British journal of Haematology, 1978, 38, 99.

Identlfication of the Hairy Cells of Leukaemic Reticuloendotheliosis by an Esterase Method

K. E. HIGGY, G. F. BURNS AND F. G. J. HAYHOE

Department of Haematological Medicine, University of Cambridge, Cambridge

(Received 14 April 1977; accepted for publication 3 August 1977)

SUMMARY. A distinctive pattern of alpha-naphthyl butyrate esterase positivity was observed in all of a series of 14 patients with HCL. This pattern was not observed in a range of other haematological malignancies including nine cases of CLL, two cast‘s of Skzary’s syndrome, six cases of null cell ALL, two cases of Schilling-type monocytic leukaemia and one case of lymphosarcoma cell leukaemia. The potential diagnostic value of this simple cytochemical test is discussed in relation to existing methods.

Hairy cell leukaemia (HCL) or leukaemic reticuloendotheliosis is an uncommon chronic leukaemia which, despite much recent interest (reviewed by Catovsky, 1977) is frequently misdiagnosed. It may be mistaken for acute lymphocytic or undifferentiated leukaemia or malignant lymphoma with bone marrow and peripheral blood involvement (LoBuglio, 1976) with disastrous consequences since the vigorous chemotherapy appropriate for these disorders may be lethal to the patient with HCL (Katayama & Finkel, 1974). In addition, HCL may be confused with chronic lymphocytic leukaemia or chronic myelofibrosis and even here the distinction is important since splenectomy has been shown to be the treatment of choice for HCL (Yam et a l , 1972; Catovsky et al, 1974; Catovsky, 1977).

There are several distinctive features of HCL which have been used as diagnostic aids. These include: the presence of tartrate-resistant acid phosphatase in a proportion of hairy cells (Yam et al , 1971); distinctive cytoplasmic ribosome-lamellar inclusions (Katayama el uf, 1972; Daniel 81 Flandrin, I 974) and characteristic cytoplasmic projections particularly well visualized either by phase contrast or transmission electron microscopy (Katayama et a l , 1972; Catovsky, 1977); the presence of a receptor for JgM on the surface of hairy cells (Burns et ul , 1977a); the finding of a high leucocyte alkaline phosphatase score (Katayama & Finkel, 1974; Burns et al, 1976) and a low serum lysozyme concentration (Catovsky, 1977). However, none of these features is diagnostic of HCL since one or more may be absent in any particular case and since any may occur in other pathological states.

In a previous report (Higgy et al, 1977) we have described and illustrated patterns ofesterase reaction in normal lymphocytes which discriminate between B, T and null cells. T cells usually show a dense, localized, dot-like positivity made up of one to four coarse granules, B cells are

Road, Cambridge CB2 2QL. Correspondence: Dr K. E. Higgy, Department of Haematological Medicine, University Clinical School, Hills

0007-1048/78/0100-0099 $02.00 Or978 Blackwell Scientific Publications

99

I00 K. E. Higgy, G. F . Burns and F. G.J. Hayhoe

mostly negative and null cells show a scattered finely granular positivity. These reactions are not inhibited by sodium fluoride, as is the well recognized strong granular positivity of monocytes. The purpose of the present report is to show that, while in many cases the normal B, T and null cell patterns of reactivity persist in pathological proliferations of respectively B, T and null cell origin, the cells of HCL show different and very characteristic patterns of esterase reaction.

PATIENTS

A consecutive series of 14 patients with HCL was involved in the present study; all fulfilled the usual clinical and haematological criteria of the disease (Bouroncle et a l , 1958).

