White blood cells

(WBCs)
 WBCs
• Phagocytes (granulocytes, monocytes) or immunocytes (lymphocytes, plasma cells, and monocytes).
• WBC reference range (4.0-11.0 X 103/μL). At birth: 10,000-25,000/ μL. 6,000-16,000/ μL: infant up to 1 year of
age.
• Granulocytes include neutrophils, eosinophils, and basophils.
• Agranulocytes: lymphocytes and monocytes.
1. Neutrophils are the first to reach the tissues and phagocytize (destroy) bacteria. In the process, they die.
• Diapedese into the tissues from the marginating pool in response to antigenic stimulation.
• Chemotactic factors attract the neutrophil to the site of inflammation; include complement, bacterial
products, injured tissue, hemostatic components.
• Opsonins such as IgG and complement component C3b help neutrophils recognize a substance as foreign.
• Phagocytosis involves neutrophil attachment to the foreign object, formation of a vacuole around it, and
neutrophilic degranulation to release lytic enzymes (respiratory burst) to kill the organism.
• Neutrophils are sensitive to the oxidants they secrete and are destroyed in the process.
Visible response to infection by neutrophils (toxic changes):
A. Toxic changes are associated with bacterial infection or growth factor therapy. Any combination of these
changes may be seen in some but not necessarily all of the neutrophils.
B. Toxic granulation is prominent granulation due to persistent staining of primary granules. Neutrophilic
cytoplasm normally contains only visible, small, secondary granules.
C. Toxic vacuolation: Colorless areas in the cytoplasm that indicate phagocytosis and degranulation have occurred
D. Dohle bodies: Small oval inclusions (RNA) located in the cytoplasm stain light blue
E. Shift to the left refers to an increased number of myelocytes, metamyelocytes, and/or bands in the peripheral
blood. It is associated with either increased or decreased WBC counts.
2. Eosinophils:
• They express Fc receptors for lgE in response to parasitic infections.
• They release substances that can neutralize products released by basophils and mast cells; eosinophils modulate the
allergic response.
3. Basophils:
• They express membrane receptors for IgE. Once activated, degranulation releases histamine and heparin.
• Mediate immediate hypersensitivity reactions (type I, anaphylactic).
• Basophils release a chemotactic factor that attracts eosinophils to the site.
4. T lymphocytes:
• Provide cellular immunity.
• 80% of lymphocytes in the blood
• Produce cytokines/interleukins.
5. B lymphocytes
• Develop into plasma cells in the tissue and produce antibodies.
• Humoral immunity.
• 20% of the lymphocytes in the blood.
6. NK (natural killer) lymphocytes:
• Destroy tumor cells and cells infected with viruses. They are also known as large granular lymphocytes (LGLs).
7. Monocytes:
• Differentiate into macrophages, and as such they work in the tissues to phagocytize foreign bodies. Known as
"scavenger cells" because of their ability to ingest foreign material.
8. Macrophages are named according to their location in the body.
• Monocytes -peripheral blood
• Kupffer cells-liver
• Microglial cells-central nervous system
• Osteoclasts-bone
• Langerhans' cells-skin
• Alveolar cells-lung
Benign quantitative
WBCs disorders

