cytology and electrophoretic pattern in γ1a (β2a) myeloma

16
Acta Medica Scandinavica. Vol. 170, fasc. 5, 1961 From the Department of Medicine (Head: Jan Waldenstrom, M. D.), University of Lund, Malmo General Hospital, Malmo, Sweden Cytology and Electrophoretic Pattern in ylA Myeloma BY F. PARASKEVAS,’ J. HERE MAN^ and J. WALDENSTROM Literature Ever since Dalrymple (1) in 1848 gave his microscopical report on the first case of multiple myeloma, also described by Bence-Jones (2) and MacIntyre (3), a vast literature has appeared on the offending cell. It took 54 years, however, after the “nucleated cells” forming the main part of the “gelatiniform mass of mollities ossium” were described, before the plasma cell, P.c., was connected with multiple myeloma (4). The credit for the first description of the plasma cell is given to Ramon y Cajal (5)/cited by Michels, (7)/. He called it “cianophil” because of the color of its cytoplasm, and Unna (6) first coined the name plasma cell for what is known today under this designation. The origin and function of plasma cells have been points of much controversy, and to a certain degree they still are. The present paper is not a discussion of the subject and the reader is referred to many excellent reviews : Michels (7), Bessis (8), Jordan (9), Sundberg (lo), Amano (1 I), Braunsteiner (12). Submitted for publication April 15, 1961. Theories proposed in the past involving a myeloblastic, lymphoblastic, erythro- blastic or megakaryocytic origin of plasma cells are no longer seriously considered. The first modern study of myeloma cytology was done by A. Wallgren (13), who demonstrated that all previous ideas about myeloblastic and similar myelomas had no real foundation. He demonstrated that we had to accept one myeloma cell that had the characteristics of a plasma cell. With the advent of sternal puncture we obtained much more material that could be studied. One of the present authors (14) was able to demonstrate that a certain group of patients with marked hyper-8-y-globulinemia had a peculiar clinical and biochemical as well as cytological picture. Together with Peder- sen he was able to prove that their pathological globulin had a high sedi- mentation constant of 19-20 S, cor- responding to a molecular weight of one l Present address: Dept. General Hospital, Canada. * Present address: Dept. of Louvain, Belgium. Pathology, Winnipeg, Medicine, University 575

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Acta Medica Scandinavica. Vol. 170, fasc. 5, 1961

From the Department of Medicine (Head: Jan Waldenstrom, M. D.), University of Lund, Malmo General Hospital, Malmo, Sweden

Cytology and Electrophoretic Pattern in y l A Myeloma

BY

F. PARASKEVAS,’ J. HERE MAN^ and J. WALDENSTROM

Literature

Ever since Dalrymple (1) in 1848 gave his microscopical report on the first case of multiple myeloma, also described by Bence-Jones (2) and MacIntyre (3) , a vast literature has appeared on the offending cell. I t took 54 years, however, after the “nucleated cells” forming the main part of the “gelatiniform mass of mollities ossium” were described, before the plasma cell, P.c., was connected with multiple myeloma (4).

The credit for the first description of the plasma cell is given to Ramon y Cajal (5)/cited by Michels, (7)/. He called i t “cianophil” because of the color of its cytoplasm, and Unna (6) first coined the name plasma cell for what is known today under this designation. The origin and function of plasma cells have been points of much controversy, and to a certain degree they still are. The present paper is not a discussion of the subject and the reader is referred to many excellent reviews : Michels ( 7 ) , Bessis ( 8 ) , Jordan (9), Sundberg ( lo) , Amano ( 1 I ) , Braunsteiner (12). Submitted for publication April 15, 1961.

Theories proposed in the past involving a myeloblastic, lymphoblastic, erythro- blastic or megakaryocytic origin of plasma cells are no longer seriously considered.

The first modern study of myeloma cytology was done by A. Wallgren (13), who demonstrated that all previous ideas about myeloblastic and similar myelomas had no real foundation. He demonstrated that we had to accept one myeloma cell that had the characteristics of a plasma cell. With the advent of sternal puncture we obtained much more material that could be studied. One of the present authors (14) was able to demonstrate that a certain group of patients with marked hyper-8-y-globulinemia had a peculiar clinical and biochemical as well as cytological picture. Together with Peder- sen he was able to prove that their pathological globulin had a high sedi- mentation constant of 19-20 S, cor- responding to a molecular weight of one

l Present address: Dept. General Hospital, Canada.

* Present address: Dept. of Louvain, Belgium.

Pathology, Winnipeg,

Medicine, University

575

576 F. PARASKEVAS, J. HEREMANS AND J. WALDENSTROM

million, and so a special disease could be delineated, that was called macroglo- bulinemia. It was shown that the bone marrow in this condition chiefly contained lymphocytoid cells and some plasma cells. Many authors have confirmed the original observation of Tischendorf and Hartman (15) that tissue mast cells are common. Lelbach (16) and later Dutcher and Fahey (17) demonstrated in the nuclei of pathological cells in macroglobulinemia inclusions that were PAS-positive. The latter authors showed by fluorescent antibody technic that these inclusions were macroglobulin (18). I t may be maintained that the separation of macro- globulinemia from the group of myeloma dysproteinemias has a cytological as well as a clinical and biochemical foundation.

There have also been many attempts to correlate cytological aspects with the biochemical changes and clinical course in multiple myeloma. Olhagen et al. (19) using an ultraviolet microspectro- photometric method showed that from the cytochemical point. of view there were two main types of myeloma cells. One (called type A) had a relatively small nuclear-cytoplasmic ratio, is similar to a plasma cell and has a highly differentiated cell organization. This cell was found in cases with increased p or y globulin. The other cell (called B-cell) showed a “myeloblastic” picture, large nuclear cyto- plasmic ratio similar to a malignant un- differentiated cell and was found in cases with normal electrophoretic serum protein patterns all of which had B. J. proteinuria. In their conclusions they speculate that possibly a more highly differentiated cell organization (A-type) produces the high- molecular weight proteins while a low- molecular weight protein (B. J. protein) may be found in the B-type of cell (less differentiated).

Wuhrmann et al. (20) concluded that 7-globulin myeloma is associated with mature plasma cells and a-globulin myeloma with immature, while the p-type occupies an intermediate position.

