Download Cytochemical Findings in Human Nonneoplastic Blood and Tonsillar

Cytochemical Findings in Human Nonneoplastic Blood and
Tonsillar B and T Lymphocytes
GERASSIMOS A. PANGALIS, M.D., STEPHEN R. WALDMAN, PH.D., AND HENRY RAPPAPORT, M.D.
Pangalis, Gerassimos A., Waldman, Stephen R., and Rappaport, Henry: Cytochemical findings in human nonneoplastic
blood and tonsillar B and T lymphocytes. Am J Clin Pathol 69:
314-318, 1978. The cytochemical profiles of B and T lymphocytes from the bloods of eight normal donors and the tonsils
of three normal individuals were studied. An intense and localized a-naphthyl acetate esterase (a-NAE) activity was found in
the majority of blood and tonsillar T lymphocytes, in contrast
to the very low a-NAE activity observed in the blood and
tonsillar B lymphocytes. A very low percentage of tonsillar B
lymphocytes had /3-glucuronidase (/3G) activity, while relatively normal /3G activity was observed in the tonsillar T
lymphocytes and the blood B and T lymphocytes. Acid phosphatase (AcP) activities were found to be similar in both B
and T lymphocytes from blood and tonsils.
These findings suggest that the a-NAE reaction may be useful
as a cytochemical marker for distinguishing B from T lymphocytic proliferations. They also revealed that there is no appreciable difference in AcP and /3G activity between B and T lymphocytes obtained from the blood of normal donors. (Key words:
Blood; Tonsillar B and T lymphocytes; Cytochemistry; aNaphthyl acetate esterase; /^-Glucuronidase; Acid phosphatase).
ACID PHOSPHATASE POS1TIVITY, particularly
when localized in the Golgi zone, has been considered
characteristic of T-lymphoblastic proliferations, 6 ' 719 - 20
and T-cell chronic prolymphocytic leukemia. 5 Increased /3-glucuronidase activity has been reported to
be consistently present in T-cell chronic lymphocytic
leukemia (CLL) and in Sezary's cells, 4,10 and decreased
or absent in the cells of lymph-node imprints from diffuse non-Hodgkin's lymphomas. 16 A negative periodic
acid-Schiff (PAS) reaction has been reported to occur
in T-cell chronic prolymphocytic leukemia, in contrast
to the B-cell CLL, in which the PAS reaction is usually
positive. 5 Whether these cytochemical differences reflect characteristics of neoplastic lymphoid cells independent of their B and T derviation or are properties inherent to the B or T nature of these cells is not known. It
Department of Anatomic Pathology, City of Hope
National Medical Center, Duarte, California
was for this reason that we decided to investigate
whether differences in the cytochemical profiles of normal B and T lymphocytes do in fact exist. For this purpose we prepared smears rich in either B or T lymphocytes from blood and tonsils obtained from normal
donors, on which a number of cytochemical tests were
performed.
Materials and Methods
B and T Lymphocyte
Separation
Tonsils were obtained from three individuals who
had tonsillectomy because of recurrent tonsillitis. Immediately after removal, the tonsils were finely minced
in RPMI-1640 medium. The minced tissue and supernatant were then filtered through a stainless steel mesh,
and the cells were resuspended in RPMI-1640 medium.
Blood leukocytes were obtained from eight normal
donors, using the Aminco "celltrifuge" continuousflow blood cell separator,* as described by Waldman
and associates. 23 Tonsillar and blood mononuclear cells
were then separated using the Ficoll-Hypaque technic.
The interphase layer containing lymphocytes and
monocytes was collected, washed, and resuspended in
RPMI-1640 medium. Monocytes were eliminated by
placing the cell suspensions in glass petri dishes
and allowed to stand for 2 h in a 5% C 0 2 atmosphere
at 37 C. Subsequently, lymphocytes were removed,
recovery was determined, and cell number was adjusted to 1 x 107 /ml for the rosetting procedure.
For the separation of the B and T lymphocyte population, the spontaneous sheep erythrocyte (SRBC)
rosette formation technic (E rosettes) described by
Abdou and colleagues 1 was employed. E rosettes were
formed by incubating 2-ml volumes of 1 x 107 cells,
2 ml 1% SRBC, and 1 ml human AB serum (preadsorbed with SRBC) in a 15-ml plastic tube for 15 min at
37 C. The tubes were subsequently centrifuged at 250
x g* for 10 min and allowed to stand at 4 C for 60 min.
