This document outlines a hematology slide presentation on a case of pediatric leukemia. Key details include a 13-year-old boy with low hemoglobin, low platelets, and 50% atypical cells on blood smear. The presentation examines blood and bone marrow smears under different magnifications and discusses the provisional diagnosis of acute leukemia. It then provides an outline on the discussion of pediatric leukemia covering etiology, epidemiology, clinical features, laboratory studies including morphology, cytochemistry, immunophenotyping and cytogenetics, and differential diagnosis.

1. HEMATOLOGY SLIDE
PRESENTATION
Rituraj Baral, MBBS
1st Year Resident
Department of Pathology, TUTH

2. Slide Presentation
• Single Wright stained Peripheral blood smear
13 years old boy
Hemoglobin- 9.4gm/dL
Total leukocyte count- 8100/cmm
Platelets- 30,000/cmm

3. Slide Presentation
WBC Morphology
1. Neutrophils (02%)
Segmented nucleus with pink granules in cytoplasm
2. Lymphocytes(45%)
Deep purple nucleus almost covering cell and sky blue
cytoplasm
3. Monocytes(03%)
Indented dark purple nuclei

4. Slide Presentation
WBC Morphology
4. Atypical cell (50 %)
Small to intermediate size
Increased N:C ratio
Scant to moderate amount of agranular cytoplasm
Fine nuclear chromatin
Inconspicuous nucleoli.

5. Slide Presentation
RBC Morphology
Predominantly normocytic normochromic
Few microcytic and hypochromic
Few target cells and polychromasia also seen.
Parasites not seen.
Platelets Morphology
Reduced in smear
1-2/OIF

6. Slide Presentation
100X MAGNIFICATION

7. Slide Presentation
200x MAGNIFICATION

8. Slide Presentation
ATYPICAL CELLS
OIL IMMERSION

9. Slide Presentation
OIL IMMERSION
ATYPICAL CELL
Scant agranular
cytoplasm
Fine chromatin

10. Slide Presentation
OIL IMMERSION
LYMPHOCYTE AND ATYPICAL CELL
Atypical cell
Lymphocyte

11. Slide Presentation
400x MAGNIFICATION
RBC MORPHOLOGY
Lymphocyte
Neutrophil

12. Slide Presentation
OIL IMMERSION
Lymphocyte and Neutrophil
Neutrophil
Lymphocyte

13. Slide Presentation
OIL IMMERSION
ATYPICAL CELL AND TARGET CELLS
Atypical
cell
Target cells

14. Slide Presentation
OIL IMMERSION
POLYCHROMASIA
Polychromasia

15. Provisional Diagnosis
• Acute leukemia
Differential diagnosis
• Acute lymphoblastic leukemia
• Acute myeloid leukemia

16. Pediatric Leukemia
Discussion

17. Outlines of discussion
• Introduction
• Etiology
• Epidemiology
• Clinical features
• Lab Studies
• Differential diagnosis

18. Introduction
Leukemia
Group of malignant disease in which genetic
abnormalities in hematopoietic cell give rise to an
unregulated clonal proliferation of cells.
Usually begin in bone marrow and results in high
number of abnormal blood cells.
Leukemias are the most common malignant neoplasm in
childhood.
Accounts for approx. 31% of all malignancies occurring
in children below 15yrs.

19. Pediatric leukemia
Leukemias occurring in childhood
• Acute lymphoblastic leukemia (ALL)- 77%
– B-ALL: 85% of ALL
– T-ALL: 15% of ALL
• Acute myeloid leukemia (AML) – 11%
• Chronic myelogenous leukemia (CML)- 2-3%
• Juvenile myelomonocytic leukemia (JMML)-
1-2%

20. Pediatric leukemia
Etiology
– Unknown
– Multifactorial ; genetic and environmental
influences.
– Immunologic deficiencies
– Viruses (EBV, HTLV-1)

21. Pediatric leukemia

22. Epidemiology
• Sex
– ALL is more common in boys.
– Other leukemias has no such predilection for sex.
• Age
– B lineage ALL peaks incidence at about 3 years.
– T lineage ALL peaks incidence in adolescents.
– Incidence of AML is relatively higher in neonatal
period, then drops and slight increase in adolescents.
– JMML typically affects children below 2 years.

