (Bauer; N. Stenningand Co. Pty Ltd., Sydney, NSW). A standard technique wasused for marrow aspiration (Morris 1989, Russell et al1994). Samples of stromal marrow particles, with as littleblood contamination as possible, were immediately trans-ferred to a watch glass containing approximately 15 to 20 mlof 3 per cent EDTA to prevent coagulation. While the qualityof marrow samples varied between horses for a number ofreasons, effort was made to ensure adequate marrowspicules were obtained. Consequently, multiple aspirateswere sometimes necessary using the same needle in a redi-rected position along the sternum.

Laboratory preparation of bone marrowBone marrow samples were prepared for microscopic

evaluation within 10 minutes of collection. Using a Pasteurpipette, marrow spicules were individually removed fromthe 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 madeusing a second slide placed directly over the top of thespicules and with application of light pressure the two slideswere slowly and evenly pulled apart. After air drying, theslides were fixed in alcohol and stained using aRomanovsky-stain (Diff-Quik; Labaids, Sydney, NSW).Coverslips were glued to all slides after which the slideswere stored in containers until microscopic examinationcould be performed.

From the two to three slides that were prepared from eachaspiration sample, only slides with good staining character-istics, normal haemopoietic cell distribution and minimallydamaged cells were selected for interpretation. Slides wereinitially examined at low (10X) magnification. Stainingquality was assessed on the basis of stain evenness, unifor-mity of stain uptake, cell and organelle clarity and ease ofidentification. Cellularity was determined by scanning forthe presence and number of marrow spicules or unit parti-cles as well as the proportion of fat and haemopoietic cells(Grindem 1989). A grade of hypocellular was given if lessthan 25 per cent of the unit particle was cellular marrow,normocellular if 50 per cent was cellular marrow and hyper-cellular if greater than 75 per cent of the unit particle wascellular marrow. An estimate of megakaryocyte numbers perlow power field also was made. Using 20X and 40X magni-fication, a subjective assessment of the proportions of ery-throid and myeloid cells, as well as the proportions of cellsthat were within proliferating or maturing stages was made.At high (100X) magnification, greater than 500 myeloid anderythroid cells at various stages of differentiation werecounted and the M:E ratio accurately calculated. Cells thatwere categorised as megakaryocytes or miscellaneous(including basket or damaged cells), were also counted butwere not included in the 500 myeloid and erythroid cellsused for the M:E ratio. Interpretation of M:E ratios wasmade in conjunction with the peripheral haematologicalresults. Reticulocytes were not counted as appropriate bonemarrow staining techniques (eg, new methylene blue) werenot used for identification. However, reticulocytes or poly-chromatic macrocytes were subjectively estimated in severalfields 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 generationBone marrow cells were identified and grouped according

to the classification described by Jain (1993) with minormodifications. In the erythroid series, only pronormoblasts(rubriblasts) and basophilic normoblasts (prorubricytes andbasophilic rubricytes) were classified as proliferative phasecells. Early, intermediate and late normoblasts (polychro-matophilic, normochromic rubricytes and meta-rubricytesrespectively) were classified as maturing phase cells.Individual cells of the erythroid line were counted for eachhorse on the selected day of investigation and then expressedas percentages of the total number of erythroid cellscounted. The percentage [mean (SEM)] was subsequentlycalculated. Similar percentages [mean (SEM)] were calcu-lated for individual cells of the myeloid series (as percent-ages of the total number of myeloid cells counted) andmiscellaneous cell series (as percentages of the total numberof all cells counted). By adding the individual percentages

of cells that made up the proliferating and maturing pools, aproliferating pool to maturing pool (P:M) ratio was calcu-

and myeloid cells counted, two specific details were added

Bone marrow response to blood collection 287lated for both erythroid and myeloid lines. The total numberof erythroid cells counted was expressed as a percentage ofthe total number of erythroid and myeloid cells counted. Asimilar calculation was made for the total number ofmyeloid cells counted. In addition, the total numbers of pro-liferative phase cells and maturing phase cells of the ery-throid series was expressed as a percentage of the totalnumber of erythroid and myeloid cells counted.

