1 ANEMIA And Its Laboratory Diagnosis By: Ahmad Sh. Silmi IUG, Hematologist [email protected].

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ANEMIA And Its Laboratory Diagnosis

By: Ahmad Sh. Silmi IUG, Hematologist

[email protected]

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ObjectivesAt the end of this chapter you will be able to:• Define anemia• Discuss the causes and clinical significance of different

categories of anemia• Describe the classification of anemia• Explain - Microcytic anemia

- Macrocytic anemia

- Normochromic normocytic anemia• Discuss the laboratory findings for each category of

anemia• Perform basic laboratory tests for the diagnosis of

anemia

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Chapter Outline3.1. Definition of anemias

3.2. Classification of anemias 3.2.1. Hematologic Response to Anemia3.2.2. Signs of Accelerated Bone Marrow Erythropoiesis3.2.3. Physiologic Response to Anemia 3.2.4. Methods of classification 3.2.5. Anemia Diagnosis/Cause3.2.6. Lab Investigation of Anemia

3.3. Types of anemia. 3.3.1 microcytic hypochromic anemia

3.3.2. macrocytic normocytic anemias3.3.3. normocytic anemias3.3.4. normocytic anemias due to hemoglobinopathies

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3.1. Introduction 3.1.1. Definition of Anemia• Anemia is a decrease in the RBC count, Hgb and/or

HCT values as compared to normal reference range for age and sex (Also determined by alteration in plasma volume)– ‘True’ anemia:

– decreased RBC mass and normal plasma volume

– Pseudo or dilutional anemia:

– normal RBC mass and increased plasma volume– An increase in plasma volume can occur in Pregnancy, volume

overload (IVs) congestive heart failure

– Low Hgb and HCT values

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Definition of Anemia cont’d

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3.1.1. Definition of Anemia cont’d…..

• Anemia must also relate to the level of hemoglobin the individual normally possesses. – If an adult male usually maintains a

hemoglobin level of 16g/dl, and over a period of days is noted to have decreased to 14g/dl, this must be considered significant even though both values are within the normal range for an adult male.

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3.1.1. Definition of Anemia cont’d…..• Various diseases and disorders are associated

with decreased hemoglobin levels. These include:– Nutritional deficiencies– External or internal blood loss– Increased destruction of RBCs– Ineffective or decreased production of RBCs– Abnormal hemoglobin synthesis– Bone marrow suppression by toxins, chemicals, or

radiation & replacement by malignant cells– Infection

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3.1.1. Definition of Anemia cont’d…..

Functionally anemia is defined as tissue hypoxia (inability of the body to supply tissue with adequate oxygen for proper metabolic function)– There is an abnormal hemoglobin with an

increased O2 affinity resulting in an anemia with normal or raised hemoglobin levels, hematocrit, or RBC count.

• Generally anemia is not a disease, but rather the expression of an underlying disorder or disease.

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3.1.1. Definition of Anemia cont’d…..

• Anemia may develop:– When RBC loss or destruction exceeds

the maximal capacity of bone marrow RBC production or

– When bone marrow production is impaired

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3.1.2.Hematologic Response to Anemia

• Tissue hypoxia causes increased renal release of erythropoietin (EPO) to accelerate bone marrow erythropoiesis

• The normal bone marrow can increase its activity 7-8 times normal – Marrow becomes hypercellular

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Signs of Accelerated Bone Marrow Erythropoiesis

• The marrow becomes hypercellular due to a marked increase in RBC precursors (called erythroid hyperplasia) and the M:E ratio falls.

• Nucleated RBCs may be released into the blood circulation along with the outpouring of reticulocytes– NRBC number tends to correlate with the

severity of anemia– Increased polychromasia on the Wright's-

stained blood smear is seen due to increased number of circulating Retics.

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• If demand exceeds maximal bone marrow activity, RBC production may occur in extramedullary sites, liver, spleen (hepatosplenomegaly).

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3.1.3. Physiologic Response to Anemia

• Ability to adapt to anemia depends on:– Age and underlying disease– Cardio/pulmonary function– Rate at which anemia develops (BM can

compensate easier if the onset of anemia is slow),

– Underlying disease

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3.1.4. Clinical features:

• Symptoms of hypoxia: decreased oxygen delivery to the tissues/organs

causes: – fatigue , faintness, weakness, dizziness,

headaches, dyspnea, poor exercise tolerance, leg cramps.

