1. CLINICAL LABORATORY DIAGNOSTICS OF HEMOBLASTOSIS
Krynytska Inna Yakivna

2. Today’s Lecture 1. Introduction to Leukemias. 2. Acute Leukemias.
3. Chronic Leukemias

3. Definition of Leukemia
Leukemia is a malignant disease characterized by unregulated proliferation of one cell type.(one clone of Immature cell)
It may involve any of the cell lines or a stem cell common to several cell lines.
Leukemias are classified into 2 major groups
Chronic in which the onset is insidious, the disease is usually less aggressive, and the cells involved are usually more mature cells
Acute in which the onset is usually rapid, the disease is very aggressive, and the cells involved are usually poorly differentiated with many blasts.

4. Types
Both acute and chronic leukemias are further classified according to the prominent cell line involved in the expansion:
If the prominent cell line is of the myeloid series it is a myelocytic leukemia (sometimes also called granulocytic)
If the prominent cell line is of the lymphoid series it is a lymphocytic leukemia
Therefore, there are four basic types of leukemia
Acute myelocytic leukemia – AML- (includes myeloblastic, promyelocytic, monocytic, myelomonocytic, erythrocytic, and megakaryocytic)
Acute lymphocytic leukemia – ALL- (includes T cell, B cell, and Null cell)
Chronic myelocytic leukemia – CML - (includes myelocytic and myelomonocytic)

5. Acute & Chronic Types
Chronic lymphocytic leukemia – CLL - (includes plasmocytic {multiple myeloma}, Hairy cell, prolymphocytic, large granular cell lymphocytic, Sezary’s syndrome, and circulating lymphoma)
Etiology – the exact cause is frequently not known, but predisposing factors are known:
Host factors
Some individuals have an inherited increased predisposition to develop leukemia
There is an increased incidence in those with an inherited tendency for chromosome fragility or abnormality or those with increased numbers of chromosomes (such as Down’s syndrome). Many of these diseases are characterized by chromosomal translocations.

6. Incidence of leukemia Environmental factors:
There is an increased incidence in those with hereditary immunodeficiencies.
There is an increased incidence in those with 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

7. Incidence of leukemia 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
CML has a peak age of 30-50

8. Comparison Acute Vs Chronic
Comparison of acute and chronic leukemias:
Acute Chronic
Age all ages usually adults
Clinical onset sudden insidious
Course (untreated) mo. or less years
Leukemic cells immature >30% blasts more mature cells
Anemia prominent mild
Thrombocytopenia prominent mild
WBC count variable increased
Lymphadenopathy mild present;often prominent
Splenomegaly mild present;often prominent

9. . Acute leukemia Acute leukemia – Is a result of: Clinical features
Malignant transformation of a stem cell leading to unregulated proliferation and
Arrest in maturation at the primitive blast stage. Remember that a blast is the most immature cell that can be recognized as committed to a particular cell line.
Clinical features
Leukemic proliferation, accumulation, and invasion of normal tissues, including the liver, spleen, lymph nodes, central nervous system, and skin, cause lesions ranging from rashes to tumors.

10. Evaluation of leukemia
Failure of the bone marrow and normal hematopoiesis may result in pancytopenia with death from hemorrhaging and infections.
Lab evaluation
The lab diagnosis is based on two things
Finding a significant increase in the number of immature cells in the bone marrow including blasts, promyelocytes, promonocytes (>30% blasts is diagnostic)
Identification of the cell lineage of the leukemic cells

11. Evaluation of leukemia
Peripheral blood:
Anemia (normochromic, normocytic)
Decreased platlets
Variable WBC count
The degree of peripheral blood involvement determines classification:
Leukemic – increased WBCs due to blasts
Subleukemic – blasts without increased WBCs
Aleukemic – decreased WBCs with no blasts
Classification of the immature cells involved may be done by:

12. Cell Morphology in Leukemia
Morphology – an experienced morphologist can look at the size of the blast, the amount of cytoplasm, the nuclear chromatin pattern, the presence of nucleoli and the presence of auer rods (are a pink staining, splinter shaped inclusion due to a rod shaped alignment of primary granules found only in myeloproliferative processes) to identify the blast type:
AML – the myeloblast is a large blast with a moderate amount of cytoplasm, fine lacey chromatin, and prominent nucleoli % of myeloblasts contain auer rods.

