Scand J Haematol (1977) 18, 437-448
Discrimination of B, T and Null Lymphocytes by Esterase Cytochemistry K. E. HIGGY, G . F. BURNS& F. G . J. HAYHOE Department of Haematological Medicine, Cambridge University Clinical SchooZ, Cambridge, England
A technique has been devised which optimally demonstrates non-specific esterase activity in human blood lymphocytes. The reaction is carried out on smears fixed with formalin vapour, using a-naphthol butyrate as substrate at pH 8 and at a low concentration for a short incubation period. The pattern of esterase activity revealed by this method provides a discriminating marker for mature T-lymphocytes, which show dense, localised, dot-like positivity, and a probable marker for ‘null’ cells in the form of scattered granular positivity. B cells appear to be negative. Monocytes show a clearly different pattern of granular positivity. K e y words: cytochemistry - esterase
- lymphocyte
Accepted for publication February 15, 1977 Correspondence to: Prof. F. G. J. Hayhoe, Department of Haematological Medicine, Hills Road, Cambridge CB2 2QL, England
The esterase activity in human haemic cells in health and disease has been studied cytochemically by many investigators in the past 25 years. To demonstrate this activity naphthy1 esters have generally been used as substrates, with incubation periods of 60-90 min or longer, at near neutral pH, the naphthol liberated by cytoplasmic esterase activity being coupled with a suitable diazonium salt to give an insoluble, brightly coloured, azo dye. The underlying biochemistry is complicated - Li et a1 (1973) were able to show that blood cells possessed at least two distinct esterases with 9 isoenzyme bands separable by polyacrylamide
gel electrophoresis at pH 4.0 - but, cytochemically, although the results from different centres are far from uniform in terms of detailed distribution of positivity, certain strong reaction patterns have come to be recognised as characteristic of monocytes and granulocytes when particular substrates or inhibitors are used. Thus, studies using as substrate a-naphthyl acetate (Braunstein 1959, Rozenszajn et a1 1968, Yam et a1 1971, Li et a1 1973) have shown strong positivity in normal monocytes and leukaemic monoblasts, with much weaker or negative reactions in other haemic cells. This esterase activity, which is inhibited in mono-
438
K. E. HIGGY, G. F. BURNS & F. G. J. HAYHOE
cytes by the addition of fluoride, is sometimes called ‘monocytic esterase’. Similarly with naphthol AS-D chloroacetate as substrate (Gomori 1953, Moloney et a1 1960, Rozenszajn et a1 1968, Yam et a1 1971, Li et a1 1973) strong positivity occurs in normal and leukaemic granulocytes with only weak or negative reactions in other cell types. This esterase positivity, which is not inhibited by fluoride, has been called ’granulocytic esterase’. A third substrate, naphthol AS or AS-D acetate (Wachstein & Wolf 1958, Shnitka & Seligman 1961, Hayhoe et a1 1964, Schmalz & Braunsteiner 1971, Daniel et a1 1971) gives a wider distribution of positivity among different cell types, most haemic cells showing some reaction but with the strong positivity of monocytes inhibitable by fluoride, whereas the reaction in granulocytes, lymphocytes and early erythroblasts is not inhibited. These methods have hitherto found their chief practical applications in haematology in the cytological classification of acute leukaemias and the determination of the granulocyte or monocyte precursor components in poorly differentiated leukaemic cell populations. While positivity in a proportion of lymphocytes has been noted in many reports of esterase reactions with substrates other than chloroacetate, the possibility that this enzymic reaction may serve to differentiate immunologically separate varieties of human lymphocytes has not yet been adequately explored, although Mueller et a1 (1975), using a-naphthyl acetate as substrate at acid pH and with very long incubation periods, found positive reactions to be apparently restricted to the T-cell component of mouse lymph nodes. The considerable variations in the detailed distribution of esterase positivity in haemic cells found with alterations in the
fixative procedure, the nature and concentration of the substrate, the length of incubation and the pH used had led us to undertake a comprehensive study of the effect of all these variables. In the course of this study we found that an esterase reaction pattern capable of differentiating several varieties of lymphocyte was most clearly demonstrable by the use of preparations fixed in formalin vapour and exposed to a-naphthyl butyrate as substrate in low concentration, at p H 8, with a short incubation period, and with either Fast blue RR or Fast Garnet as diazonium salt. Details of this method and studies with buffy coat smears, with enriched preparations of T, B and ‘null’ lymphocytes, with splenic lymphocyte suspensions and with lymphocytes transformed with phytohaemagglutinin are now reported.
METHODS AND MATERIALS C y tochemical met hods (a) Preliminary studies. Buffy coat smears were prepared from heparinised normal peripheral blood. Smears were fixed in (i) formalin vapour or (ii) 0.5, 1.0, 1.5, 2.0 or 2.5 % glutaraldehyde or (iii) 0.5, 1.0, 1.5, 2.0 or 2.5 % paraformaldehyde (the latter two were diluted in 0.05 M phosphate buffer pH 7.4) for periods increasing at 2 rnin intervals from 2 to 16 min in the case of formalin vapour and for periods of 5, 10, 15, 30, 45 or 60 rnin in the case of the other two fixatives. Each of the following substrates, a-naphthyl acetate, a-naphthyl butyrate, naphthol AS and AS-D acetate and naphthol AS-D chloroacetate was tested at a series of concentrations from 1.25 to 2.5, 5, 10 and 20 mg/100 ml of buffer at pH values ranging in steps of 0.5 from 5-9 for incubation times ranging at 10 rnin intervals from 5-90 rnin at room temp., 4 O C and 3 7 O C. (b) The cytochemical esierase technique selected. The technique giving best cell preservation, with strong esterase reactions and the most clearcut
ESTERASE CYTOCHEMISTRY O F LYMPHOCYTES differential patterns of positivity among granulocytes, monocytes and variosus lymphoscytes was as follows: Fix air-dried smears in formalin vapour for 4 min. Wash brieffy in distilled water and blot dry. Incubate at room temp. for 15-30 min in the following medium 10 ml 0.1 M phosphate buffer (pH 8.0) or-naphthyl butyrate (0.025 ml of a solution of 0.01 ml of the 2.33 x 10-4 M butyrate in 0.5 ml acetone) Fast blue BB salt (15 mg) 5x1e3M or Fast garnet GBC salt (3 mg) 9 x l P M Wash briefly in distilled water. Counterstain in haematoxylin for 5 min or in nuclear fast red for 10 min and blot dry. (c) Acid phosphatase and Periodic acid-Schiff( P A S ) reactions. These were carried out by the methods of Goldberg & Barka (1962) and Hayhoe et al (1964) respectively . Imniurzological methods
(a) Cell preparation for immunological testing. Spleens were collected immediately after surgical removal from (a) a patient with hereditary spherocytosis and (b) a patient undergoing radical surgery for stomach cancer. Single cell suspensions were obtained by forcing pieces of tissue through a 120 gauge stainless-steel mesh into Hepesbuffered Hanks balanced salt solution (H/H). The cell suspensions were kept on ice until their return to the laboratory, where they were washed in H/H and mononuclear cells were separated by centrifugation over Ficoll-Hypaque (F-H) gradients (Boyum 1968). The cells were washed a further three times in H/H before resuspending in the same medium 0.2 % bovine serum albumin (BSA) t o a final concentration of 2 x 106 cells/ml. Normal peripheral blood, obtained from volunteers, was collected into heparin and the mononuclear cells separated in a similar manner. In all cases cell viability as assessed by trypan-blue exclusion or by conversion of fluorescein-diacetate to fluorescein (Celada & Rotman 1967) was greater than 95 %.