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Signs of anemia• general signs include pallor of

mucous membrane, which occur if the Hgb concentration is less than 9g/dl,

• specific signs are associated with particular types of anemia, for example, jaundice in hemolytic anemia, leg ulcer in sickle cell anemia

3.1.4.Clinical features cont’d….:

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3.1.5. Diagnosis of anemia

• Before making a diagnosis of anemia, one must consider:– Age– Sex– Geographic location– Presence or absence of lung disease

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3.1.5. Diagnosis of anemia cont’d……

• How does one make a clinical diagnosis of anemia?A. Patient history

–Dietary habits–Medication–Possible exposure to chemicals and/or

toxins–Description and duration of symptoms

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3.1.5. Diagnosis of anemia cont’d……

–Tiredness–Muscle fatigue and weakness–Headache and vertigo (dizziness)–Dyspnia (difficult or labored breathing)

from exertion–G I problems–Overt signs of blood loss such as

hematuria (blood in urine) or black stools

Patient history cont………..

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3.1.5. Diagnosis of anemia cont’d….B. Physical exam

– General findings• Hepato or splenomegaly• Heart abnormalities• Skin pallor

– Specific findings • In vitamin B12 deficiency there may be signs of

malnutrition and neurological changes• In iron deficiency there may be severe pallor, a

smooth tongue, and esophageal webs• In hemolytic anemias there may be jaundice due to

the increased levels of bilirubin from increased RBC destruction

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3.1.5. Diagnosis of anemia cont’d……

C. Lab investigations

1. A complete blood count, CBC– RBC count– Hematocrit (Hct) or packed cell volume– Hemoglobin determination– RBC indices calculation– Reticulocyte count

2. Blood smear examination to evaluate:– Poikilocytosis– Leukocytes or Platelets abnormalities

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3.1.5. Diagnosis of anemia cont’d…..

Lab investigation cont’d……3. A bone marrow smear and biopsy to

observe:– Maturation of RBC and WBC series– Ratio of myeloid to erythroid series– Abundance of iron stores (ringed sideroblasts)– Presence or absence of granulomas or tumor

cells– Red to yellow ratio– Presence of megakaryocytes

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4. Hemoglobin electrophoresis

3.1.5. Diagnosis of anemia cont’d…..

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3.1.5. Diagnosis of anemia cont’d…..

Lab investigation cont’d……

5. Antiglobulin testing

6. Osmotic fragility test

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3.2. Methods of Anemia Classification• Several schemes of classifying anemias exist 1. Morphologic

– Based on RBC morphology– Anemia is divided into three groups mainly on

the basis of the MCV (RBC indices)2. Pathophysiologic

– Anemia is divided using three main causes/mechanisms I. Impaired erythrocyte formation (Aplastic

anemia, IDA, sideroblastic anemia, anemia of chronic diseases, megaloblastic anemia)– Retic count is low– The bone marrow fails to respond appropriately

due to disease or lack of essential supplies

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Methods of Classification cont’dIII. Increased blood loss (Acute, Chronic)

– Retic count is typically high– Anemia results when red cell loss exceeds

the bone marrow’s capacity to increase its activity

IV. Increased destruction of RBCs (hemolytic anemias)

– Retic count is typically high– Anemia results when red cell destruction

exceeds the bone marrow’s capacity to increase its activity

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Methods of Classification cont’d

Morphologic Categories of Anemia

1. Normocytic Normochromic anemia (normal red cell indices)• Blood loss anemia • Hemolytic anemia• Aplastic anemia• Chronic diseaes• Renal insufficciency

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Morphologic Categories of Anemia

• Microcytic hypochromic

( low red cell indices)

• Iron deficiency anemia

• Sideroblastic anemia

• Lead poisoning

• Thallassemia

• Chronic diseases

Methods of Classification cont’d

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Morphologic Categories of Anemia

• Macrocytic Normochromic

( high MCV and MCH, normal MCHC)

• Megaloblastic anemia

• Liver disease

• Post splenectomy

• Hypothyroidism

• Stress erythropoiesis

Methods of Classification cont’d

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1 Microcytic/hypochromic

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1 2

2 Macrocytic/Normochromic

3 Normocytic/Normochromic

Morphologic Categories of Anemia

N.B. The nucleus of a small lymphocyte (shown by the arrow) is used as a reference to a normal red cell size

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3.2.2. Pathophysiological classification

1.

