This document discusses leukocyte disorders and provides information about acute leukemia. It defines leukocytosis and leukopenia as increases or decreases in white blood cell count. Non-neoplastic causes of changes in white blood cells include neutrophilia, lymphocytosis, eosinophilia, monocytosis, and basophilia. Neoplastic disorders include acute myeloid leukemia and acute lymphoblastic leukemia. The document outlines the classification, pathogenesis, clinical features, diagnosis and treatment of acute leukemias. Diagnosis involves examination of blood and bone marrow smears, cytochemistry, immunophenotyping, cytogenetics and molecular analysis to determine the leukemia subtype and guide treatment.

1. Leukocyte disorders
Dr Samiyah Syeed Ahmed

2. Leukocyte disorders
1. Non Neoplastic
2. Neoplastic

3. Non neoplastic disorders
Leukocytosis- Increase in the total WBC count
Leukopenia- Decrease in the total WBC count

4. What is leukocytosis?
Leukocytosis is the increase in WBC count >11,000 cells/Ml in the peripheral blood

5. What is leukopenia?
Leukopenia is the reduction of WBCs in the peripheral blood below <4000 cells/Ml

6. Normal reference ranges
● Adults: 4000-11,000/μl
● At birth: 10,000-26000/μl
● 1 year: 6,000-16,000/μl
● 6-12 years: 5,000-13,000/μl
● Pregnancy: up to 15,000/μl

7. Causes of Leukocytosis

8. Neutrophilia
● An absolute neutrophil count (ANC) greater than 7500/μl
● On differential Leucocyte count: > 75% neutrophils in the peripheral blood
● Usually accompanied by Leukocytosis
● Usually associated with shift to left
● In the presence of Bacterial infection: Neutrophils show coarse granules and
vacuoles

10. Lymphocytosis
This is an increase in absolute lymphocyte count above upper limit of normal for age
(4000/μl in adults, >7200/ μl in adolescents, >9000/μl in children and infants)
On differential leucoycte count:
Lymphocytes are more than 45 % in the peripheral blood.

12. Eosinophilia
This refers to absolute eosinophil count (AEC) greater than 600/μl.

14. Monocytosis

15. Monocytosis
This is an increase in the absolute monocyte count above 1000/μl.

16. Basophilia
Increased in:
● Chronic Myeloid Leukemia
● Polycythemia Vera
● Ulcerative colitis
● Myxedema
● Mastocytosis

17. Differential leucocyte count- Normal
● Neutrophils: 40-75%
● Lymphocytes: 20-40% (in children between 4 months to 4 years of age,
percentage of lymphocytes is more than neutrophils; this is called as inverted
differential in children)
● Monocytes: 2-10%
● Eosinophils: 1-6%
● Basophils: 0-1%

18. Neutropenia (Agranulocytosis)
● Term used when neutrophil count is less than 1.5 x 109/L
● Mild : ANC between 1 and 1.5 x 109/L
● Moderate: ANC between 0.5-1 x 109/L
● Severe: ANC less than 0.5 x 109/L
● Agranulocytosis is a condition in which the absolute neutrophil count (ANC) is less than 100 neutrophils per microlitre
of the blood
● Agranulocytosis can be Hereditary or acquired

19. Neutropenia
a) Suppression of myelopoiesis: Selective or generalized
b) Peripheral destruction: Due to Autoantibodies
c) Peripheral pooling of white cells: Seen in hemodialysis and cardiopulmonary
bypass

21. Drug induced neutropenia
Immune mediated destruction: Aminopyrine, penicillin, gold and anti thyroid drugs
Immune complex mechanisms: Quinidine induced agranulocytosis
Dose dependent inhibition of granulopoiesis: Beta lactam antibiotics

22. Clinical picture
● Prone to recurrent infections
● Manifest with sore throat, boils, skin infections
● Paronychia
● Ulcers in the mouth
● Abscesses
● Poor wound healing

24. Diagnosis
● History: new medication or change in medication
● Recent exposure to chemical/physical agents
● Recent viral or bacterial infection is usually associated with agranulocytosis.
● CBC : Leucopenia with neutropenia
● Neutrophils 0-10% (ANC < 100 per microliter of blood)
● Relative lymphocytosis
● Bone marrow: Essential to rule out other causes of neutropenia such as alekuemic
leukemia, Megaloblastic anemia and aplastic anemia
● Cellularity : Normal
● Erythropoiesis: Normal
● Myelopoiesis: Arrests at Promyleocyte/ myelocyte stage
● Megakaryppoiesis : Normal

25. Management of Neutropenia
● Identify the cause
● Stop the offending drug
● Culture studies, to ensure appropriate antibiotic therapy
● Granulocyte transfusions
● Hematopoietic growth factors: GM CSF and G CSF

26. Acute Leukemia

29. Stages of Myelopoeisis

31. Definition
Neoplastic proliferations of hematopoietic cells
Acute leukemia is defined as neoplasms with more than 20% blasts in the
peripheral blood or bone marrow (WHO)