+
(b) Rosette tests. Sheep erythrocyte rosette tests (E)
439
for T-cell detection were carried out using aminoethylthiouronium bromide-treated sheep erythrocytes (AET-E) according to the method of Kaplan & Clark (1974). AET-E were suspended in H/H 0.2 % BSA t o 1 % and 2 drops mixed with 2 drops of leucocytes in the same medium. These were incubated for 15 min at 37OC, centrifuged for 1 min at 150 g and stored on ice for at least 1 h before scoring. Percentages of rosetting cells in this and the other rosette assays were assessed using the fluorescein diacetate method of Ramasamy (1974) scoring only the viable fluorescing cells under combined UV and phase contrast microscopy. F c rosette tests were carried out by the method described by Hallberg et a1 (1973). Ox rbcs were sensitised with rabbit LgG anti-ox rbc at a dilution 2 log2 units higher than that causing haemagglutination, washed, and re-suspended in WH 0.2 % BSA medium t o 1 % before rosetting and scoring as for E-rosettes. Mouse rosetting was performed by the method of Gupta et al (1976) but modified by using AETtreated mouse erythrocytes to produce more stable rosettes (Worman, personal communication) rather than fixing the formed rosettes with glutaraldehyde before reading.
+
+
(c) Preparation of enriched populations (i) T-cells. Preparations rich in T-cells were obtained by AET-E rosetting the mononuclear cell population and centrifuging the rosetted population through a Ficoll-Hypaque gradient (Smith et a1 1973). The E positive cells formed a pellet at the bottom of the tube, and the T-cells were separated from sheep cells by lysing the rbc with 0.17 M NH4Cl and removing rbc stroma by centrifuging the leucocytes through foetal calf serum. The percentage of T-cells obtained in such preparations was checked by re-rosetting with AET-E cells. (ii) B-cells. Interface cells on the F-H gradient formed after removal of AET-E rosetting cells were used as one source of B-cells. In addition B-cells were obtained by F c rosetting mononuclelar cells and isolating the F c positive (B) cells in a similar manner to that used for T-cell enrichment. Sub-populations of B-cells were also obtained by depletion of T-cells (AET-E rosettes
440
K. E. HIGGY, G . F. BURNS & F. G. J. HAYHOE
removed) and the major population of B-cells by forming mouse (M) rosettes and retaining Mpositive cells by pelleting through F-H gradients. (iii) ‘Null’ cells. Strictly cells negative for surface immunoglobulin are termed null cells, but generally these cells are also F c negative (Winchester & Ross 1976). Since the major population of Bcells are F c positive (McConnell & Hurd 1976) an enriched null cell population was obtained by first removing T-cells (AET-E rosettes), then removing B-cells (Fc rosettes) and using the interface cells after the third F-H centrifugation as being enriched in null cells. (iv) Monocytes. The propensity of rnonocytes to adhere to glass was utilised. Lab-Tek (Miles Laboratories, Inc. Illinois) tissue culture slides were employed as suitable chambers to hold 1 ml washed mononuclear cell preparations at 1 x 10V ml in 20 % homologous serum over the clean glass slides. The cultures were incubated at 37O C for 80 min before thoroughly washing in H/H to remove non-adherent cells. Materials studied cytocheniically
The cytochemical method described above was applied t o buffy coat smears prepared from heparinised normal peripheral blood, t o smears made at 24, 48 and 72 h from lymphocyte cultures stimulated by phytohaemagglutinin according t o the method of Nowell (1960) and to cytocentrifuge preparations of enriched T-cell, B-cell and ‘null’ cell concentrates, mouse-rosetted lymphocyte suspensions and splenic lymphocyte suspensions, prepared as described above.
RESULTS
The preliminary study, in which various fixation procedures, different substrates at varying concentrations, and a range of temp., time and pH of incubation was investigated, showed (i) that exposure to formalin vapour for 4 min allowed optimum demonstration of enzyme activity with a clearly defined granular
disposition, while providing adequate cytological fixation. Shorter fixation was cytologically unsatisfactory and longer fixation reduced esterase reactivity. Glutaraldehyde and paraformaldehyde gave less satisfactory preservation of cell structure with a reduction in enzyme reaction product, especially in lymphocytes. (ii) high concentrations of substrate, prolonged incubation (60 min or more), and pH levels below 7.0 all tended to give mixed diffuse and granular reactions involving to variable degrees most cell lines in bufYy coat preparations. (iii) the most selective reaction patterns in terms of cytological differentiation among lymphocytes and monocytes were found with a-naphthyl butyrate as substrate, at a concentration of 2.33 x M, at pH 8. The reaction gave optimal results after 1530 min incubation, when monocytes showed a strong granular positivity scattered all over the cytoplasm (Figures 1 and 3). This reaction could be inhibited by sodium fluoride (0.04 M). Granulocytes were negative or only very faintly positive (Figure 2), and a number of distinct reaction patterns were seen in lymphocytes which were resistant to sodium fluoride. The reaction patterns in lymphocytes 1. Buffycoat smears from normal peripheral blood
As shown in Table 1, some 67 % of normal peripheral blood lymphocytes displayed a dense localised positivity made up of 1 to 4 coarse granules (Figures 1 and 2), the majority of lymphocytes with this type of positivity having only a single granule. About 13 % of lymphocytes showed a scat-
ESTERASE CYTOCHEMISTRY OF LYMPHOCYTES
44 1
tered granular reaction in the cytoplasm (Figure 3), while about 20 % were quite negative (Figures 2 and 3). Comparison of these findings with the results of immunological marker studies carried out on the peripheral blood samples (Table 3) indicates that the percentage of lymphocytes showing the lwalised reaction product is nearly the same as the percentage of T-lymphocytes in the samples. In AET-E rosetted preparations, rosetting cells usually showed localised esterase reaction (Figure 4).
positivity predominated in the null-cell enriched preparations (Figure 7).
