1. INVESTIGATION OF LEUKOCYTES. CHANGES IN LETSKOTYC FORMULAS IN VARIOUS PATHOLOGICAL CONDITIONS. CLINICAL AND LABORATORY DIAGNOSIS OF HEMOBLASTOSIS

2. The WBC count determines the number of leukocytes per cubic millimeter of whole blood. The counting is performed very rapidly by electronic devices. The WBC may be performed as part of a CBC, alone, or with differential WBC count. An elevated WBC count is termed leukocytosis; a decreased count, leukopenia. In addition to the normal physiological variations in WBC count, many pathological problems may result in an abnormal WBC count. The WBC count determines the number of leukocytes per cubic millimeter of whole blood. The counting is performed very rapidly by electronic devices. The WBC may be performed as part of a CBC, alone, or with differential WBC count. An elevated WBC count is termed leukocytosis; a decreased count, leukopenia. In addition to the normal physiological variations in WBC count, many pathological problems may result in an abnormal WBC count.

3. Leukemia A group of malignant disorders affecting the blood and blood-forming tissues of A group of malignant disorders affecting the blood and blood-forming tissues of Bone marrow Bone marrow Lymph system Lymph system Spleen Spleen Occurs in all age groups Occurs in all age groups

5. Leukemia Results in an accumulation of dysfunctional cells because of a loss of regulation in cell division Results in an accumulation of dysfunctional cells because of a loss of regulation in cell division Fatal if untreated Fatal if untreated Progressive Progressive

6. Leukemia Often thought of as a childhood disease Often thought of as a childhood disease The number of adults affected with leukemia is 10 times that of children The number of adults affected with leukemia is 10 times that of children

8. Leukemia Etiology and Pathophysiology No single causative agent No single causative agent Most from a combination of factors Most from a combination of factors Genetic and environmental influences Genetic and environmental influences

9. Leukemia Etiology and Pathophysiology Associated with the development of leukemia Associated with the development of leukemia Chemical agents Chemical agents Chemotherapeutic agents Chemotherapeutic agents Viruses Viruses Radiation Radiation Immunologic deficiencies Immunologic deficiencies

10. Development of Leukemia in the Bloodstream Stage 1- NormalStage 2- SymptomsStage 3- Diagnosis Stage 4- Worsening Stage 5a- Anemia Stage 5b- Infection Legend White Cell Red Cell Platelet Blast Germ Sources from Leukemia, by D. Newton and D. Siegel

11. Leukemia Classification Acute versus chronic Acute versus chronic Cell maturity Cell maturity Acute: clonal proliferation of immature hematopoietic cells (the formation of blood or blood cells ) Acute: clonal proliferation of immature hematopoietic cells (the formation of blood or blood cells ) Chronic: mature forms of WBC; onset is more gradual Chronic: mature forms of WBC; onset is more gradual Nature of disease onset Nature of disease onset

12. Leukaemogenesis

13. Leukaemogenesis Philadelphia chromosome is an acquired cytogenetic anomaly that is characterizes in all leukaemic cells in CML Philadelphia chromosome is an acquired cytogenetic anomaly that is characterizes in all leukaemic cells in CML 90-95% of CML pts have Ph chromosome 90-95% of CML pts have Ph chromosome Reciprocal translocation of chromosome 22 and chromosome 9 Reciprocal translocation of chromosome 22 and chromosome 9

15. Acute Leukemia Characterized by immature or undifferentiated hematopeitic cells, usually a blast cell. Characterized by immature or undifferentiated hematopeitic cells, usually a blast cell. Onset is rapid and abrupt Onset is rapid and abrupt

16. Chronic lymphocytic leukemia Is characterised by the accumulation of nonproliferating mature-appearing lymphocytes in the blood, marrow, lymph nodes, and spleen Is characterised by the accumulation of nonproliferating mature-appearing lymphocytes in the blood, marrow, lymph nodes, and spleen In most cases, the cells are monoclonal B lymphocytes that are CD5+ In most cases, the cells are monoclonal B lymphocytes that are CD5+ T cell CLL can occur rarely T cell CLL can occur rarely

