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Hematologic examination
Jan Živný, Martin Vokurka, Stanislav
Matoušek
Department of Pathophysiology
Clinical signs of hematology diseases
• Anemia → hypoxia signs – tiredness, weakness, lack of
breath, paleness
→ cardiovascular symptoms – palpitation
• Polycythemia → hyperviscose blood → risk of thrombosis
• Bleeding, spontaneous bleeding, continuous bleeding
• Thrombosis → embolia – symptoms depend on the
localisation – DVT, pulmonary embolism
•Frequent infections
Hematologic disease
Leukemias etc.
•Myelo-(proliferative)
Disorders
of Red Cells
•Lympho-
• Anemias
•(granulo-, mono-)
Hemostatic
disorders
•Primary hemostasis
•Coagulation
MAIN REASONS OF HYPOXIA
1.
2.
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)
Laboratory Tests
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 analysis
Immunophenotyping of BM or PB cells
Complete Blood Count (CBC)
•
•
•
•
•
•
•
•
Hb concentration
Hct
RBC count
RBC parameters
WBC count
WBC differential count
Platelet count and parameters
Description of blood smear
When to do CBC?
• suspected hematologic, inflammatory,
neoplastic, or infectious disease
• screening of infants (<1 yr.), pregnant
women, elderly patients, and patients with
nutritional abnormalities
• routine patient evaluation, admission to
hospital
Red blood cell (RBC) count
• F: 3.9 – 5.0 x 1012 erythrocytes / L
• M: 4.5 –5.7 x 1012 erythrocytes / L
Number of RBC and quantity
• decrease - anemia
• increase - polycythemia = polyglobulia
• Properties of RBC
• * membrane
• * hemoglobin
• * metabolism
ANEMIA
• WHO criteria: Hb < 125 g/L in adults
• US criteria:
–M: Hb < 135 g/L
–F: Hb < 125 g/L
ANEMIA
General sequlae of anemia
less of hemoglobin
impaired delivery of oxygen to the tissues
tissue hypoxia
fatigue, dyspnea, paleness...
tachycardia
hyperkinetic circulation
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
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
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
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)
stem cells, growth factors
erythropoetin
BONE MARROW
cell division: vitamin B12, folic acid
hemoglobin synthesis: globin, porphyrin, Fe
other factors
PRODUCTION
hemolysis
PERIPHERAL BLOOD
LOSSES
bleeding
RED BLOOD CELL CBC
hemoglobin, number or RBC,
hematokrit
MCV, MCH, MCHC
shape etc.
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 to increased loss
- due to bad distribution (hyperslenism, pooling
in spleen)
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.
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.)
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)
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)
HEMOLYSIS
INTRAVASCULAR
Hb
haptoglobin
kidneys
EXTRAVASCULAR
spleen, bone marrow, liver
(macrophages)
SYMPTOMS OF HEMOLYSIS
loss of red blood cells
loose Hb
intravascular
hemoglobinemia,
hemoglobinuria
hemosiderinuria
anemia
BM activation
damage to the kidneys
reticulocytosis
extravascular
increased prodution of bilirubin
jaundice (icterus)
splenomegaly
TESTS FOR HEMOLYSIS
immune mechanisms – direct Coombs (antiglobulin) test
Search for antibodies
against proper RBC
Antibodies other than AB0
These Abs are responsible
for hemolysis
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
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
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
Acid hemolysis (Hams’) test
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
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)
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)
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
Manifest
anemia
Latent
serum iron
Tf saturation
sTfR
iron deficiency
erythropoiesis
Prelatent
no stores
serum ferritin
TIBC
iron in BM
