Hematological Examination

Jan Živný

jzivny@LF1.cuni.cz

22496-5865 / -5934

Department of Pathophysiology

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Blood / Lymph nodesBone marrow

Hematopoietic Cells

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Clinical signs of hematopoietic diseases

• Anemia→ hypoxia → cardiovascular signs

• Polycytemia → hyperviscose blood → thrombosis → embolia

→ hypoxia• Bleeding → shock → hypoxia • Trombosis → embolia / hypoxia• Frequent infections

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Laboratory Tests Basic:

• Complete blood count

Specialized:• Tests for iron metabolism• Erythropoietin measurements• Cytogenetic and known mutation analysis• Immunophenotyping of BM or PB cells• Detection of antibodies to self antigens (e.g. RBC)• Histochemical analysis of cell enzymatic activity• Functional tests (Clonogenic assay)

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Complete Blood Count (CBC)

• Hemoglobin concentration (Hb)• Hematocrit (Hct) • RBC count• RBC parameters• WBC count • WBC differential count • Platelet count • Platelet parameters• Description of blood smear

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• Hematology AnalyzerHematology Analyzers

FFirst automated cell counters came out in the 1950sirst automated cell counters came out in the 1950s

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How the analyzers workHow the analyzers work ? ? • EElectrical impedance principle lectrical impedance principle ((CoulterCoulter))

– cells break an electric circuitcells break an electric circuit as they pass though as they pass though the aperture between electrodesthe aperture between electrodes

– indicatindicatee the presence of a cell the presence of a cell (number) (number) and the and the size of size of aa cell cell

• Optical principalOptical principal – – light scatter property of the light scatter property of the cell cell

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When to do CBC?

• Suspected hematologic, inflamatory, neoplastic, or infection disease

• Screening of infants (<1yr.), pregnant women, elderly patients, and patients with nutritional abnormalities

• Controversial values during routine patient evaluation

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Hemoglobin concentration (Hb) and Hematocrit (Hct)

• Depends on age and sex of the patient

• Depends on hydratation of the patient (e.g. pregnancy)

• F: Hb 121-151 g/L Hct 36-44%

• M: Hb 138-170 g/L Hct 41-50%

• Less then 70 g/L usually symptomatic tissue hypoxia

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ANEMIA

• WHO criteria: Hb < 125 g/L in adults

• US criteria:

–M: Hb < 135 g/L

–F: Hb < 125 g/L

• Clinical sign

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General consequence of anemia

hemoglobin loss

impaired delivery of oxygen to the tissues

tissue hypoxia

Symptoms: fatigue, dyspnea, paleness...Signs: tachycardia

Anemia

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Causes of anemia

• Insufficient RBC production: deficient erythropoiesis

– Complete loss of erythropoiesis results in Hb decline of about

10% / wk

• Excessive RBC loss

– Bleeding

• Acute: shortly after massive blood loss Hb normal due to

vasoconstriction (normochromic - normocytic)

• Chronic – leads to depletion of iron which results in

insufficient RBC production

– Hemolysis

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Red blood cell (RBC) count

• F: 3.9 – 5.0 x 1012 erythrocytes / L

• M: 4.5 –5.7 x 1012 erythrocytes / L

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RBC parameters (indices) - 1

MEAN CORPUSCULAR VOLUME = MCV– MCV (fL) = Hct / RBC count – Histological classification of anemias

• microcytic anemia ( < 80 fL)

• normocytic anemia (80 – 95 fL)

• macrocitic anemia (> 95 fL)

– Not useful to detect anisocytosis = variation in cell size• Red Cell Distribution Width (RDW 11- 15 %)

– Reticulocytosis may increase MCV

Differential diagnosis of anemias

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MEAN CORPUSCULAR HEMOGLOBIN = MCH– MCH (pg/cell) = Hb / RBC count– MCH 32.7 – 33.7 pg / cell– Hypochromia MCH < 27 pg / cell

MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION = MCHC– MCHC (g/L of RBC)= Hb / Hct – MCHC: 267 – 355 g / L

RBC parameters (indices) - 2

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RDW – RDW – rred cell ed cell ddistribution istribution wwidthidth• Measure the variation of red blood cell (RBC) size

(volume) • Normal RDW = 11 – 14 %• Higher RDW values indicate greater variation in RBC size =

anisocytosis

3 5 7 9 11 13 um

num

ber

of c

ells

normamikrocytární anémiemakrocytární anémie

Calculated by dividing the standard deviation (SD) of MCV by MCV and then multiplying that result by 100 [%]

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hypochromic microcytic anemia

macrocytic anemia hypersegmented neutrophil

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Reticulocyte count• Daily RBC replacement 40,000 – 50,000 /mL

– 0.5 – 1.5% of RBC count– Maturate within 1 day in peripheral blood

• Criteria of marrow activity – Reticulocytosis

• response to blood loss (hemolytic anemias, severe bleeding) • response to therapy of anemia (e.g. B12 or Fe def.)