17. Chronic lymphocytic leukemia Is the most common form of leukemia in North America and Europe, but is extremely rare in the Orient Is the most common form of leukemia in North America and Europe, but is extremely rare in the Orient Typically occurs in older patients, with the highest incidence being in those aged 50 to 55 years Typically occurs in older patients, with the highest incidence being in those aged 50 to 55 years Affects men twice as often as women Affects men twice as often as women

18. Etiology (1) The cause of CLL is unknown The cause of CLL is unknown There is increased incidence in farmers, rubber manufacturing workers, asbestos workers, and tire repair workers There is increased incidence in farmers, rubber manufacturing workers, asbestos workers, and tire repair workers Genetic factors have been postulated to play a role in high incidence of CLL in some families Genetic factors have been postulated to play a role in high incidence of CLL in some families

19. Etiology Cytogenetics Cytogenetics clonal chromosomal abnormalities are detected in approximately 50% of CLL patients clonal chromosomal abnormalities are detected in approximately 50% of CLL patients the most common clonal abnormalities are: the most common clonal abnormalities are: trisomy 12 trisomy 12 structural abnormalities of chromosomes 13, 14 and 11 structural abnormalities of chromosomes 13, 14 and 11 patients with abnormal karyotypes have a worse prognosis patients with abnormal karyotypes have a worse prognosis Oncogenes Oncogenes in most cases of CLL is overexpressed the proto- oncogene c-fgr 9a member of the src gene family of tyrosine kinases in most cases of CLL is overexpressed the proto- oncogene c-fgr 9a member of the src gene family of tyrosine kinases

20. Clinical findings Approximately 40% of CLL patients are asymptomatic at diagnosis Approximately 40% of CLL patients are asymptomatic at diagnosis In symptomatic cases the most common complaint is fatigue In symptomatic cases the most common complaint is fatigue Less often the initial complaint are enlarged nodes or the development of an infection (bacterial) Less often the initial complaint are enlarged nodes or the development of an infection (bacterial)

21. Clinical findings Most symptomatic patients have enlarged lymph nodes (more commonly cervical and supraclavicular) and splenomegaly Most symptomatic patients have enlarged lymph nodes (more commonly cervical and supraclavicular) and splenomegaly The lymph nodes are usually discrete, freely movable, and nontender The lymph nodes are usually discrete, freely movable, and nontender Hepatomegaly may occure Hepatomegaly may occure Less common manifestation are infiltration of tonsils, mesenteric or retroperitoneal lymphadenopathy, and skin infiltration Less common manifestation are infiltration of tonsils, mesenteric or retroperitoneal lymphadenopathy, and skin infiltration Patients rarely present with features of anemia, and bruising or bleeding Patients rarely present with features of anemia, and bruising or bleeding

22. Laboratory findings The blood lymphocyte count above 5,0 G/L The blood lymphocyte count above 5,0 G/L In most patients the leukemic cells have the morphologic appearance of normal small lymphocytes In most patients the leukemic cells have the morphologic appearance of normal small lymphocytes In the blood smears are commonly seen ruptured lymphocytes (“basket” or “smudge” cells) In the blood smears are commonly seen ruptured lymphocytes (“basket” or “smudge” cells) Careful examination of the blood smear can usually differentiate CLL, and the diagnosis can be confirmed by immunophenotyping Careful examination of the blood smear can usually differentiate CLL, and the diagnosis can be confirmed by immunophenotyping

25. Laboratory findings Clonal expansion of B (99%) or T(1%) lymphocyte Clonal expansion of B (99%) or T(1%) lymphocyte In B-cell CLL clonality is confirmed by In B-cell CLL clonality is confirmed by the expression of either  or light chains on the cell surface membrane the expression of either  or light chains on the cell surface membrane the presence of unique idiotypic specificities on the immunoglobulins produced by CLL cells the presence of unique idiotypic specificities on the immunoglobulins produced by CLL cells by immunoglobulin gene rearrangements by immunoglobulin gene rearrangements typical B-cell CLL are unique in being CD19+ and CD5+ typical B-cell CLL are unique in being CD19+ and CD5+ Hypogammaglobulinemia or agammaglobulinemia are often observed Hypogammaglobulinemia or agammaglobulinemia are often observed 10 - 25% of patients with CLL develop autoimmune hemolytic anemia, with a positive direct Coombs’ test 10 - 25% of patients with CLL develop autoimmune hemolytic anemia, with a positive direct Coombs’ test The marrow aspirates shows greater than 30% of the nucleated cells as being lymphoid The marrow aspirates shows greater than 30% of the nucleated cells as being lymphoid

