Lymphoid Follicles in Bone Marrow Aspirates KOICHI MAEDA, M.D., BONG H. HYUN, M.D., AND JOHN W. REBUCK, M.D., P H . D .

Department of Pathology, Muhlenberg Hospital and Department of Pathology, Henry Ford Hospital, Detroit, Michigan

patient. Lymphoid follicles are generally thought to be physiologic, 1,5 ' 16,2 ° or of unknown clinical significance, 6 but some have regarded them as pathologic, 6,7,10 particularly when they are excessive in number. 1,6,11 This report presents an analysis of the lymphoid follicles observed in 1,450 consecutive bone marrow aspirates. It is demonstrated that lymphoid follicles are closely related to age and sex of the patient, are found twice as frequently as has previously been reported, and are associated with reactive changes of the bone marrow, such as plasmacytosis and lipid granulomas.

LYMPHOID FOLLICLES in the bone marrow are well recognized anatomic structures. They are usually present in red marrow and are frequently associated with vascular channels. They consist mainly of mature lymphocytes and rarely contain germinal centers. Although frequently round or oval, their shape is variable, and they range in size from about 80 to 500 /am. Conflicting opinions have been expressed regarding their incidence and clinical significance. The reported incidences of lymphoid follicles in the bone marrow vary greatly, depending upon the types of specimens and the extents to which the specimens are subjected to examination. As expected, lymphoid follicles are found in autopsy specimens in higher frequencies (26.1—62.3%)5,19,20 than in bone marrow aspirates (3.3-9.1%). 9 - 10 - 14 This is probably due to the fact that bone marrow samples available from autopsies are more abundant than those obtained by aspiration. The incidence is also related to age and sex of the

Materials and Methods Sections of 1,450 consecutive bone marrow aspirates examined over a 6-year period from 1967 to 1972 in the Department of Pathology of the Muhlenberg Hospital were reviewed for lymphoid follicles. The marrows are aspirated from the sternum or posterior iliac crest, or the tibia in the case of infants. Smears were prepared in the usual fashion from several drops of the marrow aspirated into the first syringe and were stained with Giemsa and Gomori's iron stains. Approximately 3 ml of aspirate obtained with the second syringe were fixed in Zenker's solution or B-5 fixative and then filtered through coarse filter paper. The units were collected with the back edge of a scalpel blade. The specimen was wrapped in lens paper, placed in a cassette, and washed in running water in the case of Zenker's solution or in 75% alcohol in the case of B-5 fixative. It was then processed in the Autotechnicon. After paraffin embedding, sections were cut at 6 /u-m and routinely stained with hematoxylin and eosin, Giemsa, and Gomori's iron technics.

Received December 22, 1975; received revised manuscript March 3, 1976; accepted for publication March 3, 1976. Presented in part for the New Jersey Society of Pathology; received the Casilli Memorial Award on May 14, 1973. Address reprint requests to Dr. Maeda, Department of Pathology, Henry Ford Hospital, Detroit, Michigan 48202.

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Maeda, Koichi, Hyun, Bong H., and Rebuck, John W.: Lymphoid follicles in bone marrow aspirates. Am J Clin Pathol 67: 41-48, 1977. Lymphoid follicles were observed in 260 of 1,450 consecutive bone marrow aspirates (17.9%). As expected, the incidence of lymphoid follicles was less than those reported from autopsies (26.1-62.3%), but was twice as great as those in previous reports based upon study of aspirations (3.3-9.1%). The number of lymphoid follicles is also related to age and sex of the patient: they are rare in childhood and common after the fourth decade of life, particularly in women. Lymphoid follicles are found in higher incidence in bone marrow aspirates with plasmacytosis and/or lipid granulomas than in those without these reactive changes. This relationship has not been described previously and suggests that the frequent occurrence of lymphoid follicles in the older age group may be a manifestation of a response to chronic immunologic stimulation. (Key words: Lymphoid follicles, Bone marrow aspirates.)

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FIG. 1 (upper). Five scattered lymphoid follicles indicated by arrows. Hematoxylin and eosin. x60. FIG. 2 (lower). A lymphoid follicle consisting of mature lymphocytes. Hematoxylin and eosin. x390.

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FIG. 5 (upper). Mature and immature lymphocytes in the germinal center shown in Fig. 4. Hematoxylin and eosin. x 1,430. FIG. 6 (lower). Lipid granuloma adjacent to a lymphoid follicle. Hematoxylin and eosin. x390.