13. Myeloid maturation MATURATION myeloblast promyelocyte myelocyte
metamyelocyte
band
neutrophil
MATURATION
Adapted and modified from U Va website

14. Myeloblasts with auer rods

15. Lymphoblasts
ALL – in contrast to the myeloblast, the lymphoblast is a small blast with scant cytoplasm, dense chromatin, indistinct nucleoli, and no auer rods

16. Lymphoblast

17. AML

18. Auer rods in AML

19. ALL

20. Cytochemistry
Cytochemistry – help to classify the lineage of a leukemic cell (myeloid versus lymphoid)
Myeloperoxidase – is found in the primary granules of granulocytic cells starting at the late blast stage. Monocytes may be weakly positive.

21. Sudan black
Sudan black stains phospholipids, neutral fats and sterols found in primary and secondary granules of granulocytic cells and to a lesser extent in monocytic lysosomes. Rare positives occur in lymphoid cells

22. Nonspecific Esterase
Nonspecific esterase – is used to identify monocytic cells which are diffusely positive. T lymphocytes may have focal staining

23. Acid phosphatase
Acid phosphatase may be found in myeloblasts and lymphoblasts. T lymphocytes have a high level of acid phosphatase and this can be used to help make a diagnosis of acute T-lymphocytic leukemia.

24. Leukocyte Alkaline phosphatase
Leukocyte alkaline phosphatase – is located in the tertiary granules of segmented neutrophils, bands and metamyelocytes. The LAP score is determined by counting 100 mature neutrophils and bands. Each cell is graded from 0 to 5. The total LAP score is calculated by adding up the scores for each cell.

25. Leukocyte alkaline phosphatase

26. Immunologic markers
Immunologic markers (immunophenotyping) – these are used mainly for lymphocytes, i.e., for determining B cell or T cell lineage. These tests rely on antibodies made against specific surface markers. (Fluorescent Antibody Tagging)
flow cytometer that will determine the number of cells that have a fluorescent tag and which are thus positive for the presence of the surface marker to which the primary antibody was made.

27. Direct versus indirect labeling of antigens

28. Flow cytometer

29. Terminal deoyxtidyl transferase
This is a unique DNA polymerase present in stem cells and in precursor B and T lymphoid cells. High levels are found in 90% of lymphoblastic leukemias. It can also be detected using appropriate antibodies and flow cytometry.

30. Cytogenetics leukemia
Cytogenetics – cytogenetics studies can now be used for diagnosis and for prognosis of hematologic malignancies.
Many leukemias (and lymphomas) are characterized by specific chromosomal abnormalities, including specific translocations and aneuploidy. The specific type of malignancy can be identified based on the specific abnormality or translocation. These may be identified by
Looking at the karyotypes of the chromsomes from the abnormal cells
DNA based tests – these tests are very useful for following the course of the disease
A normal karyotype is usually associated with a better prognosis.

31. Chromosomal translocation

32. Chromosome karyotyping

33. Acute leukemias FAB Classification
Acute lymphoblastic leukemia –
They may be classified on the basis of the cytological features of the lymphoblasts into;
L1 - This is the most common form found in children and it has the best prognosis. The cell size is small with fine or clumped homogenous nuclear chromatin and absent or indistinct nucleoli. The nuclear shape is regular, occasionally clefting or indented. The cytoplasm is scant, with slight to moderate basophilia and variable vacuoles.
L2 – This is the most frequent ALL found in adults. The cell size is large and heterogenous with variable nuclear chromatin and prominent nucleoli. The nucleus is irregular, clefting and indented. The cytoplasm is variable and often moderate to abundant, the basophilia is variable and may be deep, and vacuoles are variable.

34. ALL-L1

35. ALL-L2

36. Acute leukemias
L3 – This is the rarest form of ALL. The cell size is large, with fine, homogenous nuclear chromatin containing prominent nucleoli. The The nucleus is regular oval to round. The cytoplasm is moderately abundant and is deeply basophilic and vacuolated.

