Approach to a case of lymphocytosis:

1. APPROACH TO A
CASE OF
LYMPHOCYTOSIS
PRESENTOR: DR. POOJA DWIVEDI
MODERATOR: DR. GEETA YADAV
DEPARTMENT OF PATHOLOGY
KING GEORGE’S MEDICAL UNIVERSITY, LUCKNOW

2. LYMPHOCYTE AND LYMPHOPOIESIS
• Lymphopoiesis refers to the process by which the cellular
components of the immune system (i.e. T cells, B cells, NK cells
and certain dendritic cells) are produced during hematopoietic
differentiation
• This process begins with the hematopoietic stem cell and
continues through progenitor stages

4. LYMPHOID ORGANS

5. LYMPHOPOIESIS ( T CELLS)

6. LYMPHOPOIESIS (B CELLS)

7. LYMPHOBLAST
• Earliest recognizable cell of lymphoid series
• Size : 13-15 um
• The nucleus is round but may be indented or
convulated with dense nuclear membrane
• Chromatin is usually finely stippled but may
be coarsely granular
• One or more nucleoli are present in the
nucleus
• Cytoplasm is basophilic and may contain
vacuoles

8. PROLYMPHOCYTE
• Intermediate stage between lymphoblast and
mature lymphocyte
• Size : 9 to 18 um
• Nucleus is round, oval or slightly indented
with slightly stippled coarse chromatin having
0 to 1 nucleoli
• Cytoplasm is scant and non granular

9. LYMPHOCYTE
• Size : 12 - 16 uM large
7-10 uM small
• Nucleus is single round filling up the cell
almost completely stains deep blue
(ink spot appearance )
• Chromatin in dense clumps
• Cytoplasm:
Large lymphocytes – abundant sky blue
Small lymphocytes –scant

11. T LYMPHOCYTES
• Arise in the bone marrow but migrate to the thymus gland to
mature
• Cannot recognize antigen alone, T cell receptors can recognize only
antigen bound to cell membrane proteins ( MHC molecules)
• CD4 – TH , CD8 – TC
CRUCIAL STEPS:
• A naïve T Cell encounters antigen combined with a MHC molecule
on a cell
• T cell proliferates
• Differentiates into memory T cells and various effector T cells

12. STAGES OF T-CELL MATURATION: 5 STAGES
• STAGE ONE : THYMIC MIGRATION
• Multipotent lymphoid progenitors (MLP) enter the T cell pathway as
they immigrate to thymus
• Early thymocyte progenitors (ETP):
Most primitive cells in thymus
Retain lymphoid and myeloid potential
Transient existence
Rapidly differentiating into T and NK lineages

13. STAGE TWO: PROLIFERATIVE EXPANSION AND T LINEAGE
COMMITMENT
• The most primitive T cells retain pluripotency and differentiate into
cells of myeloid or lymphoid lineages (B cells , DC cells, T cells or
NK cells)
• DN2 cells have limited potentiality but are not yet fully restricted to
the T cell lineage (they can still develop into DC, T or NK cells)
• Final commitment to the T cell lineage –
Thymic microenvironment
When thymocytes expressing Notch1 receptors engage thymic
stromal cells expressing Notch1 ligands

14. STAGE THREE : ß-SELECTION
• Formation and rearrangement of α,ß polypeptides ( αßTCR or
conventional T cells)
• TCR ß gene rearranges followed by TCR α gene in association with
CD3 on surface of cells
• Grants the ability to distinguish self from foreign antigens

15. STAGE FOUR : T CELL RECEPTORS SELECTION (POSITIVE
SELECTION)

16. STAGE FIVE : NEGATIVE SELECTION
• Important component of central tolerance & prevents formation of
self reacting T cells producing autoimmune diseases