Differential and absolute leukocyte count

 Relative and Absolute Blood Cell Counts

• Relative count is the amount of a cell type in relation to other blood components.
Relative lymphocytosis is an increase in the percentage of lymphocytes; this is
frequently associated with neutropenia. In relative polycythemia, RBCs appear
increased due to a decreased plasma volume.
• Absolute count is the actual number of each cell type without respect to other
blood components. Absolute lymphocytosis is a true increase in the number of
lymphocytes. Absolute polycythemia is a true increase in red cell mass.
Pseudoneutrophilia: increase in the total WBC count and in the absolute number of neutrophils caused by
Caused by exercise, stress, pain, pregnancy.
• Pathologic neutrophilia: in response to bacterial and other infections, tissue destruction, drugs or toxins.
• Neutropenia: Hypersplenism, aplastic anemia, chemotherapy, defect in DNA synthesis (B12 and folate),
viral infection.
Eosinophilia
• Increase in the absolute number of eosinophils
• Associated with:
1. Parasitic infections, allergic reactions, chronic inflammation.
2. Chronic myelogenous leukemia, including early maturation stages, Hodgkin disease, tumors.
Eosinopenia
• Seen in acute inflammation and inflammatory reactions that cause release of glucocorticosteroids and
epinephrine. Also elevated in parasitic infection (helminths).
Basophilia
• Increase in the absolute number of basophils
• Associated with:
• Type I hypersensitivity reactions.
• Chronic myelogenous leukemia, including early maturation stages, polycythemia vera.
• Relative transient basophilia can be seen in patients on hematopoietic growth factors.
Basopenia: Decrease in the absolute number of basophils associated with inflammatory states and following immunologic
reactions. Difficult to diagnose because of their normally low reference range.
Monocytosis
1) Recovery stage from acute bacterial infections.
2) Tuberculosis, syphilis, subacute bacterial endocarditis.
3) Autoimmune disorders (systemic lupus erythematosus, rheumatoid arthritis)
Monocytopenia (rare):
1) Aplastic anemia.
2) Lymphocytic leukemia.
Lymphocytosis:
Viral: hepatitis, influenza, mumps, measles, rubella, and varicella.
Non-viral: Bordetella pertussis (whooping cough), brucellosis, toxoplasmosis.
Infectious mononucleosis .1
• Epstein-Barr virus (EBV) infects B lymphocytes.
• Common in the 14-24 age group with symptoms ranging from malaise and fever to pharyngitis,
lymphadenopathy, and splenomegaly.
• Transmitted through nasopharyngeal secretions.
• Positive heterophile antibody test.
:Cytomegalovirus .2
• Transmission is by blood transfusions and saliva exchange.
• Lymphocytopenia:
• AIDS (CD4).
Benign qualitative WBCs
disorders
Chediak-Higashi syndrome:
• Rare autosomal recessive.
• Recurrent bacterial infection (defective
chemotaxis) impaired motility, defect in
granulocyte degranulation.
• Patients will present with photophobia and
skin hypopigmentation.
• Oculocutaneous albinism.
• Abnormal fusion of primary and secondary
neutrophilic granules
• Giant lysosomal granules in granulocyte,
monocyte, lymphocyte, melanocyte, tissue
macrophage and platelets.
• Moderate neutropenia and thrombocytopenia.
 Inherited cytoplasmic anomalies
1. May-Hegglin anomaly (congenital defect):
• Autosomal dominant.
• Mutation in MYH9 gene in chromosome 22, a cytoskeletal protein in platelets that may be responsible for the platelet's
abnormal diameter.
• Morphologically abnormal, but functionally normal.
• Thrombocytopenia.
• Purpura and bleeding.
• Giant platelets.
• Large basophilic cytoplasmic crystallin (neutrophil) inclusion body called Dohle-like body (spindle-cigar shape).
2. Alder-Reilly anomaly:
• Autosomal recessive disorder of mucopolysaccharidosis.
• Morphologically abnormal, but functionally normal.
• Dark staining coarse cytoplasmic granules (azurophilic) in neutrophils, eosinophils, basophils, monocytes and
lymphocytes.
• Granules contain degraded mucopolysaccharides due to an enzyme defect. Precipitation of mucopolysaccharides
(Hunter and Hurler syndromes).
• Must differentiate from toxic granulation present in neutrophils only in infectious conditions.
Neutrophil nuclear abnormalities:
A. Hyposegmentation:
:Pelger-Huet anomaly .1
• Autosomal dominant disorder of neutrophil morphology, bilobed nucleus or a nucleus with no lobulation.
• 70-90% of the neutrophils are bilobed (sunglasses, pince-nez) or have no lobulation of the nucleus.
• Morphologically abnormal, but functionally normal.
• Must differentiate from a shift to the left associated with an infection (toxic changes); infection requires
treatment, but Pelger Huet anomaly (no toxic changes) does not.
:Pseudo Pelger-Huet .2
• Acquired abnormality associated with myeloproliferative disorders and myelodysplastic syndromes;
can also be drug induced.
• Nucleus is usually round instead of the dumbbell shape that is seen in the anomaly.
B. Hypersegmentation:
• Neutrophil with 6 or more nuclear lobes.
• Seen in anemia such as megaloblastic anemia due to B12 or folate deficiency.
Malignant disorders of
WBCs
 Introduction
• A malignant clone of cells proliferate that do not respond to normal regulatory mechanisms.
• Leukemia originates in the bone marrow and is initially systemic.
• Lymphoma originates in lymphoid tissue and is initially localized.
• Etiology remains unclear. Multiple theories exist about oncogene activation, which most likely includes multiple factors:
• Viral-Viruses can suppress immune function or activate oncogenes (HTLV-1, II, V) and HIV-1.
• Bone marrow damage-Radiation, chemicals, and malignancies secondary to cancer treatments
• Chromosome defects-Some chromosomal abnormalities are diagnostic for leukemic subtypes; t(15;17) is diagnostic for
acute promyelocytic leukemia.
• Genetic factors-Increased incidence in Down syndrome, Fanconi, and others.
• Immune dysfunction-Hereditary and acquired defects in the immune system.
• Treatment:
.a. Chemotherapy used is dependent on type of leukemia. Proper diagnosis is crucial
b. Radiation
c. Bone marrow/stem cell transplant
d. Supportive with transfusions of red blood cells and platelets, antibiotics, growth factors
 French-American-British (FAB) and World Health Organization (WHO)