The cell pellets were then resuspended carefully and a 4-ml
Received July 28, 1977; received revised manuscript October 24,
1977; accepted for publication October 24, 1977.
Supported by Grant Number R 10 CA 18044-01 awarded by the
National Cancer Institute, DHEW and by Hematopathology
Tutorials, Inc.
Dr. Pangalis was in receipt of a fellowship from the State Scholarships Foundation of Greece.
Address reprint requests to Dr. Rappaport, Department of
Anatomic Pathology, City of Hope National Medical Center,
Duarte, California 91010.
* American Instrument Co., Silver Springs, Maryland.
0002-9173-78-0300—0314 $00.75 © American Society of Clinical Pathologists
314
vol. 69 . No. 3
315
CYTOCHEMISTRY OF B & T LYMPHOCYTES
Table I. Surface Marker Studies of B-rich and T-rich Lymphocyte Populations
B-lymphoc yte Rich
A. Normal blood
Donor 1
Donor 2
Donor 3
Donor 4
Donor 5
Donor 6
Donor 7
Donor 8
MEAN ±
SE
B. Tonsilar
Donor 1
Donor 2
Donor 3
E-Rosettes
(% of Positive Cells)
Immunofluorescence
(% of Positive Cells)
E-Rosettes
(% of Positive Cells)
Immunofluorescence
(% of Positive Cells)
10
7
4.5
3
10
7
13
10
8.1 ± 1.2
80
85
81
85
90
87
86
85
84.9 ± 1.1
94.5
94
90
97
94
96
89
91
93.2 ± 1.0
8
9
1
0
3
1
4
2
6.0 ± 2.5
9
10
3
85
86
90
94
97
95
7
10
4
volume of Ficoll-Hypaque precooled to 4 C, was underlayed. Following centrifugation at 700 x g* for 35 min
at 10 C, the cells of the interface layer and the pellet
were collected separately. The cells of the pellet were
incubated in 0.84% NH..CI for 5 min at 37 C to eliminate
the SRBC. Both interphase layer and pellet lymphocytes were subsequently washed twice and resuspendedin RPMI-1640 medium. Viability exceeded 90%
in all cases as determined by trypan blue stain. Determinations of the composition of lymphocyte populations in the interphase layer and the pellet were performed by enumerating E rosettes 18 and detecting surface immunoglobulins by the immunofluorescence
technic described by Pernis and co-workers, 17 using a
polyvalent rabbit antihuman immunoglobulin labeled
with fluorescein isothiocyanate.t
Cytochemical
T-lymphocyte Rich
Study
Smears of the B and T lymphocyte-rich populations
from the tonsils and the blood were made on glass slides
and the following cytochemical determinations were
performed: Acid phosphatase (AcP)2 and acid phosphatase with tartaric acid, 12 /3-glucuronidase (J3G),11
esterase with a-naphthyl acetate as substrate (a-NAE), 26
esterase with naphthol AS-D acetate as substrate
(NASDA), NASDA with sodium fluoride (NASDA
+ NaF),°and periodic acid-Schiff (PAS) reaction. The
end products of AcP, /3G, and NASDA reactions were
semiquantitatively estimated according to the extent of
reaction positivity in each of at least 100 consecutively
graded lymphocytes.3-21-27 a-NAE positivity was
graded zero (0) when the reaction was entirely negative;
1+ when a focal positive material was present; 2 +
t Cappel Laboratories, Downingtown, Pennsylvania.
when 2 - 8 granules were present; 3+ when more than
eight positive granules were found. One hundred lymphocytes were consecutively graded according to
a-NAE reaction positivity.
Results were expressed as percentages of positive
cells and as scores of positivity per 100 lymphocytes.
Statistical analysis of our findings was performed using
the t test.
Results
Surface
Markers
The results of surface marker studies of B lymphocyte-rich and T lymphocyte-rich populations of the
blood and the tonsillar specimens are enumerated in
Table \,A, B. Purities of B lymphocytes of the B cellrich preparations ranged from 80 to 90%, while purities
of T lymphocytes of the T cell-rich preparations
ranged from 89 to 97%. The B cell-rich lymphocyte
populations contained small percentages (3-13%) of
T lymphocytes, presumably a few lymphocytes that
were classified as B because they had bound IgG on
their surfaces through a complement receptor, 25 and a
small number of "null" lymphocytes that were separated with the B cell population due to their inability to
form E rosettes with SRBC.
