Tohoku J. exp. Med., 1969, 97, 1-9

Histochemical Study on Plasma Cells

Atsuo Suzuki, Akira Shibata, Seiju Onodera , Akira B. Miura, Shinobu Sakamoto and Chiyuki Suzuki

Department of Internal Medicine (Prof. T. Torikai), Tohoku University School of Medicine, Sendai

Histochemical observations of plasma cells and lymphoid cells were carried out

on the bone marrow smears from 39 patients with non-hematological disorders,

polyclonal gammopathies of various causes, multiple myeloma, and macroglobulin-

emia Waldenstrom. The techniques applied were : May-Giemsa, peroxidase, PAS, .

sudan black B, acid phosphatase, alkaline phosphatase, ƒÀ-glucuronidase, non-

specific esterase, LDH, SDH and GDH stainings.

Plasma cells showed intense staining reactions to acid phosphatase, fl-glu-

curonidase and non-specific esterase, but lacked peroxidase, sudan black B and

alkaline phosphatase reactions. LDH, SDH and GDH were positive. PAS reac-

tion was diffusely and weakly positive or negative.

Plasma cells of multiple myeloma disclosed more intense acid phosphatase

activity than those of polyclonal gammopathies. There was no difference in

the acid phosphatase activity of plasma cells among yG, yA, yD and Bence-Jones

type myeloma. The relationship between this enzyme activity of plasma cells

and serum paraprotein concentration could not be clarified.

The lymphoid cells of macroglobulinemia Waldenstrom were negative for

peroxidase, sudan black B, alkaline phosphatase, and ASD-chloroacetate ester-

ase. A considerable variability was found in PAS reaction: from intense

granular positive to negative reaction. Except for a case of moderately intense

acid phosphatase and f3-glucuronidase activities, no difference in histochemical

reactions was found from normal lymphocytes.

A considerable amount of new information has accumulated in recent years

concerning the formation, structure and function of immunoglobulins. There are

growing knowledge and understanding of physiological and chemical nature of

paraproteinemia,1,2 but morphological and particularly histochemical characteristics of plasma cells or the cells proliferating in the bone marrow in paraproteinemia

are not yet satisfactorily clarified.3-7

Histochemical demonstration of acid phosphatase, non-specific esterase, ATP-

ase activities of plasma cells attracted attention of some workers because of their

usefulness in the differential diagnosis of multiple myeloma from polyclonal

gammopathies of various causes.3,4 Few reports are concerned with morphological and histochemical differences among the plasma cells appearing in yG myeloma,

yA myeloma, and yD myeloma.6,7 Very little is known about the histochemical characteristics of lymphoid cells

Received for publication, September 9, 1968. 1

2 A. Suzuki et al.

appearing in macroglobulinemia Waldenstriim except for their behavior to PAS

reaction.8,9

The aim of this report is to describe the results of histochemical studies at the

light microscopic level and to define more precisely the chemical characteristics of

the plasma cell series in normal condition, polyclonal gammopathies of various

causes and multiple myeloma, and lymphoid cells in macroglobulinemia Walden-

strom.

MATERIALS AND METHODS

Bone marrow blood was taken from 11 patients with no hematological diseases, 8

patients with polyclonal gammopathies of various causes, 16 patients with multiple myeloma

and 4 patients with macroglobulinemia Waldenstrom. Twenty-five of the patients were

male and others female. Their ages ranged from 14 to 77 years. The smears were pre-

pared from the bone marrow aspirate and submitted to staining process.

The techniques used were: May-Giemsa staining, peroxidase reaction of Mc. Jurtkin,

PAS reaction,10 sudan black B reaction,11 azo dye methods for acid phosphatase,12 alkaline

phosphatase,13 ƒÀ-glucuronidase,14 and non-specific esterase,15,16 and stainings for succinic,17

lactic and glutamic dehydrogenases" using Nitro BT as hydrogen-receptor.

Intensity of neutrophil alkaline phosphatase activities was expressed according to

Tomonaga's scoring method,13 and acid phosphatase activity of plasma cells or lymphoid

TABLE 1. Histochemistry of plasma celld

Histochemical Study on Plasma Cells 3

Fig. 1 1) Acid phosphatase activity in plasma cells. The enzyme activity shows

tendency to gather in Golgi area. 2) Acid phosphatase activity in plasma cell.

