Introduction
The primary purpose of an abnormal peripheral lymph node examination is to decide whether surgical excision for histological examination is indicated or not. In conjunction with a good clinical history and physical examination, FNAC, allows the clinician to triage the patient and decide about whether to carry out further investigations or to try a course of conservative treatment, for example with antibiotics.
Thus, any enlarged, abnormal lymph node constitutes an indication for FNA biopsy. As a rule, the cytological examination can decide whether the lymphadenopathy is due to reactive hyperplasia, metastatic malignancy, or malignant lymphomas. Several important caveats should be kept in mind when evaluating lymph nodes. In general, any lymph node that remains enlarged for an extended period should be excised, even if the cytology of such aspirates suggests a benign lesion.
One important clinical question is whether the lymphadenopathy is localized or generalized. In general, a localized process favors a benign diagnosis, whereas malignancies, especially the non-Hodgkin's lymphomas; are likely to present with generalized lymph node enlargement. As also, the palpatory quality of the lymph node often provides useful clinical data.
The lymphoid system consists of the peripheral lymphoid organs and the thymus. They are closely linked with the functions of the bone marrow. The peripheral lymphoid organs consist of the lymph nodes, the spleen, the mucosa associated lymphoid tissue (MALT), and the pharyngeal lymphoid tissue. The B and the T lymphocytes are formed after differentiation from the lymphopoietic precursor cell in the bone marrow, which undergoes final maturation in the peripheral lymphoid organs and the thymus respectively.
Anatomy of the Lymph Node
The normal lymph node comprises of three major zones viz;
- Cortex
- Paracortex
- Medulla
The cortex is made up of primary follicles composed of B lymphocytes, as are the surrounding mantle and the marginal zones. The secondary follicles arise within these primary follicles as a result of antigenic stimulation. They are characterized by the presence of germinal center cells comprising of the centrocytes, centroblasts, immunoblasts and the germinal center histiocytes.
The region between the follicles is the paracortex, in which the T lymphocytes predominate. Interdigitating dendritic cells and the post-capillary venules (also called as the high endothelial venules) predominate in the paracortical area.
The medulla, which lies deep to the cortex, is less cellular than the cortex and the paracortex. It consists mainly of plasma cells, lymph sinuses, veins, arteries and the B lymphocytes mainly.
A systematic assessment of the cytological asperate is essential to arrive at the correct diagnosis. It includes the following:
- Evaluation of the overall smear cellularity
- Pattern of cell arrangement
- The predominant cell type
- The nature of any material present in the smear background.
Lymphadenopathy can be a primary or a secondary manifestation of numerous disorders as shown below.
Infectious Diseases
- Viral—Infectious mononucleosis syndromes (EBV, CMV), infectious hepatitis, herpes simplex, herpesvirus-6, varicella-zoster virus, rubella, measles, adenovirus, HIV, epidemic keratoconjunctivitis, vaccinia.
- Bacterial—Streptococci, staphylococci, cat-scratch disease, brucellosis, tularemia, plague, chancroid, meliodosis, glanders, tuberculosis, atypical mycobacterial infections, primary and secondary syphilis, diphtheria and leprosy.
- Fungal—Histoplasmosis, coccidioidomycosis, para-coccidioidomycosis.
- Chlamydial—Lymphogranuloma venerum, trachoma.
- Parasitic—Toxoplasmosis, leishmaniasis, trypanosomiasis, filariasis.
- Rickettsial—Scrub typhus, rickettsial pox.
Immunological Diseases
- Rheumatoid arthritis
- Juvenile rheumatoid arthritis
- Mixed connective tissue disease
- Systemic lupus erythematosus (SLE)
- Dermatomyositis
- Sjögren's syndrome
- Serum sickness
- Drug hypersensitivity—diphenylhydantoin, hydralazine
- Angioimmunoblastic lymphadenopathy
- Primary biliary cirrhosis
- Graft-vs-host disease
Malignant Diseases
- Hematologic—Hodgkin's disease, non-Hodgkin's disease, acute or chronic lymphocytic leukemia, amyloidoses
- Metastatic—from numerous primary sites.
Lipid Storage Diseases
Gaucher's disease, Niemann-Pick disease, Fabry's disease, Tangier disease.