17. STAGES OF T CELL MATURATION

18. TYPES OF T CELLS
• Helper CD4+ T cells
 Assist other lymphocytes, including maturation of B cells into
plasma cells and memory B cells, and activation of cytotoxic T cells
and macrophages
 Become active when presented with peptide antigens by MHC
class II expressed on the surface of APCs and produce cytokines
• Cytotoxic T cells
 Recognise antigen in association with MHC class I molecules
 Play an important role in cell mediated immunity
• T Regulatory cells
 Tregs comprise about 5% of circulating CD4+ T cells
 Regulate autoreactive T cells in the periphery
• Unconventional T cells or γδ T cells
 Abundant in the mucosal immune system and skin
 These ‘non-MHC restricted’ T cells are involved in
specific primary immune responses, tumor
surveillance, immune regulation and wound healing
• Memory T cells
 Long- lived
 Quickly expand to large numbers of effector T cells
upon re-exposure
 Either CD4+ or CD 8+ and usually express CD45RO

19. B LYMPHOCYTES
• Approximately 3 to 21% of circulating lymphocytes are B cells
• B lymphocytes are named so as they were first studied in chicken’s
bursa of Fabricus
• Mature within the bone marrow and when they leave it , each
expresses a unique antigen–binding receptor on its membrane
• Plasma cells live for only a few days , they secrete enormous
amounts of antibody (2000/sec)

20. B CELL LYMPHOPOEISIS
• B cells are formed and mature in bone marrow and spleen
• HSC MPP CLP pro-B cell pre-B cell immature B cell
• The relative proportion of precursor B cells in the bone marrow
remains constant throughout the life :
Pro-B cells (5 to 10% of the total)
Pre-B cells (60 to 70%)
Immature B cells (20 to 25%)

21. B CELL DEVELOPMENT

22. B CELL DEVELOPMENT

23. NEGATIVE SELECTION
• Occurs through binding of self antigen with BCR
• If the BCR can bind strongly to self antigen , then the B cell undergoes one
of the following:
Clonal deletion
Receptor editing
 Anergy
Ignorance
• This negative selection process leads to a state of central tolerance in
which the mature B cells don’t bind with self antigens present in the bone
marrow

24. B CELL DEVELOPMENT

25. REARRANGEMENT OF IMMUNOGLOBULIN GENES AND
IMMUNOGLOBULIN EXPRESSION
• Rearrangement of heavy chain genes followed by light chain genes
• In pre-B cell, rearrangement of heavy chain genes causes appearance of µ
heavy chain in cytoplasm followed by rearrangement of light chain genes
• Light chains associate with µ heavy chain in cytoplasm IgM expression on
cell surface
• Mature B cells express both IgM and IgD
• In activated B cells, class switching of heavy chains occurs ( IgM to
IgG/IgA/IgE)
• Plasma cells donot have surface expression of Ig but synthesize and
secrete large amount of Ig of one class

26. B CELL TYPES
• Memory B cells
• Dormant B cells arising from B cell differentiation
• Circulate through the body and initiate a secondary antibody response if they detect the antigen
that had activated their parent B cell
• Can be generated from both T cell dependent and independent activation of B cells
• CD27+
• Follicular (FO) B cells
• Most common type
• Found mainly in lymphoid follicles of secondary lymphoid organs
• Generate majority of high affinity antibodies during an infection
• T cell dependent activation

27. PLASMA CELLS
• Large lymphocytes with abundant
basophilic cytoplasm
• Eccentric nucleus with heterochromatin
in a characteristic cartwheel or clock
face arrangement
• Show expression of CD138, CD78, IL-6,
CD 27
• Memory B cells are CD 27+ , plasma
cells are CD 27++

28. NATURAL KILLER CELLS
• 10-15% of peripheral blood lymphocytes
• Cytotoxic CD8+ cells that lack the TCR
• Large cells with cytoplasmic granules
• Express surface molecules CD16, CD56, CD57
• Donot require previous exposure or sensitization
for their cytotoxic action
• Provide host defense against tumor cells and virally
infected cells which have low level of expression of
HLA class I molecules

29. NATURAL KILLER CELLS: MECHANISM

30. LYMPHOCYTOSIS: DEFINITION
• Lymphocytosis is defined as an absolute lymphocyte count exceeding 4
× 109 /L, although somewhat higher threshold values (e.g., >5.0 × 109
/L) are sometimes used.
• The normal absolute lymphocyte count is significantly higher in
childhood.
• Lymphocytosis in young children is defined as an absolute lymphocyte
count >10.03 109/L.
• The reference range for relative lymphocytes is approximately 20% to

