A. Lymphocyte development
1. Development involves three major lymphoid compartments.
a. The pool of undifferentiated stem cells is located in the bursa-equivalent tissue of the bone marrow, where proliferation and maturation occurs.
b. Primary lymphoid tissues are the location of antigen-independent lymphopoiesis.
(1) T-lymphocyte development occurs in the thymus (Figure 4–9).
(2) B-lymphocyte development occurs in the bone marrow.
c. Peripheral or secondary lymphoid development is antigen dependent. Sec- ondary lymphoid tissues contain mixed T-cell and B-cell populations; these tissues are found in the spleen, lymph nodes, and gut-associated lymphoid tissue.
2. B-lymphocyte development (see Chapter 6, Section II B)
3. T-lymphocyte development is centered in the thymus gland (see Chapter 6, Section II A).
B. Lymphocyte structure
1. From a morphologic basis, lymphocyte development consists of three recognized stages.
a. Lymphoblasts are the youngest recognizable form of B lymphocytes or T lympho- cytes based on staining characteristics.
(1) Blast size is larger than a mature lymphocyte, averaging 16–24 mm.
(2) Blasts have 1 to 2 easily recognizable large nucleoli.
(3) Chromatin in these blasts is finely divided into a smooth grainy texture.
(4) Lymphoblasts have the highest N:C ratio of any blast cell.
(5) Cytoplasm is scant and highly basophilic in staining.
b. The prolymphocyte is a middle stage in the development between the blast and mature lymphocyte.
(1) The size of this mid-stage cell is the same or slightly larger than the blast, averaging 18 to 28μm.
(2) Nucleoli can still be easily recognized.
(3) The nuclear chromatin pattern is slightly more condensed than the blast stage.
(4) Prolyphocytes have a high-to-moderate N:C ratio.
(5) The cytoplasm is more abundant than the blast but still deeply basophilic.
c. Mature lymphocytes in the peripheral blood represent non-dividing B lymphocytes or T lymphocytes between the primary and secondary development.
(1) Size. Small lymphocytes are just slightly larger than an RBC (i.e., 8–12μm).
(2) Organelles. These cells have a sharply defined, round nucleus that contains heavy concentrations of dense chromatin appearing blocked or “smudgy.”
The nucleus stains deep blue to purple and is sometimes indented at one side.
The cytoplasm stains a pale blue. Normally, lymphocytes do not demon- strate specific cytoplasmic granules.
(3) In a small lymphocyte, the nucleus occupies most of the cell area, and there may be just a thin perinuclear zone of cytoplasm.
(4) Large lymphocytes may be found in the peripheral blood, especially in children. These lymphocytes demonstrate the following morphologic char- acteristics:
(a) 12 to 15μm in diameter (b) Abundant cytoplasm (c) Nuclei less densely staining (d) Irregular borders and shape (e) May appear as a small monocyte
(f) Few bluish purple cytoplasmic granules
(5) Lymphocytes average 25% to 45% of all circulating WBCs with a total absolute count range of 1,000 to 4,800/mm3.
(6) T and B lymphocytes cannot be distinguished from each other morpholog- ically.
d. Plasma cells originate from B lymphocytes. They are cells designed for the synthesis of immunoglobulin.
(1) Plasmablasts, or immunoblasts, have a large blast-like nucleus with nucleoli, but have other plasma cell characteristics.
(2) Plasma cells are identified by their eccentrically placed nucleus and the following morphologic characteristics:
(a) The nucleus is round with a small indentation on the cytoplasm side.
(b) Nuclear chromatin is distributed more regularly in a pattern resembling the spokes of a wheel.
(c) The cytoplasm, except for one small area, stains deeply bluish green.
(d) The unstained portion of cytoplasm corresponds to the indented portion of the nucleus. A large golgi apparatus exists here because of the large amount of protein synthesis.
2. Lymphocytes demonstrate a heterogeneity of functional types.
a. B lymphocytes function as precursors for plasma cells. They also synthesize and release immunoglobulin, which is easily detectable on their surface membranes.