B.

2.

A.

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1. Microcytic- Hypochromic Anemia

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Microcytic- Hypochromic Anemia

• Many RBCs smaller than nucleus of normal lymphocytes

• Increased central pallor.• Includes

– Iron deficiency anemia– Thalassemia– Anemia of chronic disease – Sideroblastic anemia– Lead poisoning

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Iron Protoporphyrin

HemeHeme GlobinGlobin+

HemoglobinHemoglobin

• Iron deficiency

• Chronic inflammation or malignant (ACD)

• Thalassemia ( or )

• Sideroblastic anemia

The Cause of Microcytic-Hypochromic Anemia

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RBC maturation in microcytic anemias

Normoblastic RBC maturation normocytic red cells

Abbott Manual

Microcytic/Hypochromic Anemias

Normal RBC maturation is shown for comparison

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A. Iron Deficiency Anemia (IDA) • Is a condition in which the total body iron

content is decreased below a normal level

• This results in a reduced red blood cell and hemoglobin production

• More than half of all anemias are due to iron deficiency.

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Iron Deficiency Anemia (IDA)

• Causes:– Nutritional deficiency– Malabsorption (insufficient or defective

absorption)– Inefficient transport, storage or utilization of

iron– Increased need– Chronic blood loss (GI bleeding, ulcer, heavy

menstruation, etc)

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DIETARY SOURCES OF IRONDIETARY SOURCES OF IRON

Inorganic Iron eg lentils

Organic iron eg beef

DAILY IRON REQUIREMENT 10-15mg/day (5-10% absorbed)

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Daily Iron cycle (Fig)

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Adult men 0.5-1

Post menopausal female 0.5-1

Menstruating female 1-2

Pregnant female 1.5-3

Children 1.1

Female (age 12-15) 1.6-2.6

Estimated daily iron requirementsUnits are mg/day

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Iron absorption, Transport and storage

• Iron absorbed from duodenum and jejunum in the GIT

• Moves via circulation to the bone marrow

• Incorporated with protoporpyirin in mitochondria of the erythroid precursor to make Heme

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• There are three proteins important for transporting and storage of iron:

–Transferrin, –Transferrin receptors and –Ferritin

Transport: • Transferrin: transports iron from the

plasma to the erythroblasts in the marrow for erythropoiesis

• The transferrin will bind to transferrin receptor on the erythrocyte membrane

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Storage • Hgb contains about two third of the body iron

• At the end of their life, RBCs are broken down in the macrophage of reticuloendothelial system and then iron is released from Hgb enters plasma and provided to transferrin.

• Some stored in reticuloendothelial cell as ferritin soluble protein – iron complex) and hemosiderin (37%) (degraded form of ferritin insoluble)

• iron is also found in muscles as myoglobin and in other cells as iron containing enzymes

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Amount of iron Male Female %

in average adult (g) (g) of total

Hb 2.4 1.7 65

ferritin & hemosiderin 1.0 0.3 30

Myoglobin 0.15 0.12 3.5

Heme enzyme 0.02 0.15 0.5

Transferrin-bound 0.004 0.003 0.1

iron

The distribution of body iron

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Iron Deficiency Anemia (IDA) • Sequence of iron depletion

When iron loss or use exceeds absorption, there is a sequence of iron depletion in the body:

1.Storage iron decreases/ low serum ferritin; serum iron & TIBC are normal, no anemia, normal red cells.

2.Serum iron decreases/TIBC increases (increased transferrin); no anemia, normal red cells.

3.Anemia with microcytic/hypochromic red cells = IDA.