Streicher et al. (21) refer to a “reti- kulare” plasma cell with /?-myeloma and small plasma cells with y-myeloma. The total protein was below 9.0 g yo in the first group and above 0.5 to 17.0 g in the second. No exact data are given.

Chadbourn-Zinneman (22) found that the small cell type is associated with a #?-pattern and large plasma cells with the y-pattern. They also found that statisti- cally the cell diameter, N/c ratio, and relative area of cytoplasm are of signif- icance but not the nuclear diameter. They studied 27 cases. R. di Guglielmo (23) found no sure correlation using both normal cytological technics and electron microscopy, and van Dommelen (24) came to similar conclusions.

Later, Kubota et al. (25) distinguished five different groups of myeloma cells: I) the most primitive cell; 11) a round large cell with condensed chromatin; 111) a lymphocytoid small cell; IV) the classical myeloma cell ; V) degenerating large cells (27-30 p) with pinkish-blue cytoplasm. They could not find any correlation between the electrophoretic mobility and any of the cell types as grouped by them. Waldenstrom (21) could find no correlation between cell type and globulin type.

Harboe (26), referring to the work of Janssen and Gormsen, also claims that there was no correlation between cell type and electrophoretic mobility.

Le-Xuan-Chat (27) finds certain corre- lations and describes series I : a small “reticulolymphocytic cell in multiple myeloma with absence of hyperprotein- emia”. In series 11 #?-myeloma = im-

CYTOLOGY AND ELECTROPHORETIC PATTERN IN YIA (p2*) MYELOMA 577

mature plasma cells or plasmoblasts with central nucleus and series I11 : y-myeloma = abundant cytoplasm with eccentric nucleus. Like Chadbourn he found cyto- metric data to be of significance. His material comprises 11 cases.

Another series of 50 cases investigated by Hansen et al. (28) gave no correlation between serum protein and morphology of p. c., when studied by ordinary methods. They refer only to the size of the p. c. (large or small) and have classified their cases in 5 groups on the basis of electrophoretic mobility as: y , y x , P, Py and “normal protein myeloma” groups. B. J. proteinuria appeared in every group and was irregularly distri- buted within large- and small-cell myelo- matosis.

Mandema (29), taking as criterion the degree of anaplasia of the p. c., found that the cases with more anaplastic cell types had a shorter survival, more frequently had an a- or P-globulinemia and usually ran an acute course.

The divergence of opinion and the confusion of the results stem, we think, from the fact that so far, only the electro- phoretic mobility has been used as the central criterion for grouping myelomas. In our opinion, however, this property of the protein molecule is not the most imporiant one biologically. The recent work of Heremans in collaboration with C.-B. Laurel1 and Waldenstrom seems to have established in patients with hyper- gamma globulinemia the presence of three natural types, all belonging to the y system, namely immunological y ( 7 S) with low carbohydrate content, y macro- globulin (Y].,~; 19-20 s) and immuno- electrophoretic PZA ( Y j A j both with high carbohydrate. In these papers the clinical, biochemical and immunologic findings in nearly 300 patients with M type i. e.

narrow-banded hyperglobulinemia were discussed and the patients classified. The reader is referred to these papers. Numbers of patients are the same in all these publications.

With these facts in mind we have attempted to reinvestigate the cytological aspects of multiple myeloma, using as classification criteria the immunological properties of the respective proteins particularly as we see them in immuno- electrophoresis. Such immunological pro- perties of protein molecules might be expected to reveal significant biological differences referable to specific cellular processes of protein synthesis and secre- tion, and therefore also to certain cyto- logical characteristics.

Material The material comprises slides from 72

patients with a complete analysis of the serum proteins and a few slides from patients where immunoelectrophoresis could not be performed. The patients may be divided into the following groups. I. Increase in immuno- logically typical y-globulin. 30 bone marrow preparations from such cases could be examined. 11. Patients where the chief finding was a decrease in the y-fraction without development of any distinct band. 6 such patients were present in the material. No cytological differences could be found be- tween these 2 groups and we have therefore treated them together. This group I + I1 was compared with the third group: 111 marked increase in the yia-fraction. It must be remem- bered that among these 12-13 different sera, 3 showed a marked increase in a,-globulin on ordinary paper electrophoresis and 1 had a mobility corresponding to a yl--yz-myeloma. All the others represented either rapid or slow B-mobility and were mostly classified as 8 2 .

The two materials I + I1 and 111 are usually very well defined immunoelectrophoretically and a comparison of their cytological charac- teristics seemed worth-while. Seven of the bone marrow smears from patients with definite multiple myeloma have been kindly sent to us by various physicians or hospitals

578 F. PARASKEVAS, J. HEREMANS AND J. WALDENSTROM

as is gratefully acknowledged at the end of this paper.

Finally, we have examined the bone marrows from 12 cases of macroglobulinemia and from a number (1 1) of patients who have been variously diagnosed as having some other disease of the lymphatic or reticulum cell system. All of them had electrophoretic abnormalities of the M-type.

All bone marrows had a complete differen- tial count of 500 cells (except for some cases with markedly reduced cellularity, where a 200 or 250 cell count was made).

We have followed the terminology of the Committee for the classification of the nomenclature of the cells in disease of the blood and bloodforming organs 1949.

Methods I. Staining: Routine May-Griinwald-Giem-

sa staining with buffered solution a t p H 7.0.

11. Cytochemical methods: 1 ) The McManus PAS-staining method was

used as outlined in Lison’s manual (31). Our periodic acid solution was 0.5 9 , with a p H = 1.9. The time of action of Schiff’s reagent has varied from 30 to 45 minutes. We have used as counterstain methyl-green prepared in acetate buffer according to Lison (page 283) (pyronin of course omitted) ad- justed to p H 4.7 to give a better contrast between the nucleus and the purple-red color of the intranuclear bodies.

2) Toluidine blue for metachromasia as given by Lison.

3) Feulgen reaction as given by Lillie (32). 4) Methyl-Green-Pyronin according to Li-

son (31).

III. Cytometric studies’. Ten cases from the y-group more or less

equally distributed between y3-y~ and Y J were taken. The diagnosis of myeloma was confirmed either by autopsy or by usual diagnostic procedures. Ten cases of ~ J A myeloma were also examined. One of them (L. W.) although not confirmed as ~ J A by immunoelectrophoresis was included because he had a marked increase in the a, fraction,

The measurements have been performed with a Leitz. microcytometer.