2. T , B and null cell enriched
4. Mouse-rosetted lymphocyte
preparations
3 . Spleen lymphocytes The cytochemical findings in splenic lymphocyte suspensions are shown in Table 1 and the surface marker studies on the same suspensions in Table 3. The correlation again suggests that localised esterase positivity may be a property of T-cells in the spleen as in the peripheral blood.
preparations
The results of cytochemical studies ofi these enriched populations are shown in Table 2. Cells with localised positivity predominated in the T-cell enriched preparations (Figure 5), whereas negative cells predominated in the B-cell enriched preparations (Figure 6), and cells with scattered granular
The results of esterase cytochemistry on a lymphocyte preparation enriched in the cells which rosette with mouse red cells are shown in Table 2. Negative cells are numerous, but less so than in the B-cell enriched preparations prepared by Fc rosetting.
Figure 1. A monocyte with generalised granular esterase positivity and two lymphocytes with dense localised reactions.
Figure 2. Buffy coat preparation, with 4 esterase negative polymorphs, 1 negative lymphocyte, and 4 lymphocytes with from 1 to 4 dots of localised positivity.
2
-
*
of
17.6 +_ 6.2 16.4 50.8
4 2
2
B-cell enriched preparations
‘Null cell’ enriched preparations
Mouse rosetting subpopulation
This sample expressed 85 % AET-E rosettes.
22.0 f 5.8
19.5
42.0-59.6
40.8
15.6k6.0
70.3-84.6 12.2-20.6
68.8 k 5.5
14.2-27.4
meankSD
132.2-49.4
8.4
64.5
134 2 5 . 6
8.2- 8.6
62.7-66.2
6.1- 7.6
6.6 k 0.1
range
9.3-22.6
I
6.2-14.6
meankSD
granular
range
9.Ok 3.8 60.2-79.6“
range
I
range
I
meankSD
localised ‘dot like’
I
granular meanfSD
Reaction product of esterase activity (%) negative
4
samples
T-cell enriched preparations
Lymphocyte preparations
Number
TABLE 2 Non-specific esterase activity in enriched T , B and ‘null‘ cell preparations and also in mouse rosetted lymphocytes
-
(1) (2) 26.4-49.2
range
1
meanfSD
range
1
meankSD
localised ‘dot like’
Reaction product of esterase activity (%) negative
(1) From patient with hereditary spherocytosis. (2) From patient undergoing radical surgery for stomach cancer.
Splenic lymphocyte suspensions
samples
of
Number
TABLE 1 a-naphthyl butyrate csterase activity in lyniphocytcs in normal buffy coat stnears (whole blood) and splenic lymphocyte suspensions
P P
4
0
En
F m .
ra
ESTERASE CYTOCHEMISTRY OF LYMPHOCYTES
5 . Lymphoblasts in PHA-stimulated cultures
The esterase reaction in these cells from 24 h onwards was generally negative, with weak scattered positivity in only a few cells. Non-transformed lymphocytes appeared predominantly negative with a few showing the scattered granule pattern. 6. Acid phosphatase and Periodic acid-Shiff ( P A S ) reactions in T- and B-cell enriched preparations
The results of this study are shown in Table 4. Neither reaction appeared to have much discriminative value between T and B cells.
Figure 3. Buffy coat preparation, with a monocyte showing generalised granular esterase positivity, 1 lymphocyte with localised block reaction, 1 lymphwyte with scattered granular reaction, and 1 negative lymphocyte.
443
DISCUSSION
Following a comparative study of different fixative procedures, a range of substrates and substrate concentrations, and of different periods of incubation at a range of temperatures and pH values, a technique has been devised which optimally demonstrates non-specific esterase activity in human blood lymphocytes. The reaction is carried out on smears fixed with formalin vapour, using a-naphthol butyrate as a substrate at pH 8 and at a low concentration (2.33 x M) for a short incubation period. The results show that the pattern of esterase activity revealed by the method we have used provides a discriminating marker for mature T-lymphocytes, and a probable
Figure 4. AET-E rosetted preparation: block localised reaction in a rosette-forming lymphocyte, negative reaction in a non-rosetted lymphocyte.
444
K. E. HIGGY, G. F. BURNS & F. G. J. HAYHOE TABLE 3 Immunological marker studies on lymphocytes in 6 of the normal peripheral blood samples studied cytochemically, and in splenic lymphocytes f r o m a presumed normal spleen and a spleen from a patient with hereditary spherocytosis
Fc receptor
Number of
meankSD
1
range
AET-E rosettes meankSD
1
range
marker for ‘null cells’. B-cells appear to be negative. Cytochemically, peripheral blood lymphocytes showed three different patterns of reaction product for non-specific esterase enzyme activity. Correlating these results with
immunological marker studies on the lymphocyte populations studied, peripheral b l d lymphocytes appear t o be classifiable cytochemically into three groups:
Figure 5. T-cell enriched population: 5 cells showing 1 to 4 blocks of localised reaction: 1 cell with scattered granules.
Figure 6. B-cell enriched population: 7 negative cells, 3 with localised granules and 1 with scattereld granular reaction.
(1) A first group which had positive T-cell
445
ESTERASE CYTOCHEMISTRY OF LYMPHOCYTES TABLE 4 Acid phosphatase and P A S reactions in separuted normal lymphocytes
Enriched lymphocyte preparations
Number of samples
Acid phosphatase reaction (% of positive cells) mean ? SD
I
PAS reaction
(% of positive cells)
range
mean k SD
1
range
T-cell enriched preparations
7
41.8 k 4 . 5
37.2-50.6
34.9k 3.2
30.1-39.2
9-cell enriched preparations
7
51.54k 6.3
44.2-59.6
38.0+ 3.4
32.2-42.3
immunological markers contained a high number of cells showing dense, localised ‘dot-like’ esterase positivity. (2) A second group having B-cell immunological markers showed negative esterase reaction in the vast majority of the cells.
(3) A third population where the cells had neither T- nor B-cell immunological markers - ‘null cells’ - showed scattered granular positivity in most of the cells.
Figure 7. Null cell enriched preparation: 3 cells with fine scattered cytoplasmic granules and 1 with localised coarsely granular reaction.