23. Epidemiology
• Race
– ALL is more common in whites than in black
– AML is more common in Hispanics and African
American children.
• Acute leukemia occurs 15-20 times more
frequently in children with Down syndrome
– 1st 3 years of life- AML>ALL
– ALL is more common thereafter.

24. Epidemiology
• Among identical twins, risk if one twin develops
leukemia is greater than in general population
– If 1st twin develop leukemia in 1st year of life or
monozygotic twins- risk is >70%
– If first twin develops leukemia in 5-7 years- risk is
>50%
• JMML is commonly associated with
Neurofibromatosis type 1 and Noonan syndrome.

25. Clinical features
Presentations of acute leukemia relate to three
main pathological processes:
– Bone marrow failure due to extensive infiltration
by blast cells,
– Infiltration of other tissues by blasts, and
– Systemic effects of cytokines released by tumor
cells.

26. Clinical feature

27. Clinical feature

28. Clinical feature
• ALL
– Bone and joint pain is common in ALL.
– Testes and CNS are common extramedullary sites
for ALL involvement.
– Patient with T cell ALL often have anterior
mediastinal mass (thymic infiltration) and may
present with respiratory distress.

29. Clinical features
• Other than symptoms similar to ALL due to bone
marrow infiltration, AML may present with
– Subcutaneous nodules (“blueberry muffins” lesions-
especially in infants)
– Signs of DIC (Common in M3 )
– Chloromas (granulocytic sarcomas)
• JMML may present with rashes,
lymphadenopathy, splenomegaly
and hemorrhagic manifestations.

30. Lab studies
• Primary aim: to establish the diagnosis of
leukemia by peripheral blood smear and bone
marrow aspirate and demonstration of
atypical cells (blasts)
• Followed by investigations to identify the type
of leukemia.

31. Lab studies
Diagnostic Approach
• Morphological examination of PBS and Bone
marrow aspirate
• Cytochemistry
• Immunophenotyping
• Cytogenetic analysis
• Molecular genetic analysis

32. Lab studies
PBS
– Normocytic normochromic anemia
– Thrombocytopenia and neutropenia
– Total leukocyte count is usually increased,
however it may be normal or even low.
– T lineage ALL usually presents with high leukocyte
count (>100000/cmm)
– Diagnosis of leukemia requires >20% of blasts in
PBS.

33. Lab studies
PBS
– Sub leukemic leukemia: TLC is normal but blasts
are demonstrable in peripheral blood
– Aleukemic leukemia: Blasts are not demonstrable
in peripheral blood but are present in bone
marrow.
– Leukemic hiatus: Occurs in AML (M0, M1).
Peripheral blood shows blasts cells and mature
cells, intermediate cells like promyelocyte,
myelocyte and metamyelocyte are not seen.

34. Lab studies
Bone marrow aspiration
Both ALL and AML
– Cellularity : Usually hypercellular
– Shows monotonous population of leukemic cells.
– Normal hematopoietic cells are reduced.
In CML, Bone marrow shows markedly increased
M:E ratio, <10% blast and increased reticulin and
fibrosis
JMML shows myelodysplastic pattern with blasts
cells <20%.

35. Lab studies
Morphology of blasts:
Morphology ALL AML
Size Small to intermediate Large
Cytoplasm Scant Moderate
Cytoplasmic granules Absent May be present
Nuclear chromatin Relatively coarse Fine
Nucleoli Inconspicuous, 0-2 Prominent, >3
Auer rod Absent Pathognomic, if present

36. Morphology of blast
Myeloblast Lymphoblast

37. Lab studies
Cytochemistry
– Comprises of techniques for identification of
enzymes, fat or other substances in cytoplasm of
blood cells
– Mainly used for differentiating blasts of lymphoid
or myeloid origin
– Also helps in identifying various subgroups.

38. Lab studies
Cytochemistry
Important cytochemical studies are:
• Myeloperoxidase (MPO)
– MPO enzyme located in primary (azurophilic) and
secondary granules in all stages of neutrophilic
series and eosinophils.
– Reaction product is brown and granular.
– Positive in AML M1, M2, M3, and M4
– Lymphoblasts are usually negative for MPO

39. Cytochemistry
MPO Positive

40. Lab studies
Cytochemistry
• Sudan Black B
– Stains phospholipids and neutral fats in
membranes of both primary and specific granules
in granulocytes.
– Results are similar to MPO
– Reaction product is black and granular

41. Cytochemistry
SBB positive in myeloblast

42. Lab studies
Cytochemistry
• Non specific Esterase (NSE) reaction
– Strongly positive in monocytic series (Monocyte,
monoblast and promonocytes)
– Usually demonstrated by alpha-naphthyl acetate
esterase.
– It allows the diagnosis of AML M4 and M5
– The reaction product is brown and granular.