– Reticulocytopenia• deficient erythropoiesis (nutrient , hormonal, etc.)

• Reticulocyte index = RI corrects for the severity of anemiaRI < 2% indicates hypoproliferative component of

anemiaRI = Reticulocyte Count x (HCT / normal HCT)

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Flow cytometryReticulocyte

count

Unstained

0 % 0.6 – 2.7 %

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Platelet count

• 140 – 440 K /L

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WBC count

• 4,3 – 10,8 x 109 / L

• WBC differential count– Segmented neutrophils: 34-75%; – Band neutrophils < 8%; – Lymphocytes: 12 – 50%; – Monocytes: 3-15%; – Eosinophils < 5%; – Basophils < 3%.

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Blood smear

• In case of pathologic values in automated analysis of blood count

• Morphology of blood elements– Anisocytosis = variation in size– Poikilocytosis = variation in shape

(schistocytes=RBC fragments; ovalocytes; spherocytes)

– Atypical leucocytes (e.g. blasts)

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← sférocyty

schistocyty →

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Sicle Cell Disease

Hemoglobin (Hb) S: záměna valinu za glutamin v pozici 6 Hb beta řetězce

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Peripheral blood film chronic myeloid leukemia

basophils

blast cell

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CML

blood film at 400X

leukocytosis with the presence of precursor cells of the myeloid lineage

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CML

blood film at 1000X magnification

whole granulocytic lineage, including an eosinophil and a basophil

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Specialized hematological tests

• Tests for iron metabolism• Erythropoietin measurements• Detection of antibodies to self antigens (e.g. RBC)• Cytogenetic and known mutation analysis• Immunophenotyping of BM or PB cells• Histochemical analysis of cell enzymatic activity• Functional tests (Clonogenic assay)

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Anemia caused by hemolysis (RBC destruction)

Extrisic cause (normocytic-normochromic RBC )• Immunologic abnormalities (AIHA, PNH)

• Mechanical injury (trauma, infection)

Intrinsic cause • Membrane alterations

– congenital (spherocytosis, elliptocytosis)

– Aquired (hypophosphatemia)

• Metabolic disorders (G6PD deficiency)

• Hemoglobinopaties (Sicle cell disease, Thalassemia)

reticulocytosis, LDH is increased, unconjugated bilirubin accumulate

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Hemolysis caused by immune mechanisms – Direct Coombs (antiglobulin) test

• Detection of antibodies to erythrocyte surface antigens

• Antibodies other than to AB antigens

• These Abs are responsible for hemolysis

Antiglobulin serum (anti-hu Ig) is added to washed patients RBC: agglutination indicates presence of immunoglobulins bound to RBC

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Acid hemolysis (Hams’) test• Diagnostic test for paroxysmal nocturnal

hemoglobinuria (PNH)• Acidification of blood (HCl) result in

hemolysis due to complement activation in PNH patients (Ham) test

Urine

Blood

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Diagnosis of Paroxysmal nocturnal hemoglobinuria

(PNH) flow cytometry

• Acquired hemolytic anemia due to a hematopoietic stem cell mutation defect

• Glycosyl-phosphatidylinositol anchor abnormality caused by the PIG-A gene mutation

• Clinical manifestation result from the lack of GPI dependent proteins on the surface of a portion of leukocytes and erythrocytes

• Flow cytometry analysis for CD55 and CD59 is used to diagnose PNH

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FFlow cytometrylow cytometry

• Technique which allows quantitative and qualitative analysis of cells in suspension.

• FACS (Fluorescence-activated-cell-sorting) – analysis and sorting of cells in suspension based on the

differences in light scattering and fluorescence of the cell or cell label.

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Měření exprese CD59 průtokovou cytometrií u pacientů s PNH

Erytrocyty

Granulocyty

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Evaluation of hemoglobin metabolismEvaluation of hemoglobin metabolism

• S-bilirubinS-bilirubinee – concentration reflects hem degradation concentration reflects hem degradation ((increase in hemolytic increase in hemolytic

anemiaanemia))

• S-haptoglobinS-haptoglobin ((binds binds hemoglobin)hemoglobin)• S-hemopexinS-hemopexin ((binds binds hem)hem)

((decrease in decrease in hemolytic anhemolytic anemiaemia))

• HHemoglobinemoglobin electrophoresis electrophoresis – identification of abnormal identification of abnormal hemoglobinhemoglobinss ( (e.g. e.g. hemoglobin S)hemoglobin S)

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Anemia caused by deficient erythropoiesis