31. CAUSES OF LYMPHOCYTOSIS
I. Primary lymphocytosis
A. Lymphocytic malignancies
1. Acute lymphocytic leukemia
2. Chronic lymphocytic leukemia and
related disorders
3. Prolymphocytic leukemia
4. Hairy cell leukemia
5. Adult T-cell leukemia
6. Leukemic phase of B-cell lymphomas
7. Large granular lymphocytic leukemia
a. Natural killer (NK) cell leukemia
b. CD8+ T-cell large granular
lymphocytic leukemia
c. CD4+ T-cell large granular
lymphocytic leukemia
d. γ/δ T-cell large granular
lymphocytic leukemia
B. Monoclonal B-cell lymphocytosis1
C. Persistent polyclonal B cell
lymphocytosis2,3

32. II. Reactive lymphocytosis
A. Mononucleosis syndromes
1.Epstein-Barr virus4
2. Cytomegalovirus
3. HIV
4. Herpes simplex virus type II
5. Rubella virus
6. Toxoplasma gondii
7. Adenovirus
8. Infectious hepatitis virus
9. Dengue fever virus
10. Human herpes virus type 6 (HHV-6)
11. Human herpes virus type 8 (HHV-8)
12. Varicella zoster virus
B. Bordetella pertussis
C. NK cell lymphocytosis
D. Hypersensitivity reactions
1.Insect bite
2. Drugs

33. E. Stress lymphocytosis (acute)
1.Cardiovascular collapse
a. Acute cardiac failure
b. Myocardial infarction
2. Staphylococcal toxic shock syndrome
3. Drug-induced
4. Major surgery
5. Sickle cell crisis
6. Status epilepticus
7. Trauma
F. Persistent lymphocytosis (subacute or
chronic)
1.Cancer
2. Cigarette smoking
3. Hyposplenism
4. Chronic infection
a. Leishmaniasis
b. Leprosy
c. Strongyloidiasis
5. Thymoma

34. EVALUATION OF LYMPHOCYTOSIS
1. BLOOD FILM : Evaluated for a predominance of
Reactive lymphocytes associated with Infectious mononucleosis
Large granular lymphocytes associated with Large granular lymphocytic leukemia
Smudge cells associated with Chronic lymphocytic leukemia
Blasts of Acute lymphocytic leukemia
2. CHARACTERIZATION OF CELL-SURFACE MARKERS: distinguish primary
lymphocytosis (leukemic) from secondary lymphocytosis (reactive).
3. FLOW CYTOMETRY: distinguish benign from neoplastic lymphoproliferative disease.
4. ANALYSIS FOR IMMUNOGLOBULIN: provide evidence for monoclonal B-cell
5. T-CELL RECEPTOR GENE REARRANGEMENT: provide evidence for T-cell
proliferation

35. REACTIVE / SECONDARY LYMPHOCYTOSIS
• An increase in the absolute number of lymphocytes secondary to a
physiologic or pathophysiologic response to infection, toxins, cytokines,
or unknown factors.
• Reactive lymphocytes: Large lymphocytes with an increased proportion
of cytoplasm with basophilic cytoplasmic edges, often engaging
neighboring red cells.
• Nucleoli may occasionally be evident.
• This variation in lymphocyte appearance can occur in a variety of
disorders that provoke an immunologic response, including viral
illnesses.
• They are indistinguishable in appearance by light microscopy from the
reactive lymphocytes seen in infectious mononucleosis, viral hepatitis, or
other conditions such as Dengue fever

36. MONONUCLEOSIS SYNDROMES
• The most common cause of reactive lymphocytosis is infectious mononucleosis.
• In cases of mononucleosis secondary to infection with Epstein-Barr virus (EBV),
the atypical lymphocytes commonly consist of :
• polyclonal populations of CD8+ T cells which are stimulated in
response to
• γ/δ T cells EBV-infected B cells
• CD16+CD56+ NK cells