We are grateful to Assoc. prof. GUNNAR BLOM Ph. D. for advice in the statistical treatment ofour data.

and we now know that such cases should be considered as ~ I A myelomas. Two cell dia- meters were measured (long and short) on 50 cells and the average was calculated as C.

Also two of the nuclear diameters were measured and the average taken as representa- tive of N. Nucleoli were measured when present.

The N/C was calculated and also the rela- tive cytoplasmic area by using the formula: Cz-N2 as given by Chadbourn-Zinneman (22) and Le-Xuan-Chat (27).

The following values were calculatedz : 1 ) Mean value for cell (= C) and nucleus

(= N), diameter and N/C and Cz-Nz for each patient.

2) Mean values of the above parameters for each group and also their standard devia- tion and standard error. These we call the “mean group values” for the cell (C), nucleus (N) and (N/C).

3) Probability was calculated only for the (C), (N) and (N/C), because, as we shall discuss later, the Cz-Nz value conveyed no more information than the (C) (N) and (N/C) had given. This was because the distribution of the values Cz-N2 turned out to be more assymetrical than C and N and hence the usual t test for comparison of the two groups could not be applied to this value.

Results We have classified our results in three

categories as they have been obtained by : I . Routine staining methods

11. Cytochemical methods 111. Cytometrical methods.

I. Routine staining mcthods A. T h e hypergammaglobulin group

30 cases with immunoelectrophoretic as well as chemical and ultracentrifugal exa- mination were included in this group (see table I ) . Plasma cell percentages

% of p. c. No. of pats. 0- 5 .................... 3 6-10 .................... 9

11-20 .................... 6 21-40 .................... 7 41- .................... 5

Total 30

CYTOLOGY AND ELECTROPHORETIC PATTERN IN YIA (b2A) MYELOMA 5 79

Table I . - Total Serum

No. Name Ycai Data on plasma cells in y myeloma Percentage

- Deg. p. c.

- Ho

-

57.0 32.7 29.1 42.4

7.3 14.8 25.4 33.0 46.7 29.9 12.5 70.2 16.7 91.7 25.0 32.0 12.4 54.0 44.1 40.7 66.7 50.0 37.6 29.7 30.0 70.7 25.3 64.9 63.6 50.8 51.1 68.1 60.0 71.4 26.7 0.9

57.7

29.a

-

=. c.

Y Pe Vacu. oles

Eccentr nucleus Yo

Poly- doid)

Lym- phocytes Total

Size. Large: Small: s

30.7 27.4 64.9 25.7 70.4 55.1 47.6 54.5 33.0 63.9 26.0 21.6 84.4 33.3 25.0

100.0 56.0 37.4 14.9 30.5 82.8 57.6

35.4 66.0

3.3 81.0 54.2 45.6 37.8 3.4 6.7

-

-

-

45.7 33.3 0.9

11.5

E. A.

A. A. K. J. H. M. s. 0. E. w. V. J. 0. H. s. s. E. H. A. 0. K. J. A. S. N A. J. A. s.

0. K. G. L. G. M. A. T. H. S. M. F. M. R. H. F. s. L. M. A. M. A. A. N.

G . M.

H. P. E. H. F. K. I . N. M. S.

1953 1954 1957 1956 1957 1956 1955 1953 I955 1957 1958 1955 1952 1954 1954 1958 1959 1954 1953 1956 1956 1956 1958 1957 1957 1950 I957 1957

1960 I958 1959 1960 1958 1958 1958 1959 1959

I 958

-

60.0 31.2 53.8 32.0 11,6 45.9 70.0 8.0 1.6

12.4 25.2 56.0 45.6

2.5 17.5 4.0 9.5

38.6 14.4 10.6 11.6 37.2

3.4 34.8 91.8 5.2

21.8 28.2 11.2 17.8 11.0 8.8

26.6 1.8 6.2 5.2

21.6 5.2

3.6 2.4 2.0 3.2

13.2 1.2 1 .o 2.6

2.6

2.4 1.2 0.5

2.0 3.0

16.4 1.8 1.2 0.2 0.5 0.4 0.2 5.8 0.6 0.6 0.2 0.2

0.8 0.2 0.8 0.2 0.4 0.6

-

-

-

-

- - -

63.8 33.6 57.0 35.8 24.8 51.1 71.0 11.0

I .8 15.0 25.4 59.2 47.2

3.0 18.0 6.0

12.5 56.2 17.4 11.8 12.8 38.4

3.6 35.2 47.8

6.0 23.2 28.4 11.4 17.8 11.8 9.0

27.6 2.0 7.2 6.0

21.6 5.2 -

16.0 67.8

3.5 2.2

13.7 2.4

12.7 52.7 55.5

5.3 7.1

16.9 22.3 33.3

58.3 16.0 8.2

17.2 28.8 23.4

8.3 11.1 9.1 8.8

13.3 23.3 47.2

5.3 5.5 8.5

11.1

10.0 42.8 20.0

7.7

-

-

-

-

5.2 12.4 3.4

12.4 9.4 6.4

12.5 11.0 9.8 5.2

11.8 8.0

12.5 31.0 27.0 18.0 17.0 8.6

12.2 10.6 34.2

9.0 13.6 14.0 11.2 10.4 5.4

13.0 11.6 21.4 18.2 15.0 9.6 8.4

23.6 15.6 24.2 10.6

I s I s I s I s 1 I s 1 1 1

I S

I s I s 1 1 1 1 1 1 1 1 1

-

S

S

I

1 I S

I s I

S

s S

I s

1 I s

S

s

s

49.5 62.6 36.8 55.9 93.5 6.1

95.9 85.4 44.0 42.7 55.9 58.8 58.2 66.7 52.8 91.7 72.0 78.3 55.2 83.0 48.4 30.7 77.8 31.2 33.5 26.7 71.5 33.1 64.9 71.1 66.1 77.8 55.8 60.0 77.1 80.0

2.8 65.4

1

1

47 1

I

1

12 13

117 226

I 2 5 6 8

9 14 20 64 65 94

100 126 141 163 195 196

225

247 276 288 290 33 1

Hypogamma globulinemia.