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CLINICAL FEATURES IRON DEFICIENCYCLINICAL FEATURES IRON DEFICIENCY

• Symptoms eg. fatigue, dizziness, headache

• Signs eg. pallor, Tongue atrophy/ glossitis -

raw and sore, angular cheilosis (Stomatitis)

Koilonychia Glossitis

Angular Cheilosis or Stomatitis

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• Clinical signs and symptoms– Spoon‑shaped nails (koilonychia), brittle

nails and hair.

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Lab Investigation of IDA

• Iron tests►Used to differentiate microcytic

hypochromic anemia's or detect iron overload (hemochromatosis)– Iron circulates bound to the transport protein

transferrin• Transferrin is normally ~33% saturated with iron

• Iron tests include serum iron, Total Iron Binding Capacity (TIBC), serum ferritin

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Lab Investigation cont’d• Serum iron level

– measures the amount of iron bound to transferrin– Does not include the free form of iron

• Total Iron Binding Capacity (TIBC)– Is an indirect measure of the amount of transferrin

protein in the serum– Inversely proportional to the serum iron level

• If serum iron is decreased, total iron binding capacity of transferrin increased (transferrin has more empty space to carry iron)

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Lab Investigation cont’d• Serum ferritin

– indirectly reflects storage iron in tissues – found in trace amount in plasma – It is in equilibrium with the body stores– Variation in the quantity of iron in the storing compartment is

reflected by plasma ferritin concentratione.g. Plasma ferritin is decreases in IDA

Plasma ferritin increases in ACD

Limitation: During infection or inflammation Serum Ferritin increases like other acute phase proteins, and then it is not an accurate indicator in such situations.

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Bone marrow iron (Tissue iron)

• Tissue biopsy of bone marrow

• Prussian blue stain

• Type of iron is hemosiderin

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ABSENT IRON STORES IN BONE ABSENT IRON STORES IN BONE MARROW IN IRON DEFICIENCYMARROW IN IRON DEFICIENCY

Iron deficiencyNormal control

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Iron Deficiency Anemia

• Lab findings– Low RBC, Hgb, Hct– Low MCV, MCH, MCHC– Normal WBC and PLT

Blood smear

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Iron Deficiency Anemia• RBC morphology

– Hypochromia– Microcytosis– Anisocytosis– Poikilocytosis

• Pencil cells (cigar cells)• Target cells

– no RBC inclusions

• Iron parameters– Low serum iron, – High TIBC, – Low serum ferritin

Blood smear

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Wright’s stained blood smear

Ovalocytes - Pencil forms No RBC inclusions

Iron Deficiency

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IDA cont’d

• Treatment

– Identify the underlying cause– Oral iron is given; see increased Retic

count post-therapy.– May see dimorphism following treatment

• a dual red cell population with older microcytic red cells along with the newly produced normocytic red cells.

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B. Sideroblastic Anemia (SA)

• This group of anemias are characterized by defective protoporphyrin synthesis (blocks) resulting in iron loading and a hypochromic anemia due to deficient hemoglobin synthesis.

• Block(s) in protoporphyrin synthesis leads to iron overload and microcytic/hypochromic anemia

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Sideroblastic Anemia (SA)

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Terms:• Siderocytes are mature RBCs in the blood

containing iron granules called Pappenheimer bodies....abnormal.

• Sideroblasts are immature nucleated RBCs in the bone marrow containing small amounts of iron in the cytoplasm....normal.

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SA• Sideroblastic anemia is characterized by the

– Accumulation of iron in the mitochondria of immature nucleated RBCs in the bone marrow;

– Iron forms a ring around the nucleus these are called ringed sideroblasts....abnormal.

• The iron accumulation in the mitochondria is the result of blocks in the protoporphyrin pathway.

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SA cont’dLab findings:• Microcytic/hypochromic red cells, low MCV and MCHC;

variable anemia, low retic.• RBC inclusions: Basophilic stippling and Pappenheimer

bodies (siderocytes). (May see target cells).• High serum iron and high serum ferritin (stores); low

TIBC and high % saturation. • *Decreased transferrin synthesis occurs in iron

overload states.• Bone marrow: ringed syderoblasts (Hall mark of

Sideroblastic Anemia)

6161

RBC with iron Wright’s stain

NRBC with iron Prussian blue stain

NRBC with ring of iron Prussian blue stain

Pappenheimer bodies

Blood Bone marrowBone marrow

SideroblastRinged Sideroblast

Sideroblastic Anemia (SA)

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Pappenheimer bodies Wright’s stain

Blood

Basophilic stippling/stippled RBCs

Blood

Pappenheimer bodies Prussian blue iron stain

Blood

Sideroblastic Anemia (SA)

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100x

Ringed Sideroblasts Prussian blue iron stain

Bone marrow 10x

Increased stainable iron Prussian blue iron stain

Bone marrow

Sideroblastic Anemia (SA)Bone marrow findings (if done):

1. *Ringed sideroblasts demonstrated with Prussian blue stain.