43. Lab studies
Cytochemistry
• PAS (Periodic acid- schiff)
– Stains glycogen in the cytoplasm
– Lymphoblasts show block positivity
– PAS is positive in 70% of ALL L1 and L2, however it
is negative in ALL L3.
– Erythroblasts AML M6 may also show granular PAS
positivity.

44. Cytochemistry
PAS: Block positivity in ALL

45. Cytochemistry
Diffuse granular PAS positivity in M6 (erythroblast)

46. Cytochemistry
• Acid Phosphatase
– Main diagnostic use is in diagnosis of T cell ALL
and hairy cell leukemia
– Tartrate-resistant acid phosphatase stain (TRAP) is
used for confirmation of hairy cell leukemia.
– Reaction product is red with mixture of granular
and diffuse positivity.

47. Cytochemistry

48. Lab studies
Immunophenotyping
– Technique for identification of antigens present on
leukemic cells in blood or bone marrow with
fluorescently labeled monoclonal antibodies.
– As blood and bone marrow are in fluid
suspension, flow cytometry is the method of
choice.
– Specific antigens are expressed on cells of
different lineages at different stages of
development.

49. Immunophenotyping
Lineage Markers
Primitive stem cell markers CD34, CD117,TdT
Myeloid lineage CD13, CD33
Monocytic lineage CD14, CD64
Erythroid lineage Glycophorin A
Megakaryocytic lineage CD41, CD42, CD61
B- ALL CD19, CD20, CD21, CD22, CD79a, CD10
T-ALL CD2, CD3, CD5, CD7

50. Lab studies
Immunophenotyping
– It is essential in those cases that cannot be
diagnosed as Lymphoblastic or Myeloid on the
basis of morphology and cytochemistry.
– It is also used for detection of minimal residual
disease following therapy.

51. Lab studies
Cytogenetic analysis
– Structural or numerical abnormalities of
chromosomes are detected by cytogenetic
analysis or karyotyping.
– Variety of gross alteration can be detected such as
translocation, deletion and duplication.
– Philadelphia chromosome resulting from t(9;22)
translocation is detectable in 95% of cases of CML
by routine cytogenetic studies.

52. Cytogenetic analysis
– Cytogenetic or chromosomal abnormalities are
linked to pathogenesis and prognosis of disease.
– It is also helpful in detection of remission and
relapse.

53. Cytogenetics

54. Lab studies
Molecular genetic studies
Methods used are
– Southern blot
– Polymerase chain reaction- based techniques
– Fluorescent in situ hybridisation (FISH)
• Used for detection of clonality by gene
arrangement studies

55. Differential Diagnosis
Features
Reactive lymphocytosis Seen in EBV or CMV infection with fever,
lymphadenopathy and leucocytosis.
Absence of blast, anemia and thrombocytopenia helps
to differentiate reactive lymphocytosis from ALL
Leukaemoid reaction Should be differentated from AML
Presense of immature WBC in peripheral blood due to
infection, acute hemolysis or other infilterative disease
of bone marrow.
Leucocytosis is moderate and blasts usually <5%
Metastatic tumors Metastasis of neuroblastoma in children and ewings
sacroma in adolescents
Occurs as clumps than diffuse sheets
Demonstration of primary tumor and
immunocytochemical studies helps to differentiate
Hematogones Normal B lymphocyte precursors which increases during
marrow regenerative states and immune cytopenia.
Morphologically similar to lymphoblast.
MDS Depends on proportion of myeloblasts. <20% in MDS

56. Take home message
• Leukemia is the most common malignancy of
childhood
• Acute leukemia > Chronic leukemia
• ALL > AML
• B-ALL > T-ALL
• Demonstration of blasts >20% in peripheral blood or
bone marrow aspiration is diagnostic of leukemia
• Cytochemistry and immunophenotyping is required for
confirmation and distinguishing the lineage and
subgroups of leukemia.

57. References
• Nelson’s Textbook of pediatric
• Essential of Hematology
• Dacie and Lewis Practical Hematology
• Robbin’s Pathological basis of disease
• Anderson’s Atlas of Hematology

58. Thank you