(1) Approximately 10% to 15% of circulating blood lymphocytes are B cells.
(2) Diversity
(a) Individual B cells are limited to the one type of antibody they can synthesize and release.
(b) IgM or immunoglobulin D (IgD) are found on most circulating B cells.
(c) B lymphocytes with surface IgM or IgG are found mainly in organized secondary lymphoid tissue.
(d) Immunoglobulin A (IgA)- and IgE-bearing B cells are predominately at sites of external Ig secretion (e.g., the GI and respiratory tracts, saliva).
(3) B-lymphocyte surface receptors include the following identifying markers:
(a) Fc receptors (i.e., CD7) that recognize the Fc portion of immunoglobulins (b) C3 receptors (i.e., CD21) for complement fixation
(c) Specific receptor for the Epstein-Barr virus antigen (d) Mouse RBC receptor
b. T lymphocytes are responsible for reactions of cellular immunity and modulation of humoral immunity (Figure 4–9).
(1) T cells interact with macrophages for the proper delivery of antigens to B lym- phocytes.
(2) T cell-mediated immunity is antigen directed; therefore, T cells must have surface receptors to recognize antigens.
(a) T lymphocytes display a specific receptor for sheep RBCs (i.e., CD2), forming E-rosettes.
(b) Fc receptors are present but not abundant.
(c) T cell-specific antigens CD1 to CD8 are also abundant.
c. Null lymphocytes are lymphocytes that cannot be classified as either T or B types on the basis of surface properties. These cells constitute approximately 10% of the lymphocyte population.
(1) Null cells possibly represent undifferentiated stem cells, immature T or B cells, or those lymphocytes that have lost their recognizable surface receptors.
(2) L cells are lymphocytes that do not proliferate in response to antigens but are capable of enhancing the responses of T lymphocytes in the presence of monocytes.
(a) These lymphocytes bear surface IgG and Fc receptors.
(b) L cells do not develop into antibody-producing cells.
(3) Large granular lymphocytes (LGLs) mediate antibody-dependent cytotox- icity.
(a) LGLs include natural killer (NK) and killer (K) cells.
(b) NK cells mediate cytotoxic reactions without prior sensitization in defensive mechanisms against tumors and virally transformed cells.
C. Function and regulation of the immune response
1. Monocyte-macrophages secrete IL-1, which activates the helper-inducer T lympho- cytes. IL-1 also has pyogenic effects on the central nervous system (CNS) in raising body temperature.
2. Helper-inducer T lymphocyte lymphokines, which are secreted in an immune reaction (see Chapter 6, Section II A), consist of the following substances:
a. Once activated by IL-1, the helper-inducer T lymphocyte secretes IL-2, which stimulates other helper-inducer T lymphocytes to multiply.
b. B-cell growth factor (BCGF) is released to stimulate B-cell proliferation.
c. B-cell differentiation factor (BCDF) is released to halt replication of the im- munospecific B cells and stimulates antibody production.
d. γ-interferon is released to stimulate B-cell antibody production, activate killer T lymphocytes, and localize macrophages at the site of infection.
3. B lymphocytes (see Chapter 6, Section II B) produce and secrete an antigen-specific antibody.
D. Lymphocyte pathophysiology
1. The normal circulating concentration of lymphocytes is 1,500 to 4,000/mm3. a. An absolute circulating lymphocyte count below 1,500/mm3 is considered clini-
cally to be a lymphocytopenia.
b. The reduction in circulating lymphocytes affects mostly T cells, because they rep- resent the greatest percentage of circulating lymphocytes.
2. Nonmalignant disorders of lymphocytopenia often include a hypogammaglobuline- mia (Figure 4–10). A lymphocytopenia can be found in the following disorders or conditions:
a. A blockage of the lymphatic thoracic duct results in a lymphocytopenia.
b. Radiation overexposure is highly toxic to lymphopoiesis and results in a reduction of lymphocytes.
c. Acute stress results in a reduction of lymphocytes in the circulating pool.
d. Therapy with corticoids shifts distribution of lymphocytes from the blood into the extravascular spaces. Cell lysis and an inhibition of cell proliferation are minor drug-induced mechanisms of lymphopenia.
e. Chemotherapeutic alkylating drugs interfere with lymphocyte proliferation.
f. Acquired immunodeficiency syndrome (AIDS) is a virally induced lymphocy- topenia.