In patients with more than one slide the smear with the highest percentage was considered. In this same group were also included the 6 cases without any distinct

band but with definite hypogammaglo- bulinemia.

In many cases we thought that the classification according to different matu-

580 F. PARASKEVAS, J. HEREMANS AND J. WALDENSTROM

Legends

1 ) E. J. Two typical flaming plasma cells with the characteristics of thesaurocytes (pycnotic nucleus, large cytoplasm in compartments).

2 ) E. J. Cell right as in 1 ) . Two plasma cells different degrees of flaming. One lymphocyte.

3) E. J. Normal sized plasma cell with some degree of flaming.

4) A. F. Very big cell with peripheral flaming. 5) L. W. Normal plasma cell, some degree of

flaming. Big plasma cell, more marked flaming. 6 ) L. H. Big cell with flaming in the periphery

(thesaurocyte). 7) G. L. Normal plasma cell with some flam-

ing. Typical intranuclear inclusion bodies. 8) H. S. and H. E. Large inclusion bodies in

plasma cell nuclei. 9) A. N. and M. M. Cell with two inclusion

bodies and nucleolus between them above; lower, very large inclusion body.

10) M. M. PAS stained plasma cells. Lower: bright red intranuclear inclusion body, upper: PAS positive perinuclear cytoplasm.

1 1 ) A. N. Bright red big intranuclear i. b. (PAS stain)

12) A. N., M. M. and M. 0. Three cells with i. b. of the same type in one nucleus. One smear from macroglobulinemia case (M. 0.).

13) G. M. Case with clinical myeloma but macroglobulinemia. One cell PAS stained i. b. + neutrophilic leucocyte, another large i. b. Giemsa stain (for comparison).

14) J. L. L. and L. H. Big thrsaurocytes with pycnotic nucleus.

15) J. D. Typical large thesaurocytes with pycnotic nucleus.

16) J. D. Two typical th. c. with “compart- ments” in cytoplasm.

1

5

9

13

4

8

12

16

CYTOLOGY AND ELECTROPHORETIC PATTERN IN YIA MYELOMA 58 1

ration stages was not really possible, because of a marked nucleocytoplasmic asynchronism in which a very immature nucleus was found in cells which, ac- cording to other conventional criteria, had to be considered as mature. This nucleocytoplasmic asynchronism consti- tutes one of the few characteristic dif- ferences between m. m. and plasmocytosis due to other causes.

Shedding of cytoplasm was found to be a good criterion for abnormality of p. c. especially when marked, and is one of the main factors in contributing to the variability of the cell size. Frequently, shedding was noted to be more marked in lightly stained large p. c., but otherwise differences in color have not been found to have any significant correlation with other cytological characteristics.

Cytoplasmic inclusions The following have been seen : 1) Typical Russel bodies (eosinophilic

large masses) in 1 case (hypergamma (23) (in 1.7 yo of p. c.)

2) Globules of violet color in 2 (22 + H. M. hypogamma).

3) Coarse violet granules in 1 (5, hypergamma). 4) Eosinophilic granulation (rather

fine) in 2 (7, hyper + 15, hypo). 5) Crystals in 1 (418 S. N., hyper-

gamma). 6) In only one case (E. A. hypo) were

there found very large vacuoles, arranged in the periphery of the cell.

In two patients (196 A. N. + 25), very small, multiple vacuoles were found in the nucleus, completely unstained and some- times arranged in clusters at different levels appearing as bunches of grapes. Most of them were perfect circles, sharply defined and more or less of the same size.

Nucleoli: Such were present in almost all cases in varying percentages but only in a few were they outstanding because of their size.

Megaloblastoid changes. Changes in the nuclear structure of the erythroid elements similar to megaloblasts were found in different degrees: Minimal in 6 cases and marked in 1 (6) (an instance of combina- tion with pernicious anemia; for this interesting problem see S. 0. Larsson 32 a).

B. The Y1A group We had 12 cases, all confirmed as

p A by immunoelectrophoresis. One more case is included in the series although no immunoelectrophoresis was performed because this patient (10) had a specific increase of a,- globulin by paper electro- phoresis and this may now be considered immunoelectrophoretically as y I A . Patient E.I. is also discussed in this connection even though the exact nature of his B-globulin could not be determined im- munoelectrophoretically. His cytology is very characteristic.

Among the 21 different bone marrow smears from' these patients, two (both from patient A. F.) were diluted and therefore unreliable for counting. How- ever, even in this case we were able by extensive review of all available slides to observe a number of atypical plasma cells at the end of the smear and we will refer to this case later (fig. 4).

Plasma cell percentages One case (I. L.) had a percentage just

below 5 yo, but in this patient the p. c. were of such an abnormal nature (the- saurocytes up to 80 %) that no doubt existed about the diagnosis.

582

Table II . The qlology of y i A myeloma

F. PARASKEVAS, J. HEREMANS AND J. WALDENSTROM

9.5 9.2

19.0 6.2

19.0 12.8 4.4

16.8 54.0 38.4

64.6 6.6 8.2

13.2 49.4 23.5 14.0

0.5 1.6

2.5 2.2

0.4 1.8 0.4 1.4 2.8 1.0

0.8 1.0 1.6 2.0 2.0 -

0.6 1956 302 M. I. 1.0 I /

10.5 11.2

21.5 8.6

19.4 15.0 4.8

17.2 57.4 39.4

66.0 7.6

10.0 16.6 51.4 24.0 15.6

Percent- age

-t

+ + + f t i t '

-' t t I

i

.L

1958 1957

1957 1957

1956 1960 1958

1960

1960 1958

1957

1956 1959

To-l Cytological data on plasma cells

269 H. E. - 186 L. H. 0.2

186 L. H. - 186 I,. H. 0.4 421 I. L. L. - 53 G. L.

443 M. M. 0.6 289A. E. N. -

289A.E. N. 0.2 287 H. S. -

L. w. 0.2 E. J. 1.2

303 E. H. - 302 M. I. 0.5

tal - I Size

Small

-

+

-+

The following distribution was found :

0- 5 "" . . . . . . . . . . . . . . . . 1

11-20 0" . . . . . . . . . . . . . . . . 4 6-10 yr, . . . . . . . . . . . . . . . . 2

21-40 ",, . . . . . . . . . . . . . . . . 2 above 41 q,, . . . . . . . . . . . . . . . . 2

Total 11

In most cases the cells were often very big. Many cells had very abnormal shapes and from the structure of the cyto- plasm and other cytological characteristics gave an appearance of reticulum cells. This we think is much more common in this group than in the y-group.

