Introduction to Hematology and Anemia

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Dr. Kalpana MallaMD Pediatrics

Manipal Teaching Hospital

Blood

• Blood volume is about 8% of body weight• 45 % is formed elements• 55% plasma

PLASMA

90 % Water10 % Solutes - Plasma proteins –

Albumins(58 %) - maintain osmotic (oncotic) pressure

Globulins (38 %) - antibodies synthesized by plasma cells

Clotting factors – fibrinogen – 4 %

FORMED ELEMENTS

• Three types:Erythrocytes – red blood cellsLeukocytes – white blood cellsThrombocytes – platelets – cell fragments

Development of hemopoietic system:3 anatomic stages:

Mesoblastic: in extraembryyonic structures - yolk sac (10- 14 days of gestation till 10-12 wks)

Hepatic: liver 6-8wks gestation - 20-24wks-primary site

of blood cell production (continues till remainder of gestation)

Myeloid: bone marrow (10-12 weeks)

Exception: lymphocytes –bone marrow+ other organs

Developmental changes:

• 2nd to 3rd trimester: circulating erythrocytes and granulocytes increase

• 2nd trimester - Haematocrit levels rise 30-40 % & at term rise is 50-63 % • Platelet concentration remains constant from 18th

wks till term • Life-span of RBCs ~60- 90 days in newborns vs 120

days

• Fetal bone marrow space develops - 8th wk of gestation • Neutrophils first observed ~ 5 wks of gestation • 14th wk to term: most common cell found in bone

marrow is neutrophil • Red marrow:• Newborns- in all cavities of bones• Older children & adults - in upper shaft of femur,

humerus, pelvis, spine, skull and bones of thorax• Erythropoiesis: - In-utero controlled by erythroid growth factors

produced by monocyte-macrophages of fetal liver - After birth controlled by erythropoietin from kidneys

The Red Cell

• Average life span = 120 days (60-90 days NB)• Cleared by RES (spleen, liver primarily)• Homeostasis daily loss = daily production• Otherwise anemia

Hemoglobin:

• Is complex protein - Made up of heme which contains an atom of iron and 4 polypeptide globin chains – reversible transport of Oxygen without expenditure of metabolic energy– Oxygen binds to iron in heme (also CO)– 23 % of CO2 is bound to globin portion

• If there is a problem with any part of the molecule it may not be functional

Types of Hb:

• Embryonic Hb: Gower-1: ζ2 ε2

Gower-2: α2 ε2

Portland: ζ2 2ץ

• Fetal Hb: Hb F: α2 2ץ

• Adult Hb:

Hb A : α2 β2

Hb A1: α2 δ2

Developmental changes in Hb:

• 4-8 wks gestation: Gower Hb predominates; disappears by 3rd month

• > 8th wk of gestation- Hb F predominant Hb• ~ 24 wks gestation 90 % of total Hb• At birth declines to 70 %• 6-12 months postnatal life < 2 %

Developmental changes in Hb:

• 16-20 wks gestation- some Hb A detectable• 24th wk gestation: 5-10 % , At term ~ 30 %Hb A

present• Hb A2 - < 1 % - At birth

- 2-3.4 % At 12 months (normal level)• Throughout life ratio of Hb A: Hb A2 is ~ 30:1

Hgb Norms• Normal values vary by age and gender

– High at Birth 20g/dl since HbF has high affinity for oxygen , by 3 months HbA replaces HbF

– Falls to lower-than-adult values by 3-6 months – Rises gradually to adult value by the early teenage

years– On average, Adult male Hb 2g/dl > female

counterpart due to effect of androgen .