For comparison, a number of disease states that might be confused with HCL were studied. These comprised: chronic lymphocytic leukaemia (CLL), nine cases; SPzary’s syndrome, two cases; null cell acute lymphoblastic leukaemia (ALL), six cases; Schilling-type monocytic leukaemia, two cases; and one case of lymphosarcoma cell leukaemia. In all these cases the clinical picture, cytology and cytochemistry was typical of the disorder in question. In all the cases of CLL the absolute lymphocyte count was > I 5 x 109/l., none showed T-cell features by rosetting with sheep erythrocytes (E) and other immunological markers were fully compatible with this diagnosis. Both cases of SPzary’s syndrome had skin involvement, the cells showed the typical nuclear ultrastructure and a high percentage formed E-rosettes. The six cases of null cell ALL were negative for surface immunoglobulin (SmIg), and did not possess receptors for the Fc portion of IgG or for C3, nor form E-rosettes. In both cases of monocytic leukaemia the majority of the cells possessed a receptor for the Fc portion of IgG, but lacked SmIg and did not form E-rosettes. In the lymphosarcoma cell leukaemia the circulating cells were larger and possessed abundant cytoplasm with a strong granular PAS positivity, but lacked receptors for the Fc portion of IgM (Burns et a l , 1977a) and for mouse erythrocytes (Catovsky et a l , 1975).

MATERIALS AND METHODS

For most of the study, air-dried smears of peripheral blood buffy coats were used. Sometimes, however, cytocentrifuge preparations of Ficoll-Triosil (F/T)-separated mononuclears from peripheral blood or spleen were employed. In addition, occasional imprint preparations of spleen and lymph node were used. In all cases, the type of preparation is indicated in the relevant Table.

In one case (L.H.), cytochemistry was performed on cytocentrifuge preparations made after rosetting the mononuclear cell suspension with EA(1gM); see Methods (Table IV).

Demonstration of a-naphthyl butyrate esterase. This method has been used already in this laboratory to distinguish normal B, T and iiull lymphocytes and the full details are given elsewhere (Higgy et a l , 1977). In brief, fresh air-dried preparations are fixed in formalin vapour and exposed to a-naphthyl butyrate in low concentration (pH 8), with a short incubation period, and with either Fast blue BB N or Fast Garnet GBC as diazonium salt capture agents. In all cases the test was also performed in the presence of sodium fluoride (0.04 M).

Demonstration of acid phosphatase. The method of Goldberg & Barka (1962) was employed using fresh buffy coat smears. Tartrate inhibition was performed with L( +) tartaric acid at a concentration of 0.05 M (Li et a l , 1970).

FIG I . HCL peripheral blood buffy coat prcparation; alpha-naphthyi butyratc cstcrasc. A group ofcight hairy cells are seen together with a single mature small lymphocytc. Scvcn of the hairy cells show thc distinctive rcaction pattern with a mixture of scattcrcd fine granules together, in somc instanccs, with coarser granules Iocalised in a crescentic configuration. A single hairy cell is virtually negative. The cell or1 thc lcft shows the coarse block positivity associated with normal T lyinphocytcs. FIG 2. HCL; EA(1gM) cytocentrifuge prcparation. The rosetted hairy cell shows thc typical cstcrase- reactivity, and the granular composition ofthe crescent of positivity is clearly illustrated. Thenon-rosct- ting small lymphocyte is negative for esterasc and is probably a normal B lymphocyte.

(Facing p 100)

b

4

FIG 3 . Alpha-naphtliyl butyrate esterase in HCL : (a) without sodium fluoride; (b) with sodium fluoridc. The strong granular moiiocyte activity is largely abolished by sodium fluoridc; the hairy cell pattern remains unchanged.

Hairy Cell Leukaemia I01

Surface Markers E-rosettes. Sheep erythrocyte (E) rosettes were performed according to the method of

Kaplan & Clark ( I 974). EA(ZgM)-rosettes. Ox erythrocytes were coated with rabbit IgM (EA(1gM)) and the rosette

testing carried out according to the method of Burns et a1 (1977a). Surface immunoglobulin. Surface light chains were detected by a rosette method (Ling et at ,

1977). This method employs chromic chloride coupling of antibody to indicator red cells and the purified antisera were a kind gift of Dr N. Ling of the Department of Experimental Pathology, University of Birmingham.

