1. Leukemia/Lymphoma

2. Components and General Properties of Blood
Seven kinds of formed elements
Erythrocytes: red blood cells (RBCs)
Platelets (thrombocytes) (clotting cells)
Cell fragments from special cell in bone marrow
Leukocytes: white blood cells (WBCs)-5 kinds

3. Components and General Properties of Blood
Five leukocyte types divided into two categories
Granulocytes (with granules)
3)Neutrophils
4)Eosinophils
5)Basophils
Agranulocytes (without granules)
6)Lymphocytes
7)Monocytes

4. Components and General Properties of Blood
Monocyyte
Small
lymphocyte
Neutrophil
Platelets
Eosinophil
Small
lymphocyte
Erythrocyte
Young (band)
neutrophil
Neutrophil
Monocyte
Large
lymphocyte
Neutrophil
Basophil

5. Leukocytes Least abundant formed element
5,000 to 10,000 WBCs/L
Protect against infectious microorganisms and other pathogens
Conspicuous nucleus
Spend only a few hours in the bloodstream before migrating to connective tissue
Retain their organelles for protein synthesis
Granules
All WBCs have lysosomes called nonspecific (azurophilic) granules: inconspicuous so cytoplasm looks clear
Granulocytes have specific granules that contain enzymes and other chemicals employed in defense against pathogens

6. Types of Leukocytes Granulocytes
Neutrophils (60% to 70%): polymorphonuclear leukocytes
Barely visible granules in cytoplasm; three- to five-lobed nucleus
Eosinophils (2% to 4%)
Large red-orange granules;
Basophils (less than 1%)
Large, abundant, violet granules

7. Agranulocytes Lymphocytes (25% to 33%) Monocytes (3% to 8%)
Variable amounts of bluish cytoplasm (scanty to abundant); ovoid/round, uniform dark violet nucleus
Monocytes (3% to 8%)
Largest WBC; generally ovoid, kidney-, or horseshoe-shaped nucleus

8. Granulocytes Neutrophils—increased numbers in bacterial infections
Phagocytosis of bacteria
Release antimicrobial chemicals
Eosinophils—increased numbers in parasitic infections, collagen diseases, allergies, diseases of spleen and CNS
Phagocytosis of antigen–antibody complexes, allergens, and inflammatory chemicals
Release enzymes to destroy large parasites

9. Basophils—increased numbers in chickenpox, sinusitis, diabetes
Secrete histamine (vasodilator): speeds flow of blood to an injured area
Secrete heparin (anticoagulant): promotes the mobility of other WBCs in the area

10. Agranulocytes
Lymphocytes—increased numbers in diverse infections and immune responses
Destroy cells (cancer, foreign, and virally infected cells)
“Present” antigens to activate other immune cells
Coordinate actions of other immune cells
Secrete antibodies and provide immune memory

11. Agranulocytes
Monocytes—increased numbers in viral infections and inflammation
Leave bloodstream and transform into macrophages
Phagocytize pathogens and debris
“Present” antigens to activate other immune cells—antigen-presenting cells (APCs)

12. The Leukocyte Life Cycle
Leukopoiesis—production of white blood cells
Pluripotent stem cells (PPSCs)
Myeloblasts—form neutrophils, eosinophils, basophils
Monoblasts—form monocytes
Lymphoblasts give rise to all forms of lymphocytes

13. Leukopoiesis leaves Pluripotent stem cell Colony-forming units (CFUs)
Precursor
cells
Mature
cells
leaves
Eosinophilic
CFU
Eosinophilic
myeloblast
Eosinophilic
promyelocyte
Eosinophilic
myelocyte
Eosinophil
Basophilic
CFU
Basophilic
myeloblast
Basophilic
promyelocyte
Basophilic
myelocyte
Basophil
Neutrophilic
CFU
Neutrophilic
myeloblast
Neutrophilic
promyelocyte
Neutrophilic
myelocyte
Neutrophil
Monocytic
CFU
Monoblast
Promonocyte
Monocyte
B lymphocyte
B prolymphocyte
T prolymphocyte
T lymphocyte
Lymphocytic
CFU
Lymphoblast
NK prolymphocyte
NK cell

14. What Is Leukemia? Cancer of the white blood cells Acute or Chronic
Affects ability to produce normal blood cells
Bone marrow makes abnormally large number of immature white blood cells called blasts

15. History Means “white blood” in Greek
Discovered by Dr. Alfred Velpeau in France, 1827
Named by pathologist Rudolf Virchow in Germany, 1845