Hgb and MCV VariabilityAge Hgb mean MCV mean

birth 16.5 1082 wk 16.5 1052 mo 11.5 96

6mo-2yr 12.5 772-6 yr 12.7 80

10-12 yr 13.5 8512-17 14 85Adult 14-16 90

Contemporary Pediatrics, Vol 18, No. 9

GRANULOCYTES

• Neutrophils – phagocytes• Eosinophils – red granules, associated with

allergic response and parasitic worms• Basophils – deep blue granules - Release heparin

and histamine

Neutrophil

Eosinophil

Basophil

AGRANULOCYTES

• Granules too small to be visible• Monocytes – become macrophages• Lymphocytes – B cells and T cells = immune

functions

Monocyte

Lymphocyte

Lab Investigation

• Table: Laboratory Tests in Anemia Diagnosis• i. Complete blood count (CBC)• A. Red blood cell count• 1. Hemoglobin• 2. Hematocrit• B. Red blood cell indices• 1. Mean cell volume (MCV)• 2. Mean cell hemoglobin (MCH)• 3. Mean cell hemoglobin concentration (MCHC]• 4. Red cell distribution width (RDW)C.

RBC indices

• Part of the (CBC) - • Mean cell volume(MCV) – Quantifies average red blood

cell size • Mean cell hemoglobin (MCH) –Hb amount per red

blood cell• Mean cell hemoglobin concentration (MCHC) - The

amount of hemoglobin relative to the size of the cell (hemoglobin concentration) per red blood cell

Contd -Red Blood Cell Indices Index Normal Value• MCV = 90 ± 8 hematocrit /red cell count (80 – 100) femtoliter • (MCH) = 30 ± 3 pg

Hb /red cell count (27 - 31)picograms/cell

• (MCHC)= 33 ± 2 Hb/hematocrit or MCH/MCV (32 – 36) gm/dl

Anemias - based on cell size (MCV) and amount of Hgb (MCH)

• MCV < lower limit of normal: microcytic anemia

• MCV normal range: normocytic anemia• MCV > upper limit of normal: macrocytic

anemia• MCH < lower limit of normal: hypochromic

anemia• MCH within normal range: normochromic

anemia• MCH > upper limit of normal: hyperchromic

anemia

Mentzer index

• Calculated number to help differentiate between iron deficiency vs. thalassemia if having microcytic anemia

• MCV/RBC• >13 iron deficiency• <13 thal trait

Red Cell Volume Distribution Width (RDW)• Reflects the variability in cell size

• Aids in further differentiating between specific etiologies of microcytic, normocytic, and macrocytic

• RDW = (Standard deviation of MCV ÷ mean MCV) ×

100

Contd

• C. White blood cell count• 1. Cell differential• 2. Nuclear segmentation of neutrophils• D. Platelet count

Blood smear

• Assess the size, color, and shape of red cells– Look for abnormalities – macrocyte, leptocyte, target cell, Tear drop,

Elliptocytosis,burr cell,acanthocyte, Schistocytes,Spherocytosis,Sickle cells,Poikilocytes– Anisocytosis, Polychromasia

MACROCYTE

Larger than normal >8.5 µm diameter

LEPTOCYTE

Hypochromic cell with a normal diameter and decreased MCV

Thalassemia.

TARGET CELL

Hypochromic with central "target" of hemoglobin. Liver disease, thalassemia, hemoglobin D, postsplenectomy

TEAR DROP CELL

Drop-shaped erythrocyte, often microcytic. Myelofibrosis and infiltration of marrow with tumor. Thalassemia

ELLIPTOCYTE

Oval to cigar shaped. Hereditary elliptocytosis, certain anemias (particularly vitamin B-12 and folate deficiency)

ECHINOCYTE (BURR CELL)

Evenly distributed spicules on surface of RBCs, usually 10-30. Uremia, peptic ulcer, gastric carcinoma, pyruvic kinase deficiency

.

ACANTHOCYTE

Five to 10 spicules of various lengths and at irregular interval on surface of RBCs.

STOMATOCYTE

• Slitlike area of central pallor in erythrocyte. Liver disease, acute alcoholism, malignancies, hereditary stomatocytosis, and artifact

SCHISTOCYTE

Fragmented helmet- or triangular-shaped RBCs. Microangiopathic anemia, artificial heart valves, uremia, malignant hypertension

Microcytic and Hypochromic

Smaller than normal ( <7 µm diameter

Less hemoglobin in cell. Enlarged area of central pallor.