37. 1. EPSTEIN-BARR VIRUS: INFECTIOUS MONONUCLEOSIS
• Clinical features: Adolescent with fever, sore throat, lymphadenopathy, and enlarged
tonsils
• Splenomegaly 50%, hepatomegaly 10%
• Predominantly sinusoidal infiltrate of atypical T lymphocytes with minimal necrosis
• Atypical large peripheral blood lymphocytes known as DOWNEY CELLS.
• Downy cells are activated CD8 T lymphocytes,
• Confirm by serologic testing: Paul-Bunell Test, Heterophile tests
• Best test for diagnosing and monitoring EBV infections in the immunocompromised
host is the blood viral load (or quantitative EBV DNAemia assay) by PCR

38. DOWNY CELLS

40. • Expansion is mixture of B and T-cells Usually CD8 > CD4 Many
CD30 positive immunoblasts EBV positive cells (in situ
hybridization) include both small and large cells.
• Immunoblasts resembling R-S cells that are also CD30 positive
be misdiagnosed as classical Hodgkin lymphoma.
• Increased CD30+, CD20+ immunoblasts can be misdiagnosed as
diffuse large B-cell lymphoma, especially in cases with necrosis.
• So clinical history is extremely important .

41. EBV ASSOCIATED DISEASES
EBV infection is associated with Neoplasia of lymphoid and epithelial origins including-
• Endemic Burkitt’s lymphoma (eBL)
• Hodgkin’s lymphoma (HL)
• Nasopharygeal carcinoma
• Gastric carcinoma
Several immunodeficiences involving T and NK cells result in severe EBV related
outcomes includes-
• Familial hemophagocytic lymphohistiocytosis 2 (FHL2),
• X-linked lymphoproliferative syndrome (XLP),
• X-linked immunodeficiency with Mg+2 defect (XMEN) disorders

42. 2. CYTOMEGALO VIRUS: ACUTE INFECTION LYMPHOCYTOSIS
• Disorder that occurs in children usually between the ages of 2 and 10 years.
• Characterized by an increase in blood lymphocytes, often to 20 to 30 × 109 /L and
occasionally as high as 100 × 109 /L, which might be mistaken for acute leukemia.
• Lymphocytes may vary in size.
• Patients usually are asymptomatic but may have fever, abdominal pain, or diarrhea.
• Lymph node enlargement and splenomegaly do not occur.
• Patient’s serum usually is negative for heterophile antibodies found in patients with infectious
mononucleosis caused by EBV
• Disease resembles infectious mononucleosis caused by viruses other than EBV, such as
cytomegalovirus.
• Typical small lymphocyte with dense chromatin pattern and scant rim of cytoplasm and
somewhat two larger lymphocytes with less-dense chromatin pattern.

43. BORDETELLA PERTUSSIS
• Bordetella pertussis : Gram-negative bacterium
• Absolute lymphocyte counts range from 8 to 70 × 109 /L,
with a mean of approximately 30 × 109 /L.
• Involve all lymphocyte subsets.
• Lymphocytosis primarily results from failure of
lymphocytes to leave the blood because of pertussis
toxin, which is released by the bacteria.
• A notable proportion of lymphocytes have cleaved
nuclei, characteristic of the cells in cases of pertussis

44. STRESS LYMPHOCYTOSIS
• Both trauma and nontraumatic stress have been associated with lymphocytosis.
• Elevated lymphocyte count, often greater than 5 × 109 /L, which may revert to
normal or below-normal levels within hours.
• Trauma, surgery, acute cardiac failure, septic shock, myocardial infarction, sickle
cell crisis, or status epilepticus may be associated with an elevated lymphocyte
count.
• A transient lymphocytosis can be induced by the redistribution of leukocyte
subsets after both physical and psychological stress.
• Characteristically, two phases are recognized after catecholamine administration:
a quick (<30min) mobilization of lymphocytes, followed by an increase in
granulocyte numbers with decreasing lymphocyte numbers.