Separated populations of uncontaminated T-cells, B-cells or null cells are not readily obtainable, but studies with lymphocyte populations enriched in one or other of these components provided strong confirmation of the specificity of the cytochemical findings. Thus the majority of lymphocytes in the B-cell enriched populations studied had negative esterase reactions, the minor component with localised or granular reactions being attributable to retained T or null cells. Similarly, the majority of lymphocytes in the T-cell enriched populations had localised positivity, the minor component with negative or granular reactions being attributable to retained B- or null-cells. Again, the null cell enriched populations showed a predominance of cells with scattered granular esterase positivity, with minor components of negative cells or cells with localised positivity, attributable t o retained B- or T-cells. Indeed since no cognizance is made of SmIg+ Fc- B-cells, such cells may account for the residual B-cells. No account was taken of the various minor subclasses of lymphocytes such as E+ Fc+ or of cells bearing various combinations of markers including surface immunoglobulin, FC and the fixed component of C3 (C3b). However, such cell types as SmIg+ Fc- C3b+ and SmIg+ Fc- C3- represent very small proportions of circulating cells (McConnell &
446
K. E. HIGGY, G. F. BURNS & F. G. J. HAYHOE
Hurd 1976) and their classification and nomenclature remains obscure. Since it has been suggested that mouse RBCs rosette with a sub-population of Blymphocytes (Stathopoulos & Elliot 1974, Forbes & Zalewski 1976) it was of interest to determine whether any difference could be observed cytochemically between the Mrosetting cells and other B-lymphocytes. Our findings confirm the B-nature of the Mrosetting cells in that the M-rosette preparation was predominantly esterase negative (Table 2), and we were unable to show any particular cytochemical marker on these cells. Normal peripheral blood monocytes showed a strong and extensive granular reaction with this esterase technique which allowed them to be differentiated very easily from lymphocytes and myeloid cells. The latter were almost negative in this reaction. Weak esterase activity in lymphocytes has been described before, with a-naphthyl acetate, a-naphthyl butyrate and naphthol AS and AS-D acetate as substrates (Wachstein & Wolf 1958, Braunstein 1959, Moloney et a1 1960, Rozenszajn et a1 1968, Li et a1 1973). However, the a-naphthyl butyrate esterase activity demonstrated with the technique described here appears to be more intense and more discriminating than that described in earlier reports. Mueller et a1 (1975), using a-naphthyl acetate as a substrate at pH 5.8 and with a prolonged incubation period of 21 h, reported a dotlike localised positivity in the cytoplasm of lymphocytes in the paracortical areas of lymph nodes in mice, presumed to be Tcells. Fixation in Baker’s formolcalcium was used. The method we have used gives a more clearcut positivity at pH 8 than at acid pH levels. Air-dried smears are used,
and give satisfactory esterase reactions even after storage for up to 4 weeks. Moreover, the short incubation period gives better discriminative results than does prolonged incubation. There have been previous attempts to differentiate cytochemically between T- and B-lymphocytes. Acid phosphatase is known to be strong in PHA activated blast cells and in lymphoid cells of infectious mononucleosis (Quaglino et a1 1962, Li et a1 1970, Biberfeld 1971). Again, the blast cells of T-cell leukaemias (T-ALL) show strong localised acid phosphatase activity more frequently than do the cells of non-T lymphoblastic leukaemias, while the PAS reaction tends to show an opposite pattern of positivity, being stronger in unmarked ALL than in T-ALL (Catovsky et a1 1974). The lysosomal enzymes N-acetyl-p-glucosaminidase and p-glucuronidase as well as acid phosphatase have been reported to be more prominent in T-cells and T-dependent parts of peripheral lymphoid organs than in B-cells in rodents and in man (Tamaoki & Essner 1969). However, neither acid phosphatase nor PAS reactions in normal peripheral blood lymphocytes show major differences between the T- and B-cells (Table 4). Farid et 21 (1975) using a-naphthy1 acetate as substrate, demonstrated a difference in the esterase activity between T- and B-cells, with 17.7 % k 8.6 positive cells in the sheep RBC rosette-forming lymphocytes (T-cells) and 11.7 % .t 4.3 positive cells in the B-rich fraction. Their method is clearly less discriminatory than the one we have used. The negative a-naphthyl butyrate esterase reaction in PHA-transformed lymphocytes contrasts with the strong acid phosphatase activity in such cells. Both esterase and acid phosphatase are lysosomal enzymes,
ESTERASE CYTOCHEMISTRY O F LYMPHOCYTES
447
REFERENCES but they appear to be active at different phases of T-cell maturation and prolifera- Biberfeld P (1971) Morphogenesis in blood lymtive activity. Ferluga et a1 (1972) demonphocytes stimulated with phytohaemagglutinin (PHA). Acta Pathol et Microbiol Scand, Suppl strated that killing effects mediated by T223, 1-65. cells against target cells were inhibited by Boyum A (1968) A one-stage procedure for isolaesterase inhibitors, thus suggesting that tion of granulocytes and lymphocytes from huesterases may contribute to the cytotoxic man blood. Scand J Clin Lab Invest 21, Suppl function of T-cells. 97, 51-76. Work on the application of thls cyto- Braunstein H (1959) Esterase in leukocytes. ,'Histochem Cytochem 7 , 202. chemical reaction in leukaemic and lymCelada F & Rotman B (1967) A fluorochromatic phomatous states is currently in progress, test for immunocytotoxicity against tumor cells and will be reported shortly. Preliminary and leucocytes in agarose plates. Proc Nut1 Acad results suggest that the pattern of a-naphSci USA 57, 630-36. thy1 butyrate esterase reactivity, as a marker Catovsky D, Galetto J, Okos A, Miliani E & Galton A G (1974) Cytochemical profile of B and for T, B and 'null' cells is maintained in T leukaemic lymphocytes with special reference pathological proliferations. This simple techto acute lymphoblastic leukaemia. J Clin Path nique may therefore prove of great value 27, 767-71. in estimating the relative proportions of dif- Daniel M Th, Flandrin G, Lejeune F, Liso P & Lortholary P (1971) 1. Les estkrases spkcifiques ferent lymphocyte populations in whole monocytaires. Utilisation dans la classification blood preparations in cases where viable des leuchmies aigues. Nouv Rev Fr Hematol 11, cells are not available or where immuno233-39. logical studies are otherwise impracticable. Farid N R, Noel E P & Kutas C (1975) A populaThe existence of abnormal proportions of tion of human lymphocytes staining for esterases. Experientia 32, 340-41. T and B cells in peripheral blood may have importance in many pathological conditions Ferlenga J, Asherson G L & Becker E L (1972) The effect of organophosphorus inhibitors, pin addition to lymphoproliferative states, nitrophenol and cytochalasin-B on cytotoxic and this test provides a simple method for killing of tumour cells by immune spleen cells differential assessment. More elaborate and the effect of shaking. Immunology 23, 57790. methods for T and B cell evaluation in whole blood smears have been described, Forbes I J & Zalewski P D (1976) A subpopulation of human B lymphocytes that rosette with such as that of Pepys et a1 (1976) who used mouse erythrocytes. Clin E x p Immunol 26, 99a combination of IgG immunofluorescence 107. and peroxidase staining, but the test de- Gomori G (1953) Chloroacylesters as histochemical substrates. J Histochem Cytochem 1, 469scribed here is easy to perform and re70. quires only one cytochemical stain on a Goldberg A F & Barka T (1962) Acid phosphasingle slide. tase activity in human blood cells. Nature 195, ACKNOWLEDGEMENTS We are grateful to Mr. R. J. Flemans for technical and photographic assistance and to Miss I. Thompson for typing the manuscript. KEH and GFB hold Research Studentships from the United Arab Republic of Egypt and the Medical Research Council, respectively.