LYMPHOID FOLLICLES IN MARROW

Vol. 67 • No. I

Results

Table I. Incidence of Lymphoid Follicles in Bone Marrow Aspirates in Relation to Age and Sex Male Age (years) 0-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81Unknown Total

Female

Total

No.

%

No.

%

No.

%

2/73 1/44 2/41 6/44 5/81 18/117 20/117 22/115 8/34

2.7 2.3 4.7 13.6 6.2 15.4 17.1 19.1 23.5

0/59 3/40 2/55 9/77 19/119 34/128 31/125 53/107 24/59

0 7.5 3.6 11.7 16.0 26.6 24.8 49.5 40.7

2/132 4/84 4/96 15/121 24/200 52/245 51/242 75/222 32/93 1/15

1.5 4.8 4.2 12.4 12.0 21.2 21.1 33.8 34.4 6.7

84/666

12.6

175/769

22.8

260/1450

17.9

Table 2. Underlying Disease in Cases with Lymphoid Follicles in Bone Marrow Aspirates Iron-deficiency anemia Megaloblastic anemia Malignancy (non-hematopoietic) Chronic hepatic disease Diabetes mellitus Hypertensive and/or arteriosclerotic cardiovascular disease Leukemia and lymphoma* Multiple myeloma Nonspecific inflammation Chronic renal disease Arthritis, rheumatoid and/or gouty Fractures, traumatic Myeloproliferative disorder Idiopathic and drug-induced thrombocytopenic purpura Viral infection Congestive heart failure Cerebral vascular accident Chronic pulmonary disease Pneumonia Hemoglobinopathy Anemia, unclassified Marrow aplasia Peptic ulcer Gastrointestinal bleeding Agranulocytosis Fever of unknown etiology Pancreatitis Others

37 31 22 18 18 14 13 8 8 7 6 5 5 4 4 4 4 4 3 3 3 2 2 2 2 ?

2 27

* Excluding lymphocytic lymphoma involving bone marrow and chronic lymphocytic leukemia.

of plasmacytes in the marrow is less than 2%) was studied in relation to lymphoid follicles (Table 3). Of the 260 aspirates, 86 (34.2%) showed plasmacytosis, averaging 3.5%. Examination of a control series of 260 consecutive bone marrow aspirates without lymphoid follicles revealed plasmacytosis in 66 cases (25.4%). The average age of control series is somewhat younger. Details regarding this point are described in the discussion. There was no significant difference between the group with lymphoid follicles containing germinal centers (37.5%) and those without germinal centers (34.2%). Table 4 summarizes the incidence of plasmacytosis in relation to the number of lymphoid follicles. There appears to be a greater incidence of plasmacytosis in the aspirates with large numbers of lymphoid follicles. The incidence of lipid granulomas in the aspirates showing lymphoid follicles is also of interest (Table 5). Thirty-seven (14.2%) of 260 aspirates with lymphoid follicles were found to have lipid granulomas, compared with the overall incidence of lipid granulomas of approximately 4% in the bone marrow aspirates in our laboratory (Fig. 6). Eosinophilia was not quantitated in this study, but appeared to be a frequent accompaniment of lymphoid follicles, although this was neither a consistent nor a prominent finding (Fig. 7).

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Two hundred sixty (17.9%) of the 1,450 consecutive bone marrow aspirates showed lymphoid follicles, including 184 aspirates from the sternum and 67 from the posterior iliac crest. In nine cases the site of aspiration was not stated. The numbers of lymphoid follicles in individual aspirates to ranged from one to 32, with an average of 3.3, and sizes ranged from 50 to 1,000 /xm (Figs. 1 to 3). The lymphoid follicles were frequently associated with small vessels and were occasionally found near venous sinuses. Germinal centers were seen in only 16 cases, four in iron-deficiency anemia, two in multiple myeloma, one each in megaloblastic anemia, thalassemia major, acquired thrombocytopenia, and myeloproliferative disorder, and six in other miscellaneous conditions (Figs. 4 and 5). The distribution of lymphoid follicles among age and sex groups is summarized in Table 1. They were found more frequently in elderly people, with the highest incidence (45%) in women more than 70 years of age. In children less than 10 years of age lymphoid follicles were extremely rare, and were found in only two of 132 aspirates (1.5%). The incidence of lymphoid follicles was analyzed according to the underlying primary diseases (Table 2). It was obvious that many patients had multiple diseases, and the analysis was based upon what was thought to be the most important clinical diagnosis in each case. Thirty-seven patients had iron-deficiency anemia; 31, megaloblastic anemia; 22, nonhematologic malignancies (seven with metastasis); 18, chronic hepatic diseases; 18, diabetes mellitus; 14, hypertensive and/or arteriosclerotic cardiovascular disease; 13, leukemias and lymphomas other than the welldifferentiated lymphocytic type. Patients known to have lymphocytic lymphoma involving the bone marrow and chronic lymphocytic leukemia were excluded from this series. The incidence of plasmacytosis (our normal range