45. DRUGS AND HYPERSENSITIVITY REACTIONS
INDUCED LYMPHOCYTOSIS
• Certain medications such as allopurinol, carbamazepine, vancomycin, and sulfa drugs
may have correlated Drug reactions with eosinophilia and systemic symptoms
(DRESS), and this can be related to lymphocytosis.
• Dasatinib used for chronic myelogenous leukemia (CML) have correlation to
lymphocytosis.
• Dasatinib cause expansion of highly differentiated CD8+ T lymphocytes or NK
cells
• Ibrutinib used for CLL is associated with lymphocytosis: just one dose of
ibrutinib, increases in the absolute lymphocyte count of up to 66 percent

46. CONTD…
• An infectious mononucleosis-like syndrome can be induced in some patients by
salazosulfapyridine or sulfasalazine.
• Idiosyncratic drug reactions also may be associated with subacute
lymphocytosis, typically developing 2 to 8 weeks after initiating administration
of the responsible drug.
• Delayed hypersensitivity reactions to insect bites, especially mosquitos, may be
associated with a large granular lymphocytic lymphocytosis and adenopathy.
• These delayed hypersensitivity reactions can be associated with EBV-NK
lymphocytosis.

47. PLASMACYTOID LYMPHOCYTES
• This is a type of reactive lymphocytosis.
• Lymphocytes are large and have deep blue-colored cytoplasm, approaching the
coloration of plasma cell cytoplasm
• They retain the nuclear appearance, cell shape, and cell size of a medium-size
lymphocyte
• They do not develop a prominent paranuclear clear zone or markedly eccentric
nuclear position as do most plasma cells
• They may be seen in a variety of situations including infections, drug hypersensitivity,
and serum-sickness-type reactions.

48. PERSISTENT LYMPHOCYTOSIS
• Patients may have subacute or chronic lymphocytosis, termed persistent
lymphocytosis.
• Patients with lymphocytosis may have underlying neoplastic disease such as
malignant thymoma may have a polyclonal T-cell lymphocytosis thought to be
secondary to the aberrant release of thymic hormones by the neoplastic thymic
epithelium.
• Patients may develop polyclonal lymphocytosis following splenectomy.
• An absolute lymphocyte count ranging from 4.0 to 8.7 × 109 /L often is noted 4 to
242 months after splenectomy and can persist for prolonged periods.
• Chronic Infections: A reactive lymphocytosis commonly is associated with many viral
and certain bacterial infections, which, if protracted, can result in subacute or
chronic lymphocytosis

49. PRIMARY LYMPHOCYTOSIS
• An increase in the absolute number of lymphocytes secondary to
an intrinsic defect in the expanded lymphocyte population.
• These conditions also are referred to as lymphoproliferative
disorders.
• Most commonly are secondary to the neoplastic accumulation of
monoclonal B cells, T cells, natural killer (NK) cells, or less fully
differentiated cells of the lymphoid lineage.

50. 1. ACUTE LYMPHOBLASTIC LEUKEMIA
• >25% marrow blasts define leukemia.
• ALL- primarily in children.
• A neoplastic disease results from multistep somatic mutations in a single lymphoid
progenitor cell at one of several discrete stages of development.
• Proliferation and accumulation of clonal blast cells in the marrow result in
suppression of hematopoiesis and, thereafter, anemia, thrombocytopenia, and
neutropenia.
• Lymphoblasts can accumulate in various extramedullary sites, especially the
meninges, gonads, thymus, liver, spleen, and lymph nodes.

51. CLINICAL FEATURES: SIGNS AND SYMPTOMS
• The clinical presentation of ALL is highly variable.
• Approximately half of patients present with fever, fatigue, lethargy, arthralgia and
bone pain
• Fever can be caused by either neutropenia-induced infection or leukemia-released
cytokines (e.g., interleukin-1, interleukin-6, and tumor necrosis factor) released from
leukemia cells.
• Fever resolves within 72 hours after the start of antileukemia therapy.
• Intracranial hemorrhage occurs mainly in patients with an initial leukocyte count
greater than 400 × 109 /L.
• Pallor, petechiae, and ecchymosis in the skin and mucous membranes, and bone
tenderness are most common signs.

52. LABORATORY FEATURES
• Anemia, neutropenia, and thrombocytopenia are common in patients with newly
diagnosed ALL.
• The severity reflects the degree of marrow replacement lymphoblasts.
• Presenting leukocyte counts range widely, from 0.1 to 1500 × 109 /L.
• Liver dysfunction as a result of leukemic infiltration.
• Chest radiography is needed to detect enlargement of the thymus or mediastinal
nodes and pleural effusions.
• Examination of the cerebrospinal fluid (CSF) is an essential diagnostic procedure.
• Traditionally, CNS leukemia is defined by the presence of at least 5 leukocytes/μL of
CSF (with leukemic blast cells apparent in a cytocentrifuged sample) or by the
presence of cranial nerve palsies.