Cytochemical Findings in Blood B and T
Lymphocyte-rich
Populations
The end product of the cytochemical reactions was
in the form of granules, varying in number from cell
to cell. The percentages of cells containing AcP and
j8G, the overall AcP and /3G activities expressed as
scores, and the patterns of positivity (scattered gran-
316
PANGALIS, WALDMAN, AND RAPPAPORT
A.J.C.P. . March 1978
Table 2. Results of Cytochemical Studies of Normal Blood B-rich and T-rich Lymphocyte Populations
B Lymphocyte-•rich Population
%* of Positive
Cells
Acid phosphatase
/3-Glucuronidase
a-Naphthyl acetate esterase
Naphthol AS-D acetate esterase
45
49
30
98
±
±
±
±
17
23
15
2
T Lymphocyte- rich Population
Score* per 100
Cells
70
137
31
168
±
±
±
±
P Values of B
vs. T lymphocyte-rich
Populations
%* of Positive
Cells
33
76
15
10
61
72
71
99
Score* per 100
Cells
± 17
± 18
± 6
± 2
100
138
74
171
±
±
±
±
47
59
10
5
%
Score
.068
.049
.0001
.3
.14
.95
.0001
.4
* Mean * SE.
a-NAE and NASDA activities with and without NaF
inhibition in the tonsillar B and T lymphocyte-rich
populations were found to be similar to those observed
in the blood lymphocytes.
In both blood and tonsillar lymphocytes, the PAS
reaction was very difficult to interpret because the
end product of the reaction was in a weak diffuse form
rather than granular. This is probably due to the rapid
change in the cytoplasmic glycogen content that occurs
during incubation. 5,24
ules) were similar in the two cell populations (Table 2).
Acid phosphatase activity was entirely inhibited in
both B and T lymphocyte-rich populations after addition of tartaric acid.
In all specimens rich in B lymphocytes, a-NAE
activity was found to be present in only a small percentage of cells (Fig. 1). In contrast, in all specimens
rich in T lymphocytes, a-NAE positivity was found
to be present in the majority of the cells; the end
product of the reaction was very intense and was localized in a small paranuclear area (Fig. 2). Statistical
analysis of our findings in the blood lymphocytes
showed that the difference in the a-NAE reactions
between B and T lymphocyte-rich populations was
highly significant (P = .0001) (Table 2). NASDA positivities were similar in the two lymphocyte populations,
and the reaction was not influenced by sodium fluoride.
Discussion
One of the most striking findings of our study was the
difference in a-NAE activities between the B and T
lymphocytes. Alpha-naphthyl acetate esterase activity
was found to be consistently present in an intensely
localized form in blood and tonsillar T lymphocytes,
in contrast to a low activities in blood and tonsillar B
lymphocytes. Similar differences have been observed
by Mueller and associates 13 in experimental animals.
In an excellent study in which they combined immunologic, cytochemical and histochemical methods, these
investigators found very low a-NAE activity in mouse
B-lymphoid cells. They postulated that a-NAE might
be used as a simple marker for differentiating B and T
lymphocytes. The consistency, frequency and pattern
of positivity with which the a-NAE reaction was observed in the T human lymphocytes indicates that
further study of this reaction should be carried out in
the lymphoid malignancies in conjunction with im-
Cytochemical Findings in Tonsillar B and T.
Lymphocyte-rich
Populations
AcP activities of the tonsillar B and T lymphocyterich populations were similar to those of the blood B
and T lymphocyte-rich populations (Tables 2 and 3);
the enzymatic activity was entirely inhibited by
tartaric acid. /3-Glucuronidase activity of the tonsillar
B lymphocyte-rich population was found to be present
in a very small percentage of the cells, in contrast to the
high percentages of positive cells in the tonsillar T
lymphocyte-rich and the blood B and T lymphocyterich populations (Tables 2 and 3, and Figs. 3 and 4).