RESULTS

a-Naphthyl Butyrate Esterase Most of the hairy cells of HCL showed a highly distinctive reaction pattern with a mixture

of scattered fine granules, together with coarser granules localized in a crescentic configuration (Fig I ) , although a variable but usually small percentage showed no positivity; the positivity was not inhibited by sodium fluoride (Fig 3 ) . The percentage of mononuclear cells in each sample giving this characteristic reaction pattern is shown in Table I, where the percentages of mononuclear cells with B cell, T cell, null cell and monocytic patterns of esterase reaction, as previously described and illustrated by Higgy et a1 (I977), are also shown. Apart from the high incidence of the hairy cell pattern in all samples, except for the single node imprint which showed a predominance of the negative, B cell, reaction, the Table illustrates the virtual absence of monocytes in HCL and the variable depression, sometimes amounting to almost complete absence, of both T and B cell reaction patterns. In the case of patient L.S., a negative reaction was found in some cells with distinctively hairy cell morphology.

Table I1 shows the distribution of cytochemical esterase reaction patterns in a number of other lymphoproliferative states. In only two of these 20 cases were small numbers of mononuclear cells with the hairy cell type of positivity encountered. In general the esterase cytochemistry reflected the cell lineage of each condition as determined by surface marker characteristics. Thus six of the nine cases of CLL, a B cell neoplasm, showed chiefly the negative B cell pattern, both cases of Sizary’s syndrome, T cell by surface markers, showed a predominance of the T cell esterase pattern and all six null cell ALLs showed a predominance of the null cell esterase pattern. The presence of predominating null cell positivity patterns in the cells of two cases of otherwise typical CLL and the presence of I 1 ~ 5 0 % of cells with the pattern in three other cases indicates the need for further study of the possible cytochemical variations in this disease.

(:orrelation .f Esterase Method with other Diagnostic Methods Acidphosphatase. In order to compare the possible diagnostic value of the esterase with that

of tartrate-resistant acid phosphatase, this was performed in 10 of the cases of HCL and the results are summarized in Table 111. Although five of the cases showed definite tartrate-resistant acid phosphatase activity, in four the enzyme activity was completely inhibited and in two almost completely inhibited by tartrate. Furthermore, considerable variation was sometimes observed in specimens from the same case when sampled at different times.

I 0 2 K . E. Hi'y, G . F. Burns and F. G. J . Hayhoe

TABLE I. a-Naphthyl butyrate esterase patterns in the mononuclear cells of HCL*

Pattern of esterase activity observed (%)

T y p e 1-4 coarse Scattered Crescent form H e a v y overall of No. of No reaction dots f ine granules granularity granularity

Patient prepara- times ( B cell ( T cell (null cell (hairy cell (monocyte t i o n t tested pattern) pattern) pattern) pattern) pattern)

D.C.

M.B.$ L.S.

H.D. W.H. R.H. P.S. F.W.

N.W.

L.H.§

R.J.

F.B.

L.P.

K.D.

BC Spleen cyto Node

imprint BC BC

BC BC BC BC BC BC

Spleen cyto

Spleen imprint

BC Spleen cyto

Spleen cy to BC BC

Spleen imprint

PB cyto

I I

I

3 2

3 I

2

2

I

I

I

I

I

I

I

2

3 I

1

16 14

70

0

32(+8) (many HC)

S ( f S ) 15 28

2

I

17 I

< I

7 2

0

7(&2) 2

< I

9

42 6

22

< I

0

6(*4)

38(+_3) 20

I 0

20

33 3

< I

28 3

2

25 d + 6 )

1

34

I

0

I

< I

0

< I

0

0

0

0

2

0

0

0

4

0

< I

< I

0

2

0

0

0

I (* I ) I

0

0

0

0

< I

0

0

0

0 0

0

0

0

0

0

* 500 mononuclear cells scored on each occasion. Results on each occasion averaged and expressed as percentage

t BC = Peripheral blood buffy coat smear. Spleen cyto = cytocentrifuge preparation from a suspension of splenic

$ This patient was atypical in a number of regards (see case 6 , Burns et 01, 1977a) § This undoubted case of HCL was unique in that hairy cells formed E-rosettes. This patient has been extensively

( + I SD).

cells. PB cyto =centrifuge preparation of peripheral blood leucocytes after separation over ficoll-triosil.

investigated and is reported elsewhere (Burns et a l , 1977b).