53. DIAGNOSIS AND CELL CLASSIFICATION
B-ALL:
• Microscopy:
• Paracortical lymph node involvement (less
commonly diffuse)
• Numerous mitotic figures
• Focal starry-sky appearance
• Cytochemistry:
• MPO-ve
• PAS +ve
• Immunohistochemistry:
• CD19, cCD79a, cCD22, CD10, pax5, TdT= +
MORPHOLOGIC AND CYTOCHEMICAL ANALYSIS:

54. T-ALL: MORPHOLOGIC AND CYTOCHEMICAL ANALYSIS
• Microscopy:
• Complete nodal architecture effacement
• Capsule involvement
• Multinodular pattern mimic follicular lymphoma
• Starry sky mimic Burkitt lymphoma (nucleoli and cytoplasm less prominent
in T-LBL)
• Mitotic figures numerous
• Infiltration in surrounding structures
• Immunohistochemistry:
• TdT+
• Variable= CD1a, CD2, CD3, CD4, CD5, CD7, CD8

56. • Lymphoblasts tend to be relatively small
(ranging from the same size to twice the
size of small lymphocytes) with scanty,
often light-blue cytoplasm; a round or
slightly indented nucleus; fine to slightly
coarse and clumped chromatin; and
inconspicuous nucleoli.
• In some cases, the lymphoblasts are
large, with prominent nucleoli, moderate
amounts of cytoplasm, admixed with
smaller blasts

57. • Cytoplasmic granules are found in the
lymphoblasts of some patients with ALL.
• Granules are negative for myeloperoxidase and
myeloid-pattern Sudan black B staining.
• Granules are amphophilic, readily distinguishable
from primary myeloid granules (which stain deep
purple), and demonstrated to be mitochondria by
electron microscopy.
• B-cell blasts in Burkitt-type ALL are characterized
by intensely basophilic cytoplasm, prominent
nucleoli, and cytoplasmic vacuolation.

59. 2. CHRONIC LYMPHOCYTIC LEUKEMIA AND
RELATED DISORDERS
• Chronic lymphocytic leukemia is a malignancy of mature B cells
• Characterized by progressive lymphocytosis, lymphadenopathy,
splenomegaly, and cytopenias.
• The diagnosis of CLL requires the presence of at least 5000 circulating B
cells/μL with clonality demonstrated by flow cytometry according to
International Workshop on Chronic Lymphocytic Leukemia (IWCLL)
criteria.
• Most common leukemia of adults in western countries
• Accounts for 7% of NHL

60. • Clinical features:
• Asymptomatic subjects- routine analysis
• Drenching night sweats, fevers, and weight loss (B symptoms)
• Lymphadenopathy(not fixed or tender), anemia, splenomegaly or thrombocytopenia- less
often seen
• Recurring infectious complications, especially upper respiratory tract infections
• Autoimmune cytopenias
• Hypogammaglobulinemia
• Progression and transformation to high grade lymphoma
• 2-8%- DLBCL
• <1% Hodgkin lymphoma

61. EVALUATION OF THE PATIENT WITH CLL
Microscopy: (LN & Spleen)
• Diffuse effacement of architecture
• Proliferation of small lymphocytes- pseudofollicles
• Low mitotic activity
• Proliferation centres- c/o small lymphocytes, prolymphocytes and paraimmunoblasts
• Prolymphocytes:
• Medium sized, clumped chromatin, small nucleoli
• Paraimmunoblasts:
• Large, round to oval nuclei, dispersed chromatin, central eosinophilic nucleolus, basophilic
cytoplasm

62. Microscopy (BM & Blood):
• Small lymphocytes- clumped chromatin and
scant cytoplasm
• Smudge cells
• Prolymphocytes:
• Usually- <15%
• Atypical CLL- >15% & <55% (a/w trisomy
12)
• B- cell prolymphocyic leukemia- >55%
• BM- interstitial, nodular, mixed or diffuse
Immunophenotype:
• Leukemic cells- CD19, dim surface
IgM/IgD, CD20, CD22 & CD79b
• CD23 & CD200 – strong +
• Aberrant expression of LEF1- used
to identify infiltration in tissues