The enzyme activity is also demonstrated in vacuoles. 3) ƒÀ—glucuronidase act-

ivity in plasma cell. 4) LDH activity in plasma cells. 5) Non-specific esterase

in plasma cell. 6) Plasma cell with granular PAS positive reaction seen in ƒÁA

myeloma. 7) Lymphoid cell with granular PAS positivity in macroglobulinemia

Waldenstrom. 8) Acid phosphatase activity in lymphoid cell of macroglobulin-

emia Waldenstrom.

cells according to Rozenszajn's method." The immunoglobulin concentration was determined by the antibody—agar plate method"

in some cases.

RESULTS

Table 1 shows the histochemical nature of plasma cells.

Peroxidase was constantly negative in the cells, whether normal or malignant.

As a general rule plasma cells showed faint diffuse or negative PAS reaction, but

4 A. Suzuki et al.

the reaction was variable from case to case. Some of the plasma cells appearing in the bone marrow of yA myeloma, however, disclosed positive reaction in the form of granular staining (Fig. 1). Sudan black B was negative in plasma cells, whether normal or pathological.

It was found that plasma cells showed intense histochemically demonstrable acid phosphatase activity as red granules of various sizes. In some plasma cells, the granules tended to gather in the chromophobic zone. More detailed descrip-tion concerning this enzyme will be given later. Alkaline phosphatase activity was

demonstrated neither in nucleus nor in cytoplasm of plasma cells. The activity of fl-glucuronidase was shown as diffuse red tinge, or red granules

of various sizes and the protoplasm was almost entirely occupied by the granules in some cells. The tendency to accumulation of positive granules in the chromo-

phobic zone was also found in some plasma cells. After incubation in a medium containing naphthol-AS acetate as the substrate,"

non-specific esterase activity was elicited as brown needle-shaped or rod-like

granules in the cytoplasm of plasma cells. It seemed that occasionally they had tendency to gather in the Golgi area. Incubation in the medium using naphthol-ASD chloroacetate as substrate16 did not bring about non-specific esterase staining reaction in the plasma cells, contrary to intense staining reaction in neutrophils.

By LDH staining strong activities of LDH were demonstrated all over the

protoplasm as blue granules of various sizes or diffuse staining. SDH and GDH activities were shown as several blue granules scattered in

the protoplasm of plasma cells. The histochemical findings of lymphoid cells appearing in the bone marrow

of macroglobulinemia Waldenstrom were considerably variable from case to case as shown in Table 2.

They lacked peroxidase, sudan black B reaction, alkaline phosphatase, and non-specific esterase activity using naphthol-ASD chloroacetate as substrate. Variable staining behaviors of lymphoid cells were seen when PAS technique was applied to bone marrow smears. In the first case almost all of cells showed negative PAS reaction, but in the second case the cells with granular positivity

TABLE 2. Histochemistry of lymphoid cells in macroglobulinemia Waldenstrom

Histochemical Study on Plasma Cells 5

Fig. 2. Neutrophillalkaline phosphatase activity of monoclonal and polyclonal gam - mopathies. Bone marrow blood. Myeloma o Macroglobulinemia

Fig. 3. Acid phosphatase activity of plasma cells.

were seen as shown in Fig. 1. In the first case moderately intense acid phos-

phatase and ƒÀ-glucuronidase activities were demonstrated as red granules distributed

in cytoplasm.

As indicated in Fig. 2, alkaline phosphatase scores of bone marrow neutrophils

6 A. Suzuki et al.

Fig. 4. Acid phosphatase activity of myeloma cells and lymphoid cells in macroglobulinemia Waldenstrom.

expressed according to Tomonaga's method were considerably variable in poly-clonal gammopathies. On the other hand, it may be worthy of notice that the scores were high in monoclonal gammopathies almost without exception.

Acid phosphatase activity of plasma cells was very intense. Only reticulo-endothelial cells and megakaryocytes could be compared with plasma cells on this point. The general mode of distribution of positive granules was already described. In a case of multiple myeloma with myeloma cells having many vacuoles, acid phosphatase activity was demonstrated in the vacuoles. When the intensity of acid phosphatase activity of plasma cells was expressed accord-ing to Rozenszajn's method, it became evident, as shown in Fig. 3, that myeloma cells had stronger enzyme activity than plasma cells appearing in polyclonal

gammopathies or other hematological disorder. In order to evaluate acid phosphatase scores as a means for morphological

differentiation of plasma cells in association with different immunoglobulin types, the scores were plotted against each type. It is clearly seen in Fig. 4 that acid

phosphatase scores of plasma cells are not related to immunoglobulin type. Acid phosphatase activity of lymphoid cells of macroglobulinemia Waldenstrom was apparently weaker than plasma cells of multiple myeloma as shown simultaneously in Fig. 4.