Sickle cellElongated cell with pointed ends.

Hemoglobin S and certain types of hemoglobin C

Spherocyte Loss of central pallor, stains more densely, often microcytic. Hereditary spherocytosis and

certain acquired hemolytic anemias.poikilocytosis &

anisocytosisvariation in shape and variation in size

Contd

• II. Reticulocyte count• III. Iron supply studies• A. Serum iron• B. Total iron-binding capacity• C. Serum ferritin, marrow iron stain

Contd

• IV. Marrow examination• A. Aspirate• 1. E/G ratio• 2. Cell morphology• 3. Iron stain• B. Biopsy• 1. Cellularity• 2. Morphology E/G ratio, ratio of erythroid to

granulocytic precursors.

Reticulocyte Count

• Reticulocyte production index • RPI= Retic ct x Hb(obsv)/ Hb(normal)

x0.5• Indicates whether the BM is

appropriately responding to anemia• RPI >3 : inc prod = blood

loss/hemolysis• RPI <2 : dec prod / ineffective prod

CLASSIFICATION

INADEQUATE RESPONSE (RPI <2)• Hypochromic, microcytic• Normocytic normochromic• Macrocytic

ADEQUATE RESPONSE (RPI >3)• Hemolytic anemia• Blood loss

ANEMIA

What is Anemia?

• Anemia is defined as a reduction of the red blood cell (RBC) volume or hemoglobin concentration below reference level for the age and sex of the individual

• Hb < - 2SD or 95th centile for age and sex

Anemia Basics

All anemias are either due to….

1. Ineffective RBC productionor

2. Accelerated destruction of the RBC

• By RBC morphology and By Etiological factors responsible for anemia

Classification

Microcytic hypochromic anemia

1. Iron deficiency anemia – nutritional, posthemohragic2. Ineffective Erythropoiesis - Abnormal hemoglobinopathies, Thalassemia

syndrome, - Lead poisoning, Cu deficiency,

- Pyridoxine responsive -chronic ds - infection, inflammations , renal ds

MICROCYTIC

TAILS P: • T - Thalassemia

A - Anemia of chronic disease • I - Iron deficiency anemia • L - Lead toxicity associated anemia • S - Sideroblastic anemia • P – Pyridoxine deficiency

• Megaloblastic Erythropoiesis a) Nutritional - Folate deficiency, B12 deficiencyb) Toxic – Treatment with antifolate compound – methotrexate,, and drugs that inhibit DNA

replication – zidovudine, phenytoinc) Congenital disorders of DNA synthesis like Orotic aciduria etc. d) Malabsorption - liver ds - normal newborns, reticulocytosis

Macrocytic anemia

Macrocytic anemia• Non - Megaloblastic Erythropoiesisa) Chronic hemolytic anemia b) Liver dsc) Hypothyroidismd) Diamond blackfan syndrome

1. Impaired cell production (low reticulocyte count) - aplastic anemia - pure red cell aplasia - physiological anemia of infancy - infections - Systemic diseases like endocrinal, renal and hepatic diseases - bone marrow replacement – leukemia, tumors, starage ds, myelofibrosis, osteopetrosis2 Hemolytic anemia ( reticulocyte count high)

Normocytic, Normochromic anemia

DIMORPHIC ANEMIA

• When two causes of anemia act simultaneously, e.g : macrocytic hypochromic due to hookworm infestation leading to deficiency of both iron and vitamin B12 or folic acid

• following a blood transfusion

ETIOLOGICAL CLASSIFICATION OF ANEMIA • Blood loss Acute

Chronic

• Decreased iron assimilation - Nutritional deficiency - Hypoplastic or aplastic anemia - Bone marrow infiltration like leukemia & other malignancies, - Myelodysplastic syndrome

- Dyserythropoietic anemia

• Increased physiologic requirement - Extracorpscular - alloimmune & isoimmune hemolytic

anemia, microangiopathic anemias, infections, hypersplenism,

- Intracorpsular defect – Red cell membranopathy i.e. congenital

spherocytosis,elliptocytosis

– Hemoglobinopathy like HbS, C,D,E etc. Thalassemia syndrome

– RBC enzymopathies like G6PD deficiency, PK deficiency etc.