32. Blast equivalents
In a few cases, cells other than blast cells are counted as blast cells and are known as
blast equivalents
● Promonocytes in monocytic leukemia and myelomonocytic leukemia
● Promyelocytes in acute promyelocytic leukemia
● Erythroblasts in acute erythroleukemia

33. Classification
Leukemias are classified into 2 major groups:
Acute: Acute Onset is usually rapid
The disease is very aggressive
The cells involved are usually poorly differentiated with many blasts
Chronic: Onset is insidious
The disease is usually less aggressive
The cells involved are usually more mature cells

34. Classification
Both acute, chronic leukemias are further classified according to the prominent
cell line:
If the prominent cell line is of the myeloid series : MYELOCYTIC LEUKEMIA
(sometimes also called granulocytic)
If the prominent cell line is of the lymphoid series: LYMPHOCYTIC LEUKEMIA

35. Classification
Therefore, there are four basic types of leukemia
Acute myelocytic leukemia – AML Acute lymphocytic leukemia –
ALL
Chronic myelocytic leukemia – CML Chronic lymphocytic leukemia –
CLL

36. Etiological factors
● Host factors:
○ Inherited tendency for chromosome fragility or abnormality
○ Chromosomal disorder (such as Down’s syndrome)
○ Hereditary immunodeficiencies
○ Chronic marrow dysfunction such as those with myeloproliferative diseases, myelodysplastic
syndromes, aplastic anemia, or paroxsymal nocturnal hemoglobinuria
● Environmental factors:
○ Exposure to ionizing radiation
○ Exposure to mutagenic chemicals and drugs
○ Viral infections

37. Incidence
Acute leukemias can occur in all age groups
● ALL is more common in children
● AML is more common in adults
● Chronic leukemias are usually a disease of adults
● CLL is extremely rare in children and unusual before the age of 40 years
● CML has a peak age of 30-50

38. Pathogenesis
Malignant transformation of hematopoietic stem cell
Unregulated proliferation
Arrest of maturation at the blast stage

39. Pathogenesis
Leukemic proliferation, accumulation and
invasion
Invasion of normal tissues: Liver, spleen,
LNs, CNS, skin etc
Failure of the bone marrow and normal
hemopoiesis→ Pancytopenia

40. Clinical features
Symptoms due to bone marrowfailure
- Pallor, lethargy,
- Bleeding manifestations
- Fever
- Infections
Symptoms related to organ infiltration
- Pain and tenderness of bones
- Lymphadenopathy
- Hepatosplenomegaly
- Gum hypertrophy
- Chloromas
- Meningeal signs

41. Clinical manifestations
Non Specific symptoms:
- Fever
- Night sweats
- Fatigue
- Loss of appetite
- Weight loss
- Easy bruising and bleeding
- Bone pain
- Lymphadenopathy

42. Physical findings
- Hepatomegaly
- Splenomegaly
- Lymphadenopathy
- Sternal tenderness
- Evidence of infections or bleeding

45. Lab diagnosis of acute leukemia

46. Lab diagnosis - Acute Leukemia
● Currently, diagnosis of leukemias is based on combination of:
● Clinical features
● Microscopic examination of peripheral blood and bone marrow
● Cytochemistry,
● Immunophenotyping by flow cytometry, cytogenetics,
● Molecular analysis

47. 1. Morphology
● Initial step in the diagnosis of acute leukemias is examination of smears of
Peripheral blood and Bone marrow aspirate.
● Typical case: Bone marrow suppression leads to:
● Normocytic normochromic anemia
● Total leukocyte count is usually elevated; however, it may be normal or low.
● Neutropenia
● Thrombocytopenia
● Blasts> 20% is diagnostic of acute leukemia

48. Different kinds of Blast cells

49. Myeloblast
● Myeloblast is a large cell (15-20 MicroM)
● Having moderate to abundant granular cytoplasm
● Large nuclei with fine chromatic
● Prominent 0-3 nucleoli
● 10-40% of myeloblasts have Auer rods

50. Lymphoblast
● Smaller in size (10-15 Micro m)
● Scant agranular cytoplasm
● High N:C ratio
● Coarse clumped chromatin
● 0-1 Indistinct nucleoli
● No Auer rods

53. 2. Cytochemistry in acute leukemia
● Cytochemistry comprises of techniques for identification of enzymes, fats, or
certain other substances in the cytoplasm of blood cells.
● In acute leukemia, cytochemistry is mainly useful for identifying various
subtypes of AML.
● In lymphoid leukemias, cytochemistry has been replaced by
immunophenotyping.
● The results of cytochemistry should always be interpreted along with
conventional morphology and immunophenotyping.