• Iron deficiency – microcytic-anisocytosis, ↓ reticulocytes

• Vitamin B12 or Folate deficiency– macrocytes-anisocytosis

• Marrow failure - chronic diseases, aplastic anemia, myelodysplasia, leukemia– normochromatosis-normocytosis– BM hypoplasia

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Tests of iron metabolismSerum iron (SI) • F: 600-1400 g/L, 11-25mol/L; M: 750-1500 g/L, 13-27mol/L

– Low in Fe deficiency and chronic disease– High in hemolytic syndromes and iron overload

Total iron binding capacity (TIBC) • 2500 – 4500 g/L , 45-82 mol/L

– High in Fe deficiency– Low in chronic disease

Serum ferritin (30-300 ng/mL)• Fe storage glycoprotein• Closely correlates with total body Fe stores• <12 ng/mL Fe deficiency• Elevated in Fe overload, liver injury, tumors (Acute phase protein)

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Tests for iron metabolism

Serum transferin receptor

• Increase in increased erythropoiesis and early Fe deficiency

RBC ferritin

• storage status over the previous 3 month (Fe deficiency/overload)

• unaffected by liver function or acute illness

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Microcytic Hypochromic Anemia (MCV<83; MCHC<31)

Serum Iron

Total Iron-Binding Capacity

(TIBC)

Bone Marrow Iron

Comment

Sideroblastic Ring sideroblasts in marrow

Hemoglobinopaties Hemoglobin electrophoresisN N ++

N ++++

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Microcytic Hypochromic Anemia (MCV<83; MCHC<31)

Serum IronTotal Iron-

Binding Capacity (TIBC)

Bone Marrow Iron

Comment

Iron deficiency Responsive to iron therapy

Chronic inflammation

Unresponsive to iron therapy

++

0

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Serum erythropoietin (EPO)

• Enzyme-Linked ImmunoSorbent Assay (ELISA)

• Kidney - main source (but some other tissues produce as well)

• Expression increased by hypoxia (e.g. caused by anemia)

• Expression decreased – kidney disease (ClCr < 45 mL/min), other chronic diseases

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Epo

Role of Erythropoietin in RBC Production

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Sandwich ELISA

Antibody coated wellse.g. anti-EPO

Antigen binds to antibodye.g. EPO from human plasma/serum

Second monoclonal antibody binds to immobilized antigen (e.g. EPO)

A substrate is added and converted by enzyme to colored product

1

2

3 4

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Bone Marrow (BM) Analysis• Aspiration of bone marrow

– usually from posterior iliac crest or sternum 0.5-2.0 mL• Bone marrow biopsy

Direct observation of bone marrow activity

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Bone Marrow (BM) Analysis

• Indication – Unexplained anemia and other cytopenias– Unexplained leukocytosis and thrombocytosis– Suspicion of leukemia and myeloproliferative

diseases

• Histologic, cytologic, cytogenetic, and molecular biologic analysis

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BM biopsy

BM failure BM normal

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BM cell aspirate: CML

BM cells (400X) demonstrates clear dominance of granulopoiesis

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Chromosomal abnormalities - AML, CML, MDS

Cytogenetic Analysis of BM

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Philadelphia chromosome

• Chromosome 22 often referred as Ph (95% of pts with CML)

• Reciprocal translocation t(9;22) – piece of chromosome 9 (c-abl) translocate to chromosome 22 (bcr) [BCR-ABL] and piece of 22 to 9 [ABL-BCR].

• BCR-ABL - constitutively active tyrosin-kinase• target for therapy (STI571)

– prognostic value

– therapy response - Minimal Residual Disease

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CML - FISH

control bcr/abl fusion present

DNA probes: • bcr (22q11.2) in red • c-abl (9q34) in green.

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Molecular analysis of BM and PB cells

• measurement of gene expression or identification of mutations– Hemochromatosis - Hephestine– Monitoring of leukemia therapy:

• AML: e.g. AML-ETO

• CML: BCR-ABL

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Immunophenotyping of BM or PB cells

Flow cytometry (FACS): – suspected lymphoproliferative and

myeloproliferative diseases (AML, CML)

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Flow cytometry use in hematology:

• immunophenotyping of myeloproliferative and lymphoproliferative diseases using fluorescently labeled antidodies

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PV, Leukemia, MDS

– cultivation of BM cells in semisolid media with growth factors

– EPO independent BFU-E colonies P. Vera– CFU-GM/cluster ratio – decrease in leukemic

hematopoiesis

Functional tests - Clonogenic assay

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Clonogenic assay

CFU-GM BFU-E

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Sensitivity of BFU-E progenitors to Sensitivity of BFU-E progenitors to eryterythhropoetinropoetin

0%

25%

50%

75%

100%

0 30 60 125 250 3000

EPO (mU/ml)

PV (EEC)

PFCP

Normal

EEC

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Burst Forming Unit - Erythroid colony

Thank you