45

MAEDA, HYUN AND REBUCK

46

Table 3. Plasmacytosis in Relation to Lymphoid Follicles in Bone Marrow Aspirates Cases of Plasmacytosis*

Group I. Control, without lymphoid follicles Group II. Lymphoid follicles

No. Aspirates

No.

%

260

66

25.4

260

89

34.2

^ Cases with plasma-cell dyscrasies excluded from (his analysis.

were from children, the youngest of whom had tuberculous meningitis, and the other seven were from adult patients of various ages. Our 16 aspirates with germinal centers were all from adult patients, ranging in age from 29 to 82 years. This age difference in the incidences of germinal centers in the two series may be explained in part on the basis of differences in the incidences of lymphoid follicles resulting from the types of specimens examined. Askanazy1 stated that lymphoid follicles in excessive numbers in combination with many germinal centers may be a pathologic phenomenon. The presence of lymphoid follicles is associated with a variety of conditions. According to Williams,20 lymphoid follicles occur in many diseases and are frequently found in cases where no infection or significant inflammatory lesion is demonstrable. They are also present frequently in the marrow in cases of sudden death from traumatic injuries. Kabelitz10 believed that lymphoid follicles in the marrow were a compensatory reaction occurring in diseases of the lymph nodes and severe disorders of hematopoiesis. Hashimoto and colleagues6 summarized their work as follows: 1. Lymphoid follicles appear as a compensatory reaction when the lymph nodes and other lymphoid apparatus of the body are severely damaged or atrophied. 2. They appear in such diseases as aregenerative anemia and agranulocytosis. 3. They appear for unknown reasons in some cases. Their earlier work in experimental animals showed that there were no lymphoid follicles in normal marrows, but some were present in abnormal marrows.4 As described in our series, various diseases are associated with the presence of lymphoid follicles. Our observations indicate a high incidence in megaloTable 4. Number of Lymphoid Follicles in Bone Marrow Aspirates and Incidence of Plasmacytosis No. Lymphoid Follicles in Aspirate

No. Cases with Lymphoid Follicles

No. Cases with Lymphoid Follicles and Plasmac:ytosis

% with Plasmacytosis

1 2 3 4 5

97 60 39 15 16

28 24 14 5 4

28.9 40.0 35.9 33.3 25.0

6 7 8 9 10

6 5 3 2 5

3 2 2 0 2

50.0 40.0 66.7 00.0 40.0

11-15

4

2

50.0

16-20

4

0

00.0

21

4

3

75.0

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Since Dominici3 first described focal collection of lymphocytes in the bone marrow in 1902, there have been a number of reports concerning lymphoid follicles found at autopsy5'6,19,20 or in aspirated specimens9,10,14 (Table 6). The frequency of occurrence of lymphoid follicles in bone marrow has been said to be closely related to age. Generally, it is considered to be low in childhood,1,5,19,20 although Miale reported a high incidence.12 It is high in older age groups.1,5,19,2° The present series indicates an extremely low incidence of 1.5% (two in 132 aspirates) in children less than 10 years of age, compared with the overall incidence of 17.9% and with the highest incidence of 34.4% in those more than 81 years of age. One of the two children in our series with lymphoid follicles was a 3-year-old boy who had fever of unknown origin, and the other was a 4-year-old boy who had acute lymphocytic leukemia. The latter case was included in the present series because the lymphoid follicles were distinct from surrounding lymphoblasts. Hashimoto and associates reported some sex difference, with a slightly higher incidence in females. Our study, however, shows a significant sex difference, as lymphoid follicles were found in 12.6% of the aspirates from male patients, compared with 22.9% from female patients. These figures were accounted for by the difference found in those aspirates from patients more than 41 years of age. In this age group lymphoid follicles were found in 73 of 464 aspirates (16.2%) from men, compared with 161 of 538 (31.5%) from women. The higher incidence in females might be correlated with the fact that females, in general, have better immune responses than males. No sex difference in the incidence of lymphoid follicles was observed in those less than 40 years of age, aspirates from 11 of 202 males (5.4%) and 14 of 231 females (6.1%) revealing lymphoid follicles. Some have reported a close relationship between lymphoid follicles and vascular channels,6,18,21 and we have observed the same relationship. Germinal centers are rarely seen. Hashimoto and colleagues,5 in their series of 532 autopsy specimens, observed lymphoid follicles in 139, of which only 13 contained germinal centers. Six of these specimens