• FAB classification is based on cellular morphology and cytochemical stain results.

• WHO classification is based on cellular morphology and cytochemical stains, but also utilizes
information obtained from immunologic probes of cell markers, cytogenetics, molecular
abnormalities, and clinical syndrome.
• FAB defines acute leukemia as ≥ 30% bone marrow blasts.
• WHO defines acute leukemia as ≥ 20% bone marrow blasts.
• WHO classification is now the standard for diagnosis.
• FAB classification is easier to use and is still widely taught.
 55 – 60 years Acute myelogenous leukemia(AML)

2 – 10 years Acute lymphoid lukemia(ALL)

> 40 years Chronic myelogenous leukemia(CML)

> 55 years (65 – 70) Chronic lymphoid leukemia(CLL)

 Acute Lymphoproliferative Disorders
1. Acute lymphoblastic leukemia (ALL):
• FAB classification:
A. ALL 1: Most common childhood leukemia (2- to 10-year peak); also found in young adults
• Small lymphoblasts, homogeneous appearance.
• Best prognosis
• T cell lineage.
B. ALL 2:
• Most common in adults.
• Large lymphoblasts, heterogeneous appearance.
C. ALL 3: Seen in both adults and children. Leukemic phase of Burkitt lymphoma
• Lymphoblasts are large and uniform with prominent nucleoli; cytoplasm stains deeply basophilic and may show vacuoles.
• Poor prognosis
• B cell lineage.
 Chronic Lymphoproliferative Disorders
A. Chronic lymphocytic leukemia (CLL):
• B cell malignancy (CD19, CD20 positive).
• Laboratory: Bone marrow hypercellular; blood shows absolute lymphocytosis of >5.0 X 109/L;
homogeneous, small, hyperclumped lymphocytes and smudge cells.
• Anemia is not usually present unless secondary to warm autoimmune hemolytic anemia
(frequent complication).
• Small lymphocyte lymphoma (SLL) is the lymphoma phase of CLL.
B. Hairy cell leukemia (HCL)
• Found in adults over 50 years old; more common in males (7:1)
• B cell malignancy (CD19, CD20 positive)
• Massive splenomegaly.
• Laboratory: Pancytopenia; cytoplasm of lymphocytes shows hair-like projections; hairy cells are
tartrate-resistant acid phosphatase (TRAP) stain positive.
 Other Lymphoid Malignancies
Plasma cell neoplasms
 Multiple myeloma:
• Monoclonal gammopathy causes B cell production of excessive lgG (most common) or IgA, with decreased
production of the other immunoglobulins.
• Found in adults over 60 years old; incidence higher in males
• Multiple skeletal system tumors of plasma cells (myeloma cells) cause lytic bone lesions and hypercalcemia.
• Identified on serum protein electrophoresis by an "M"-spike in the gamma-globulin region.
• Excessive IgG or lgA production by myeloma cells causes increased blood viscosity.
• Abnormal immunoglobulin binds to platelets, blocking receptor sites for coagulation factor binding; this results in