297. Gupta S , Good R A & Siegal F P (1976) Rosette formation with mouse erythrocytes. 11. A marker for human B and non-T lymphocytes. Clin Exp lmmunol 25, 319-27. Hallberg T,Gurner B W & Coombs R R A (1973) Opsonic adherence of sensitized ox red cells to human lymphocytes as measured by rosette
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formation. In! Arch Allergy 44, 500-13. Hayhoe F G J, Quaglino D & Doll R (1964) The Cytology and Cytochemistry of Acute Leukaemias. M. R. C. Special Report Series No 304, H. M. S. O., London. Kaplan M E & Clark C (1974) An improved rosetting assay for detection of human T lymphocytes. J Immunol Methods 5, 131-35. Li C Y, Lam K W & Yam L T (1973) Esterases in human leucocytes. J Histochem Cytochem 21, 1-12. Li C Y, Yam L T & Lam K W (1970) Acid phosphatase isoenzyme in human leucocytes in normal and pathologic conditions. J Histochem Cytochem 18, 473-81. McConnell I & Hurd C M (1976) Lymphocyte receptors. 1. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells. Immunology 30, 825-33. Moloney W C, McPherson K & Fliegelman L (1960) Esterase activity in leucocytes demonstrated by the use of naphthol AS-D chloroacetate substrate. J Histochem Cytochem 8, 200-07. Mueller J, Brun Del Re G, Buerki H, Keller H U, Hess M W & Cottier H (1975) Non-specific acid esterase activity: a criterion for differentiation of T and B lymphocytes in mouse lymph nodes. Eur J Immunol 5, 270-74. Nowell P C (1960) Phytohaemagglutinin: an initiator of mitosis in cultures of normal human leucocytes. Cancer Res 20, 462-66. Pepys M B, Sategna-Guidetti C, Mirjah D D, Wansbrough M H & Dash A C (1976) Enumeration of immunoglobulin-bearing lymphocytes in whole peripheral blood. Clin E x p Immunol 26, 91-94. Quaglino D, Hayhoe F G J & Flemans R J (1962) Cytochemical observations on the effect of phytohaemagglutinin in short-term tissue cul-
tures. Nature 196, 338-40. Ramasamy R (1974) A fluorescent stain for viable rosette forming cells. J Immunol Methods 5, 305-06. Rozenszajn L, Leiberich M, Shoham D & Epstein J (1968) The esterase activity in megaloblasts, leukaemic and normal haemopoietic cells. Br J Haematol 14, 60-10. Schmalzi F & Braunsteiner H (1971) The application of cytochemical methods to the study of acute leukaemias. A review. Acta Haematol (Basel) 45, 209-17. Shnitka T K & Seligmann A M (1961) Role of esterase inhibition on localisation of esterase and the simultaneous cytochemical demonstration of inhibitor sensitive and resistant enzyme species. J Histochern Cytochem 9, 504-27. Smith J L, Barker C R, Clein G P & Collins R J (1973) Characterisation of malignant mediastinal lymphoid neoplasm (Sternberg sarcoma) as thymic in origin. Lancet 1, 74-77. Stathopoulos G & Elliot E V (1974) Formation of mouse or sheep red blood cell rosettes by lymphocytes from normal and leukaemic individuals. Lancet 1, 600-01. Tamaoki N & Essner E (1969) Distribution of acid phosphatase, beta-glucuronidase and Nacetyl-beta-glucosaminidase activities in lymphocytes of lymphatic tissues of man and rodents. J Histochem Cytochem 17, 238-43. Wachstein M & Wolf G (1958) The histochemical demonstration of esterase activity in human blood and bone marrow smears. J Histochem Cytochem 6, 457. Winchester R J & Ross G (1976) Methods for enumerating lymphocyte populations. In N R Rose & H Friedman (eds) Manual of Clinical Immunology, pp 64. Am SOCfor Microbiol. Yam L T, Li C Y & Crosby W H (1971) Cytochemical identification of monocytes and granulocytes. A m J Clin Pathol 55, 283-90.
Discrimination of B, T and Null Lymphocytes by Esterase Cytochemistry K. E. HIGGY, G . F. BURNS& F. G . J. HAYHOE Department of Haematological Medicine, Cambridge University Clinical SchooZ, Cambridge, England
A technique has been devised which optimally demonstrates non-specific esterase activity in human blood lymphocytes. The reaction is carried out on smears fixed with formalin vapour, using a-naphthol butyrate as substrate at pH 8 and at a low concentration for a short incubation period. The pattern of esterase activity revealed by this method provides a discriminating marker for mature T-lymphocytes, which show dense, localised, dot-like positivity, and a probable marker for ‘null’ cells in the form of scattered granular positivity. B cells appear to be negative. Monocytes show a clearly different pattern of granular positivity. K e y words: cytochemistry - esterase
- lymphocyte
Accepted for publication February 15, 1977 Correspondence to: Prof. F. G. J. Hayhoe, Department of Haematological Medicine, Hills Road, Cambridge CB2 2QL, England
The esterase activity in human haemic cells in health and disease has been studied cytochemically by many investigators in the past 25 years. To demonstrate this activity naphthy1 esters have generally been used as substrates, with incubation periods of 60-90 min or longer, at near neutral pH, the naphthol liberated by cytoplasmic esterase activity being coupled with a suitable diazonium salt to give an insoluble, brightly coloured, azo dye. The underlying biochemistry is complicated - Li et a1 (1973) were able to show that blood cells possessed at least two distinct esterases with 9 isoenzyme bands separable by polyacrylamide
gel electrophoresis at pH 4.0 - but, cytochemically, although the results from different centres are far from uniform in terms of detailed distribution of positivity, certain strong reaction patterns have come to be recognised as characteristic of monocytes and granulocytes when particular substrates or inhibitors are used. Thus, studies using as substrate a-naphthyl acetate (Braunstein 1959, Rozenszajn et a1 1968, Yam et a1 1971, Li et a1 1973) have shown strong positivity in normal monocytes and leukaemic monoblasts, with much weaker or negative reactions in other haemic cells. This esterase activity, which is inhibited in mono-
438
K. E. HIGGY, G. F. BURNS & F. G. J. HAYHOE
cytes by the addition of fluoride, is sometimes called ‘monocytic esterase’. Similarly with naphthol AS-D chloroacetate as substrate (Gomori 1953, Moloney et a1 1960, Rozenszajn et a1 1968, Yam et a1 1971, Li et a1 1973) strong positivity occurs in normal and leukaemic granulocytes with only weak or negative reactions in other cell types. This esterase positivity, which is not inhibited by fluoride, has been called ’granulocytic esterase’. A third substrate, naphthol AS or AS-D acetate (Wachstein & Wolf 1958, Shnitka & Seligman 1961, Hayhoe et a1 1964, Schmalz & Braunsteiner 1971, Daniel et a1 1971) gives a wider distribution of positivity among different cell types, most haemic cells showing some reaction but with the strong positivity of monocytes inhibitable by fluoride, whereas the reaction in granulocytes, lymphocytes and early erythroblasts is not inhibited. These methods have hitherto found their chief practical applications in haematology in the cytological classification of acute leukaemias and the determination of the granulocyte or monocyte precursor components in poorly differentiated leukaemic cell populations. While positivity in a proportion of lymphocytes has been noted in many reports of esterase reactions with substrates other than chloroacetate, the possibility that this enzymic reaction may serve to differentiate immunologically separate varieties of human lymphocytes has not yet been adequately explored, although Mueller et a1 (1975), using a-naphthyl acetate as substrate at acid pH and with very long incubation periods, found positive reactions to be apparently restricted to the T-cell component of mouse lymph nodes. The considerable variations in the detailed distribution of esterase positivity in haemic cells found with alterations in the
fixative procedure, the nature and concentration of the substrate, the length of incubation and the pH used had led us to undertake a comprehensive study of the effect of all these variables. In the course of this study we found that an esterase reaction pattern capable of differentiating several varieties of lymphocyte was most clearly demonstrable by the use of preparations fixed in formalin vapour and exposed to a-naphthyl butyrate as substrate in low concentration, at p H 8, with a short incubation period, and with either Fast blue RR or Fast Garnet as diazonium salt. Details of this method and studies with buffy coat smears, with enriched preparations of T, B and ‘null’ lymphocytes, with splenic lymphocyte suspensions and with lymphocytes transformed with phytohaemagglutinin are now reported.