Microcytic Hypochromic Anemia
(MCV<83; MCHC<31)
Iron
deficiency
Chronic
inflammation
Thalassemia
major
Thalassemia
minor
Responsive to iron
therapy
Unresponsive to iron
therapy
Reticulocytosis and
indirect bilirubinemia
Elevation of fetal
hemoglobin, target
cells, and
poikilocytosis
Lead
poisoning
Basophilic stippling
of RBCs
Sideroblastic
Ring sideroblasts in
marrow
Hemoglobinopaties
Hemoglobin
electrophoresis
Microcytic Hypochromic Anemia
(MCV<83; MCHC<31)
Serum Iron
Lead poisoning
N
Sideroblastic
Hemoglobinopaties
N
Total IronBinding
Capacity
(TIBC)
N
Bone Marrow
Iron
++
Comment
Basophilic
stippling of
RBCs
N
++++
Ring
sideroblasts in
marrow
N
++
Hemoglobin
electrophoresis
Microcytic Hypochromic Anemia (MCV<83;
MCHC<31)
Serum Iron
Total IronBinding
Capacity (TIBC)
0
Iron deficiency
Chronic
inflammation
Thalassemia
major
Thalassemia
minor
Bone Marrow
Iron
N
N
N
Comment
Responsive to
iron therapy
++
Unresponsive to
iron therapy
++++
Reticulocytosis
and indirect
bilirubinemia
++
Elevation of A of
fetal
hemoglobin,
target cells, and
poikilocytosis
Macrocytic Anemia (MCV, >95)
Deficiency of vitamin B-12
Deficiency of folic acid
Megaloblastic
bone marrow
Drugs affecting DNA synthesis
Inherited disorders of DNA synthesis
Liver disease
Hypothyroidism and hypopituitarism
Nonmegaloblastic
Accelerated erythropoiesis (reticulocytes)
bone marrow
Hypoplastic and aplastic anemia
Infiltrated bone marrow
Blood smear
• Morphology of blood elements
– Anisocytosis = variation in size
– Poikilocytosis = variation in shape
(schistocytes=RBC fragments; ovalocytes;
spherocytes)
Various Forms of RBCs
Spherocyte
Loss of central pallor, stains more densely, often
microcytic. Hereditary spherocytosis and certain acquired
hemolytic anemias.
Target cell
Hypochromic with central "target" of hemoglobin. Liver
disease, thalassemia, hemoglobin D, postsplenectomy.
Elliptocyte
Oval to cigar shaped. Hereditary elliptocytosis, certain
anemias (particularly vitamin B-12 and folate deficiency).
Schistocyte
Fragmented helmet- or triangular-shaped RBCs.
Microangiopathic anemia, artificial heart values, uremia,
malignant hypertension.
Stomatocyte
Slitlike area of central pallor in erythrocyte. Liver disease,
acute alcoholism, malignancies, hereditary
stomatocytosis, and artifact.
Sickle cell
Elongated cell with pointed ends. Hemoglobin S and
certain types of hemoglobin C and l.
Sicle Cell Disease
Hereditary sferocytosis
Peripheral blood film chronic myeloid leukemia
basophils
blast cell
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%.
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
BM Failure
BM - Cytogenetic Analysis
Chromosomal abnormalities - AML, CML, MDS
Phases of chronic myeloid leukaemia
CML
leukocytosis with the presence of precursor cells of the myeloid lineage
blood film at 400X
CML
whole granulocytic lineage, including an eosinophil and a basophil
blood film at 1000X magnification
CML
BM cells (400X) demonstrates clear dominance of granulopoiesis
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
Philadelphia chromosome (Ph)
CML - FISH
control
DNA probes:
• bcr (22q11.2) in red
• c-abl (9q34) in green.
bcr/abl fusion present
The action of Imatinib Mesylate (STI571,
Gleevec)
Goldman JM, Melo JV. N Engl J Med. 2001. 344:1084-1086.
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
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)
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)
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
Clonogenic assay
CFU-GM
BFU-E
Sensitivity of BFU-E progenitors to
erythropoetin
100%
PV (EEC)
75%
PFCP
50%
Normal
EEC
25%
0%
0
30
60
125
EPO (mU/ml)
250
3000
Scheme of normal and malignant
hematopoiesis
SRC
Progenitors
LTC-IC pluripotent determined
Diferenciated
cells
AML
SL-IC
AML
blasts
AML-CFU
Hematopoietic Cells