Hematologic examination
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Hematologic examination







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Hematologic examination. Jan Živný , Martin Vokurka , Stanislav Matou šek Department of Pathophysiology. Clinical signs of hemato logy diseases. Anemi a → hypoxi a signs – tiredness, weakness, lack of breath, paleness → c ardiovas c ul ar s ymptoms – palpitation
Hematologic examination

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Slide 1

Hematologic examination

Jan Živný, Martin Vokurka, Stanislav Matoušek

Department of Pathophysiology

Slide 2

Clinical signs of hematology diseases

  • Anemia → hypoxia signs – tiredness, weakness, lack of breath, paleness

  • → cardiovascularsymptoms – palpitation

  • Polycythemia→ hyperviscoseblood → risk of thrombosis

  • Bleeding, spontaneous bleeding, continuous bleeding

  • Thrombosis → embolia – symptoms depend on the localisation – DVT, pulmonary embolism

  • Frequent infections

Slide 3

Hematologic disease

  • Leukemias etc.

  • Myelo-(proliferative)

  • Lympho-

  • (granulo-, mono-)

  • Disorders of Red Cells

  • Anemias

  • Hemostatic disorders

  • Primary hemostasis

  • Coagulation

Slide 4

MAIN REASONS OF HYPOXIA

  • Lack of oxygen in inspired air / low oxygen partial pressure

  • Respiratory failure

    • (= hypoxic hypoxia)

      3. Lack of hemoglobin – oxygen transporter

  • (= anemic, transport hypoxia)

    4. Circulation disorders (= circulatory hypoxia)

  • „lack of oxygen to the organs, tissues“

    5. Cell metabolism disorders (= histotoxic hypoxia)

Slide 5

  • Basic:

    • Complete blood count

  • Specialized:

    • Tests for iron metabolism

    • Erythropoietin measurements

    • Detection of antibodies to self antigens (e.g. RBC)

    • Histochemical analysis of cell enzymatic activity

    • Cytogenetic and known mutation analysisImmunophenotyping of BM or PB cells

Laboratory Tests

Slide 6

Complete Blood Count (CBC)

  • Hb concentration

  • Hct

  • RBC count

  • RBC parameters

  • WBC count

  • WBC differential count

  • Platelet count and parameters

  • Description of blood smear

Slide 7

When to do CBC?

  • suspected hematologic, inflammatory, neoplastic, or infectious disease

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

  • routine patient evaluation, admission to hospital

Slide 8

Red blood cell (RBC) count

  • F: 3.9 – 5.0 x 1012 erythrocytes / L

  • M: 4.5 –5.7 x 1012 erythrocytes / L

Slide 9

Number of RBC and quantity

  • decrease - anemia

  • increase - polycythemia = polyglobulia

  • Properties of RBC

  • * membrane

  • * hemoglobin

  • * metabolism

Slide 10

ANEMIA

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

  • US criteria:

    • M: Hb < 135 g/L

    • F: Hb < 125 g/L

Slide 11

ANEMIA

General sequlae of anemia

less of hemoglobin

impaired delivery of oxygen to the tissues

tissue hypoxia

fatigue, dyspnea, paleness...

tachycardia

hyperkinetic circulation

Slide 12

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

Slide 13

RBC parameters (indices) - 1

Differential diagnosis of anemias

MEAN CORPUSCULAR VOLUME = MCV

  • MCV (fL) = Hct / RBC count

  • Histological classification of anemias

    • microcytic anemia ( < 80 fL)

    • normocytic anemia (80 – 95 fL)

    • macrocytic anemia (> 95 fL)

  • Not useful to detect anisocytosis = variation in cell size

    • Red Cell Distribution Width (RDW 11- 15 %)

  • Reticulocytosis may increase MCV

Slide 14

RBC parameters (indices) - 2

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

Slide 15

Types of anemias due to RBC parameters

  • size:

  • normocytic

  • microcytic - smaller

  • macrocytic (megaloblastic) – greater

  • Hb content(„colour“):

  • normochromic

  • hypochromic (decreased amount of Hb)

  • hyperchromic (increased amount of Hb)