Hof

- -

44.6

44.2 32.5

66.0 32.0 12.5 56.0 71.1 55.5

39.5 59.2 77.1 41.6 35.4 23.5

uoles

- -

16.1

-

7.0

2.1 6.7

29.2 7.7

12.2 9.0

13.1 2.0

1.5 6.2 6.9

-

cen- tr. -

14.3 82.1

13.9 62.8

71.1 86.7 54.2 60.4 47.7 54.5

94.7 67.3 67.5 74.7 31.2 40.7

Poly-

dY. 310-

-i- + + -c

t + + +

-

7-

7- -1 + -t

+

-1 -

Nu- cleol !L

9.5 58.9

11.6 20.9

16.5 36.0

7.7 37.6 75.5

-

36.8 14.0 27.7 37.8 6.2

13.8

LYm FhO. cyte

tal TO-

40.0 11.4

11.0 8.0

4.4 7.4

19.0 27.0

5.4 26.4

12.0 7.6

11.2 11.0 6.6 7.5 6.0

Other elements

1.3 abn. lymph.

0.5 abn. lymph.

0.2 baso 0.2 baso 0.2 bas0

(P. c.)

(P. c.)

1.4 abn. lymph.

1.4 abn. lymph.

(P. c.1

(P. c.)

13.4 abn. lymph. (P. c.)

Polyploidy was present in all cases except I . L. with a low p. c. count.

Nucleoli: Most of the cases (except one) had very large, rather outstanding nu- cleoli. Often they are very irregular and bluish in color. Large nucleoli may also be seen in cells from patients with y- myeloma, however.

Besides the above general cytological results we have been able to observe the following morphological properties to occur in the plasma cells of thisgroup but so far not in any other of the 30 examined cases of definite y-myeloma : 1. Intranu- clear inclusions. 2. Flaming cytoplasm. 3. Thesaurocytes.

CYTOLOGY AND ELECTROPHORETIC PATTERN IN YI* MYELOMA 583

1. Intranuclear inclusion bodies (i. b.)

(figs. 7-13)

This phenomenon was observed in 2 of our patients as the outstanding property of the cells (A. E. N. and M. M.) and in three more cases (in one combined with “flaming” cytoplasm) in a smaller number of cells. One of the two first named cases had in addition “thesaurocytes” in small percentages.

The percentage of plasma cells with i. b. was 2.5 in A.E.N. and 3.5 in M.M. The other three cases although of the same nature showed nuclei with i. b. in smaller numbers. From the picture presented in fig. 9 it is evident that this inclusion is different from the nucleolus and should not be confused with it.

Nucleoli, found in both y- and yla- myelomas, are sometimes very large usually of equal size, somewhat irregular or serrated at their perimetry, are found very frequently in many cells, and - most important - have a bluish tint (RNA). The i. b. are never so frequent and have a marked variability in size. We have been able to observe them from rather small to very large, occupying the greatest part of the nucleus with only a little ring of chromatin left. When the intranuclear body becomes very big we have seen destruction of the chromatin pattern of the nucleus. The chromatin accumulates in coarse, very loosely con- nected granules, with empty spaces around them. We think that this inclusion in the nucleus may interfere with its physiological function causing the death of the cell. The i. b. are usually round, sometimes look like perfect circles, but if they have irregular shapes their contour is more or less smooth and usually not serrated. The intranuclears sometimes have a slight eosinophilic hue in the

routine panoptic staining. They look smooth, and their surface as they appear in the microscope is in sharp contrast with the color of the chromatin. Some- times they do not stain (see figs. 9 and 13). Occasionally the intranuclears are mul- tiple and give a moth-eaten appearance to the nucleus.

At times they are numerous, arranged concentrically and thus give an onion- like appearance. In some cells the nuclear membrane is disrupted completely and the intranuclear material appears to be coming out (see fig. 9). Intranuclears have been observed in both small and large plasma cells, sometimes in only one nucleus of a binucleated cell, sometimes in both.

We have also found a typical i. b. and a nucleolus in the same nucleus (fig. 9 upper). In 3 of the 4 cases, where the intranuclears were the main pathological feature, some nuclei were markedly distorted in contour and presented mul- tiple constrictions (even clover-leaf shapes) with polar bodies hanging from them by little stalks.

2 . Flaming cytoplasm (figs. 1-7) This was present in 4 patients as the

outstanding property of the cell and in one more to a smaller degree. Among these 4 patients it was combined with intranuclear i. b. in one (G. L.) and with thesaurocytes in a second (L. H.). Flaming is usually a diffuse and very intense eosinophilic coloration to the point that obscures the normal blue color of the cytoplasm. We do not mean the slight eosinophilia present sometimes throughout in some cases of myeloma or even in a normal plasma cell, a color that has given the very characteristic violet-blue color to many plasma cells. The cell actually looks as if in “flames”

584 F. PARASKEVAS, J. HEREMANS AND J. WALDENSTROM

as Undritz has characteristically called them. Sometimes it occurs mixed with a slight blue color in the interior portions of the cytoplasm but the periphery is mostly very homogeneous and containing compact masses pure in color. The pictures show what we seek to describe.

3. lhesaurocyte (figs. 14-16) We have coined this name as more or

less descriptive and using by tradition the Greek language. This type of cell has been observed in two of the patients as the outstanding property. The percentage in the two cases is 26 in I. D. and 78 in I. L. L. In two others it was found to a smaller extent together with a second unusual property (“flaming” in one and intranuclears in the other).

By the term thesaurocyte we seek to describe a cell that has in all probability stored inside its cytoplasm large amounts of globulins. The thesaurismoses (storage maladies) where cells are also storing inside their limits some kind of material - usua!ly lipids - may be a parallel.