Endocrine Diseases
Hyperthyroidism
Other Disorders
- Castleman's disease (giant lymph node hyperplasia)
- Sarcoidosis
- Dermatopathic lymphadenitis
- Lymphomatoid granulomatosis
- Kikuchi's disease
- Rosai-Dorfman disease
- Kawasaki's disease
- Histiocytosis-X
- Familial mediterranean fever
- Severe hypertriglyceridemia
- Vascular transformation of sinuses
- Inflammatory pseudotumor of lymph node.
Soft, flat, submandibular nodes (<1 cm) are often palpable in young children and young adults. Healthy adults may have palpable inguinal lymph nodes of up to 2 cm, which are considered normal (Table 1.1). It is generally agreed that a firm lymph node, larger than 1 cm, that is not associated with a documentable infection and that persists longer than 4 weeks should be considered for biopsy.
The medical history should reveal the setting in which the lymphadenopathy is occurring. Symptoms such as sore throat, cough, fever, night sweats, fatigue, weight loss or pain in the lymph nodes should be sought. The patients’ age, sex, occupation, exposure to pets, sexual behavior and use of drugs like diphenylhydantoin should be also sought for. A thorough ENT examination and history of tobacco intake in cases of cervical lymphadenopathy must also be inquired. Children and young adults usually have benign, i.e. non-malignant disorders that account for the lymph node enlargement. In contrast, after the age of 50 years, there is an increase in the incidence of malignant disorders.4
The physical examination can provide useful clues such as:
- Extent of lymphadenopathy (localized or generalized)
- Size of nodes and texture
- Presence or absence of nodal tenderness
- Signs of inflammation over the nodes
- Skin lesions (for dermatopathic lymphadenopathy)
- Splenomegaly (for hematologic malignancies).
Localized or regional adenopathy implies involvement of a single anatomic area. Generalized lymphadenopathy has been defined as an involvement of three or more noncontiguous lymph node areas. Nodes < 1.0 cm2 in area are usually secondary to benign, non-specific causes.
Cervical adenopathy is by far the commonest swelling in the neck encountered in clinical practice. The causes are enumerated below.
Causes
Acute
- Acute pyogenic lymphadenitis
- Acute lymphatic leukemia
- Infectious mononucleosis.
Chronic
- Inflammatory:
- Non-specific—chronic lymphadenitis
- Specific—
- Infections
- tuberculosis
- syphilis in secondary stage
- sarcoidosis
- toxoplasmosis
- Neoplastic:
- Primary:
- lymphoma (Hodgkin's)
- lymphosarcoma
- chronic lymphatic leukemia
- Secondary—metastases from carcinoma or melanoma
- Autoimmune:
- SLE
- Juvenile rheumatoid arthritis (Still's disease)
Reactive Lymphoid Hyperplasia
The criteria by which the diagnosis of reactive lymphadenopathy and follicular hyperplasia is established are as follows:
- High cell density
- Clear polymorphic pattern of cells without malignant features
- Considerable number of ‘tingible body macrophages, i.e. (germinal center histiocytes with abundant clear cytoplasm containing phagocytozed fragments of degenerated cells).
The polymorphic cell pattern composed of lymphocytes exhibiting variable sizes and different cytoplasm and nuclear characteristics must be stressed on. The usual predominant component of small lymphocytes is accompanied by a variable number of typical follicle center cells, i.e. centrocytes, centroblasts, immunoblastic cells and of cells belonging to the plasma cell series (plasmoblasts, plasmacytoid cells, or mature plasma cells) and tingible body macrophages. Mitotic activity can be prominent.
The presence of lymphoglandular bodies is very characteristic of lymph node aspirates. These are fragments of lymphocyte cytoplasm, which stain pale greenish-gray on Papanicolaou's (Pap) stain and blue-gray to blue on Giemsa stain. These are found in both benign and malignant lymphoid processes and their presence generally supports a lymphoid process.
Capillaries may often be prominent in smears. Delicate branched capillaries are encased by lymphocytes, though vascular fragments may be seen in aspirates of malignant lymphomas, especially of T cell type. Nevertheless, they are more common in benign lymph node aspirates.
In air dried Romanowsky's stained smears, artifacts resulting from crushing and slow air-drying make it difficult to distinguish small mature lymphocytes from lymphoblasts. Therefore, the smears ought to be examined in the better preserved and the better-stained areas. Eosinophils and Reed- Sternberg cells should be looked for at the edges of the smear. Alcohol fixed Pap stained smear clearly shows the nuclear membrane and highlights clearly any nuclear membrane and chromatin irregularities.