Slide 16

stem cells, growth factors

BONE MARROW

erythropoetin

cell division: vitamin B12, folic acid

hemoglobin synthesis: globin, porphyrin, Fe

other factors

PRODUCTION

hemolysis

RED BLOOD CELL CBC

PERIPHERAL BLOOD

LOSSES

hemoglobin, number or RBC,

hematokrit

MCV, MCH, MCHC

shape etc.

bleeding

Slide 17

  • Causes of anemia

  • due to decreased production

    • Stem cell disorder

    • DNA synthesis impaled

    • Hemoglobin synthesis impaled

    • Lack of erythropoietin

      • Complete loss of erythropoiesis result in decline of

      • about 10% / wk

  • due to increased destruction

    • Erythrocyte defect

    • Extra-erythrocyte causes

  • due toincreased loss

  • due to bad distribution (hyperslenism, pooling

  • in spleen)

Slide 18

Activity of erythropoesis

  • number of reticulocytes(0.5-1.5 %)

  • serum (solubile) transferrin receptor (sTfR) – increased need for Fe

  • Low: suppression of hemopoiesis (erythropoiesis)

  • High (reticulocytosis): activization

  • after bleeding

  • after hemolysis

  • successful treatment of anemia etc.

Slide 19

Reticulocyte count

  • Daily RBC replacement

    • 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.)

Slide 20

Blood loss anemia

  • Acute blood loss

    • shortly after massive blood loss Hb normal due to vasoconstriction

    • normochromic - normocytic

  • Chronic blood loss

    • results in iron deficiency

  • Excessive hemolysis (RBC destruction)

Slide 21

Excessive hemolysis (RBC destruction)

reticulocytosis, LDH is increased, unconjugated bilirubin accumulate

Extrinsic RBC defect (normocytic-normochromic RBC )

  • Immunologic abnormalities (AIHA, PNH)

  • Mechanical injury (trauma, infection)

    Intrinsic RBC defect

  • Membrane alterations

    • congenital (spherocytosis, elliptocytosis)

    • Aquired (hypophosphatemia)

  • Metabolic disorders (G6PD deficiency)

  • Hemoglobinopaties (Sicle cell disease, Thalassemia)

Slide 22

HEMOLYSIS

INTRAVASCULAR

haptoglobin

kidneys

Hb

EXTRAVASCULAR

spleen, bone marrow, liver(macrophages)

Slide 23

SYMPTOMS OF HEMOLYSIS

intravascular

hemoglobinemia,

hemoglobinuria

hemosiderinuria

loss of red blood cells

loose Hb

anemia

BM activation

damage to the kidneys

reticulocytosis

extravascular

increased prodution ofbilirubinjaundice (icterus)

splenomegaly

Slide 24

TESTS FOR HEMOLYSIS

immune mechanisms – directCoombs (antiglobulin) test

Search for antibodies against proper RBC

Antibodies other than AB0

These Abs are responsiblefor hemolysis

Slide 25

Direct antiglobulin (Coombs’) test (DAT)

  • Detection of antibodies to erythrocyte surface antigens

  • AIHA

  • Antiglobulin serum is added to washed RBC from the patient ------ agglutination indicates presence of immunoglobulins or complement components bound to RBC

Slide 26

TESTS FOR HEMOLYSIS

Test of osmotic resistence

RBC survive only in isotonic surrounding but have some toleration to its changes

RBC in some hemolytic states have decreased tolerance

Special tests

membrane properties (electrophoresis of proteins)

properties of hemoglobin

genetic tests

Slide 27

Acid hemolysis (Hams’) test

  • Diagnostic test for paroxysmal nocturnal hemoglobinuria (PNH)

  • HCl acidification of blood = hemolysis when PNH

  • Currently Flow cytometry analysis for CD55 and CD59 is more reliable to diagnose PNH

  • Glycosyl-phosphatidyl-anchor abnormality caused by the PIG-A gene mutation = clinical manifestation result from the lack of GPA dependent proteins on the surface of cells

Slide 28

Acid hemolysis (Hams’) test

Slide 29

Deficient erythropoiesis

  • Iron deficiency

    • microcytic-anisocytosis, ↓ reticulocytes

  • Vitamin B12 or Folate deficiency

    • macrocytes-anisocytosis

  • BM failure - chronic diseases, aplastic anemia, myelodysplasia, leukemia

    • normochromatosis-normocytosis

    • BM hypoplasia

Slide 30

Tests for iron

  • iron concentration in serum (age , sex)