Table 3. Results of Cytochemical Studies of Tonsillar B-rich and T-rich Lymphocyte Populations*
T Lymphocyte-rich Population
B Lymphocyte-rich Population
/3G
AcP
a-NAE
/3G
AcP
NASDA
Donor
%
Score
%
Score
%
Score
%
Score
%
1
2
3
53
65
60
86
100
96
7
8
5
10
16
9
21
24
30
21
24
30
96
98
94
154
175
168
58
69
60
• Score
98
86
101
a-NAE
NASDA
%
Score
%
Score
%
Score
53
67
42
108
116
78
59
64
60
59
64
60
98
95
97
172
164
180
' AcP = acid phosphatase; /3G =0-glucuronidase; a-NAE = a-naphthyl acetate esterase; NASDA = naphthol AS-D acetate esterase.
317
CYTOCHEMISTRY OF B & T LYMPHOCYTES
Vol. 69 • No. 3
•
•
w
FIG. I (upper, left). B lymphocytes from blood of a normal donor, showing a negative a-naphthyl acetate esterase reaction in most cells.
An a-naphthyl acetate esterase-positive cell is present (arrow), x 1,200.
FIG. 2. (upper, right). T lymphocytes from blood of the same normal donor, showing a-naphthyl acetate esterase
positivity in an intense localized form in all cells, x 1,200.
FIG. 3 (lower, left). B lymphocytes from tonsil of a normal donor, showing a negative /3-glucuronidase reaction in most cells.
An occasional /3-glucuronidase-positive lymphocyte was observed, x 1,200.
FIG. 4 (lower, right). T lymphocyte from tonsil of the same normal donor, showing a positive /3-glucuronidase reaction in most cells.
This reaction is intense, with many well-defined granules. Cells with various degrees of positivity are present, x 1,200.
munologic markers in an attempt to investigate the
possible value of this reaction in distinguishing B from
T lymphocytic proliferations.
Catovsky and colleagues 5 were the first to demonstrate a strong localized AcP positivity in cells of
lymphocytic malignancies of T cell type. Since his
initial report, Catovsky 6 and other investigators 19,20 ' 24
have obtained similar results. According to these ob-
servers, AcP may be used as a simple and reliable
marker for identifying T-lymphocytic proliferations.
(Acid phosphatase positivity in an intensely localized
form has been observed in a few cases of acute myeloblasts leukemia by Catovsky 8 and one of the authors, 14
an observation that does not invalidate the concept of
AcP specificity for T cells in an otherwise proven neoplastic lymphoproliferative disease.)
318
PANGALIS, WALDMAN, AND RAPPAPORT
However, in our study we failed to find any meaningful differences in either patterns or degrees of AcP
positivity between B and T lymphocytes obtained
from blood and tonsils of normal donors. Similar
findings in blood samples have recently been reported
by Wehinger and Mobius24 and confirmed in our laboratory by biochemical methods.15 In 1969, Tamaoki
and Essner,22 in a study of AcP and j8G reactions on
lymph-node frozen sections obtained from man and
experimental animals, observed no enzymatic activity
in the follicular and mantle lymphocytes, while AcP
and BG activities were present in the paracortical
lymphocytes. These findings were subsequently interpreted as evidence of a cytochemical difference between B (follicle and mantle) and T (paracortical)
lymphocytes. Our results are consistent with Tamaoki
and Essner's observations22 with respect to the BG
findings, but not with respect to the AcP observations.
The very low BG activity of tonsillar B lymphocytes
in comparison with tonsillar T lymphocytes and blood
B and T lymphocytes seems to indicate a difference between the B and T lymphocyte populations of the
tonsils. This difference is probably not related to the
B orT nature of the cells, but may be related to the state
of the cell cycle of the follicular tonsillar B lymphocytes. The observation of a low BG activity in tonsillar B lymphocytes is of interest in view of the
finding of a very low BG activity in the cells of
lymph node imprints from diffuse non-Hodgkin's
lymphomas."5
In summary, our findings revealed: 1. Very intense
and localized a-NAE activity in the majority of the
blood and tonsillar T lymphocytes, in contrast to very
low a-NAE activity in the blood and tonsillar B lymphocytes; 2. Very low BG activity in tonsillar B lymphocytes, in contrast to a relatively normal BG activity in
tonsillar T lymphocytes. The significance of these findings and their possible practical application to diagnostic
hematology should be further investigated by studying
malignant lymphocytic proliferations by both cytochemical and immunologic methods.
Acknowledgments.
Mrs. Aldana Martin and Mr. Michael D.
Lockwood provided skillful technical assistance.
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