The proliferation of different cell types in reactive lymphadenopathy is dependent on many factors like:
- The basic disease of the patient
- Patient's age
- The causative agent
- The stage of infection.
More or less, a mixed polymorphic cell population is generally observed consistent with follicular hyperplasia.6
Cytomorphology of Follicular Hyperplasia
Germinal center histiocytes (tingible body macrophages), accompanied by transformed lymphocytes of follicle-center cell origin, are a distinctive feature of pronounced follicular hyperplasia. The presence of tingible bodies alone does not allow an unequivocal exclusion of malignancy. They are also seen in:
- High grade lymphomas
- Burkitt ‘s lymphoma
- Lymph nodes partially infiltrated by malignant lymphoma
- Centroblastic—centrocytic lymphomas.
It is also advisable to use low power (100X), to examine the cell pattern, which is polymorphic with a preponderance of small normal lymphocytes in benign lesions, but is rather monomorphic in cases of NHL. A diagnosis to the etiology of lymphadenopathy may be suggested by a characteristic cell population, like (Figs 1.1 to 1.10):
- in infectious mononucleosis, there is proliferation of immunoblasts and plasmacellular elements
- in toxoplasma, there is presence of histiocytes appearing as epithelioid cells, arranged in small clusters or sheets.
Fig. 1-1: Pap stained smear (100X) shows a polymorphous pattern of lymphocytes with small lymphocytes predominating
Fig. 1-2: Giemsa stained smear (100X) shows a polymorphous pattern of lymphocytes with small darkly staining lymphocytes predominating
Fig. 1-3: Giemsa stained smear (100X) shows a polymorphous pattern of lymphocytes with immunoblasts (arrow)
Fig. 1-4: Pap stained smear (400X) shows a polymorphous pattern of lymphocytes with a lymphoglandular body (arrow)
Fig. 1-7: Pap stained smear (100X) showing a predominance of small lymphocytes along with many histiocytes with abundant pale cytoplasm (arrow)
Fig. 1-8: Pap stained smear (400X) showing a plasma cell in the center with eccentric nucleus, clock face chromatin, dense opaque cyanophilic cytoplasm with a paranuclear hof (arrow)
The usual predominant component of small lymphocytes is accompanied by a variable number of typical follicle center cells, cells of the immunoblastic series and the plasma cell series (plasmoblasts, plasmacytoid cells and plasma cells).
A variable number of typical follicle center cells consisting of the following accompany the usual predominant component of small lymphocytes (Figs 1.11 to 1.22):
- Centrocytes
- Centroblasts
- Immunoblastic cells.
Centrocytes
They are somewhat larger than small lymphocytes. The nuclei are irregularly shaped with deep indentations. The chromatin is fine and even. Occasionally, a small nucleoli may be present. The cytoplasm is scant and ill-defined. It is difficult to differentiate them from small T lymphocytes.
Centroblasts
They are medium sized cells. They are 2-3 times the size of a small lymphocyte. The nuclei are round with smooth nuclear membranes. The chromatin is finely granular. Nucleoli are usually multiple, cozying to the nuclear membranes. The cytoplasm is easily discernable as a narrow rim.
Immunoblasts
They are the most prominent cells observed under low power in lymph node aspirates. They are large cells, nearly 3-4 times the size of a small lymphocyte. The nucleus is round, which is the most striking feature. The chromatin is finely granular or can have areas of parachromatin clearing 12and condensation. One or two centrally placed prominent nucleoli are usually present. The cytoplasm is abundant, deeply basophilic along with the presence of occasional cytoplasmic vacuoles.
Plasma Cells
They are approximately twice the size of the small lymphocyte. The nucleus is characteristically eccentric, with the presence of coarse clumped chromatin (also referred to as ‘cart-wheel chromatin pattern’). The presence of a paranuclear hof is very diagnostic of its identity. The cytoplasm is deeply basophilic or cyanophilic, due to the presence of abundant RNA.
Plasmacytoid Cells
They are intermediate in size. The nucleus is eccentric with coarse chromatin. The cytoplasm is distinctly outlined, basophilic or cyanophilic and often triangular.