16. Leukocyte Disorders
Leukemia—cancer of hemopoietic tissue that usually produces an extraordinary high number of circulating leukocytes and their precursors
Myeloid leukemia: uncontrolled granulocyte production
Lymphoid leukemia: uncontrolled lymphocyte or monocyte production
patient subject to opportunistic infection, anemia

17. Acute vs Chronic Leukemia
Acute leukemia: appears suddenly, progresses rapidly, death within months –blasts found in peripheral blood
Chronic leukemia: undetected for months, survival time average of 3 years
Effects: normal cell percentages disrupted; impaired clotting; opportunistic infections

18. Main Types Acute Lymphocytic Leukemia (ALL)
Acute Myelogenous Leukemia (AML)
Chronic Lymphocytic Leukemia (CLL)
Chronic Myelogenous Leukemia (CML)

19. Demographics of Leukemia Patients (2001 Data)
CLL=Chronic Lymphocytic
ALL=Acute Lymphocytic
CML=Chronic Mylogenous
AML=Acute Mylogenous
Total Reported Cases = 31,500

20. Pictures Of Blood Normal human blood Blood with leukemia White Cell
Red Cell
Platelet
Blood with leukemia
Blasts
Red Cell
Platelet
White Cell

21. Development of Leukemia in the Bloodstream
Stage 1- Normal
Stage 2- Symptoms
Stage 3- Diagnosis
Stage 5a- Anemia
Stage 5b- Infection
Stage 4- Worsening
Legend
White Cell
Red Cell
Platelet
Blast
Germ

22. Causes High level radiation/toxin exposure Viruses Genes Chemicals
Mostly unknown

23. Signs and Symptoms of AML
Insidious nonspecific onset
Pallor due to anemia
Febrile (fever) due to ineffective WBC
Petechiae (skin bruising) due to thrombocytopenia
Bone pain
Petechiae

24. Typical Labs of AML Leukocytosis Blastemia Leukemic hiatus
Auer rods – only found in myelocytic blasts
Thrombocytopenia
Anemia
>20% blasts in Bone Marrow

25. Auer Rods
Auer Rod

26. CD Markers
The cluster of differentiation (cluster of designation) (often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cells.
The CD markers can be used to identify the type of cell.

27. Other Findings CD 13 and CD 33 in flowcytometry
Cytochemistries-stains that can be used to differentiate leukemias
Myeloperoxidase
Sudan black B
Choloroacetate esterase (specific)
Nonspecific esterase

28. Flow Cytometry
Large clustering of CD 33s shows presence of blasts

29. FAB (1976) Classification M0 -- Undifferentiated AML
M1 -- AML without maturation
M2 -- AML with maturation
M3 -- Acute Promyelocytic Leukemia
M4 -- Acute Myelomonocytic Leukemia
M5 -- Acute Monocytic Leukemia
M6 -- Erythroleukemia (DiGuglielmo’s)
M7 -- Megakaryoblastic Leukemia

31. Myeloperoxidase
(MPO)
p-Phenylene diamine + Catecol + H2O2
MPO > Brown black deposits
Brown deposits considered to be a positive test-differentiates AML from other leukemias

32. M1 and M2

33. M3 M4 M5

34. Chloracetate (Specific) Esterase
Myeloid Cell Line
Naphthol-ASD-chloracetate
CAE > Free naphthol compounds
+ Stable diazonium salt (eg, Fast Corinth)
> Red deposit

35. Non-Specific Esterase
Monocytic Line
 Naphthyl acetate
ANAE > Free naphthyl compounds
+Stable diazonium salt (eg, Fast blue RR)
> Brown deposits

36. FAB vs WHO Classifications of Hematologic Neoplasm
FAB criteria
Morphology
Cytochemistry
WHO criteria
Morphology
Immunophenotyping
Genetic features
Karyotyping
Molecular testing
Clinical features

37. WHO Classification of AML
AML with recurrent cytogenic translocations
AML with multi-lineage dysplasia
AML and myelodysplasia, therapy related
AML, not otherwise categorized

38. AML with Recurrent Cytogenetic Translocations (WHO 1995)
t(8;21) -- some maturation of neutrophilic line; rare in older patients; AML1/ETO fusion protein; >90% FAB M2
t(15;17) -- APL (granular and microgranular variants); retinoic acid receptor (RAR) leukemias; middle aged adults; DIC
inv(16) or t(16;16) -- monocytic and granulocytic; abnormal eosinophilic component
11q23 -- monocytic; children; most common is t(9;11)