prolonged bleeding (pseudothrombocytopenia).
• Laboratory: Bone marrow plasma cells > 30%, marked rouleaux, increased erythrocyte sedimentation rate (ESR),
blue background to blood smear, plasma cells and lymphocytes on blood smear.
• Bence Jones proteins (free light chains-kappa or lambda) found in the urine; toxic to tubular epithelial cells; cause
kidney damage.
• Waldenstrom macroglobulinemia: excessive lgM (macroglobulin).
 Acute Myeloproliferative Disorders
• Platelets, erythrocytes, granulocytes, and/or monocytes can be affected.
Acute myelogenous leukemia (AML):
 FAB classification (M0-M7).
A. AML-M0:
• Blasts positive for CD13, CD33 & CD34.
• Negative with MPO, & SBB stains.
B. AML-M1 (AML without maturation):
• 90% myeloblasts in BM.
C. AML-M2 (AML with maturation):
• <90% myeloblasts.
• Positive for CD13 & CD33. Chromosome abnormality t(8;21).
 Both M1 & M2 are SBB, MPO & specific esterase positive.
D. AML-M3 (Acute promyelocytic leukemia):
• >30% promyelocytes in BM.
• Bundles of Auer rods (faggot cells).
• Procoagulant activity (DIC)
• Positive with SBB, MPO & specific esterase stains.
• Positive for CD13, CD33. Chromosomal abnormality: t(l5;17).
E. AML-M4 (Acute myelomonocytic leukemia):
• >20% monocytic origin.
• Increased urine/serum lysozyme.
• Positive with SBB, MPO, & non-specific esterase.
• Positive for CD13, CD33 & CD14 (monocytes).
• M4Eo, AMML subclass, presents with eosinophilia.
F. AML-M5 (Acute monocytic leukemia, AMoL):
• Monoblasts.
• Positive with nonspecific esterase.
• Positive for CD14.
• 2 variants:
• M5a: in children. >80% monoblasts in BM.
• M5b: in adults. <80% monoblasts in BM.
G. AML-M6 (Acute erythroleukemia, AEL):
• >50% dysblastic marrow normoblasts.
• PAS positive, positive CD45, CD71 (glycophorin A): malignant normoblasts.
• CD15 positive.
H. AML-M7 (Acute megakaryocytic leukemia, AMegL):
• Megakaryoblasts and megakaryocytes.
• Pancytopenia.
• Positive for CD41, CD42 & CD61.
• Down syndrome.
 Chronic Myeloproliferative Disorders
1. Chronic myelogenous leukemia (CML):
• Proliferation of granulocytes.
• Splenomegaly.
• Increased M:E ratio.
• WBC between 50 and 500 X 109/L (full spectrum of myeloid series), Myelocytes predominate.
• Basophilia & eosinophilia.
• Low LAP (NAP) score.
• BCR-ABL1 fusion gene, (p210BCR/ABL), Philadelphia chromosome, t(9;22).
2. Essential thrombocythemia (ET):
• proliferation of megakaryocytes.
3. Polycythemia vera (PV):
• Malignancy of multipotential myeloid stem cell.
• High blood viscosity.
• Increased RBC (7-10 X 10 12/L), hemoglobin (>20 g/dL), and hematocrit (>60%) along with
leukocytosis and thrombocytosis indicate polycythemia.
 JAK2 (Janus kinase) oncogene, JAK2(V617F) mutation.
• Polycythemia vera & Essential thrombocythemia.
 Leukemoid reaction

• Benign leukocytic proliferation.