Statistical analysisStatistical analysis was performed using a repeated meas-

ures, one-way analysis of variance (ANOVA; Statistica,StatSoft Inc. Tulsa, Oklahoma, USA) to determine the effectof removal of 20 ml kg1 of blood on different erythroid celltypes over time. Post hoc determination of least significantdifference was performed if the F value indicated a signifi-cant difference (P < 005) over time after blood collection.All results are expressed as mean (SEM) unless otherwiseindicated. While statistical analysis was performed on theother cell types described, emphasis was placed on the ery-throid series only. However, general trends were noted, inparticular regarding descriptive analyses of the myeloidseries and miscellaneous cells.

RESULTSVital signs and results of haematology were consistently

within reference range (Jain 1986, Lumsden et al 1980) inall horses 2 days prior to blood removal and before eachbone marrow collection. Body weight ranged from 455 to580 kg [516 (51) kg] and volume of blood collected rangedfrom 9 to 11 L [102 (10) L].

Smear quality and subjective microscopic evaluationIn general, the staining quality of all smears was good,

with clear cellular and organelle characteristics and easyidentification of cell types. Nevertheless, occasional smearswere moderately understained while others had uneven staindistribution between different fields. Also, most smears hadmoderate to high spicule density (>10 to 30 spicules) andwere classified as normocellular. However, while severalindividual marrow aspirates were hypocellular, there was noparticular time during the period of bone marrow investiga-tion 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 miscel-laneous 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, pinkishstructures). As a group, miscellaneous cells constituted lessthan 12 per cent of the total number of erythroid, myeloidand miscellaneous cells counted. The average number ofpolychromatic macrocytes was 5 per field in smears beforeblood collection although >5 per field in all smears up to 21days after blood collection. By day 31, the average numberswere only marginally >5 per field.

Subjective assessment of smears at low power showed

profound increases in cells of the erythroid series 3 to 9 daysafter blood removal. While increased numbers of prolifera-tive erythroid cells were seen during this time, numbers ofmaturing cells predominated 14 to 31 days post-collection,which tended to maintain high overall erythroid cell num-bers. Concomitantly, less myeloid cells were noted andcounted over the study period and subjectively the M:E ratioremained low between 3 and 31 days post blood collection.

Objective microscopic evaluationMean values and ranges for all individual erythroid and

myeloid cells as well as for the proliferating and maturingpools of the erythroid and myeloid series, the total numberof erythroid and myeloid cells counted, the M:E ratio andthe P:M ratio of the erythroid and myeloid series, prior toblood removal, are presented in Table 1. For comparison,previously reported ranges and values also are shown(Archer 1954, Calhoun 1954, Franken et al 1982a, Jain1986, Tschudi et al 1975). In general, most of the means andranges for the erythroid series generated from the five nor-mal horses in this investigation prior to blood removal, werecomparable to values reported elsewhere. A summary of alldata generated over the 31 days of investigation is shown inTable 2. In addition, Figs 1 to 4 illustrate the regenerativeresponses of cells of the erythroid series over 31 days andthe times at which results were significantly different fromtime zero.

Erythroid seriesAfter the removal of 20 ml kg1 of blood, the total ery-

throid cell compartment expanded initially by 137 per centin the first 3 days, peaking by 9 days with a further 135 percent increase. Total erythroid cell numbers were greater 3 to31 days post-collection, than at time zero (P < 001). Whilenumbers declined after 9 days, values remained higher up to31 days than at time zero and 3 days post-collection (P 005].

When cells of the proliferating and maturing pools wereconsidered as a percentage of the total number of erythroidto the above results. First, proliferative erythroid cells

N. Malikides, A. Kessell, J.L. Hodgson, R.J. Rose, D.R. Hodgson288

TAB

LE1:

Bon

e m

arro

w d

iffer

entia

l cel

l cou

nts

(%) f

rom

norm

al ho

rses

in th

e cur

rent

stud

y and

repo

rted i

n lite

ratu

re

Cell T

ype

Curre

nt S

tudy

Arch

er (1

954)

Calh

oun

(1954

)Ts

chud

i et a

l (197

5)Fr

anke

n et

al (1

982a

)Ja

in (1

993)

(n = 5

horse

s)(12

ponie

s)(7

horse

s)(15

horse

s)(24

horse

s)(4

horse

s)

Mea

n (se

m)%

Min

-Max

rang

e%R

efer

ence

rang

e%R

ange

%*

Mea

n%R

ange

%M

ean%

Ran

ge%

Mea

n%R

ange

%

Mea

n%R

ange

%

Eryt

hroi

d Se

ries

Pron

orm

obla

sts

26

(09)

07

54

05

70

087

034

04

34

16b

06

04

218

0 0

20

07

06

11

Baso

ph. n

orm

obl.

