Clin. lab. Haemat. 1983, 5, 353-360

The evaluation of neutropenia: the use of the granulocyte mobilization test

C A T H E R I N E BALDWIN & S . ROATH Department of Haematology, Faculty of Medicine, University of Southampton School of Medicine, General Hospital, Southampton

Accepted for publication 11 August 1983

Summary Ten individuals with idiopathic neutropenia and similar numbers of normal and abnormal controls were tested for mobilization of their marginal granulocyte pools and bone marrow reserve by using epinephrine and hydrocortisone intravenously. Individuals with ‘benign’ idiopathic neutropenia appeared to have a normal response while half the abnormal controls responded poorly. It is suggested that granulocyte mobilization tests are valuable in the assessment of individuals with neutropenia.

Keywords: neutrophils, neutropenia, granulocyte kinetics, bone marrow failure

Many individuals presenting with neutropenia tend either to have or develop some obvious underlying cause such as leukaemia or aplastic anaemia or are known to have received drugs which depress neutrophil production; others appear to have no significant associated disorders and are usually termed idiopathic neutropenics. These form a group which is difficult to assess; &examination of the bone marrow is usually inconclusive-the pattern being within normal limits-and repeated visits for blood counts and to out-patient clinics may cause considerable anxiety in those so monitored. Usually in this group there is no history of life-endangering infections, although a history of minor, presumably infectious, disorders can usually be elicited. The question arises whether these are individuals who in fact have adequate numbers of granulocytes available when needed but in whom circulating counts are low, or do they have difficulty in granulocyte mobilization in response to infections? The granulocyte mobilization test is designed to carry out such an evaluation. In this study patients with ‘idiopathic neutropenia’ were so studied, as were a number of normal individuals and some who were known or suspected to have depressed granulocyte production.

Materials and methods

Ten individuals with idiopathic neutropenia were studied. All had persistent neutropenia (less than 2 x lo9 cells on at least four occasions during 1 year).

Correspondence and reprint requests: S. Roath, Department of Haematology, South Laboratory and Pathology Block, Southampton General Hospital, Tremona Road, Southampton SO9 4XY.

0141-9854/83/1200-0353$02.00 0 1983 Blackwell Scientific Publications 353

Tab

le 1

. R

esul

ts in

con

trols

and

idi

opat

hic

neut

rope

nia

Incr

ease

s in

CG

P A

ge &

B

ase

<2

in

Ih

2-

5 h

MG

P af

ter

Dia

gnos

is

sex

SPL

INF

BM

ct

t2

pa

st

AB

S %

A

BS

YO

resp

. 1

h B

MR

IN

IN

IN

IN

IN

IN

IN

IN

INS

INS

M61

F5

3 F6

4 M

45

F62

F4 1

F68

M23

F7

4 M

38

Min

. 21

M

ax.

75

N

0.31

+

N

0.91

+

N

1.45

+

I

1.5

+ N

1.

65

+ -

1.68

+

- 2.

8 -

-

3.88

-

N

1.6

+ -

2.6

-

+ 1.

44

465

1.69

56

0 N

+

0.76

85

3.

78

420

N

+ 2.

35

162

5.08

35

0 N

+

2.96

19

7 7.

05

470

N

+ 1.

31

80

2.97

18

0 N

+

1.4

83

3.44

20

5 N

+

0.55

20

4.

06

145

1 +

1.47

38

12

.32

317

1 +

2.35

14

7 2.

36

148

N

+ 1.

12

43

4.6

177

1

1 1

1 N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Con

trol

s (m

ean

of 1

1 su

bjec

ts)

2.4

1.51

43

4.

76

150

N(a

l1)

N(a

l1)

N(a

l1)

5.7

4.32

15

5 14

.8

435

IN =

idio

path

ic n

eutro

peni

a; I

NS

= id

iopa

thic

neu

trope

nia

with

spl

enom

egal

y; M

GP

= m

argi

nal

gran

uloc

ytic

poo

l; B

MR

= bone

mar

row

re

serv

e; C

GP

= ci

rcul

atin

g gr

anul

ocyt

ic p

ool;

SPL

= sp

leno

meg

aly;

IN

F =

infe

ctiv

e ep

isod

es;

BM =

mar

row

cel

lula

rity;

< 2

= n

eutro

phil

coun

t >

2 x

109/

l; A

BS

= a

bsol

ute

neut

ropi

l cou

nt.

Tabl

e 2.

Res

ults

in p

atie

nts

with

‘ex

plai

ned’

neu

trop

enia

Incr

ease

s in

C

GP

Age

8c

Bas

e <

2 in

l

h

2-5

h M

GP

af

ter

Dia

gnos

is

sex

SPL

INF

BM

ct

<

2

past

A

BS

%

AB

S YO

re

sp.