• WBCs count more than 50,000/ μL.
• Few immature cells (band, metamyelocytes and myelocytes).
• Blood picture closely resembles leukemia (CML).
• Caused by severe bacterial infection, severe burns.
 Myelodysplastic Syndromes (MDSs)
• Affecting the pluripotential stem cell.
• Dyspoiesis affects erythroid, myeloid, and megakaryocytic cell lines.
• MDS development can be triggered by chemotherapy, radiation, and chemicals.
 Hematologic evidence of dyspoiesis:
• Erythroid: Variable anemia; erythrocytes can be macrocytic (with oval macrocytes) or microcytic
and hypochromic; dimorphic erythrocytes, poikilocytosis, Howell-Jolly bodies, basophilic
stippling, Cabot rings, nucleated RBCs.
• Myeloid: Neutropenia, hypogranulation, hyposegmentation of neutrophils, shift to the left.
• Thrombocytes: Variable platelet count, giant platelets, hypogranulation, micromegakaryocytes.
 Cytochemical Stains-Used in Diagnosis of Hematologic Disorders

1. Myeloperoxidase (MPO):
• Cells of the granulocytic series and to a lesser degree the monocytic series contain the enzyme peroxidase in their
granules that is detected by this stain.
• Auer rods also stain positive; lymphocytic cells are negative for this stain.
• Used to differentiate blasts of acute myelogenous leukemias (AMLs) from acute lymphoblastic leukemias (ALLs).
2. Sudan black B:
• Stains phospholipids and lipoproteins.
• Granulocytic cells and Auer rods stain positive (blue-black granulation); lymphocytic cells are negative for
Sudan black B.
• Used to differentiate blasts of AML from ALL.
3. Esterases
A. Specific esterase stain (naphthol AS-D chloroacetate esterase stain):
• Detects esterase enzyme present in primary granules of granulocytic cells; monocytic cells negative for this stain.
B. Nonspecific esterase stains (alpha-naphthyl acetate and alpha-naphthyl butyrate):
• Detects esterase enzyme present in monocytic cells; granulocytic cells negative for these stains.
• The esterase stains may be useful in distinguishing acute leukemias that are of myeloid origin (FAB Ml, M2, M3, M4)
from those leukemias that are primarily cells of monocytic origin (FAB M5).
4. Periodic acid-Schiff (PAS):
• PAS stains intracellular glycogen bright pink.
• Immature lymphoid cells, malignant erythroblasts, and megakaryocytic cells stain positive with this stain; myeloblasts
and normal erythrocytic cells are negative with this stain.
• Useful in diagnosis of erythroleukemia (FAB M6) and acute lymphoblastic leukemia.
5. Leukocyte alkaline phosphatase (LAP):
• Detects alkaline phosphatase enzyme activity in primary granules of neutrophils
• A positive stain will show dark precipitate when alkaline phosphatase activity is present; color is dependent on dye used.
• Used to differentiate chronic myelogenous leukemia (CML) from a neutrophilic leukemoid reaction (NLR)
LAP score:
• 100 neutrophils are graded on a scale from 0 to 4+ based on stain intensity and size of granules.
Results are added together.
• Reference range is 13-130.
• Clinical significance:
• Decreased LAP score: CML, paryoxysmal nocturnal hemoglobinuria
• Normal LAP score: CML in remission or with infection, Hodgkin lymphoma in remission,
secondary polycythemia
• Increased LAP score: Neutrophilic leukemoid reaction, polycythemia vera, CML in blast crisis,
late trimester pregnancy.
6. Tartrate-resistant acid phosphatase stain (TRAP):
• Almost all blood cells contain the acid phosphatase enzyme and show positivity with acid
phosphatase stain. Once tartrate is added, staining is inhibited in most cells.
• Only hairy cells from hairy cell leukemia are resistant to inhibition with tartrate and continue to
stain positive; all other cells stain negative.
7. Perl's Prussian blue stain
• Free iron precipitates into small blue/green granules in mature erythrocytes;
cells are called siderocytes. Iron inclusions are called siderotic granules or
Pappenheimer bodies when visible with Wright's stain.
• Sideroblasts are nucleated RBCs in bone marrow that contain iron granules.
These are normal. Ringed sideroblasts contain iron that encircles the nucleus.
These are abnormal.
• Increased percentage of siderocytes is seen in severe hemolytic anemias
(e.g., beta-thalassemia major), iron overload, sideroblastic anemia, and post-
splenectomy; ringed sideroblasts are seen in bone marrow of myelodysplastic
syndrome (refractory anemia with ringed sideroblasts [RARS]) and
sideroblastic anemias.
This has been a presentation of Alyazeed Hussein
Thanks for your attention and kind patience

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Alyazeed Hussein, BSc, SUST