■ Figure 4–10 Algorithm demonstrating the differential diagnosis of the nonmalignant lymphocytic disorders. AIDS=acquired immune deficiency syndrome; IgA=immunoglobulin A; SCID=severe combined immunodeficiency.
(1) The virus that causes AIDS has been named human T-cell lymphotrophic virus-3 (HTLV-3) or human immunodeficiency virus (HIV).
(2) Patients who have AIDS have a high risk of infections because of a significant decrease in helper-inducer T lymphocytes.
(a) The AIDS virus invades the helper-inducer T cells and renders them inca- pable of functioning.
(b) The T lymphocyte helper-suppressor (H-S) ratio is decreased below the normal range of 0.9% to 2.9%.
g. Primary immunodeficiency disorders include several disorders caused by a de- velopmental defect in either B or T lymphocytes.
(1) X-linked agammaglobulinemia (Bruton type) is a developmental defect of B cells that primarily affects male infants.
(a) B-cell zones of lymph nodes and spleen are depleted of B cells.
(b) Blood lymphocyte counts are normal, but the serum immunoglobulin con- centrations are very low, and patients suffer from recurrent infections.
(2) Hypogammaglobulinemia of infancy is a decrease in immunoglobulins caused by delayed immune development in the first years of life.
(a) Normally, following the gradual disappearance of maternal IgG, an infant’s own IgG and IgM levels increase to approximately 75% of the adult level by 1 year of age.
(b) Infants who have this disorder have a delayed onset of immunoglobulin synthesis and are subject to recurrent infections.
(c) The disorder usually self-corrects by 2 years of age.
(3) Late-onset variable primary hypogammaglobulinemia occurs in adults, usually by 30 years of age.
(a) The pattern of immunoglobulin deficiency varies among individuals.
(b) There is a defect in the differentiation of B lymphocytes into plasma cells.
(c) Patients have an increased occurrence of infections and an increased inci- dence of autoimmune disorders.
(4) Selective immunoglobulin deficiency is an acquired decrease in one subtype of immunoglobulin. A lack of IgA is most common.
(5) Thymic aplasia (Di-George’s syndrome) is a developmental defect of the thymus gland.
(a) T-cell zones of the lymph nodes are depleted.
(b) Serum immunoglobulin levels are normal.
(6) Severe combined immune deficiency is a defect in the common stem cell that leads to a deficiency of both T and B lymphocytes.
3. Lymphocytosis is clinically defined as an absolute circulating increase in the lymphocyte count >4,000/mm3 in adults, 7,000/mm3 in children, and 9,000/mm3 in infants.
Disorders that demonstrate lymphocytosis include the following characteristics:
a. Infectious lymphocytosis occurs mainly in children.
(1) The disorder is believed to be caused by viruses (e.g., coxsackievirus A and B6, echoviruses, adenovirus).
(2) Symptoms include vomiting, fever, and abdominal discomfort.
(3) Laboratory findings
(a) A leukocytosis of 20,000 to 50,000/mm3is one of the earliest findings.
(b) The blood differential shows 60% to 95% normal lymphocytes.
(c) Atypical lymphocytes are not seen with this disorder.
(d) Eosinophilia is common.
(e) The lymphocytosis usually lasts only 3 to 5 weeks and, in most cases, is acute and self-correcting. Rarely, some patients demonstrate a chronic course.
(f) Serologically, patients have a negative test for infectious mononucleosis (IM).
(g) Bone marrow of these patients is normal.
b. Pertussis (i.e., whooping cough) is a childhood inflammatory reaction of the res- piratory system.
(1) Cause. The etiologic agent is the bacterium Bordetella pertussis.