65. 3. PROLYMPHOCYTIC LEUKEMIA
• Prolymphocytes must be >55%
• Cases of CLL with t(11;14)(q13;q32) or SOX11 expressing are excluded- Mantle cell
lymphoma with leukemic expression
• Most common among >60yrs
• Clinical Features:
• B- symptoms
• Massive splenomegaly
• +/- peripheral lymphadenopathy
• Lymphocyte count >100x109/L
• Anemia, thrombocytopenia

66. • Medium sized, round nucleus,
moderately condensed
chromatin, prominent central
nucleoli and scant faint
basophilic cytoplasm
• D/D- mantle zone lymphoma-
detect Cyclin D1 over
expression or t(11;14)(q13;q32)

67. 4. HAIRY CELL LEUKEMIA
• Hairy cell leukemia (HCL) is an uncommon form of adult chronic B-cell leukemia.
• Cell of origin is uncertain, at diagnosis the characteristic leukemic cells are found in the marrow, the blood, and the
spleen.
Clinical features:
• Fatigue, infections, fever, bleeding, pancytopenia, hepatomegaly, splenomegaly.
• Monocytopenia- characteristic
• Small mature lymphoid cells with oval indented nuclei, homogenous chromatin, absent nucleoli,
abundant cytoplasm with so-called hairy projections
Immunophenotypic profile:
• Annexin A1positive- specific
• CD20, CD22, CD11c, CD25, CD123, cyclin D1- positive
• CD5 and CD10- mostly negative

70. 5. ADULT T-CELL LEUKEMIA
• ATL is an uncommon lymphoproliferative neoplasm of mature CD4+CD25+ T-cells.
• Caused by infection with the retrovirus, HTLV-1.
• Cells classically have a leukemic “flower-cell” appearance.
• At least 5 percent of circulating abnormal T lymphocytes are required to diagnose ATL.
• Mean age is 62 years
Clinical features:
• Hepatosplenomegaly, lymphadenopathy
• Elevated LDH, hypercalcemia, visceral and cutaneous lesions
• Leukemic presentation, lymphoma variant characterized by lymphadenopathy

71. • Neoplastic cells pleomorphic,
highly lobulated nuclei (“clover
leaf ” or “flower cell”
appearances), condensed nuclear
chromatin, inconspicuous nucleoli
Immunophenotype:
• Cells express the surface T-cell
lymphocytic markers CD2, CD4
and CD5, CD45RO, CD29, TCR-αβ
• Negative for CD7, CD8, and CD26
• Reduced CD3 expression

72. 6. LARGE GRANULAR LYMPHOCYTIC LEUKEMIA
• LGLL is a heterogeneous disorder characterized by an increase in the number of
blood large granular lymphocytes between 2 and 20 × 109 /L for more than 6
months without a clearly identified cause.
• Large granular lymphocytosis can result from expansions of NK cells, CD8+ T
cells, or, more rarely, CD4+ T cells.
Natural killer (NK) cell large granular lymphocytic leukemia: most common
form, termed NK lymphocytosis
• NK cell counts typically approximate 4 × 109 /L, but can sometimes exceed 15 ×
109 /L.
• Patients with NK lymphocytosis frequently have recurrent cutaneous lesions, such
as livedoid vasculopathy, urticarial vasculitis, or complex recurrent aphthous
stomatitis.

73. CD8+ T-cell large granular lymphocytic leukemia
CD4+ T-cell large granular lymphocytic leukemia
• T-cell large granular lymphocytosis may be secondary to an
exaggerated cellular immune response to infection with human
CMV.
• Large granular lymphocytosis also may be associated with
rheumatoid arthritis.
• It is almost invariably is associated with neutropenia in the absence
of splenomegaly and thus may represent a subset of Felty
syndrome.