55

(07)

40

79

25

85

016

43

51

882

09

05

781

09

53

6 5

513

1c

Prol

ifera

ting

Pool

81

(06)

63

94

59

103

Early

nor

mob

last

s19

9 (1

6)14

52

47

130

26

22

213

0a

634

832

a20

910

23a

162

145

44a

282

147

26

Inte

rm. n

orm

obla

sts

373

(19)

322

43

129

74

49

07

43

Late

nor

mob

last

s35

0 (0

9)31

93

71

322

38

84

239

217

65

242

137

254

534

914

36

232

107

15

4M

atur

ing

Pool

919

(06)

906

93

889

79

41

P:M

ratio

009

(001

)0

060

10

050

13

Tota

l ery

thoi

d ce

lls54

8 (1

5)52

26

07

486

61

026

16

346

660

48

559

332

56

2M

yelo

id S

erie

sM

yelo

blas

ts0

8 (0

1)0

51

10

41

20

066

024

03

20

119

00

50

10

03

15

Prom

yelo

cyte

s3

2 (0

5)1

74

41

05

40

140

99

05

05

18

00

30

127

05

35

1 7

10

19

Mye

locy

tesi

205

(35)

110

32

84

536

511

83

09

194

266

56

381

10

53

3 3

31

07

53

22

14

1Pr

olife

ratin

g Po

ol24

4 (3

0)16

53

45

110

37

8M

etam

yelo

cyte

sii26

3 (2

7)18

93

27

143

38

319

14

17

263

511

68

11

515

5

62

39

1Ba

ndsii

i38

6 (1

5)33

54

21

320

45

26

265

157

74

159

Segm

ente

rsiv

105

(20)

47

172

15

195

83

316

174

222

414

511

31

120

74

3 2

25

105

114

25

4M

atur

ing

Pool

754

(31)

647

83

561

48

94

P:M

ratio

03

(006

)0

20

50

060

54

Tota

l mye

loid

cel

ls45

2 (1

5)39

34

78

386

52

063

756

74

345

337

928

14

84

M:E

ratio

08

(005

)0

650

92

06

10

11

102

243

09

38

164

03

08

06

05

09

071

052

14

5M

isce

llane

ous

Serie

sM

isce

llane

ous

cells

v8

8 (1

3)4

211

92

814

82

522

610

2

32

112

67

10

106

21

515

55

22

114

Bask

et c

ells

64

(13)

28

104

04

124

23

90

i. m

yelo

cyte

s in

clude

neu

troph

ilic, b

asop

hilic

and

eos

inop

hilic

form

sii.

met

amye

locy

tes

inclu

de n

eutro

philic

, bas

ophi

lic a

nd e

osin

ophi

lic fo

rms

iii. b

ands

inclu

de n

eutro

philic

, bas

ophi

lic a

nd e

osin

ophi

lic fo

rms

iv. se

gmen

ters

inclu

de n

eutro

philic

, bas

ophi

lic a

nd e

osin

ophi

lic fo

rms

v. m

isce

llane

ous

cells

incl

ude

mon

ocyt

es/m

acro

phag

es, m

itotic

figu

res,

pla

sma

cells

, lym

phoc

ytes

and

unc

lass

ified

cells

a. ra

nge

for e

arly

and

iterm

edia

te n

orm

obla

sts

(clas

sified

as ru

bricy

tes by

Jain,

1986

).b.

def

ined

by

Calh

oun

as s

tem

cel

ls a

nd n

ot in

clude

d in

tota

l ery

thro

id c

ount

.c.

cla

ssifie

d as

pro

rubr

icyte

s a

nd b

asop

hilic

rubr

icyte

s b

y Ja

in, 1

986.

* m

ea

n (2

sd).