(2) Laboratory findings. Patients demonstrate a significant lymphocytosis as high as 30,000/mm3. Lymphocytosis is highest in the first 3 weeks of the disease, then decreases after 4 weeks. Lymphocytosis is caused by the release of lymphocytosis-promoting factor (LPF) from B. pertussis. LPF induces an acute release of lymphocytes from lymph nodes and also in- hibits the migration of lymphocytes from the circulation into the lympha- tics.
(3) Morphologic characteristics. The lymphocytes in this disorder are small and mature.
c. IM (see Chapter 6, Section XIII B)
(1) Serious complications of IM may require hospitalization.
(a) AIHA is reported in 1% to 3% of patients, which is related to the develop- ment of an autoimmune anti-i.
(b) Mild thrombocytopenia (i.e., approximately 5,000 to 100,000/mm3) is re- ported in 50% of patients who have IM.
(c) Liver involvement can occur, which leads to mild jaundice and hepatitis.
(d) Splenomegaly can also be a dangerous complication.
(2) Recognizing hematologic features of IM can result in a rapid diagnosis.
(a) Leukocytosis between 12,000/mm3and 25,000/mm3persists for the first 3 weeks of infection with a differential showing 60% to 90% lymphocytes.
(b) A neutrophilia occurs during the first week of infection with a left shift, metamyelocytes, toxic granulation, and D¨ohle’s bodies.
(c) Abundant atypical lymphocytes are found on the peripheral blood smear.
These are active or transformed lymphocytes involved in an immune reac- tion. Atypical lymphocytes are not exclusive to IM and are also found in the following disorders:
(i) Cytomegalovirus infections (ii) Toxoplasmosis
(iii) Infectious hepatitis
(iv) Viral pneumonia and mumps
d. Cytomegalovirus infection (CMV) demonstrates clinical and laboratory symp- toms that are in some ways identical to those of IM.
(1) Patients receiving massive blood transfusions are in a high-risk category for contracting CMV.
(2) Presentation. Patients have a leukocytosis involving a lymphocytosis in which 20% or more of lymphocytes may be reactive.
(3) Serologic tests. Patients are negative for heterophil antibodies and Epstein- Barr virus (EBV) antibodies.
(4) Diagnosis is made by the demonstration of CMV antibodies by complement fixation or hemagglutination techniques.
e. Toxoplasmosis also produces clinical and laboratory symptoms similar to those of IM.
(1) Presentation. Patients have an absolute lymphocytosis and atypical lympho- cytes on the peripheral blood smear.
(2) Serologic tests for EBV and heterophil antibodies are negative.
(3) Diagnosis is made by the demonstration of toxoplasmosis antibodies by the Sabin-Feldman dye test, fluorescent antibody, or hemagglutination.
f. Miscellaneous causes of lymphocytosis (1) Syphilis
(2) Smallpox
(3) Para-aminosalicylic acid hypersensitivity
(4) Phenytoin (i.e., Dilantin) and Mesentoin hypersensitivity
4. Hypergammaglobulinemias are related disorders in which the levels of one or more serum immunoglobulins are increased above normal levels. Hypergamma-globulinemias may or may not be accompanied by an absolute lymphocytosis.
a. Multiple myeloma (MM) is a monoclonal gammopathy (i.e., an abnormality of only one B-cell clone) in which only one type of gamma-globulin is increased.
(1) MM involves a neoplastic proliferation of plasma cells primarily in the bone marrow. Plasma cell proliferation may be either nodular or diffuse.
(2) Clinical symptoms are found primarily in persons older than 40 years.
(a) Bone pain is the most common symptom, and MM patients are often first seen with bone fractures.
(b) Tumor growth in the marrow increases bone destruction, and the serum calcium level is high.
(c) Patients have a high susceptibility to infection.
(d) Renal insufficiency is also a common symptom with MM.
(3) The MM laboratory profile includes the following results:
(a) Normocytic/normochromic anemia (b) Normoblasts on the peripheral blood smear
(c) Rouleaux formation and increased erythrocyte sedimentation rate (ESR) (i.e., due to increase in serum globulins)
(d) Possible shift to the left with metamyelocytes (e) Increased serum calcium level
(f) Circulating plasma cells
(g) An increase in one of the serum gamma-globulins as demonstrated by protein electrophoresis
(4) Bone marrow examination shows the presence of plasma cells varying from 1% to 90%.