METHODS AND MATERIALS C y tochemical met hods (a) Preliminary studies. Buffy coat smears were prepared from heparinised normal peripheral blood. Smears were fixed in (i) formalin vapour or (ii) 0.5, 1.0, 1.5, 2.0 or 2.5 % glutaraldehyde or (iii) 0.5, 1.0, 1.5, 2.0 or 2.5 % paraformaldehyde (the latter two were diluted in 0.05 M phosphate buffer pH 7.4) for periods increasing at 2 rnin intervals from 2 to 16 min in the case of formalin vapour and for periods of 5, 10, 15, 30, 45 or 60 rnin in the case of the other two fixatives. Each of the following substrates, a-naphthyl acetate, a-naphthyl butyrate, naphthol AS and AS-D acetate and naphthol AS-D chloroacetate was tested at a series of concentrations from 1.25 to 2.5, 5, 10 and 20 mg/100 ml of buffer at pH values ranging in steps of 0.5 from 5-9 for incubation times ranging at 10 rnin intervals from 5-90 rnin at room temp., 4 O C and 3 7 O C. (b) The cytochemical esierase technique selected. The technique giving best cell preservation, with strong esterase reactions and the most clearcut
ESTERASE CYTOCHEMISTRY O F LYMPHOCYTES differential patterns of positivity among granulocytes, monocytes and variosus lymphoscytes was as follows: Fix air-dried smears in formalin vapour for 4 min. Wash brieffy in distilled water and blot dry. Incubate at room temp. for 15-30 min in the following medium 10 ml 0.1 M phosphate buffer (pH 8.0) or-naphthyl butyrate (0.025 ml of a solution of 0.01 ml of the 2.33 x 10-4 M butyrate in 0.5 ml acetone) Fast blue BB salt (15 mg) 5x1e3M or Fast garnet GBC salt (3 mg) 9 x l P M Wash briefly in distilled water. Counterstain in haematoxylin for 5 min or in nuclear fast red for 10 min and blot dry. (c) Acid phosphatase and Periodic acid-Schiff( P A S ) reactions. These were carried out by the methods of Goldberg & Barka (1962) and Hayhoe et al (1964) respectively . Imniurzological methods
(a) Cell preparation for immunological testing. Spleens were collected immediately after surgical removal from (a) a patient with hereditary spherocytosis and (b) a patient undergoing radical surgery for stomach cancer. Single cell suspensions were obtained by forcing pieces of tissue through a 120 gauge stainless-steel mesh into Hepesbuffered Hanks balanced salt solution (H/H). The cell suspensions were kept on ice until their return to the laboratory, where they were washed in H/H and mononuclear cells were separated by centrifugation over Ficoll-Hypaque (F-H) gradients (Boyum 1968). The cells were washed a further three times in H/H before resuspending in the same medium 0.2 % bovine serum albumin (BSA) t o a final concentration of 2 x 106 cells/ml. Normal peripheral blood, obtained from volunteers, was collected into heparin and the mononuclear cells separated in a similar manner. In all cases cell viability as assessed by trypan-blue exclusion or by conversion of fluorescein-diacetate to fluorescein (Celada & Rotman 1967) was greater than 95 %.
+
(b) Rosette tests. Sheep erythrocyte rosette tests (E)
439
for T-cell detection were carried out using aminoethylthiouronium bromide-treated sheep erythrocytes (AET-E) according to the method of Kaplan & Clark (1974). AET-E were suspended in H/H 0.2 % BSA t o 1 % and 2 drops mixed with 2 drops of leucocytes in the same medium. These were incubated for 15 min at 37OC, centrifuged for 1 min at 150 g and stored on ice for at least 1 h before scoring. Percentages of rosetting cells in this and the other rosette assays were assessed using the fluorescein diacetate method of Ramasamy (1974) scoring only the viable fluorescing cells under combined UV and phase contrast microscopy. F c rosette tests were carried out by the method described by Hallberg et a1 (1973). Ox rbcs were sensitised with rabbit LgG anti-ox rbc at a dilution 2 log2 units higher than that causing haemagglutination, washed, and re-suspended in WH 0.2 % BSA medium t o 1 % before rosetting and scoring as for E-rosettes. Mouse rosetting was performed by the method of Gupta et al (1976) but modified by using AETtreated mouse erythrocytes to produce more stable rosettes (Worman, personal communication) rather than fixing the formed rosettes with glutaraldehyde before reading.
+
+
(c) Preparation of enriched populations (i) T-cells. Preparations rich in T-cells were obtained by AET-E rosetting the mononuclear cell population and centrifuging the rosetted population through a Ficoll-Hypaque gradient (Smith et a1 1973). The E positive cells formed a pellet at the bottom of the tube, and the T-cells were separated from sheep cells by lysing the rbc with 0.17 M NH4Cl and removing rbc stroma by centrifuging the leucocytes through foetal calf serum. The percentage of T-cells obtained in such preparations was checked by re-rosetting with AET-E cells. (ii) B-cells. Interface cells on the F-H gradient formed after removal of AET-E rosetting cells were used as one source of B-cells. In addition B-cells were obtained by F c rosetting mononuclelar cells and isolating the F c positive (B) cells in a similar manner to that used for T-cell enrichment. Sub-populations of B-cells were also obtained by depletion of T-cells (AET-E rosettes
440
K. E. HIGGY, G . F. BURNS & F. G. J. HAYHOE
removed) and the major population of B-cells by forming mouse (M) rosettes and retaining Mpositive cells by pelleting through F-H gradients. (iii) ‘Null’ cells. Strictly cells negative for surface immunoglobulin are termed null cells, but generally these cells are also F c negative (Winchester & Ross 1976). Since the major population of Bcells are F c positive (McConnell & Hurd 1976) an enriched null cell population was obtained by first removing T-cells (AET-E rosettes), then removing B-cells (Fc rosettes) and using the interface cells after the third F-H centrifugation as being enriched in null cells. (iv) Monocytes. The propensity of rnonocytes to adhere to glass was utilised. Lab-Tek (Miles Laboratories, Inc. Illinois) tissue culture slides were employed as suitable chambers to hold 1 ml washed mononuclear cell preparations at 1 x 10V ml in 20 % homologous serum over the clean glass slides. The cultures were incubated at 37O C for 80 min before thoroughly washing in H/H to remove non-adherent cells. Materials studied cytocheniically
The cytochemical method described above was applied t o buffy coat smears prepared from heparinised normal peripheral blood, t o smears made at 24, 48 and 72 h from lymphocyte cultures stimulated by phytohaemagglutinin according t o the method of Nowell (1960) and to cytocentrifuge preparations of enriched T-cell, B-cell and ‘null’ cell concentrates, mouse-rosetted lymphocyte suspensions and splenic lymphocyte suspensions, prepared as described above.