Bone marrow response to blood collection 289

ll

9)1)3)5)6)6)3)0)6)8)4)7)9)9)0)5)9)0)6)1)

3)8)TABLE 2: Results (mean [sem]) of bone marrow aspirate percentage cecounts from two horses after 6 weeks.

Day 0 Day 3 Day 5

Erythroid Series1Pronormoblast 26 (01) 87 (18) 73 (0Basoph. normoblast 55 (07) 73 (15) 94 (2Proliferating pool (P) 81 (06) 160 (14) 167 (2Early normoblast 196 (16) 136 (19) 126 (1Interm-normoblast 373 (19) 330 (17) 373 (2Late normoblast 350 (09) 374 (35) 334 (2Maturing pool (M) 919 (06) 840 (15) 833 (2P:M ratio (erythroid) 01 (00):1 02 (00):1 02 (0Total erythroid cells2 548 (15) 685 (35) 730 (2Myeloid Series1Myeloblasts 08 (01) 30 (02) 23 (0Promyelocytes 32 (05) 52 (16) 46 (1Myelocytes 205 (35) 108 (16) 105 (1Proliferating pool 244 (30) 190 (31) 174 (1Metamyelocytes 263 (27) 147 (37) 127 (1Bands 386 (15) 471 (32) 448 (4Segmenters 105 (20) 191 (56) 251 (3Maturing pool 754 (31) 818 (31) 826 (1P:M ratio (myeloid) 03 (01):1 02 (00):1 02 (0Total myeloid cells2 451 (15) 315 (35) 270 (2M:E ratio 08 (00):1 05 (01):1 04 (0MiscellaneousSeries3Miscellaneous cells 88 (13) 83 (20) 38 (1Baskets cells 64 (14) 65 (18) 20 (0Comparativeincreased from values prior to blood collection of 44 (02)per cent to 122 (17) per cent, (P < 001) 5 days after collec-tion. Numbers subsequently declined, reaching values notsignificantly 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 phaseafter blood collection, maturing phase cells also concomi-tantly increased from time zero percentages of 505 (17) to710 (35) per cent, (P < 001) 9 days after collection, main-taining this level up to 31 days.

Fig 3 shows the variable changes of the early, intermedi-ate and late normoblast cells of the maturing pool during thepost-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 anda reference range [mean (2 sd)] of 06 to 10 (Table 1).Although numbers of horses in this study were few, theseresults were similar to published values (Franken et al1982a, Jain 1993, Latimer and Andreasen 1992). The M:Eratio was lower at all days after blood collection than at timezero (P < 001). The lowest M:E ratio occurred after 9 days,beyond which the M:E ratio remained significantly lower upto day 31, than at time zero and day 3 (P < 001) (Fig 4). Incomparison, the P:M ratio increased sharply after bloodremoval with values higher after 3 days (P < 005), 5 days (P

Haematology4PCV 033 (00) 032 (00) 031 (00)RCC 71 (03) 69 (03) 66 (03)HB 119 (38) 116 (49) 111 (42)MCV 465 (21) 463 (19) 467 (13)RDW 180 (02) 190 (04) 182 (02)WCC 82 (07) 89 (09) 97 (07)

1. Results [mean (sem)] of individual cells of the erythroid and myeloid series arespectively.2. Results [mean (sem)] of total erythroid or total myeloid cells are expressed as3. Results [mean (sem)] of the total miscellaneous (all miscellaneous cells inclunumber of all cells counted.4. All results [mean (sem)] of selected haematological variables were calculated counts taken from five horses on selected days over 4 weeks and cell

Day 9 Day 14 Day 21 Day 31

69 (18) 38 (08) 53 (12) 39 (03)65 (05) 85 (15) 84 (09) 53 (13)

134 (18) 123 (20) 137 (17) 93 (11)200 (28) 173 (26) 112 (15) 118 (09)304 (15) 358 (30) 286 (18) 390 (29)362 (33) 346 (34) 465 (27) 399 (22)866 (18) 877 (20) 863 (17) 907 (11)

:1 02 (00):1 01 (00):1 02 (00):1 01 (00):1820 (16) 795 (22) 783 (36) 784 (38)