74. MONOCLONAL B-CELL LYMPHOCYTOSIS
• Multiparameter flow cytometric and molecular diagnostic techniques has identified a
syndrome in patients who have expanded populations of monoclonal B cells without
other associated clinical signs or symptoms.
• An absolute B-cell count of less than 5.0 × 109 /L rather than the absolute
lymphocyte count is used to distinguish MBL from CLL.
• MBL could be diagnosed in two situations:
1. Screening MBL/ low-count MBL: In subjects with a normal lymphocyte count via a
screening assay
<500 monoclonal B cells per μL
diagnosed when high-sensitivity flow cytometric techniques are used in
unaffected sibling families with a genetic predisposition to CLL

75. 2. Clinical MBL: Clinical MBL is more commonly encountered in clinical practice
when patients are evaluated for lymphocytosis.
• This condition is biologically indistinguishable from CLL.
• Individuals with clinical MBL should be followed with a physical examination and
complete blood counts by a hematologist every 6 to 12 months, while longer
followup intervals of 12 to 18 months are recommended in screening MBL.

76. PERSISTENT POLYCLONAL B CELL LYMPHOCYTOSIS
• Defined as a chronic, moderate increase in absolute lymphocyte counts (>4 × 109 /L)
without evidence for infection or other conditions that can increase the lymphocyte
count.
• It is a rare disorder that mostly affects young to middle-age women who often are
human leukocyte antigen (HLA)-DR7 positive and is associated with smoking.
• It is characterized by the persistent expansion of CD27+immunoglobulin (Ig) M+
IgD+ B cells and increased IgM serum levels.
• Presence of circulating binucleated lymphocytes and have an unusual binucleated
appearance on the blood film.
• Specific morphologic features: basophilic vacuolated cytoplasm and monocytoid
changes.
• These lymphocytes typically have low-to-negligible expression of CD5 or CD23
found in patients with CLL and are polyclonal with respect to light-chain expression
and immunoglobulin heavy-chain gene rearrangements

77. • Patients can have features resembling those of patients with various monoclonal B-
cell malignancies.
• Patients may have mild splenomegaly
• Histologic examination of marrow and secondary lymphoid tissues from patients with
progressive splenomegaly can reveal features resembling marginal zone B-cell
lymphoma.
• Although the lymphocytosis generally is not progressive, most patients have small
numbers of blood B cells with chromosomal abnormalities, include
• An additional isochromosome +i(3q)
• Premature chromosome condensation
• t(14;18) translocation involving the BCL-2
• Immunoglobulin heavy-chain loci

79. REFERENCES
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81. MCQ’S
1. The final activated and differentiated cell during T cell development is:
A) Prolymphocyte
B) Large lymphocyte
C) Small lymphocyte
D) Lymhoblast
Ans: C) Small lymphocyte

82. 2. Which of the following cells are ‘non MHC’ restricted ?
A) γδ T cells
B) T regulatory cells
C) Cytotoxic T cells
D) Memory T cells
Ans: A) γδ T cells

83. 3. Which of the following is not a T cell marker ?
A) CD45
B) CD3
C) Tdt
D) CD10
Ans: D) CD10

84. 4. Earliest event during B cell development :
A) PAX 5 expression
B) Heavy chain gene rearrangement
C) Light chain gene rearrangement
D) CD79a expression
Ans: B) Heavy chain gene rearrangement

85. 5. Which of the following is a NK cell marker :
A) PAX-5
B) CD19
C) CD56
D) Tdt
Ans: C) CD56

86. 6. Atypical lymphocyte seen in Infectitious mononucleosis known as:
A) R-S cell
B) Downy cells
C) Popcorn cell
D) Ballet cells
Ans: B) Downy cells

87. 7. Causes of reactive lymphocytosis are all except:
A) Mononucleosis syndromes
B) Stress lymphocytosis
C) Monoclonal B- cell lymphocytosis
D) Persistent lymphocytosis
Ans: C) Monoclonal B- cell lymphocytosis

88. 8. Lymphocytosis in adults defined as:
A) Absolute lymphocyte count >3.0 109/L
B) Absolute lymphocyte count >10.0 3 109/L
C) Absolute lymphocyte count >12.0 109/L
D) Absolute lymphocyte count exceeding 4 × 109 /L
Ans: D) Absolute lymphocyte count exceeding 4 × 109 /L

89. THANKYOU!