54. Cytochemistry
● Myeloperoxidase (MPO)
● Sudan black B (SBB)
● Non specific esterase (NSE)
● Periodic Acid Schiff (PAS)
● Acid phosphatase
● MPO rules out acute lymphoblastic leukemia, confirms myeloid lineage
● In B Cell ALL: presence of block positivity on PAS stain
● In T cell ALL : Acid phosphatase (localised positivity)

56. Stain AML ALL
Myeloperoxidase + -
Sudan Black B + -
Non specific esterase In M4, M5 and M7 -
Periodic acid schiff (PAS) Fine positivity in M6 ,M7 + Block positivity
Acid phosphatase - T ALL

57. 3. Immunophenotyping in acute leukemia
● This technique consists of identification of antigens present on leukemic cells in
blood or bone marrow
● Use of fluorescently-labeled monoclonal antibodies.
● As blood and bone marrow cells are in fluid suspension, flow cytometry is the
method of choice.
● Cell surface antigens are named according to the cluster of differentiation (CD)
system.
● Specific antigens are expressed on cells of different lineages at different stages of
development.
● Panel of specific antibodies is employed to determine the immunophenotype

58. 3. Immunophenotyping of acute leukemia
● Flow cytometer should be used in conjunction with Morphology, cytochemistry,
cytogenetics etc
● Immunophenotyping results can be obtained the same day and helps in early
treatment
● It helps to know the lineage whether Myeloid or Lymphoid
● If lymphoid- whether B cell or T cell
● To diagnose AML M0- undifferentiated
● To diagnose specific antigens on leukemic cells and institute specific targeted
therapy_ Eg Rituximab in CD20 Positive leukemia

59. Markers required for assigning lineage
Myeloid Markers: CD 13, CD33, CD117, MPO
B cell Lymphoid markers: CD10, CD19, CD20, CD22, CD79a
T cell Lymphoid markers: CD3, CD2, CD4, CD5, CD7
Monocytic markers: CD14, CD68, CD64
Megakaryoctyic markers: CD41, CD61

60. 4. Cytogenetic analysis
● Structural or numerical abnormalities of chromosomes are detected by
cytogenetic analysis or karyotyping
● Translocations, deletions, and duplications can be detected

61. 5. Molecular analysis
● Molecular methods are used for detection of chromosomal translocationsthat
generate fusion transcripts and chimeric proteins.
● The commonly used methods are:
● Reverse transcription-polymerase chain reaction (RT-PCR)
● Fluorescent in situ hybridization (FISH).
● Eg: Detection of t(15;17) in acute promyelocytic leukemia that generates
PML/RARα fusion gene,
● t(9;22) in B-ALL that generates bcr/abl fusion gene.
● Detection of these translocations is also helpful for determining prognosis and
response to treatment

62. Classification of acute leukemia
● Division of acute leukemia into Acute myeloid leukemia and acute lymphoid
leukemia is important
● With recent advances in molecular biology and treatment modalities, it is
essential to subtype the leukemia to assess prognosis and institute a specific
chemotherapy
2 classification systems are presently in use:
- FAB classification
- WHO classification

63. FAB classification of Acute leukemia
Acute Lymphoblastic Leukemia
Subtype of ALL Characteristics
L1 Small, homogeneous blasts, scanty
cytoplasm, indistinct nucleoli
L2 Large, heterogeneous blasts, indented
nuclei, one or more nucleoli, abundant
cytoplasm, minimal cytoplasmic vacuolation
L3 Large, homogeneous blasts, abundant
basophilic cytoplasm with prominent
cytoplasmic vacuolations

64. FAB classification of Acute leukemia
Acute myeloid leukemia
Subtype of AML Characteristics
M0 AML- Undifferentiated
M1 AML without maturation
M2 AML with maturation
M3 AML promyelocytic
M4 AML- Myelomonocytic
M5 AML Monoblastic/monocytic
M6 AML Erythtoid
M7 AML Megakaryocytic

65. Criticism of FAB classification
● It does not take into account cytogenetics and molecular characteristics which
have a prognostic role
● Immunological subtypes of ALL are of prognostic significant, but are not
defined in FAB
● It does not recognize biphenotypic leukemia
● It has limited relevance to therapeutic or prognostic implications

66. WHO classification of acute leukemia

67. Classification of acute myeloid leukemia
● Acute myeloid leukemia with recurrent genetic abnormalities
● Acute myeloid leukemia with myelodysplastic-related changes
● Therapy-related myeloid neoplasms
● Acute myeloid leukemia , NOS
● Myeloid sarcoma
● Myeloid proliferation related to Down syndrome
● Blastic plasmacytoid dendritic cell neoplasms

68. Acute myeloid leukemia with recurrent genetic
abnormalities
- AML with t(8,21)
- AML with Inv 16 or t(16,16)
- AML with t (15,17)-Acute promyelocytic leukemia with PML-RARA
- AML with t (9,11)
- AML with t (6,9)
- AML with Inv 3
- AML with t (1,22)
- AML with BCR ABL 1
- AML with Mutated NPM1
- AML with biallelic mutation of CEBPA
- AML with Mutated RUNX1

69. Acute myeloid leukemia, NOS
● AML with minimal differentiation (M0)
● AML without maturation (M1)
● AML with maturation (M2)
● Acute myelomonocytic leukemia (M4)
● Acute monoblastic and monocytic leukemia (M5)
● Pure erythroid leukemia (M6)
● Acute megakaryoblastic leukemia (M7)
● Acute basophilic leukemia
● Acute panmyelosis with myelofibrosis

71. Thank You