26. The diagnostic criteria for CLL 1) A peripheral blood lymphocyte count of greater than 5 G/L, with less than 55% of the cells being atypical 2) The cell should have the presence of Bcell- specific differentiation antigens (CD19, CD20, and CD24) and be CD5(+) 3) A bone marrow aspirates showing greater than 30% lymphocytes

27. Differential diagnosis Infectious causes Infectious causes bacterial (tuberculosis) bacterial (tuberculosis) viral (mononucleosis) viral (mononucleosis) Malignant causes Malignant causes B-cell B-cell T-cell T-cell leukemic phase of non-Hodgkin lymphomas leukemic phase of non-Hodgkin lymphomas Hairy-cell leukemia Hairy-cell leukemia Waldenstrom macroglobulinemia Waldenstrom macroglobulinemia large granular lymphocytic leukemia large granular lymphocytic leukemia

29. Investigations Pretreatment studies of patients with CLL should include examination of: Pretreatment studies of patients with CLL should include examination of: complete blood count complete blood count peripheral blood smear peripheral blood smear reticulocyte count reticulocyte count Coomb’s test Coomb’s test renal and liver function tests renal and liver function tests serum protein electrophoresis serum protein electrophoresis immunoglobulin levels immunoglobulin levels plasma  2 microglobulin level plasma  2 microglobulin level If available immunophenotyping should be carried out to confirm the diagnosis If available immunophenotyping should be carried out to confirm the diagnosis Bone marrow biopsy and cytogenetic analysis is not routinely performed in CLL Bone marrow biopsy and cytogenetic analysis is not routinely performed in CLL

30. Staging Rai Classification for CLL Rai Classification for CLL 0 - lymphocytosis (>5 G/L) 0 - lymphocytosis (>5 G/L) I - lymphocytosis + lymphadenopathy I - lymphocytosis + lymphadenopathy II - lymphocytosis + splenomegaly +/-lymphadenopathy II - lymphocytosis + splenomegaly +/-lymphadenopathy III - lymphocytosis + anemia (Hb <11g%) +/- lymphadenopathy or splenomegaly III - lymphocytosis + anemia (Hb <11g%) +/- lymphadenopathy or splenomegaly IV - lymphocytosis + thrombocytophenia (Plt <100G/L) +/- anemia +/-lymphadenopathy +/- splenomegaly IV - lymphocytosis + thrombocytophenia (Plt <100G/L) +/- anemia +/-lymphadenopathy +/- splenomegaly

33. Prognosis Rai classification Rai classification stagemedian survival (years) 0>10 I> 8 II 6 III 2 IV< 2 Binet classification Binet classification stagemedian survival (years) A> 10 B 7 C 2

35. Markers of poor prognosis in CLL Advanced Rai or Binet stage Advanced Rai or Binet stage Peripheral lymphocyte doubling time <12 months Peripheral lymphocyte doubling time <12 months Diffuse marrow histology Diffuse marrow histology Increased number of prolymphocytes or cleaved cells Increased number of prolymphocytes or cleaved cells Poor response to chemotherapy Poor response to chemotherapy High  2 - microglobulin level High  2 - microglobulin level Abnormal karyotyping Abnormal karyotyping

36. Treatment Treatment is reserved for patients with low- or intermediate risk disease who are symptomatic or have progressive disease (increasing organomegaly or lymphocyte doubling time of less than 12 months) and patients with high -risk disease Treatment is reserved for patients with low- or intermediate risk disease who are symptomatic or have progressive disease (increasing organomegaly or lymphocyte doubling time of less than 12 months) and patients with high -risk disease Alkylating agents (chlorambucil, cyclophosphamide) Alkylating agents (chlorambucil, cyclophosphamide) Nucleoside analogs (cladribine, fludarabine) Nucleoside analogs (cladribine, fludarabine) Biological response modifiers Biological response modifiers Monoclonal antibodies Monoclonal antibodies Bone marrow transplantation Bone marrow transplantation And systemic complications requiring therapy And systemic complications requiring therapy antibiotics antibiotics immunoglobulin immunoglobulin steroids steroids blood products blood products