  • TIBC (total iron binding capacity for Fe)

  • transferrin saturation (N 20-55 %)

  • serum ferritin

  • serum (solubile) transferrin receptor (sTfR)

Slide 31

Tests of iron metabolism

Serum iron ( SI)

  • F: 600-1400 mg/L, 11-25mmol/L; M: 750-1500 mg/L, 13-27mmol/L

  • Low in Fe deficiency and chronic disease

  • High in hemolytic syndromes and iron overload

    Total iron binding capacity (TIBC)

  • 2500 – 4500 mg/L , 45-82 mmol/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)

Slide 32

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

    Free RBC porphyrin

  • increased when heme synthesis altered

Slide 33

anemia

Manifest

Latentiron deficiency

erythropoiesis

serum iron

Tf saturation

sTfR

Prelatentno stores

serum ferritin

TIBC

iron in BM

Slide 34

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

Slide 36

N

N

++

N

++++

N

N

++

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

Slide 37

0

++

N

++++

N

N

++

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

Slide 38

Macrocytic Anemia (MCV, >95)

Slide 40

Blood smear

  • Morphology of blood elements

    • Anisocytosis = variation in size

    • Poikilocytosis = variation in shape (schistocytes=RBC fragments; ovalocytes; spherocytes)

Slide 41

Various Forms of RBCs

Slide 42

Sicle Cell Disease

Slide 43

Hereditary sferocytosis

Slide 44

Peripheral blood film chronic myeloid leukemia

basophils

blast cell

Slide 45

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%.

Slide 46

Bone Marrow (BM) Analysis

Aspiration usually from posterior iliac crest 0.5-2.0 mL

Direct observation of bone marrow activity

  • Indication

    • Unexplained anemia and other cytopenias

    • Unexplained leukocytosis and thrombocytosis

    • Suspicion of leukemia and myeloproliferative diseases

Slide 47

BM Failure

Slide 48

BM - Cytogenetic Analysis

Chromosomal abnormalities - AML, CML, MDS

Slide 49

Phases of chronic myeloid leukaemia

Slide 50

CML

leukocytosis with the presence of precursor cells of the myeloid lineage

blood film at 400X

Slide 51

CML

whole granulocytic lineage, including an eosinophil and a basophil

blood film at 1000X magnification

Slide 52

CML

BM cells (400X) demonstrates clear dominance of granulopoiesis

Slide 53

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

Slide 55

Philadelphia chromosome (Ph)

Slide 56

CML - FISH

bcr/abl fusion present

control

  • DNA probes:

    • bcr (22q11.2) in red

    • c-abl (9q34) in green.

Slide 57

The action ofImatinib Mesylate (STI571, Gleevec)

Goldman JM, Melo JV. N Engl J Med. 2001. 344:1084-1086.

Slide 58

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

Slide 59

Immunophenotyping of BM or PB cells

Flow cytometry (FACS):

  • suspected lymphoproliferative and myeloproliferative diseases (AML, CML)

  • diagnosis of PNH:

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

    • Harris test: GPI-anchored antigens are lacking from a portion of leukocytes, and usually erythrocytes in patients with PNH (e.g. CD59)

Slide 60

Histochemical analysis of enzymatic activity in BM blasts

  • ALP – high in normal blasts / low in leukemia blasts

  • Peroxidase activity – leukemic blasts

  • Nonspecific esterase – monocytes (leuk. phenotype)

Slide 63

Functional tests - Clonogenic assay

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

Slide 64

Clonogenic assay

BFU-E

CFU-GM

Slide 65

Sensitivity of BFU-E progenitors to erythropoetin

100%

PV (EEC)

75%

PFCP

50%

Normal

EEC

25%

0%

0

30

60

125

250

3000

EPO (mU/ml)

Slide 66

Scheme of normal and malignant hematopoiesis

Progenitors

SRC

LTC-IC

pluripotent

determined

Diferenciated

cells

AML

AML

blasts

SL-IC

AML-CFU

Slide 68

Hematopoietic Cells


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