18 (05) 27 (11) 16 (09) 11 (06)21 (10) 34 (14) 35 (11) 43 (13)86 (16) 99 (33) 65 (32) 78 (12)

125 (25) 159 (47) 115 (37) 13 3 (28)135 (41) 188 (21) 168 (27) 135 (38)412 (71) 402 (31) 417 (60) 375 (62)331 (75) 250 (66) 300 (83) 357 (87)877 (26) 841 (47) 885 (37) 867 (28)

:1 01 (00):1 02 (01):1 01 (01):1 02 (00):1180 (16) 205 (22) 217 (36) 216 (38)

:1 02 (01):1 03 (01):1 03 (01):1 03 (01):1

85 (34) 54 (08) 101 (14) 116 (52)57 (29) 39 (07) 72 (09) 55 (26)< 001) and 9 days (P < 005), than at time zero.Subsequently, the P:M ratio gradually declined to valuescomparable to time zero in a manner similar to the responseof the proliferating pool (Fig 4).

Myeloid and miscellaneous seriesIn general, the proliferative phase cells (myeloblasts,

promyelocytes and neutrophilic, eosinophilic and basophilicmyelocytes) of the myeloid series tended to decline over the31 days after blood removal despite early increases inmyeloblasts and promyelocytes. In contrast, the cells of thematuring myeloid pool (neutrophilic, eosinophilic andbasophilic metamyelocytes, bands and segmenters) tendedto increase over the 31 days, due mostly to increases in neu-trophilic, eosinophilic and basophilic segmenters. Themyeloid P:M ratio, therefore, tended to decrease over the 31days after collection.

The numbers of individual cells classified as miscella-neous (including lymphocytes, monocytes/macrophages,mitotic figures, unclassified and basket cells) fell withinsimilar ranges to those reported (Jain 1993). The total num-ber of miscellaneous cells (not including basket cells)increased as time progressed beyond 14 days and as morebone marrow aspirations were taken. However, average

033 (00) 030 (00) 032 (00) 031 (00)72 (04) 65 (03) 70 (04) 69 (04)

120 (64) 109 (55) 119 (72) 118 (69)462 (16) 457 (14) 455 (12) 448 (19)181 (02) 182 (02) 183 (01) 180 (03)

97 (07) 81 (06) 80 (05) 82 (05)

re expressed as a percentage of the total erythroid and myeloid cells counted a percentage of the sum of the erythroid and myeloid cells counted.ding basket cells) and basket cells are expressed as a percentage of the totalfrom blood samples collected prior to bone marrow aspiration.

N. Malikides, A. Kessell, J.L. Hodgson, R.J. Rose, D.R. Hodgson290

12

10

8

6

4

2

12

10

8

6

4

2

2 3 5 9 14 21 31

Time (days)

Bas

oph

ilic

nor

mob

last

s (p

erce

nta

ge)

Pro

nor

mob

last

s (p

erce

nta

ge)

*

*

* *

*

*

FIG 1: Bone marrow regenerative response over 31 days after 20 ml kg1blood removal: Values [mean (sem)] for pronormoblasts and basophilic nor-moblasts expressed as a percentage of the total number of erythroid cellscounted.*significantly different than time zero (P < 005).

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

2 3 5 9 14 21 31

Time (days)M

atu

rin

g po

ol (

perc

enta

ge)

Pro

life

rati

ng

pool

(pe

rcen

tage

)

**

*

*

*

*

*

FIG 2: Bone marrow regenerative response over 31 days after 20 ml kg1blood removal: Values [mean (sem)] for the erythroid proliferative pool(pronormoblasts and basophilic normoblasts) and maturing pool (early, inter-mediate and late normoblasts) expressed as a percentage of the total numberof erythroid cells counted.*significantly different than time zero (P < 005).

24

20

16

12

8444036322824484440363228

Ear

ly (

%)

Inte

rmed

iate

(%

)L

ate

(%)

2 3 5 9 14 21 31

Time (days)

**

**

d

# #

FIG 3: Bone marrow regenerative response over 31 days after 20 ml kg1blood removal: Values [mean (sem)] for early, intermediate and late nor-moblasts expressed as a percentage of the total number of erythroid cellscounted.*significantly different than time zero and 9 days after blood collection (P