1 h

BM

R

AA

M

54

-

+ I*

0.

05

+ +

0 0

0 0

1 1

1 A

L M

69

-

+ 1

0.12

+

+ 0.

11

92

0.51

42

5 N

1

1 PR

M

42

-

-

1 0.

63

+ +

1.89

30

0 5.

79

920

N

N

N

AL

M57

+

+ l*

1.33

+

+ 0.

4 30

1.

98

149

1 1

1 A

L

M54

+

+ 1

1.51

+

+ 0

0 1.

07

71

1 1

1 C

A

F73

-

+ 1

1.63

+

+ 1.

43

83

5.07

31

0 N

N

N

z C

LL

M

67

+ +

CL

L*

0.91

+

+ 2.

24

246

3.98

43

7 N

N

N

+

M

1.02

+

+ 1.

32

130

1.04

10

2 N

N

1

MM

F6

9 -

%

MM

M

63

-

-!- M

2.

2 -

+ 1.

14

52

3.02

13

7 N

N

N

M

M

M69

-

-

M

3.0

-

+ 0.

87

29

3.35

11

0 1

N

N

E B

AA

= a

plas

tic a

naem

ia; P

R =

pre

leuk

aem

ia; C

LL

= c

hron

ic ly

mph

atic

leuk

aem

ia;

AL

= a

cute

leuk

aem

ia;

CA

= c

arci

nom

atou

s inv

olve

men

t

Y

m

e

0 3

of m

arro

w;

MM

= m

ultip

le m

yelo

ma;

AB

S =

abs

olut

e ne

utro

pil

coun

t. *A

sses

sed

by b

iops

y: o

ther

wis

e by

spi

cule

cel

lula

rity.

356 Catherine Baldwin and S. Roath

Details are shown in Table 1. Only one complained of infection-a series of upper respiratory infections. Two had persistent palpable splenomegaly. None has developed any associated disorder in the 2 years of observation following this study (giving at least 3 years of observation in all patients). In addition, a similar number of hospital patients with ‘bone marrow failure’ were also studied, their details are given in Table 2. Normal control volunteers’ values are recorded in Table 1.

The granulocyte mobilization test

The method used is a composite one based mainly on that of Deinard and colleagues (1974). All normal volunteer controls and patients were kept in a hospital ward or day ward for the duration of the test. Informed consent was obtained in all cases. An indwelling plastic cannula was placed in a forearm vein and a blood sample taken for the base line white blood cell count and differential. Pulse rate was monitored and the patient’s blood pressure taken before and at intervals during the test.

Epinephrine, 1 : 1000, 0.4 ml/m2 surface, was administered by subcutaneous injection. At the same time, 100mg of hydrocortisone succinate was given intravenously through the indwelling cannula. Sodium citrate solution 3.9% was used to maintain potency. Serial blood samples were taken at 10, 20 and 30 min and 1, 2, 3, 4, 5 and 6 h from the indwelling cannula. White blood cell counts were obtained from each sample using a Coulter S Counter, andfferentials were carried out by one of the authors. The individuals taking part in the test were free to move around and were allowed food and drink normally during the period of test. In two of the volunteers a sham procedure was undertaken. The volunteers understood that two of them would be so processed (the cannula was inserted but normal saline was given in the subcutaneous injection and through the cannula).

Results

Side eJtrects

Most of the individuals in the study noted no untoward reaction and in no one was the test abandoned or corrective treatment needed. Some pallor of the face and hands was observed occasionally; palpitations, sensation of fullness of the throat and apprehension were also recorded and persisted for between 30 min and 2 h. None of these was severe.

Figure 1 shows the results in the controls and in the patients with idiopathic neutropenia. Figure 2 shows the percentage increase in cell counts in the first hour and the absolute increase in 2-5 h is shown in Figure 3. The group of hospital patients with presumed bone marrow failure-‘explained neutr0penia’- is also shown in these histograms.

The volunteers subjected to sham processing showed no increase in their neutrophil counts.

The evaluation of neutropenia 357

500 -

400 -

300 - %

200 -

0 -. ,--- / --.----7L- / ----IN

----_____ -=<%-..- %.- IN S

AC

'. *------__ AC

Figure 1. Mean neutrophil count for _---.. each group at each sample time.

C = control; I N = idiopathic neutro- penia, S = splenomegaly; AC =

,.----- -- -. c

1 I abnormal control, for lower groups; 0 1 2 3 4 5 6 the lower of the two with damaged

Hours after drug administration marrow.

Discussion

Patients with idiopathic neutropenia have a small circulating granulocyte pool size. This may be unimportant if they are able to muster adequate granulocytes on demand from the marginal granulocyte pool (MGP) or the bone marrow reserve (BMR). The observed response to epinephrine should generally reflect the former (Athens et al. 1961) and the response to hydrocortisone the latter (Dale et a/ . 1975), although there is some overlap.