RESULTS
The preliminary study, in which various fixation procedures, different substrates at varying concentrations, and a range of temp., time and pH of incubation was investigated, showed (i) that exposure to formalin vapour for 4 min allowed optimum demonstration of enzyme activity with a clearly defined granular
disposition, while providing adequate cytological fixation. Shorter fixation was cytologically unsatisfactory and longer fixation reduced esterase reactivity. Glutaraldehyde and paraformaldehyde gave less satisfactory preservation of cell structure with a reduction in enzyme reaction product, especially in lymphocytes. (ii) high concentrations of substrate, prolonged incubation (60 min or more), and pH levels below 7.0 all tended to give mixed diffuse and granular reactions involving to variable degrees most cell lines in bufYy coat preparations. (iii) the most selective reaction patterns in terms of cytological differentiation among lymphocytes and monocytes were found with a-naphthyl butyrate as substrate, at a concentration of 2.33 x M, at pH 8. The reaction gave optimal results after 1530 min incubation, when monocytes showed a strong granular positivity scattered all over the cytoplasm (Figures 1 and 3). This reaction could be inhibited by sodium fluoride (0.04 M). Granulocytes were negative or only very faintly positive (Figure 2), and a number of distinct reaction patterns were seen in lymphocytes which were resistant to sodium fluoride. The reaction patterns in lymphocytes 1. Buffycoat smears from normal peripheral blood
As shown in Table 1, some 67 % of normal peripheral blood lymphocytes displayed a dense localised positivity made up of 1 to 4 coarse granules (Figures 1 and 2), the majority of lymphocytes with this type of positivity having only a single granule. About 13 % of lymphocytes showed a scat-
ESTERASE CYTOCHEMISTRY OF LYMPHOCYTES
44 1
tered granular reaction in the cytoplasm (Figure 3), while about 20 % were quite negative (Figures 2 and 3). Comparison of these findings with the results of immunological marker studies carried out on the peripheral blood samples (Table 3) indicates that the percentage of lymphocytes showing the lwalised reaction product is nearly the same as the percentage of T-lymphocytes in the samples. In AET-E rosetted preparations, rosetting cells usually showed localised esterase reaction (Figure 4).
positivity predominated in the null-cell enriched preparations (Figure 7).
2. T , B and null cell enriched
4. Mouse-rosetted lymphocyte
preparations
3 . Spleen lymphocytes The cytochemical findings in splenic lymphocyte suspensions are shown in Table 1 and the surface marker studies on the same suspensions in Table 3. The correlation again suggests that localised esterase positivity may be a property of T-cells in the spleen as in the peripheral blood.
preparations
The results of cytochemical studies ofi these enriched populations are shown in Table 2. Cells with localised positivity predominated in the T-cell enriched preparations (Figure 5), whereas negative cells predominated in the B-cell enriched preparations (Figure 6), and cells with scattered granular
The results of esterase cytochemistry on a lymphocyte preparation enriched in the cells which rosette with mouse red cells are shown in Table 2. Negative cells are numerous, but less so than in the B-cell enriched preparations prepared by Fc rosetting.
Figure 1. A monocyte with generalised granular esterase positivity and two lymphocytes with dense localised reactions.
Figure 2. Buffy coat preparation, with 4 esterase negative polymorphs, 1 negative lymphocyte, and 4 lymphocytes with from 1 to 4 dots of localised positivity.
2
-
*
of
17.6 +_ 6.2 16.4 50.8
4 2
2
B-cell enriched preparations
‘Null cell’ enriched preparations
Mouse rosetting subpopulation
This sample expressed 85 % AET-E rosettes.
22.0 f 5.8
19.5
42.0-59.6
40.8
15.6k6.0
70.3-84.6 12.2-20.6
68.8 k 5.5
14.2-27.4
meankSD
132.2-49.4
8.4
64.5
134 2 5 . 6
8.2- 8.6
62.7-66.2
6.1- 7.6
6.6 k 0.1
range
9.3-22.6
I
6.2-14.6
meankSD
granular
range
9.Ok 3.8 60.2-79.6“
range
I
range
I
meankSD
localised ‘dot like’
I
granular meanfSD
Reaction product of esterase activity (%) negative
4
samples
T-cell enriched preparations
Lymphocyte preparations
Number
TABLE 2 Non-specific esterase activity in enriched T , B and ‘null‘ cell preparations and also in mouse rosetted lymphocytes
-
(1) (2) 26.4-49.2
range
1
meanfSD
range
1
meankSD
localised ‘dot like’
Reaction product of esterase activity (%) negative
(1) From patient with hereditary spherocytosis. (2) From patient undergoing radical surgery for stomach cancer.
Splenic lymphocyte suspensions
samples
of
Number
TABLE 1 a-naphthyl butyrate csterase activity in lyniphocytcs in normal buffy coat stnears (whole blood) and splenic lymphocyte suspensions
P P
4
0
En
F m .
ra
ESTERASE CYTOCHEMISTRY OF LYMPHOCYTES
5 . Lymphoblasts in PHA-stimulated cultures
The esterase reaction in these cells from 24 h onwards was generally negative, with weak scattered positivity in only a few cells. Non-transformed lymphocytes appeared predominantly negative with a few showing the scattered granule pattern. 6. Acid phosphatase and Periodic acid-Shiff ( P A S ) reactions in T- and B-cell enriched preparations
The results of this study are shown in Table 4. Neither reaction appeared to have much discriminative value between T and B cells.
Figure 3. Buffy coat preparation, with a monocyte showing generalised granular esterase positivity, 1 lymphocyte with localised block reaction, 1 lymphwyte with scattered granular reaction, and 1 negative lymphocyte.