37. ALL-L3

38. Acute leukemias
ALL may also be classified on the basis of immunologic markers into:
Early pre-B ALL
Pre-B ALL
B ALL
T ALL
Null or unclassified ALL (U ALL) - lack B or T markers and may be the committed lymphoid stem cell)

39. B cell maturation

40. T cell maturation

41. Acute leukemias
Incidence – ALL is primarily a disease of young children (2-5 years), but it can also occur in adults
Clinical findings – pancytopenia with resulting fatigue, pallor, fever, weight loss, irritability, anorexia, infection, bleeding, and bone pain.
L1 occurs in children, L2 in adults, and L3 is called Burkitts leukemia

42. Acute leukemias
Prognosis – age, WBC count, and cell type are the most important prognostic indicators
Patients younger then 1 and greater than 13 have a poor prognosis
If the WBC count is < 10 x 109/L at presentation, the prognosis is good; If the WBC count is > 20 x 109/L at presentation the prognosis is poor
T cell ALL (more common in males) has a poorer prognosis than any of the B cell ALLs which have a cure rate of 70%

43. Acute leukemias
Acute leukemias with mixed lineage – there are occasionally acute leukemias that are biphenotypic and display phenotypes for two different lineages
B lymphoid/myeloid
T lymphoid/myeloid
B/T lymphoid
Myeloid/Natural killer
A rare trilineage leukemia has also been seen (was B/T lymphoid/myeloid!)

44. Acute leukemias
Acute myeloid leukemia (also called acute granulocytic leukemia) – classification depends upon
Bone marrow blast morphology
Degree of cell maturation
Cytochemical stains
Immunophenotyping
AML is divided into 7 different classifications:
M1 – myeloblastic without maturation
The bone marrow shows  90% blasts and < 10% promyelocytes
The disease occurs in older adults

45. AML – M1
Note the myeloblasts and the auer rod:

46. Acute leukemias M2 – myeloblastic with maturation
The bone marrow shows 30-89% blasts and > 10% promyelocytes;
This is characterized by an 8,21 chromosomal translocation
This occurs in older adults
M3 – hypergranular promyelocytic
This form of AML has a bone marrow with >30% blasts
Is more virulent than other forms
Occurs with a medium age of 39
The WBC count is decreased
Treatment causes a release of the granules and may send the patient into disseminated intravascular coagulation and subsequent bleeding
It is characterized by a 15,17 chromosomal translocation

47. AML – M2
Note myeloblasts and hypogranulated PMNs:

48. AML – M3
Note hypergranular promyelocytes:

49. Acute leukemias M3m – hypogranular promyelocytic –
The bone marrow has > 30% blasts
The WBC count is increased.
Like the M3 type, treatment causes a release of the granules and may send the patient into disseminated intravascular coagulation and subsequent bleeding and
It is characterized by a 15,17 translocation
M4 – acute myelomonoblastic leukemia
Both myeloblasts and monoblasts are seen in the bone marrow and peripheral blood
Infiltration of extramedullary sites is more common than with the pure granulocytic variants

50. AML – M3m
Note hypogranular promyelocytes:

51. AML – M4
Note monoblasts and promonocytes:

52. Acute leukemias M5 – acute monoblastic leukemia M6 – erythroleukemia
>80% of the nonerythroid cells in the bone marrow are monocytic
There is extensive infiltration of the gums, CNS, lymph nodes and extramedullary sites
This form is further divided into
M5A - Poorly differentiated (>80% monoblasts)
M5B - Well differentiated (<80% monoblasts)
M6 – erythroleukemia
This is rare and is characterized by a bone marrow having a predominance of erythroblasts
It has 3 sequentially morphologically defined phases;
Preponderance of abnormal erythroblasts
Erythroleukemia – there is an increase in both erythroblasts and myeloblasts
Myeloblastic leukemia – M1, M2, or M4
Anemia is common

53. AML – M5A
Note monoblasts:

54. AML-M5B
Note monoblasts, promonocytes, and monocytes:

55. AML – M6
Note M1 type monoblasts

56. Acute leukemias Treatment of leukemias – There are 2 goals:
M7 - Acute megkaryoblastic leukemia
This is a rare disorder characterized by extensive proliferation of megakaryoblasts, atypical megakaryocytes and thrombocytopenia
Treatment of leukemias –
There are 2 goals:
Eradicate the leukemic cell mass
Give supportive care
Except for ALL in children, cures are not common but complete remission (absence of any leukemia related signs and symptoms and return of bone marrow and peripheral blood values to within normal values) is

