WHITE BLOOD CELLS

• All WBCs (leukocytes) have a nucleus and no hemoglobin • Granular or agranular classification based on presence of cytoplasmic granules made visible by staining

• granulocytes are neutrophils, eosinophils or basophils • agranulocytes are monocyes or lymphocytes

• Neutrophil (Granulocyte) • Polymorphonuclear Leukocytes or Polys

• Nuclei = 2 to 5 lobes connected by thin strands • older cells have more lobes

• young cells called band cells because of horseshoe shaped nucleus (band)

• Fine, pale lilac practically invisible granules • Diameter is 10-12 microns • 55 - 75% of circulating WBCs

• Neutrophil Function : • Fastest response of all WBC to bacteria

• Direct actions against bacteria • release lysozymes which destroy/digest bacteria

• release defensin proteins that act like antibiotics & poke holes in bacterial cell walls destroying them • release strong oxidants (bleach-like, strong chemicals ) that destroy bacteria

• Eosinophils (Granulocyte) • Nucleus with 2 or 3 lobes connected by a thin strand • Large, uniform-sized granules stain orange-red with acidic dyes

• do not obscure the nucleus • Diameter is 10 to 12 microns

• 1 - 3% of circulating WBCs • Eosinophil Function : • Leave capillaries to enter tissue fluid • Release histaminase

• slows down inflammation caused by basophils • Attack parasitic worms

• Phagocytoze antibody-antigen complexes

• Basophils (Granulocyte) • Large, dark purple, variable-sized granules stain with basic dyes

• obscure the nucleus

• Irregular, s-shaped, bilobed nuclei • Diameter is 8 to 10 microns • 0 - 1% of circulating WBCs • Basophil Function :

• Involved in inflammatory and allergy reactions • Leave capillaries & enter connective tissue as mast cells • Release heparin, histamine & serotonin

• heighten the inflammatory response and account for hypersensitivity (allergic) reaction

• Lymphocyte (Agranulocyte) • Dark, oval to round nucleus

• Cytoplasm sky blue in color • amount varies from rim of blue to normal amount • Small cells 6 - 9 microns in diameter

• Large cells 10 - 14 microns in diameter • increase in number during viral infections • 25 - 35% of circulating WBCs

• Lymphocyte Functions : • B cells • destroy bacteria and their toxins

• turn into plasma cells that produce antibodies • T cells • attack viruses, fungi, transplanted organs, cancer cells & some bacteria • Natural killer cells • attack many different microbes & some tumor cells

• destroy foreign invaders by direct attack • Monocyte (Agranulocyte) • Nucleus is kidney or horse-shoe shaped

• Largest WBC in circulating blood • does not remain in blood long before migrating to the tissues

• differentiate into macrophages • fixed group found in specific tissues • alveolar macrophages in lungs

• Kupffer cells in liver • wandering group gathers at sites of infection

• Diameter is 12 - 20 microns • Cytoplasm is a foamy blue-gray • 2 - 8% o circulating WBCs

• Monocyte Function : • Take longer to get to site of infection, but arrive in larger numbers

• Become wandering macrophages, once they leave the capillaries • Destroy microbes and clean up dead tissue following an infection

LEUKEMIA

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.

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)

• 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.

• Incidence of leukemia • 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

• 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

• Comparison Acute Vs Chronic • Comparison of acute and chronic leukemias :

Acute Chronic Age : all ages usually adults Clinical onset : sudden insidious Course (untreated) : 6 mo. or less 2-6 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

• Acute leukemia – • Is a result of 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.

• 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

• 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 •

• Cell Morphology in Leukemia • Morphology – an experienced morphologist (pathologist and hematologist)

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;

10-40% of myeloblasts contain Auer rods

• ALL – in contrast to the myeloblast, the lymphoblast is a small blast with scant cytoplasm, dense chromatin, indistinct nucleoli, without Auer rods

• 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

• 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

• Nonspecific esterase – is used to identify monocytic cells which are diffusely positive; T lymphocytes may have focal staining

• 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

• 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.

• 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

• 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. • Cytogenetics in Leukemia :

• Cytogenetic 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.

ACUTE LEUKEMIAS and 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.

• 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.

• 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)

• 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 • 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%

• 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!)

• 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

• 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

• 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

• M5 – Acute Monoblastic Leukemia

• >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 ( Di Guglielmo’s Syndrome ) • 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

• 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

• 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

• CHRONIC LYMPHOCYTIC LEUKEMIA :

• commonest leukemia in the western world

• most common form of leukemia in older adults • patients usually in their 50’s when diagnosed • average survival time is 5 years; May take an aggressive course with only 1-2 years of survival time • many CLL patients die from infections • no known specific etiologic agent or cause for CLL • most frequently a neoplasm of B-lymphocytes (rarely T cells) • lymphocytes are small and have relatively mature, well-differentiated appearance • bare nuclei, called “smudge cells” frequently found • clonal proliferation of the B-Lymphocytes • younger patients now seen • M:F ratio = 2:1 • highly variable disorder • 75% cases, diagnosis by chance on a routine blood test • not associated with radiation or exposure to occupational hazards • among the leukemias, CLL has the strongest tendency for familial incidence

• results to altered humoral immunity resulting from suppression of all classes of immunoglobulins, leading to hypogammaglobulinemia; subsequent increase in susceptibility to infections • many patients (15-35%) develop autoimmune disorders and produce autoantibodies to neutrophils, platelets (i.e. ITP), or RBCs (WAIHA) • have significantly impaired immunologic activity

•

• Clinical Findings : • asymptomatic - incidental finding

• anaemia & thrombocytopenia • infections • weight loss, night sweats, fever • lymphadenopathy • splenomegaly • AIHA (Auto immune haemolytic anaemia)

• 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

Stage Definition 0 Absolute lymphocytosis >15x109/l. 1 Stage 0+enlarged lymph nodes.

11 Stage 0+liver or/and spleen adenopathy.

111 Stage 0+anemia organomegally or adenopathy.

1V Stage 0+thrombocytopenia organomegally or adenopathy.

• CHRONIC MYELOCYTIC LEUKEMIA :

• malignancy of the haemopoietic system

• also called chronic granulocytic leukemia • 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 • olecular and cellular therapeutics • cytogenetic Abnormality of CML - Ph Chromosome

• Molecular Methods the Ph Chromosome - Fluorescence in situ hybridisation (FISH)

Chronic phase • Stable counts

• Easily controlled • 3-4 years (without therapy)

Accelerated phase • Unstable counts

• Fatal within months Blast crisis • Lots of blasts

• Fatal within weeks