Idiopathic Abnormal neutropenia control

Figure 2. Percentage neutrophil increase in first hour. indicates 45%, above which the response is considered to be normal.

358 Catherine Baldwin and S. Roath

neutropenia control

I Figure 3. Absolute neutrophil increase in 2-5 h. --- indicates 2 x lo9 cells, above which the response is considered to be normal.

In normal subjects a 45% increment has been defined as a physiological demargination response (Deinard et al. 1974); up to 100% is often recorded since the normal MGP: CGP ratio is 1:l (Joyce et al. 1976), suggesting that there is traffic between the MGP and CGP which may be approximately equal. The results seen in our subjects with idiopathic neutropenia demonstrate a normal response in seven patients and a borderline in three. This reflects very closely the response in our controls. It is worth noting that in two of the patients with a sub-

A

A

0 A A

I I I I I I 1 I I Figure 4. Age and absolute neut-

0 10 20 30 40 50 60 70 80 rophil increase in 2-5 h. A control; Age of subjects patients normally responding.

The evaluation of neutropenia 359

optimal response on the day of testing their circulating counts were already just about within normal limits. It might be assumed that the procedures already undertaken might have elevated plasma adrenalin and adrenal corticosteroid levels (Lader 1967) and mobilization of the marginal pool may already have begun. The ‘abnormal controls’ with permanent or temporary bone marrow damage showed a wide spread of responses as might be expected; about half of them probably had subnormal responses.

Our findings in the idiopathic neutropenic patients might suggest a diminished total body granulocyte pool rather than one of normal size as proposed by Bishop et al. (1971), but other explanations have also been put forward- particularly that of ‘shift neutropenia’ (Joyce et al. 1976), where there is simply a tendency to increased margination.

The evaluation of the bone marrow reserve is somewhat more complex and absolute values tend to be used rather than percentage increases (Athens et al. 1961, Bishop et al. 1967)-an increase of 2 x lo9 cells per 1 over base line is considered to be a normal response. All idiopathic neutropenic patients gave an adequate response except one. This individual had shown no history of infection and subsequently underwent major abdominal surgery without infective complications. Unusually, he had a high monocyte count during the evaluation and following surgery.

The two individuals with splenomegaly had entirely normal responses in both phases of mobilization, and an assessment of response by age of patient did suggest that there might be some diminution in the elderly (Figure 4) (Cream 1968).

In conclusion, it would appear that the individuals with idiopathic neutropenia did not suffer obvious risk of infection during the 3 years of observation, nor did any of them develop overt haematological or other bone marrow damaging disease during this period. These individuals also showed normal or nearly normal responses to tests of mobilization of the marginal granulocyte pool and the bone marrow reserve. These tests evoked a poor response in individuals with impaired leucocyte production, which suggests that granulocyte mobilization tests are useful in the assessment of individuals with neutropenia, and that those with normal responses need not undergo further investigation or monitoring.

References

ATHENS J.W., RAAB S.O., HAAB O.P., MANER A.M., ASHENBRUCKER H., CARTWRIGHT G.E. & WINTROBE M.M (1961) Leucokinetic studies: IIIdistribution of granulocytes in the blood of normal subjects. f. din. Invest. 40, 159-164

BISHOP C.R., ATHENS J.W., BOWS D.R. & CARTWRIGHT G.E. (1967) The mechanism of cortisol induced granulocytosis. Clin. Res. 15, 130

BrsHoP C.R., RUTHSTE~N G., ASHENBRUCKER H.E. & ATHENS J.W. (1971) Leucokinetic studies: XIV. Blood neutrophil kinetics in chronic steady state neutropenia. J . d i n . Invest. 50, 1678-1 689

360 Catherine Baldwin and S. Roath

CREAM J.J. (1968) Prednisolone-induced granulocytosis. Br. J . Haem. 15, 259 DALE D.C., FAUCI A.S., DUPONT GUERRY IV & WOLFF S.M. (1975) Comparison of agents

producing a neutrophilic leucocytosis in man. J . clin. Invest. 56, 808-81 3 DEINARD A.S., FORTUNY I.E., THEOLOGIDES A. ANDERSON G.L., BOEN J. & KENNEDY B.J. (1974)

Studies on the neutropenia of cancer chemotherapy. Cancer 33, 121&1218 JOYCE R.A., BOGGS D., HASIBA U. & SRODES C. (1976) Marginal neutrophil pool size in normal

subjects and neutropenic patients as measured by epinephrine infusion. J. lab. Clin. Med. 88, LADER M.H. (ed.) (1967) Studies of Hospital Admission. p p . 40-52