39. Lymphocytic Leukemias
Can involve T or B lymphocytes
B lymphs mature in bone marrow
Responsible for making antibodies
T lymphs go to thymus to mature
Are cytotoxic cells of immune system

43. FAB Classification of ALL
L1: Small homogeneous blasts; mostly in children
L2: Large heterogeneous blasts; mostly in adults
L3: “Burkitt” large basophilic B-cell blasts with vacuoles

44. L2 L3

46. Periodic Acid Schiff
Periodic acid + Glycogen
oxidation > Aldehyde + Schiff reagent
(para-rosaniline, Na metabisulfite)
> Red deposit

47. ALL Cytochemistries Oil Red O: stains L3 vacuoles
Terminal deoxynucleotidyl transferase (Tdt): DNA polymerase in early lymphoblasts
Cell surface markers (CD’s)
Cytoplasmic and surface immunoglobulins: B-cell line
T-cell receptor (TCR)

48. WHO Classification of Lymphoproliferative Syndromes
Precursor B Lymphoblastic Leukemia/Lymphoma (ALL/LBL) -- ALL in children (80-85% of childhood ALL); LBL in young adults and rare; FAB L1 or L2 blast morphology
Precursor T ALL/LBL -- 15% of childhood ALL and 25% of adult ALL
Burkitt Leukemia/Lymphoma (FAB L3)

49. Prognosis Indicators Favorable Poor WBC < 50,000/L  50,000/L Age
1 - 10
< 1 or 10
Gender
Female
Male
Blast
B-cell
T-cell and mixed
Karyotype
Hyperploidy
Trisomy 4, 10, 17
t(12;21) (TEL/AML1)
Hypoploidy
Trisomy 5
t(1;19 (E2A/PBX1)
Mixed lineage leukemia
T(9;22) (Ph)
BM blast count during induction
Mkd reduction at day 7
Mild reduction at day 7

50. Burkitt’s Lymphoma

51. CML

52. Typical Labs in CML Leukocytosis with blastemia Thrombocytosis
Basophilia
Micro-megakaryocytes
Low LAP score (intermediate if infected)
About 10% blasts in BM
Philadelphia chromosome

53. Bone marrow aspirate
and biopsy

54. Conventionally, a leukocytosis exceeding 50,000 WBC/mm3 with a significant increase in early neutrophil precursors is referred to as a leukemoid reaction.

55. Serum leukocyte alkaline phosphatase is normal or elevated in leukemoid reaction, but is depressed in chronic myelogenous leukemia.

56. Leukemoid reactions are generally benign and are not dangerous in and of themselves, although they are often a response to a significant disease state

57. Historically, various clues including the leukocyte alkaline phosphatase score and the presence of basophilia were used to distinguish CML from a leukemoid reaction. However, at present the test of choice in adults to distinguish CML is an assay for the presence of the Philadelphia chromosome, either via cytogenetics and FISH, or via PCR for the BCR/ABL fusion gene.

58. Leukocyte Alkaline Phosphatase (LAP)
Naphthol AS-MX phosphate LAP at pH8.6 >
Naphthol AS-MX + Diazonium salt
(eg, Fast blue RR)
> Insoluble pigment

59. LAP Score Count 100 consecutive segmented neutrophils and bands Score:
0 = no granules
1+ = occasional diffuse granules
2+ = moderate number of granules
3+ = many strongly positive granules
4+ = confluent strongly positive granules

60. 1+ 2+ 3+ 4+

61. LAP Score Example: 0 x 35 cells = 0 1+ x 30 cells = 30

62. Philadelphia Chromosome
9 ;22 translocation almost specific to CML
Karyotype to visualize Ph chromosome
Produces BCR/c-abl fusion oncogene
Gene product p190 is a hyperactive tyrosine kinase
Ph chromosome seen in ALL produces p210 and chronic neutrophilic leukemia produces p230

63. Karyotype 46,XX,t(9;22)(q34;q11.2) -- Ph chromosome

65. FISH showing the BCR (green), ABL (orange), and BCR-ABL fusion signals (arrow): A=positive (contains a residual ABL signal), B=normal

66. Chronic Lymphocytic Leukemia
Exclusive in elderly
Lymphocytosis unrelated to viral infection
Hyper-mature lymphocytes with highly condensed nuclei
Smudge cells

67. CLL
PB and BM
Smudge cell

70. Bone Marrow