(5) Serum immunoglobulins are increased in a variety of electrophoresis pat- terns among different patients. Serum protein electrophoresis usually shows a homogeneous band in the gamma or beta region of the electrophoretic tracing known as an “M-spot.”
(a) Most patients with MM are hypergammaglobulin producers, but in 25% of patients, only the light chains of the globulin are produced (i.e., Bence Jones proteins) by the abnormal plasma cells. A serum hypogammaglobulinemia is found in these patients, because light chains are cleared by the kidney into the urine.
(b) Half of reported MM patients show an increase in IgG only.
(c) A monoclonal increase in IgA is found in approximately 20% of MM pa- tients.
(d) IgD is increased in approximately 1% of patients.
(e) IgE is rarely found to demonstrate a monoclonal increase.
(f) Proteinuria with Bence Jones proteins can also be demonstrated in more than 50% of MM patients.
(6) Course. MM usually has a chronic course with the median survival approx- imately 3 years.
(a) In 5% of MM patients, AMML develops as a secondary manifestation of the malignancy.
(b) In some patients, plasmacytosis may be found in the blood and bone marrow, and the disease is then classified as plasma cell leukemia.
b. Waldenstr¨om’s macroglobulinemia (WM) is a hypergammaglobulinemia variant of chronic lymphocytic leukemia, in which there is a greater degree of maturation of the B lymphocytes into plasma cells.
(1) WM is characterized by a generalized proliferation of B cells and plasma cells and an increase of monoclonal IgM in the serum that amounts to at least 15% of the total serum protein.
(2) Clinical symptoms of WM are primarily found in individuals older than 40 years. Symptoms are caused by the cellular proliferation and increased blood viscosity caused by the increased IgM. Patients who have WM are commonly seen with the following symptoms:
(a) Neurologic abnormalities (b) Renal insufficiency (c) Heart failure
(d) Clotting abnormalities (e.g., DIC)
(3) The laboratory profile includes the following results:
(a) IgM in excess of 1.0 g/dL
(b) Normocytic-normochromic anemia (i.e., occasionally hemolytic with a pos- itive direct Coombs’ test)
(c) Thrombocytopenia caused by IgM platelet clumping or pancytopenia caused by marrow infiltration
(d) Lymphocytosis
(e) Marked rouleaux and increased ESR (f) Bence Jones proteinuria in 10% of patients
(4) The bone marrow often cannot be aspirated easily. A biopsy demonstrates an increase in small normal lymphocytes and plasma cells.
c. Heavy-chain diseases are disorders related to the production and excretion of the immunoglobulin heavy chains without the light chains.
(1) Gamma heavy-chain disease (γ-HCD) resembles a malignant lymphoma rather than a myeloma.
(a) The patient has a blood picture of anemia, leukopenia, thrombocytopenia, atypical lymphocytes, and plasma cells.
(b) A broad serum protein “spike” is found in the beta-gamma region of the electrophoretic pattern, accompanied by serum hypogammaglobulinemia.
(2) Alpha heavy-chain disease (α-HCD) is more common thanγ-HCD and oc- curs in a younger age group.
(a) Common symptoms include intestinal involvement with mal- absorption, diarrhea, and a massive lymphoplasmacytic infiltration in the intestinal mu- cosa.
(b) The bone marrow and lymph nodes are not usually involved.
(c) Protein electrophoresis is normal, but small amounts ofα chain may be detected in serum and urine on immunoelectrophoresis.
(3) Mu heavy-chain disease (μ-HCD) can be diagnosed only by a serum immu- noelectrophoresis that demonstrates an increase inμ-chains.
5. Lymphoproliferative diseases
a. These disorders represent a group of clonal disorders originating from cells of the lymphoreticular system (Table 4–11).