ETIOLOGICAL CLASSIFICATION OF ANEMIA

Differential of Anemia

lead poisoning

chronic d isease

thalssem ia

iron def

Hypochrom ic, m icrocytic

Renal d isease

Transient erythroblastopeniaof childhood

Ca/BM failure

chronic d is

Norm ochrom ic,norm ocytic

Drugs (etoh)

Down Syndrom e

Liver d isease

B12/fo late def

Macrocytic

Inadequate response (RPI<2)

Im m une Hem olytic anem ia

extrinsic factors(D IC ,HUS,TTP)

m em branopathy

enzym opathy

hem oglobinopathy

Adequate response (RPI>3)r/o b lood loss/hem olytic d is

Hgb, ind ices, retic count and sm ear

Follow-up

• Re-check CBC 4-6 weeks (to confirm response)• Continue iron 3-4 months (to replace stores)• Generally, should not need treatment for more than

5 months unless there are ongoing losses• If no improvement on adequate iron therapy,

consider evaluating the child for lead poisoning or thalassemia

PHYSIOLOGIC ADJUSTMENTS

• increased cardiac output • increased oxygen extraction (increased

arteriovenous oxygen difference)• shunting of blood flow toward vital organs

and tissues• the concentration of 2,3-

diphosphoglycerate (2,3-DPG) increases within the RBC

• The resultant “shift to the right” of the oxygen dissociation curve, reducing the affinity of hemoglobin for oxygen, results in more complete transfer of oxygen to the tissues

CLINICAL FATURES

• weakness • tachypnea • shortness of breath on exertion• tachycardia• cardiac dilatation• congestive heart failure • ultimately result from increasingly severe

anemia, regardless of its cause.

Serum Iron TIBC BM Iron Comment

Iron deficiency D I 0 Responsive to iron therapy

Chronic inflammatio

n

D D ++ Unresponsive to iron therapy

Thalassemia major

I N ++++ Reticulocytosis and indirect bilirubinemia

D/D of microcytic anemia:

Thalassemia minor N N ++

Elevation of A of fetal hemoglobin, target cells, and poikilocytosis

Lead poisoning N N ++ Basophilic stippling of RBCs

Sideroblastic I N ++++

Ring sideroblasts in marrow

Serum iron

TIBC BM Iron

Comment

PYROPOIKILOCYTE

• RBCs w/c are extremely sensitive to heat

HEMOLYTIC ANEMIA- INTRACORPUSCULAR

Hereditary spherocytosisHereditary elliptocytosisHemoglobinopathiesThalassemiasCongenital dyserythropoietic anemiasHereditary RBC enzymatic deficienciesParoxysmal nocturnal hemoglobinuriaSevere iron deficiency

HEMOLYTIC ANEMIA- EXTRACORPUSCULAR

Physical agents: Burns, cold exposureTraumatic: Prosthetic heart valves, graft rejectionChemicals: Drugs and venomsInfectious agents: Malaria, toxoplasmosis, leishmaniasisHepatic and renal diseaseCollagen vascular diseaseMalignanciesTransfusion of incompatible bloodHemolytic disease of the newbornCold hemagglutinin d/sAutoimmune hemolytic anemia

Thrombotic thrombocytopenic purpura (TTP)Hemolytic uremic syndrome (HUS)

DIC

Alterations of Hbs by disease:• Gower Hb in few newborns: Trisomy 13/15• Hb Portland: stillborns with homozygous α-thalassemia• Elevated HbF (>2 %): β-thalassemia trait homozygous thalassemia Hb SS, Hb SC preterm infants treated with human recombinant

EPO others: hemolytic anemias leukemia aplastic anemia

• Hb A2 > 3.4 %: β-thalassemia trait

megaloblastic anemia• Decreased Hb A2 :IDA

α-thalassemia

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