We have been able to observe pic- tures suggesting an accumulation of this material progressively from very small amounts to the formation of the large typical cell. In rather advanced stages the basophilia of the cytoplasm noticeably disappears, and the accumu- lated material appears in compartments separated by thin granular basophilic trabeculae. In later stages there are not even signs of compartments but one continuous, homogeneous picture from the coalescence of all the smaller compart- ments. Due to the accumulation of this material, the cell becomes very large and distorted, the nucleus is gradually pushed away and compressed and eventually appears pycnotic (fig. 15), while in the other cells of the same patient it is usually

very immature, with large nucleoli. Finally, we have seen such cells with disruption fo the cytoplasmic membrane and pouring of the stored material into the surroundings.

In one bone marrow puncture from one of our cases (L. H.) the accumulated material was strongly eosinophilic, i. e. flaming. In this patient the flaming property is the most characteristic while the flaming “thesaurocytes” occur only in small numbers. In patient I. J. it was only known that the pathological globulin was of ,!?-mobility. His cells are, however, very typical.

There are two more cases belonging to the YIA-group in which we could not find any of the above properties even in first rate preparations. In one of them (M. I.) there was a significant variability of lymphocytic elements with high per- centages of an abnormal lymphoid cell or a plasmocytic lymphocyte. The other patient (E. H.) has a reticular type of plasma cell with a very immature nucleus and an abnormally large nucleolus. We have not been able to find any structure that could satisfy us as being a typical intranuclear i. b., but frequently the chromatin structure has a very peculiar appearance with clear irregular spaces between the chromatin granules making them appear very loose. Somewhat similar changes in the nucleus have been observed in another case (H. E.), who however has typical large intranuclear i. b. also. In one case (A. F.) reliable bone marrow counts could not be performed because of a scarcity of cellular elements. We found a few cells with strongly eosino- philic “flaming” peripheral cytoplasm (fig. 4). Here we may refer once more to a case which has an outstandingly abnormal type of plasma cell. This is patient E. I. who has a p-globulin which

CYTOLOGY AND ELECTROPHORETIC PATTERN IN YIA (&) MYELOMA 585

has not been classified immunoelectro- phoretically. His cells are shown in picture 1-3. He has characteristic cells of unusual dimensions and moreover the stored material stains intensely red by Giemsa, i. e. the cells are flaming thesau- rocytes present in great numbers. In many cells very small perinuclear areas are not occupied by the red material and stain blue as normally. Cells that are not thesaurocytes have “flaming” cytoplasm and a very immature nucleus with a large nucleolus.

II. Cytochemical methods A. T h e y-group

We have performed periodic-acid- Schiff staining (PAS) in a number of myeloma patients with hypergamma- globulinemia and have never been able to find any PAS-positive material inside the nucleus or in the cytoplasm, either as granules or as other inclusions.

Unfortunately we had no unstained slides available to do any cytochemical work in the two patients with Russell bodies or in patients with the violet cytoplasmic globules. The crystalline intracytoplasmic inclusions in patient S. N. (24) are PAS-negative and have no metachromatic properties as revealed by toluidine blue nor do they stain with pyronin.

The coarse granules (red-violet) found in abundance in p. c. from E. B. ( 5 ) were also PAS-negative. They did not stain metachromatically with toluidine blueand also did not take up pyronin.

B- ylA-gr0up 1. Intranuclear i. b . We have been able

to investigate the cells from two patients (8, A. E. N. and 7, M. M.). Both have given the same results. The inclusions

stain positively by the periodic acid Schiff method with a varying intensity even after diastase treatment. This speaks against the i. b. containing glycogen and in favor of a glycoprotein with high carbo- hydrate content. They do not stain meta- chromatically with toluidine blue, which indicates that they do not contain any acid mucopolysaccharides nor do they take either methyl green or pyronin. Finally they give no Feulgen reaction.

We have also found that in these two patients a fairly intense red Schiff colora- tion occurs diffusely in the cytoplasm of the plasma cell. In one of them especially (7 , M. M.), this diffuse intensely PAS- positive material seems distributed rather in the usually large Hof area of the Giemsa stained smear (see fig. 10 upper).

The cytoplasmic area of the ordinary y- group plasma cell is not stained to any appreciable degree with the PAS-stain- ing method. We think that the y lA protein with its much higher carbohydrate content might give this staining property to both the intranuclear inclusion and the cytoplasm.

The cytoplasm of these two patients gave a very strong color with pyronin and toluidine blue in more or less peripheral areas of the cell.

There is only one other patient (No. 20 - G. M. in the y group), who has been included in this group because clinically she is a myeloma. She has however y lM protein i. e. macroglobulin in her blood, and in the last bone marrow smear (from the three we have) she has typical in- tranuclear i. b. They were first observed in the routine Giemsa stain and were later proven by periodic acid Shiff staining to be PAS-positive (fig. 13). They have the same staining properties as the i. b. of the ylM-group. We have observed with the PAS-staining method

586

Table III. Mean values for each patient

F. PARASKEVAS, J. HEREMANS AND J. WALDENSTROM

C . . . . . . . . N . . . . . . . . N / C . . . . n . . . . . . . .

yiA- group

17.50 0.66 9.58 & 0.30 0.55 -+ 0.018 2.87rt0.17

15.18 0.54 9.88 & 0.30 0.656 & 0.023 2.90 * 0.11

2.32 & 0.86 -0.30 & 0.43 -0.106 f 0.033

-

- N

- C

- C n Patient N N / C -

0.76 0.50 0.68 0.72 0.71 0.65 0.68 0.58 0.64 0.64

0.65

0.074 -

n - 3.5 2.5 3.1 2.8 3.3 2.5 2.7

2.9 2.8

2.9

-

0.33

Patient

1 2 3 4 5 6 7 8 9

10

Mean

Standar devia- tion

19.5 17.7 19.8 19.5 16.1 16.6 14.0 19.7 15.0 17.1

17.50

2.098

10.4 10.0 8.3 9.6 9.2

10.4 8.0

11.1 9.4 9.4

9.58

0.95

0.53 0.56 0.42 0.49 0.58 0.62 0.57 0.56 0.62 0.55

0.55

0.073

3.2 2.2 2.7 3.7 2.4 3.3 2.4 2.6

3.33

2.87

-

0.52

0.3 1 0.22 0.33 0.38 0.26 0.32 0.30 0.23

0.35

0.30

-

1 2 3 4 5 6 7 8 9

10

Mean

Standar devia- tion

14.6 19.2 14.4 13.6 15.1 15.1 15.3 13.0 16.6 14.9

15.18

1.72

11.0 9.5 9.8 9.7

10.7 9.9

10.4 7.6

1C.6 9.6

9.88

0.95

0.32 0.26 0.32 0.29 0.3 1 0.25 0.26

0.27 0.29

0.29

-

Table I E Mean values f o r the groupJ

P < 0.02 Not significant P < 0.01

some coarse globular granules of PAS material in the cytoplasm of her plasma cells. For a detailed description of this patient see Waldenstrom 1961 (35).