A.J.C.P. • January 1977

Vol. 67 • No. 1

LYMPHOID FOLLICLES IN MARROW

47

blastic anemia. This association was previously reported by Ziegler,22 who observed a large number of lymphoid follicles in pernicious anemia. Dameshek and Valentine2 found a striking increase in the numbers of mature lymphocytes in bone marrows of pernicious anemia, with marked reductions of these cells after appropriate therapy. We observed a high incidence of plasmacytosis in the aspirates with lymphoid follicles compared with those without them. Lymphocytes and plasma cells often are found together in lymphatic tissue, in bone marrow smears and in the connective tissues, but to our knowledge no specific study has been done previously to suggest a relationship between lymphoid follicles and plasmacytosis. Plasmacytosis is known to be related to an immunologic response. From our study we realize that plasmacytosis may also be related to age. The importance of the age factor is suggested by our data regarding the correlation between lymphoid follicles and plasmacytosis. An increased incidence of plasmacytosis with lymphoid follicles may be significant in the understanding of the nature of lymphoid follicles, that is, their greater incidence in older individuals may be a manifestation of a response to certain states of immunologic stimulation. Lipid granulomas are known occasionally to be

present near lymphoid follicles.816 Our study indicates a high incidence of lipid granulomas in the bone marrow aspirates with lymphoid follicles, a phenomenon that has not been reported thus far. Eosinophilia is occasionally encountered around lymphoid follicles. Rywlin and co-workersls reported a remarkable case of an eosinophilic fibrohistiocytic lesion associated with lymphoid follicles. Rebuck and colleagues13 observed a transformation from lymphocyte to macrophage in the exudate in their skinwindow studies. These findings also suggest that lymphoid follicles may be reactive, as is the case with lymphocytosis, plasmacytosis, eosinophilia, and histiocytosis. Nodular aggregates of lymphocytes in the bone Table 5. Lipid Granulomas in Relation to Lymphoid Follicles

At Muhlenberg Hospital (consecutive aspirates) At Muhlenberg Hospital (with lymphoid follicles) Reported by Rywlin et al.1"

No. Aspirates

Incidence of Lipid Granulomas (%)

1.000

4.0

260 400

14.2 9.0

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FIG. 7. Aggregate of eosinophils (arrow) with a small lymphoid follicle. Hematoxylin and eosin. x390.

48

MAEDA, HYUN AND REBUCK

A.J.C.P. . January 1977

Table 6. Reported Incidences of Lymphoid Follicles in Bone Marrow

Reference

Sites Examined

Total Number of Cases

Cases with Lymphoid Follicles

Incidence of Lymphoid Follicles (%)

20

3.3

A. Data from aspirations Rohr, 194014

Sternum 10

600

Kabelitz, 1950

Sternum

150

12

8.0

Johnstone, 19549

Sternum

230

21

9.1

Author, 1975

Sternum, posterior iliac crest

1,450

260

17.9

Humerus, vertebra, rib

61

38

62.3

Williams, 1939

Femur, humerus, tibia, sternum, vertebra

230

67

29.1

Hashimoto et al., 19575

Femur, humerus, ilium, rib, sternum, vertebra

532

139

26.1

B. Data from autopsies Von Fischer, 191719 20

References 1. Askanazy M: Uber die Lymphfollikel im menachlichen Knochenmark. Virchows Arch Pathol Anat 220:257-275, 1915 2. Dameshek W, Valentine EH: The sternal marrow in pernicious anemia. Arch Pathol 23:159-189, 1937 3. Dominici H: In Manuel d'Histologie Pathologique, by Comil. Ranvier L. Paris, 2:581, 1902 4. Hashimoto M, Hamaguchi H, Iwamoto A, et al: Lymph nodules appearing in pathologic rabbit bone marrow. J Kyushu Hematol Soc 5:128-131, 1955