Fig. 1-11: Pap stained smear (400X) showing a binucleated tingible body macrophage (arrow) with abundant cytoplasm and engulfed debris along with centrocytes
Fig. 1-12: Giemsa stained smear (400X) showing a tingible body macrophage (arrow) with abundant cytoplasm and intracytoplasmic apoptotic debris
Fig. 1-13: Pap stained smear (400X) showing a mononuclear tingible body macrophage (arrow) and lymphocytes
Fig. 1-14: Giemsa stained smear (400X) showing a binucleated tingible body macrophage (arrow) with abundant cytoplasm with engulfed debris along with small lymphocytes
Fig. 1-15: Pap stained smear (400X) showing centroblasts in the center (arrow) and centrocytes (double arrow)
Fig. 1-16: Pap stained smear (400X) showing centroblasts in the center (double arrow), large cleaved lymphocyte (arrow) and centrocytes
Fig. 1-17A: Pap stained smear (400X) showing centroblasts with multiple nucleoli (arrow), lymphoglandular body (arrowhead) and centrocytes
Fig. 1-17B: Pap stained smear (400X) showing a polymorphous population of centrocytes (arrow), large lymphocyte and centroblasts
Fig. 1-18A: Pap stained smear (400X) showing an immunoblast with abundant cytoplasm and a prominent nucleoli along with a polymorphous population of lymphocytes
Fig. 1-18B: Pap stained smear (400X) showing an immunoblast (arrow) with abundant cytoplasm and a prominent nucleoli and a plasma cell along with lymphocytes
Fig. 1-18C: Pap stained smear (400X) showing an immunoblasts (arrow) with a large round nucleus, abundant cytoplasm and a prominent nucleoli
Fig. 1-18D: Pap stained smear (400X) showing an immunoblasts (arrow) surrounding small and large lymphocytes; the polymorphism being very evident
Fig. 1-19: Giemsa stained smear (400X) showing an immunoblast (double arrow) with abundant basophilic vacuolated cytoplasm and a prominent nucleoli and a lymphoglandular body (arrow)
Fig. 1-20: Giemsa stained smear (400X) showing an immunoblast (arrow) with abundant basophilic vacuolated cytoplasm and a prominent nucleoli
Histiocytes
These are large cells. Their nuclei can be round, reniform (kidney shaped) or sometimes epithelioid like with smooth nuclear membranes. The chromatin is vesicular. The cytoplasm is abundant and foamy due to the presence of vacuolations.
Differential diagnosis of reactive lymphoid hyperplasia:
- Hodgkin's lymphoma
- Toxoplasmosis
- Lymph nodes only partially involved by neoplasia, including non-Hodgkin's lymphoma
- Small lymphocytic lymphoma.
A useful cytologic criterion in the identification of lymphoreticular cells is their lack of cellular cohesion; however, the tendency of lymphocytes to cluster has been reported in aspiration smears of non-Hodgkins lymphoma.
Lymphohistiocytic Aggregates
The lymphohistiocytic aggregates are seen as sheets or syncytia of histiocytes admixed with a polymorphic population of lymphocytes. They have a diameter of 50-100 micrometers and are easily discernible with 10X objective. They appear as loosely cohesive clusters with an irregular border and spatial separation between their nuclei. The relative proportion of the two cell types varies from aggregate 21to aggregate. When lymphocytes are relatively sparse, the abundant pale histiocytic cytoplasm stands out distinctly. When lymphocytes are relatively numerous, these aggregates appear relatively dark.
The lymphocytes show a spectrum of nuclear shapes, sizes and chromatin patterns of the follicular germinal-center cells. The larger histiocytic cells are interspersed among the lymphocytes.
Cytomorphology of the histiocytic cells:
- Nucleus—round, vesicular with smooth nuclear borders
- Nucleolus—small and central
- Cytoplasm—foamy, somewhat granular cytoplasm, copious and ill-defined. It is best seen along, the edges of the syncytia in air-dried Giemsa stained preparations and account for the poorly delineated margin for the syncytia as a whole.
They may also have three-dimensional or spherical shapes. The lymphocytes and histiocyte nuclei are tightly packed. Lymphocytes comprise majority of the cells, but the histiocytes are readily apparent with fine focusing. Most of the lymphocytes are small and large cleaved follicular-center cells, though a smaller percentage may be of the noncleaved type. The same features of the histiocytes seen in the syncytial aggregates are also noted in the spherical aggregates. As in the syncytial aggregates, the histiocytes are seen in the periphery of the spherical aggregates.