(1) When neoplastic cells involve mainly the bone marrow and blood, the disorder is known as a leukemia.
(2) When the disease is limited mainly to lymph nodes or organs, the disease is known as a lymphoma.
(3) Occasionally, a lymphoma develops into leukemia.
Table 4–11 Comparison and Differentiation of the Malignant Lymphoproliferative Disorders Based on Immunological Typing and Cytogenetic Abnormalities
Disorder Cell Type CD Marker Cytogenetic Abnormality
Chronic B cell; 95% CD5, CD19, CD20, CD22, CD24 +12; t(11:14)
lymphocytic leukemia T cell; 5% CD2, CD3, CD8
HCL B cell sIg, CD5, CD19, CD20, CD22, CD25
PLL B cell sIg, CD19, CD20, CD22, CD24
Acute lymphocytic leukemia t(4:11)
CALLA Early B; 70% TdT, CD10, CD19, CD20
Null cell Pre-B TdT, cIg
T cell Early T; 15%–20% TdT, CD1, CD2
Burkitt type Late B sIg, CD19, CD20, CD22, CD24
Lymphoma
NSCC B cell sIg, CD19, CD20, CD24 t(14:18)
Burkitt’s Late B cell sIg, CD19, CD20, CD24 t(8 or 2, or 22:14)
SLL, DLCL B cell sIg, CD19, CD20, CD24 t(11:14)
TCL Late T cell CD2, CD3, CD4, CD5 t(7:14; or 11 or 9)
CALLA=common acute lymphocytic leukemia antigen; CD=cluster designation; cIg=cytoplasmic immunoglobulin; DLCL=diffuse large-cell lymphoma;
HCL=hairy cell leukemia; NSCC=nodular small cleaved lymphoma; PLL=prolymphocytic leukemia; sIg=surface membrane immunoglobulin; SLL= small-cell lympocytic lymphoma; t=chromosomal translocation; TdT=terminal deoxynucleotidyl transferase;+ =trisomy.
b. Chronic lymphocytic leukemia (CLL) is a slowly progressing clonal malignancy of lymphocytes in an arrested stage of maturation.
(1) The patient’s clinical profile includes the following features:
(a) CLL is commonly seen in adults with a mean age of occurrence at 55 years.
(b) The disorder is twice as common in men as compared with women.
(c) Onset is slow, unrevealing, and is commonly discovered incidentally or only in the late stages of the disease.
(d) Patients are seen with symptoms, such as weakness, fatigue, anorexia, weight loss, enlarged lymph nodes, and abdominal discomfort caused by liver and spleen enlargement.
(2) The laboratory profile of a patient who has CLL includes the following:
(a) Patients have a leukocytosis ranging from 10,000 to 150,000/mm3 with 80% to 90% lymphocytes persistent over a period of weeks to months.
(b) Smudge cells are commonly found on blood smears.
(c) Lymphocytes in patients with CLL have a characteristic morphology.
(i) The nuclear chromatin is coarsely condensed.
(ii) Nucleoli may be demonstrable.
(iii) Lymphocytes show minimal size and shape variation.
(iv) The cytoplasm is small to moderate in amount.
(d) Occasionally, immature lymphocytes (i.e., usually fewer than 10%) may be found on the peripheral blood smear.
(e) Patients who have CLL do not usually have anemia or thrombocytopenia in early stages. As lymphocyte proliferation replaces the marrow with leukemic cells, production of other cell lines may suffer, and the symptoms will appear.
(f) AIHA develops in 10% of patients who have CLL. The patient’s blood smear demonstrates spherocytes and reticulocytosis.
(g) Hypogammaglobulinemia may also be present because of qualitative de- fects of the leukemic lymphocytes.
(3) A marrow aspirate commonly shows an increase in morphologically mature lymphocytes.
(a) If AIHA is present, then a marrow picture of increased erythropoiesis is predominant.
(b) In later stages of CLL, the lymphocytes overrun the marrow, replacing the other hemapoietic tissues.
(4) Ninety-five percent of CLL patients immunologically type as having a B-cell leukemia.