443
DISCUSSION
Following a comparative study of different fixative procedures, a range of substrates and substrate concentrations, and of different periods of incubation at a range of temperatures and pH values, a technique has been devised which optimally demonstrates non-specific esterase activity in human blood lymphocytes. The reaction is carried out on smears fixed with formalin vapour, using a-naphthol butyrate as a substrate at pH 8 and at a low concentration (2.33 x M) for a short incubation period. The results show that the pattern of esterase activity revealed by the method we have used provides a discriminating marker for mature T-lymphocytes, and a probable
Figure 4. AET-E rosetted preparation: block localised reaction in a rosette-forming lymphocyte, negative reaction in a non-rosetted lymphocyte.
444
K. E. HIGGY, G. F. BURNS & F. G. J. HAYHOE TABLE 3 Immunological marker studies on lymphocytes in 6 of the normal peripheral blood samples studied cytochemically, and in splenic lymphocytes f r o m a presumed normal spleen and a spleen from a patient with hereditary spherocytosis
Fc receptor
Number of
meankSD
1
range
AET-E rosettes meankSD
1
range
marker for ‘null cells’. B-cells appear to be negative. Cytochemically, peripheral blood lymphocytes showed three different patterns of reaction product for non-specific esterase enzyme activity. Correlating these results with
immunological marker studies on the lymphocyte populations studied, peripheral b l d lymphocytes appear t o be classifiable cytochemically into three groups:
Figure 5. T-cell enriched population: 5 cells showing 1 to 4 blocks of localised reaction: 1 cell with scattered granules.
Figure 6. B-cell enriched population: 7 negative cells, 3 with localised granules and 1 with scattereld granular reaction.
(1) A first group which had positive T-cell
445
ESTERASE CYTOCHEMISTRY OF LYMPHOCYTES TABLE 4 Acid phosphatase and P A S reactions in separuted normal lymphocytes
Enriched lymphocyte preparations
Number of samples
Acid phosphatase reaction (% of positive cells) mean ? SD
I
PAS reaction
(% of positive cells)
range
mean k SD
1
range
T-cell enriched preparations
7
41.8 k 4 . 5
37.2-50.6
34.9k 3.2
30.1-39.2
9-cell enriched preparations
7
51.54k 6.3
44.2-59.6
38.0+ 3.4
32.2-42.3
immunological markers contained a high number of cells showing dense, localised ‘dot-like’ esterase positivity. (2) A second group having B-cell immunological markers showed negative esterase reaction in the vast majority of the cells.
(3) A third population where the cells had neither T- nor B-cell immunological markers - ‘null cells’ - showed scattered granular positivity in most of the cells.
Figure 7. Null cell enriched preparation: 3 cells with fine scattered cytoplasmic granules and 1 with localised coarsely granular reaction.
Separated populations of uncontaminated T-cells, B-cells or null cells are not readily obtainable, but studies with lymphocyte populations enriched in one or other of these components provided strong confirmation of the specificity of the cytochemical findings. Thus the majority of lymphocytes in the B-cell enriched populations studied had negative esterase reactions, the minor component with localised or granular reactions being attributable to retained T or null cells. Similarly, the majority of lymphocytes in the T-cell enriched populations had localised positivity, the minor component with negative or granular reactions being attributable to retained B- or null-cells. Again, the null cell enriched populations showed a predominance of cells with scattered granular esterase positivity, with minor components of negative cells or cells with localised positivity, attributable t o retained B- or T-cells. Indeed since no cognizance is made of SmIg+ Fc- B-cells, such cells may account for the residual B-cells. No account was taken of the various minor subclasses of lymphocytes such as E+ Fc+ or of cells bearing various combinations of markers including surface immunoglobulin, FC and the fixed component of C3 (C3b). However, such cell types as SmIg+ Fc- C3b+ and SmIg+ Fc- C3- represent very small proportions of circulating cells (McConnell &
446
K. E. HIGGY, G. F. BURNS & F. G. J. HAYHOE
Hurd 1976) and their classification and nomenclature remains obscure. Since it has been suggested that mouse RBCs rosette with a sub-population of Blymphocytes (Stathopoulos & Elliot 1974, Forbes & Zalewski 1976) it was of interest to determine whether any difference could be observed cytochemically between the Mrosetting cells and other B-lymphocytes. Our findings confirm the B-nature of the Mrosetting cells in that the M-rosette preparation was predominantly esterase negative (Table 2), and we were unable to show any particular cytochemical marker on these cells. Normal peripheral blood monocytes showed a strong and extensive granular reaction with this esterase technique which allowed them to be differentiated very easily from lymphocytes and myeloid cells. The latter were almost negative in this reaction. Weak esterase activity in lymphocytes has been described before, with a-naphthyl acetate, a-naphthyl butyrate and naphthol AS and AS-D acetate as substrates (Wachstein & Wolf 1958, Braunstein 1959, Moloney et a1 1960, Rozenszajn et a1 1968, Li et a1 1973). However, the a-naphthyl butyrate esterase activity demonstrated with the technique described here appears to be more intense and more discriminating than that described in earlier reports. Mueller et a1 (1975), using a-naphthyl acetate as a substrate at pH 5.8 and with a prolonged incubation period of 21 h, reported a dotlike localised positivity in the cytoplasm of lymphocytes in the paracortical areas of lymph nodes in mice, presumed to be Tcells. Fixation in Baker’s formolcalcium was used. The method we have used gives a more clearcut positivity at pH 8 than at acid pH levels. Air-dried smears are used,
and give satisfactory esterase reactions even after storage for up to 4 weeks. Moreover, the short incubation period gives better discriminative results than does prolonged incubation. There have been previous attempts to differentiate cytochemically between T- and B-lymphocytes. Acid phosphatase is known to be strong in PHA activated blast cells and in lymphoid cells of infectious mononucleosis (Quaglino et a1 1962, Li et a1 1970, Biberfeld 1971). Again, the blast cells of T-cell leukaemias (T-ALL) show strong localised acid phosphatase activity more frequently than do the cells of non-T lymphoblastic leukaemias, while the PAS reaction tends to show an opposite pattern of positivity, being stronger in unmarked ALL than in T-ALL (Catovsky et a1 1974). The lysosomal enzymes N-acetyl-p-glucosaminidase and p-glucuronidase as well as acid phosphatase have been reported to be more prominent in T-cells and T-dependent parts of peripheral lymphoid organs than in B-cells in rodents and in man (Tamaoki & Essner 1969). However, neither acid phosphatase nor PAS reactions in normal peripheral blood lymphocytes show major differences between the T- and B-cells (Table 4). Farid et 21 (1975) using a-naphthy1 acetate as substrate, demonstrated a difference in the esterase activity between T- and B-cells, with 17.7 % k 8.6 positive cells in the sheep RBC rosette-forming lymphocytes (T-cells) and 11.7 % .t 4.3 positive cells in the B-rich fraction. Their method is clearly less discriminatory than the one we have used. The negative a-naphthyl butyrate esterase reaction in PHA-transformed lymphocytes contrasts with the strong acid phosphatase activity in such cells. Both esterase and acid phosphatase are lysosomal enzymes,
ESTERASE CYTOCHEMISTRY O F LYMPHOCYTES
447
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