There is a positive correlation between the number of aspirated lymphohistiocytic aggregates and the presence of follicles; thus providing indirect evidence that lymphohistiocytic aggregates are aspirated from lymph node follicles.
Their presence does not eliminate the possibility of small microscopic metastases, within the aspirated lymph nodes. Ultrastructural studies of both follicular non-Hodgkin's lymphoma and follicular hyperplasia have revealed an intimate association of lymphocytes and histiocytes within follicular structures.
The practical importances of the presence of lymphohistiocytic aggregates in aspiration cytology are four-fold:
- They must not be assumed to be, nor be confused with metastatic epithelial tumors because of intercellular cohesiveness alone.
- Lymphohistiocytic aggregates provide evidence of lymphoid follicles; this finding must be correlated with other aspirates and clinical findings to differentiate follicular hyperplasia from follicular lymphomas.
- The histiocytes of lymphohistiocytic aggregates must be distinguished from the epithelioid histiocytes of non-caseating granulomatous inflammation of sarcoidosis.
- Binucleated histiocytes of lymphohistiocytic aggregates, especially in the highly reactive forms must not be confused with the Reed-Sternberg cells.
Tingible bodies within histiocytes are common. Tingible body macrophages (TBM) with intracytoplasmic apoptotic debris, though characteristic, are not pathognomic of a hyperplastic process. They are also seen in high-grade lymphomas such as Burkitt's lymphoma and other malignancies with brisk proliferation rates. However, a large number of TBM, in the presence of a full spectrum of lymphoid cells with small lymphocytes predominating, leads to a benign diagnosis.
Lymphohistiocytic aggregates of dendritic reticulum cells and large and small cleaved and non-cleaved lymphoid cells have their origin in germinal centers (Figs 1.23 to 1.28).22
Fig. 1-23: Giemsa stained smear (50X) showing lymphohistiocytic aggregate (arrow) in a background of lymphocytes
Fig. 1-24: Giemsa stained smear (100X) showing lymphohistiocytic aggregate with an increased number of lymphocytes
Fig. 1-25: Giemsa stained smear (100X) showing lymphohistiocytic aggregate (arrow) with an increased number of histiocytes
Fig. 1-26: Pap stained smear (100X) showing lymphohistiocytic aggregate (arrow) with an increased number of histiocytes
Fig. 1-27: Pap stained smear (100X) showing lymphohistiocytic aggregate (arrow) with an increased number of lymphocytes
Fig. 1-28: Pap stained smear (400X) showing a lymphohistiocytic aggregate. Histiocytes show oval nucleus with a vesicular chromatin pattern, ill-defined cytoplasmic borders and a prominent nucleolus (arrow)
Suggested Reading
- Behm FG, O'Dowd CJ, Frable WJ. Fine needle aspiration effects on benign lymph node histology. Am J Clin Pathol 1984;82:195–98.
- Frable WJ, Kardos TF. Fine needle aspiration biopsy. Applications in the diagnosis of lymphoproliferative disorders. Am J Surg Pathol 1989;12 (Suppl 1):62-72.
- Geisinger KR, Rainer RO, Field AS. Lymph node. In: Fine needle aspiration cytology of superficial organs and body sites. Churchill-Livingstone New York. 1999;1-49.
- Leong ASY, Steven M. Fine needle aspiration biopsy for diagnosis of lymphoma: A perspective. Diagn Cytopathol 1996;15:352–57.
- Lukas PF. Lymph node smears in diagnosis of lymphadenopathy. A review. Blood 1955;10:1030–54.
- O'Dowd GJ, Frable WJ, Behm FG. Fine needle aspiration cytology of benign lymph node hyperplasia. Diagnostic Significance of Lymphohistiocytic aggregates. Acta Cytol 1985;29:554–58.
- Patrick H Henry, Dan L Longo. Enlargement of the lymph nodes and spleen. In: Fauci, Hauser, Longo, Jameson (Eds): Harrison's Principle of Internal Medicine Vol I. MacGraw Hill New York. (2nd edn) 2005;344-48.
- Stani Josefine. Cytologic diagnosis of Reactive Lymphadenopathy in fine needle aspiration biopsy specimens. Acta Cytol 1987;31:8–13.