2. “Flaming” cells and thesaurocytes. Un- fortunately we had no unstained slides available with either one of these pro- perties from our patients for a cytochemi- cal test.

cytoplasmic ratio is much lower in the yIA-group and this is highly significant. The cytometrical studies confirm an ob- servation made by studying the smears that there is a greater variation in the ylA-group, shown by the greater standard deviation. Measurements of the nucleoli were also done in both groups. In 9 cases from each group all the nucleoli were measured in 50 plasma cells and the calculated mean found to be 2.81 (S. D. 0.52) for the ylA-group and 2.90 (S. D. 0.33) for the y-group. The n/N ratio for both the two groups was 0.30 and 0.29 respectively. Thus no difference could be found between the nucleoli in the two groups.

III. Cytometric methods Both groups. The results are shown in

tables I11 and IV. We found that the cell diameter is greater in the y I A group and the difference is almost significant. The mean of the nuclear diameters in each group appears to be the same. The nucleo-

CYTOLOGY AND ELECTROPHORETIC PATTERN IN ) Y ~ A (&A) MYELOMA 587

Results and discussion

The literature contains numerous at- tempts to classify patients with multiple myeloma according to biochemical and cytological criteria. We think that our review of the literature has proved that so far such classifications have been un- successful. Many facts are in favor of the assumption that the mere velocity of migration of a pathological plasma protein is not in itself a suitable characteristic and extensive data from electrophoretic studies indicate that myeloma proteins may migrate as az back to pa globulins. The recent developments in globulin chemistry, with methods that allow the determination of carbohydrate content and also separation with immunoelectro- phoresis, seem to have given more reliable criteria for differential diagnosis. In a very large material of sera it has been found that a group of usually rapidly migrating globulins has a comparatively high content of carbohydrates without being macroglobulins. On ultracentrifu- gation they were found to be 7 S com- ponents or of somewhat higher molecular weight up to 11 S. When such sera were subjected to immunoelectrophoresis by Heremans it was found that they contained an increased amount of y I A component. Heremans has therefore in- troduced the new myeloma group ylA, where the pathological globulin seems to be an entity separate from ordinary myeloma 1) globulin (Heremans 1959

Knowing that macroglobulins (19-20 S components) contain a high percentage of carbohydrates, several authors who have found PAS positive inclusions in the nuclei of bone marrow cells in this disease have asked themselves if there is not a biological connection between these 38-613003. Acta Mcd. Scand. Vol. 170.

(36)).

two phenomena. (Lelbach, (16) Dutcher and Fahey, (17, 18) Curtain (18 a). I t seems tempting to assume that the PAS-positive intranuclear material might correspond to that found in the macro- globulin molecule. We now know that y I A globulin has a high carbohydrate content, and it was therefore logical to ask if myeloma cells connected with the production of this type of globulin might also contain intranuclear inclusions. Several bone marrow slides from patients with this type of myeloma had numerous inclusion bodies, whereas such bodies could not be found in the cells from patients with immunoelectrophoretic y (7 S) myeloma. It was also noticed that these inclusions were strongly PAS- positive both in an interesting transitional case between myeloma and macroglo- bu1inem.a that has been published else- where by one of the authors (Walden- strom 1961) and in two cases with typical yjA myeloma. From the practical point of view it seemed to be quite important that these intranuclear inclusion bodies could be well defined in ordinary Giemsa stain smears without the use of the Schiff reagent. I t is possible that such inclusions are connnected with the synthe- sis of globulins especially rich in carbo- hydrates, but only further work could confirm or refute this theory.

Such inclusions may occur in numerous plasma cells, but they are usually quite rare and they are then of little diagnostic importance. On the other hand some other features may help in the recogni- tion of y j A myeloma. The nature of so-called flaming plasma cells has never been established, but their occurrence may be quite remarkable and they are then recognised at the first look into the microscope. Flaming plasma cells were found in high percentage in several of

588 F. PARASKEVAS, J. HEREMANS AND J. WALDENSTROM

our yIA patients, and if present this is a most striking characteristic that could not be overlooked. Evidently the explana- tion of this phenomenon must be sought in the chemical or physico-chemical prop- erties of the pathological globulin. Recent investigations regarding the eosin- ophilia of so-called Russell bodies seem to indicate that such bodies are stained with eosin at a different pH. Two ques- tions therefore arise. Could it be that the whole preparation has been stained in a buffer of a lower pH, when the plasma cells came out flaming? This idea is refuted by the fact that the other marrow cells show normal coloration. At least for several slides we know that the ordinary buffer has been used for preparing the dye. When the plasma cells are flaming this is usually characteristic of all the large plasma cells, but there may at the same time be found smaller plasma cells with a strongly basophilic cytoplasm. This proves a) that flaming can not be an artefact caused by changes in the pH of the staining solution, b) that the majority of the usually very large cells are flaming. Sometimes it is clearly seen that they contain an eosinophilic as well as a slightly basophilic region in their over- abundant cytoplasm.

This leads to the next cellular change found in i / lA myeloma, i. e. the presence of cells with a small pycnotic nucleus and a large cytoplasm containing compartments. We have chosen the name thesaurocytes for these cells as they resemble some of the cells found in the bone marrow in the thesaurismoses. I t is of course a mere assumption that these cells contain pathological globulins, but the proof may be near at hand with the aid of fluorescent antibodies against y I A globulin.