EAZgM rosetteformation. Since this rosette test may be of diagnostic value in HCL (Burns et al , rg77a) the esterase test was compared with it by performing the cytochemistry on a rosette preparation from a single case of HCL (Table IV, Fig 2 ) . The results tabulated clearly show a close correlation between those cells forming EAIgM rosettes and those yielding the hairy cell-type of esterase positivity.

Hairy Cel l Leukaemia I 0 3

TABLE 11. a-Naphthyl butyrate esterase pattern in the peripheral blood mononuclear cells of CLL, SPzary’s syndrome, ALL, monocytic leukaemia and lymphosarcoma cell leukaemia (buffy coat smears).

Pattern of esterase activjty observed (%)

1-4 coarxe Scattered Crescentform H e a v y overall No reaction dots f ine granules granularity granularity

Diagnosis ( B cell (T-cell (null cell (hairy cell (monocyte pattern) pattern) pattern) pattern) pattern)

CLL CLL CLL CLL CLL CLL CLL CLL CLL Stzary’s syndrome Stzary’s syndrome Null cell ALL Null cell ALL Null cell ALL Null cell ALL Null cell ALL Null cell ALL Schilling monocytic

leukaemia Schilling monocytic

leukaemia Lymphosarcoma cell

leukaemia

> 99 6

33 58 96 99 66 18 93

8 3

39 40 23 3 0 9 0

2

I 0

87

< I < I

4 25 I

< I 2

0

0

5 1

95 8 6 0

4

I4 2

8

4

7

0

92 so I 1

< I

< I

29 80 0

I 0 2

52

54 77 66 89 86

0

0

0

0

0

0

0

0

0

0

0

0

7 0

0

0

0

0

0

0

0

0

6

0

2

I 3 6 3

3

7 24 0

< I 0

0

0

0

0

0

2

90

86

0

Since, in all the cases of HCL, the percentage of cells giving the hairy cell esterase pattern was markedly higher than the percentage of obvious hairy cells in Romanowsky preparations, it seemed important to compare estimates of hairy cell numbers by different methods. The results of such a comparison in six patients with HCL are set out in Table V. Here the number of cells with tartrate resistant acid phosphatase is seen to show no correlation with any of the other measurements of hairy cell numbers. In contrast, the percentages of cells showing the hairy cell esterase patterns and bearing a given surface light chain at their surface were broadly similar, but consistently greater than the percentage of morphologically typical hairy cells in the Romanowsky preparations.

DISCUSSION In this paper a distinctive pattern ofesterase positivity has been described in the hairy cells of all of a large series of patients with HCL. The method, which employed alpha-naphthyl butyrate esterase as substrate, gave a pattern of positivity in HCL quite different from that observed in a range of pathological states that could conceivably be confused with HCL.

K. E. Hisgy, G. F. Burns and F. G. J . Hayhoe

TABLE 111. Percentage acid phosphatase + ve mononuclear cells in HCL and effect of tartrate

Acid phosphatase

Type of I C’ithout With Patient preparation* tartrate tartrate

(Yo+) (%+)

1l.C. BC 67 0

BC 24 0

M.B. BC I00 I

BC I 0 0 0

H.D. BC 99 63 BC 51 49

R.H. BC 3 3 0

P.S. BC I00 64 BC 63 0

N.W. BC 29 3 Spleen cyto 9s 3 2

Spleen imprint I00 92

Spleen cyto 92 10 L.H. BC 41 6

J.M. BC 66 0

F.B. BC 62 2

L.P. BC 98 96 BC I00 I00

Spleen imprint 100 I00

* BC=peripheral blood buffy coat smears. Spleen cyto= cytocentrifuge preparation from a suspension of splenic cells.