57. Acute leukemias There are four general types of therapy
Chemotherapy – usually a combination of drugs is used
Bone marrow transplant
Radiotherapy
Immunotherapy – stimulate the patients own immune system to mount a response against the malignant cells

58. Chronic Lymphocytic leukemia
Commonest leukemia in the western world
Clonal proliferation of the B-Lymphocytes
Disease of the elderly
Younger patients now seen
M:F ratio, 2:1
CLL is highly variable disorder
75% cases, diagnosis by chance on a routine blood test

59. Aetiology Cause unknown
Not associated with radiation or exposure to occupational hazards
Among the leukemias, CLL has the strongest tendency for familial incidence

60. Clinical Findings Asymptomatic: incidental finding
Anaemia & thrombocytopenia
Infections
Weight loss, Night sweats, Fever (B Symptoms)
Lymphadenopathy
Splenomegaly
AIHA (Auto immune haemolytic anaemia)

61. Prognosis Late stage patients have usually progressive disease
Highly Variable for early stage patients
Significant subset of early stage eventually progress
Refractory to treatment
Infectious Complications
Autoimmune complications

62. Chronic Myeloid Leukaemia
Malignancy of the haemopoietic system
Transformation of the pluripotent stem cell
9;22 translocation giving rise to the Philadelphia (Ph’) chromosome
Creation of a leukaemia specific mRNA (BCR-ABL)
Resistance to apoptosis, abnormal signalling and adhesion
Molecular diagnostics
Molecular and cellular therapeutics

63. Cytogenetic Abnormality of CML: The Ph Chromosome1 2 3 4 5 6 7 8 9 10 11 12
[slide 12]
Cytogenetic Abnormality of CML: The Ph Chromosome
The Ph chromosome was first described in 1960 as a shortened chromosome 22 present in myeloid cells from patients with CML. This was the first report of a human cancer associated with a specific genetic abnormality.
Ninety-five percent of patients with CML have the Ph chromosome—hence, this chromosome is the hallmark of CML.
The Ph chromosome can be detected in bone marrow cells in metaphase by standard cytogenetic techniques.1,2
The Ph chromosome is present in all myeloid cell lineages, including erythrocytes, granulocytes, monocytes, and megakaryocytes, as well as some cells of lymphocytic lineage, indicating that malignant transformation to CML originates at the stem cell level.2,3
References
1. Osarogiagbon UR, McGlave PB. Chronic myelogenous leukemia. Curr Opin Hematol ;6:
2. Pasternak G, Hochhaus A, Schultheis B, et al. Chronic myelogenous leukemia: molecular and cellular aspects. J Cancer Res Clin Oncol ;124:
3. Sawyers CL. Chronic myeloid leukemia. N Engl J Med ;340: 13 14 15 16 17 18 19 20 21 22 x Y

64. Molecular Methods the Ph Chromosome
Fluorescence in situ hybridisation (FISH)
Interphase
Metaphase
[slide 18]
Molecular Methods for Detecting bcr-abl at the Ph Chromosome
FISH is the detection of highly specific DNA probes that have been hybridised to either interphase or metaphase chromosomes using fluorescence microscopy. FISH has a high rate of sensitivity (1/250 cells) and specificity.
To detect the Ph chromosome, the fluorescent probe will be designed to hybridise to the bcr-abl fusion gene.
FISH is being used experimentally to monitor remission status in Ph+ CML with greater sensitivity than conventional cytogenetics.1 Its is also used for early detection of relapse and evaluation for mixed chimerism following stem cell transplantation (SCT).2
References
1. Duba HC, Hilbe W, Mehringer A, et al. Hypermetaphase and interphase fluorescence in situ hybridisation for monitoring of remission status in Philadelphia chromosome positive chronic myeloid leukaemia. Int J Oncol. 2000;17:
2. Tamura S, Saheki K, Takatsuka H, et al. Early detection of relapse and evaluation of treatment for mixed chimerism fluorescence in situ hybridization following allogeneic hematopoietic cell transplant for hematological malignancies. Ann Hematol ;79:
Courtesy of Charles Sawyers, UCLA.