5. Hashimoto M, Higuchi M, Saito T: Lymph nodules in human bone marrow. Acta Pathol Jap 7:33-52, 1957 6. Hashimoto H, Hashimoto N: The occurrence of lymph nodules in human bone marrow with particular reference to their number. Kyushu J Med Sci 14:343-354, 1963 7. Hedinger E: Uber die Kombination von Morbus Addisonii mit Status Lymphaticus: Frankfurt Z Pathol 1:527-543, 1907 8. Hyun BH, Park YH, Ashton JK, et al: Granulomas in bone marrow aspirates. Exhibit in Atlanta, American Society of Clinical Pathologists, Annual Meeting, 1970 9. Johnstone JM: The appearance and significance of tissue mast cells in human bone marrow. J Clin Pathol 7:275, 1954 10. Kabelitz HJ: Klinische und hamatologische Boebachtungen zur Entwicklung lymphatischer Keimzentren im Knochenmark. Acta Haematol 3:347-366, 1950 11. Mayer E, Furuta S: Zur Frage der Lymph Knotchen in menschlichen Knochenmark. Virchows Arch Pathol Anat 253:574-586, 1924 12. Miale JB: Laboratory Medicine—Hematology. Third edition St. Louis, C. V. Mosby, 1967, pp 228-242 13. RebuckJW, Monto RW, Monaghan EA, et al: Potentialities of the lymphocytes with an additional reference to its dysfunction in Hodgkin's disease. Ann NY Acad Sci 73:8, 1958 14. Rohr K: Das menschliche Knochenmark. Stuttgart, George Thieme, 1960 15. Rywlin AM, Hoffman EP, Ortega RS: Eosinophilic fibrohistiocytic lesion of bone marrow: A distinctive new morphologic finding, probably related to drug hypersensitivity. Blood 40:464-472, 1972 16. Rywlin AM, Ortega RS: Lipid granulomas of the bone marrow. Am J Clin Pathol 57:457-462, 1972 17. Rywlin AM, Ortega RS, Dominguez CJ: Lymphoid nodules of bone marrow: Normal and abnormal. Blood 43:389-400,1974 18. Sundberg RD: Lymphocytes and plasma cells. Ann NY Acad Sci 59:671-689, 1955 19. Von Fischer O: Uber die Lymph Knotchen in menschlichen Humerus, Wirbel und Rippenmark. Frankfurt Z Pathol 20: 347-380, 1917 20. Williams RJ: The lymphoid nodules of human bone marrow. Am J Pathol 15:377-384, 1939 21. Yoffey JL, ConsticeFC: Lymphatics, Lymph and the Lymphoid Complex. London, New York, Academic Press, 1970, pp 642-644 22. Ziegler K: Uber die Morphologic der Blutbereitung bei pernizioser Anamie. Dtsch Arch Klin Med 99:431-467, 1910

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marrows of patients who have lymphocytic neoplasms are quite similar to lymphoid follicles, and the morphologic differentiation between the two may be extremely difficult or impossible. Not only sections but also smears with many lymphoid follicles can give rise to diagnostic problems. Rywlin and associates17 proposed a descriptive term, "nodular lymphoid hyperplasia." However, the clinical significance remains unknown. From our study, we observed the following findings that may be helpful for differentiation between benign and malignant lymphoid follicles: 1. Cytologic examination of benign lymphoid follicles almost always shows well-differentiated lymphocytes; exceptions are those with germinal centers. These well-differentiated lymphocytes tend to have a compact nuclear chromatin pattern, which is seen in the smears. 2. Benign lymphoid follicles are more frequently polymorphic and are accompanied by eosinophils, histiocytes and plasma cells. 3. Distribution of benign lymphoid follicles is generally patchy. Irregular infiltration separated from the main collection of lymphocytes, such as infiltration of the satellite type or Indian file type, is unlikely. Further comparative study of benign and malignant lymphoid follicles is needed to clarify their exact characteristics.

Lymphoid follicles in bone marrow aspirates.

Lymphoid Follicles in Bone Marrow Aspirates KOICHI MAEDA, M.D., BONG H. HYUN, M.D., AND JOHN W. REBUCK, M.D., P H . D . Department of Pathology, Muhl...
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