Summary

The authors have investigated the cytological picture of the bone marrow from 12 patients presenting typical mul- tiple myeloma with increase in the y l A (formerly so-called p z A ) fraction of serum globulins. It was found that one or several characteristic cytological features occurred in many of these patients, i. e. “flaming” of plasma cells as well as in- tranuclear Schiff-positive inclusion bodies and thesaurocytes, i. e. big cells with a peculiar structure oftheir cytoplasm and a small pycnotic nucleus. A large number of bone marrow preparations from 30 pa- tients with typical y myeloma and from 6 patients with low 1’ globulin content did not seem to show these changes.

Acknowledgements We wish to thank several colleagues for putting

sera and bone marrow smears a t our disposal. Spe- cial thanks are due to drs Elsa Segerdahl, Bollnas and Torsten Flemberg, Kristinehamn. Nurse Signe Troell deserves special gratitude for expert techni- cal help.

References 1. DALRYMPLE, I.: Dublin Quart. J. Med. Sci.

2. JONES, H. B. : Phil. Trans. B. I: 55, 1848. 3. MACINTYRE, W.: Med.-chir. Trans. 33: 211,

1850 (quoted from Snapper). 4. WRIGHT, I. H.: Trans. Ass. Amer. Physcns.

15: 137, 1900. 5. RAMON Y CAJAL (cited by Michels, 7) : Manual

de anatomia pathologica general, ed. 1. Barcelona 1894.

6. UNNA, P.: Abh. prakt. Derm. 12: 296, 1891. 7. MICHELS, N.: Arch. Path. 11: 775, 1931. 8. BESSIS, M. & SCEBAT, L.: Rev. HCmat. I:

9. JORDAN, H.: Anat. Rec. 119: 325, 1954.

2: 85, 1848.

447, 1946.

10. SUNDBERG, D.: Ann. N. Y. Acad. Sci. 59:

11. AMANO, SH.: Sang 28: 753, 1957. 671, 1955.

CYTOLOGY AND ELECTROPHORETIC PATTERN IN ylA (p2A) MYELOMA 589 12. BRAUNSTEINER, H. : Physiologie und Physio-

pathologie der weissen Blutzellen. Georg Thieme Verlag, Stuttgart 1959, p. 286.

13. WALLGREN, A. : Untersuchungen uber die Myelomkrankheit. Upsala Lak.-Foren. Fork 25: 403, 1920.

14. WALDENSTROM, J.: Acta med. scand. 117: 2 16, 1944.

15. TISCHENDORF, W. & HARTMAN, R.: Acta Haemat. 4: 372, 1950.

16. LELBACH, W. K.: Frankfurt. Z. Path. G8: 440, 1957.

17. DUTCHER, T. F. & FAHEY, I. : J. Nat. Cancer Inst. 22: 887, 1959.

18. DUTCHER, T. F. & FAHEY, I.: Proc. SOC. exp. Biol. (N. Y . ) 103: 452, 1960.

18 a. CURTAIN, C. C.: Aust. Ann. Med. vol. 8: 143, 1959.

19. OLHAGEN, B., THORELL, B. & WISING, P.: Scand. J. Clin. Lab. Invest. I: 49, 1949.

20. WUHRMANN, F., WUNDERLY, C. & HUGEN- TOBLER, F.: Dtsch. med. Wschr. 16: 481, 1949.

2 1. WALDENSTROM, J. : Snapper and Dock. Adv. int. Med. 5: 398, 1952.

22. CHADBOURN, W. & ZINNEMAN, H. H.: Blood 10: 1109, 1955.

23. DI GUGLIELMO, R.: I Plasmocitomi. Rom 1955. 24. VAN DOMMELEN, C. K. V.: Multipel Myeloom.

25. KUBOTA, C., SCHWARTZ, S. 0. & PUTNAM,

26. HARBOE, N. M. G.: Acta Haemat. 20: 27,

27. LE-XUAN-CHAT: Sang 28: 620, 1957. 28. HANSEN, H. A., ISAKSSON, B. & PAULSEN, L.:

Proc. 6th Congr. Eur. SOC. Haem. Copen- hagen 1957, p. 26.

29. MANDEMA, E.: Proc. 6th Congr. Eur. SOC. Haem. Copenhagen 1957, p. 27.

30. A series of papers by Heremans, Laurel1 and Waldenstrom in Acta Med. Scand. Suppl. 367, 1961.

31. LISON, L.: Histochimie et cytochimie animale. Gauthier-Villards, Ed., Paris 1953.

32. LILLIE, R. F.: Histopathologic technics and practical histochemistry. Blakinston Co. 1954.

Thesis. Amsterdam 1955.

F. W.: Acta Haemat. 16: 105, 1956.

1958.

32 a. LARSSON, S. 0.: Unpublished.

33. UNDRITZ, E. : Hamotologische Tafeln. Sandoz, Basel, p. 63.

34. THI~RY, I. P.: Ciba Found Symp.: Cellular aspects of immunity. J. & A. Churchill Ltd, London 1960, p. 59.

35. WALDENSTROM, J.: Tocantins. Progress in hematology 196 1 (in press).

36. HEREMANS, J.: Les globulines skriques du systeme gamma. Arscia, Bruxelles 1960.

Addendum. Table V.

Number Name

32 J. B.

101 J. D. 269 H. E.

83 A. F.

186 L. H.

303 E. H. 312 M. J.

53 G. L. I. L. L.

M. M.

289 A. E. N.

E. J.

L. w.

Electroph. per cent

5.6 P 2 - Y l

8.6 ))

6.2 a,

4.7 y,-,

4.2 j't-,Yl

6.6 iYz-Y1 5.9 8 2 2.4 jY,

5.8 /J',

0.8 ,Y2

5.3 j( l2

8

1.0 a,

Cytology

Tendency towards

Thesaurocytes I. b. Nuclear con-

tractions. Unreliable smear.

Some flaming cells? Flaming + thesauro-

cytes. Nothing typical

Thesaurocytes Flaming (i- i. b.) I. b. (3.5 94 of p. c.)

(thesaui ocytes?) I. b. (2.5 % of p. c.) +

+ thesaurocytes. Flaming + th. c. (no

immunoelec tro- phor.)

Flaming + th c. (no immunoelectro- phor.)

flaming.

)> >>

Addendum to the proofi. In three further cases we have been able to make the diagnosis YIA mye- loma already from the smears and have had it confirmed later. One further case of YIA myeloma showed an uncharacteristic cytologic picture.