TABLE IV. Pattern of a-naphthyl butyrate esterase in cell rosetting with EAIgM EAIgM

Rosette-forming cells Non-rosette-jorming cells

% EAlgM HC T-cell HC T-celt Patient rosettes pattern pattern Negative pattern pattern Negative

L.H. 34 80 I 0 I 0 3 58 39

Hairy Cel l Leukaemia 10s

TABLE V. Hairy cells as a percentage of mononuclears in peripheral blood determined by different methods

Patient

Morphological hairy cells

in Romanowsky preparations

(whole blood smears)

Hairy cell esterase pattern

(huffy coat smears)

P.S. F.W. L.H. F.B. L.P. K.D.

35 27 3 3 17 60 49

88 79 65 67 88 5s

Light chain Smlg Tartrate resistant (Fl Tl acid phosphatase cell suspension

(bufly coat smears) K 1

-54 4 62 ND 0 5 0 6-10 52 4

2 57 2 I00 3 62 ND 0 73

Others have noted variable alpha-naphthyl acetate esterase positivity in the hairy cells of HCL (Yam et al , 1972), but have not commented on the precise pattern of positivity obtained. The present paper, therefore, is the first to note a characteristic pattern of esterase positivity associated with the disease. Furthermore, it is shown here that the esterase activity of hairy cells, unlike that of monocytes, is not inhibited by sodium fluoride and this argues against the suggestion of Schnitzer & Kass (1974) that the alpha-naphthyl acetate positivity of hairy cells relates them to monocytes.

In view of the diagnostic promise of this esterase method, it was compared with the tartrate-resistant acid phosphatase method which has come to be considered diagnostic of HCL (Yam et al , 1971). In agreement with others (Catovsky, 1977). we have found that the acid phosphatase test is not by any means a completely reliable diagnostic tool since the activity is weak or absent in a proportion of cases and is often partially, or sometimes Completely, inhibited by tartrate. In contrast, the butyrate esterase method was invariably positive in a high proportion of the cells of HCL.

The finding of a persistently higher percentage of mononuclear cells giving the hairy cell pattern with the esterase method as compared with estimates of hairy cell numbers in simple Romanowsky preparations suggested that there were more cells involved in the leukaemic proliferation than was apparent by simple morphological analysis. The detection of similar high percentages of cells bearing light chain SmIg of a single class-an objective measurement of circulating monoclonal leukaemic cells in other B cell neoplasms (Brouet & Seligmann, 1977)-strongly supports this suggestion.

The signlficance of the distinctive esterase pattern detected only in hairy cells is at present not clear. However, since there are nine isoenzymes identdiable in human leucocytes (Li et al, I973), it is possible that the hairy cell esterase is a distinct isoenzyme analagous to the tartrate-resistant isoenzyme 5 of acid phosphatase possessed by some hairy cells. Whatever the precise biochemical explanation for the distinctive esterase pattern, it seems to represent yet another unique feature of the hairy cell of HCL.

The esterase method described here provides a simple differential diagnostic aid in HCL which is not only more reliable than previous cytochemical methods, but also provides an

I 06 K. E. H i a y , G. F. Burns and F. G. J . Hayhoe

estimate of circulating leukaemic cell numbers in the disease. The method may also be of diagnostic value in other lymphoproliferative disorders.

ACKNOWLEDGMENTS

W e would like to thank Drs J. K. H. Rees, M. Edwards, M. King and J. C. Cawley, who allowed us to study their cases of HCL. In addition, it is a pleasure to acknowledge the excellcnt technical assistance of M r R. J. Flemans, and Miss J. Thompson for typing the manuscript. K.E.H. and G.F.B. hold research studentships from the Arab Republic of Egypt and the Medical Research Council (U.K.) respectively.

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