Histochemical Study on Plasma Cells7

Fig. 5. The relationship between serum paraprotein concentration and acid phosphatase activity of plasma cells (yG myeloma).

In 7 cases of y-G myeloma, in which serum paraprotein concentration was meas- ured, the acid phosphatase activity of myeloma cells did not exhibit a clear cor- relation to serum para-protein concentration (Fig. 5).

DISCUSSION

The result of histochemical investigations showed that plasma cells displayed

intense activities of some hydrolases, namely acid phosphatase, ƒÀ-glucuronidase,

non-specific esterase, which were mentioned as lysosomal enzymes. 21 Moreover, it

became clear that they were generally distributed diffusely throughout cytoplasm,

though they had tendency to gather in the Golgi area occasionally. This finding

raises an interesting question concerning the localization of these enzymes in

organellas of plasma cells. By the electron microscopic observation the number

of lysosomes of: plasma cells are not so numerous as expected from optical histo-

chemical findings. One should presume extra-lysosomal localization of these

lysosomal enzymes in plasma cells. The present authors reported elsewhere on

electron microscopic histochemical observation of acid phosphatase activity of

plasma cells. The enzyme activities were demonstrated as deposition of lead

salt attaching to the rough surfaced endoplasmic reticulum in addition to lysosomes

and Golgi apparatus.22 The significance of this finding remained obscure. How-

ever, some authors stated that production of lysosomal enzyme was carried out at

the ribosomes attaching to endoplasmic reticulum.21

There have been few reports about clinical usefulness of histochemical tech-

niques to blood smears except for alkaline phosphatase and PAS reaction. The

value of histochemical demonstration of the acid phosphatase activity in plasma

cells for the differential diagnosis of multiple myeloma from secondary plasma-

8.A. Suzuki et al.

cytosis was advocated by Liffler and Schubert.3 The plasma cells of multiple

myeloma showed more intense enzyme activity in comparison with the cells of

secondary plasmacytosis. The authors' results may support their opinion.

Although precise appreciation of physiological meaning of the finding is not possible,

it is probable that physiological and chemical differences between neoplastic and

reactive cells produce such different histochemical findings.

In one case of Bence-Jones type myeloma, the cells appearing in the bone

marrow showed figures resembling lymphocytic cells rather than plasma cells.

However, their acid phosphatase activity was very intense, and the behavior was

compatible with their plasma cell nature. And it was ascertaind that these cells

were really plasma cells, because the electron microscopy revealed evident lamellar

development of rough surfaced en.doplasmic reticulum. This fact seems to

indicate that the histochemical demonstration of acid phosphatase activity on

bone marrow smears is a simple and valuable method in establishing the diagnosis

of multiple myeloma in cases where myeloma cells have atypical appearance.

The enzyme activity of the lymphoid cells in macroglobulinemia Walden.striim

was apparently weaker than that of the plasma cells in multiple myeloma as shown

in Fig. 4.

Although no conclusive result was obtained probably because of paucity of

cases examined, Fig. 5 seems to show that no correlation exists between acid

phosphatase activity of myeloma cells and serum paraprotein concentration.

It became clear that histochemical characteristics of lymphoid cell of macro-

globulinemia Waldenstrom were considerably variable according to cases. It

seems that these findings correspond to the result of observation on May-Giemsa

preparations that the morphological figures of lymphoid cells of this malady are

considerably variable.

It is said that positive PAS staining reaction is characteristic of lymphoid cells

of macroglobulinemia Waldenstrom.8,9,25 In a case (Case 2) many cells exhibited

a positive reaction in a granular pattern, but in another case (Case 1), the cells

positive for PAS scarcely appeared. It seems questionable if PAS reaction is

valuable for the diagnosis of macroglobulinemia Waldenstrom.

Except for high acid phosphatase and ƒÀ-glucuronidase activity in one case (Case

1), no different finding from normal lymphocytes was obtained by other

histochemical techniques. It does not seem that these histochemical techniques

will offer valuable clues for the differential diagnosis of macroglobulinemia

Waldenstrom from other lymphoproliferative disorders.

Acknowledgment

Grateful acknowledgment is due to Prof. T. Torikai for his guidance and review of

the manuscript, and to Dr. S. Takase for his suggestions in histochemistry.

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