2. Increased stainable iron in macrophages.

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Types of Sideroblastic anemia cont’d

• Primary ‑ cause unknown (can't identify blocks) and are not reversible....called Idiopathic or primary Sideroblastic anemia.

1.Elderly, responds to no treatment. Requires transfusion support if severe anemia.

2.Characterized by a dimorphic red cell population - micro/hypo red cells with

3.normocytic and/or macrocytic red cells....MCV is variable and RDW is high.

4.Primary type of sideroblastic anemia is one of myelodysplastic syndromes called Refractory Anemia with Ringed Sideroblasts; may terminate in leukemia

65Wright’s stained blood smear

Stippled RBCs – Lead poisoning

Secondary Types of SA• Alcohol inhibits vitamin B6/pyridoxine

• Anti-tuberculosis drugs inhibit vitamin B6

• Lead causes multiple blocks

– Inhaled or ingested

– Abnormal lead level

– Neurologic problems

– Lead line (gums)

– Chelation therapy (EDTA).

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C. Anemia of chronic disease• Anemia of chronic disease (ACD) – inability to use

iron and decreased response to EPO – Very common anemia, #2

• Associated with systemic disease, including chronic inflammatory conditions:

–Rheumatoid arthritis–Chronic renal disease –Thyroid disease –Malignancies –Tuberculosis –Chronic fungal infections etc

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ACD pathogenesis

• Lactoferrin is an iron biding protein in the granules of neutrophils

• Its avidity for iron is grater than transferrin• During infection or Inflammation, neutrophil-

lactoferrin released into plasma and Scavenges available iron

• Bind to macrophage and liver cells (because they have receptor for lactoferrin

• Cytokines: Produced by macrophages during inflammation and contribute to ACD by inhibiting erythropoiesis

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Lab Diagnosis

• Blood findings– Early stage: normocytic normochromic– Late stage: hypochromic microcytic,

– Low serum iron, low TIBC, normal or high serum ferritin

• Leukocytosis

• Abundant storage of iron in macrophage (Prusian blue)

7070Target cells/Codocytes

Beta

Alpha

D. Thalassemias • Inherited decrease in alpha or beta globin chain synthesis

needed for Hgb A; quantitative defect– All have microcytic/hypochromic RBCs and target cells

• Genetic mutations classified by:– ↓ beta chains = beta thalassemia…Greek/Italian– ↓ alpha chains = alpha thalassemia…Asian

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Haemoglobin Molecule

Hgb A = 2α & 2β Hgb A2 = 2α & 2δ Hgb F = 2α & 2γ

• Consists of 4 globin chains + 4 heme groups• Normally, each individual inherits 2α, 1β, 1γ,

and 1δ gene from each parent.....so 4α, 2β, 2γ, and 2δ genes are inherited.

97% 2% 1%

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Thalassemia • Impaired alpha or beta

globin synthesis results in an unbalanced number of chains produced that leads to:– RBC destruction in beta

Thalassemia major– Production of

compensatory Hgb types in beta thals

– Formation of unstable or non-functional Hgb types in alpha thals

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Thalassemia• Severity ranges from lethal, to severe

transfusion-dependency, to no clinical abnormalities; severity depends on the number and type of abnormal globin genes inherited.

1.Major severe anemia; no α (or β) chains are produced, so cannot make normal hemoglobin (s).

2.Minor/trait mild anemia; slight decrease in normal hemoglobin types made.

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Heinz bodies Excess alpha chains Supravital stain

Beta Thal Major (Homozygous)• Both beta genes abnormal

– Marked decrease/absence of beta chains leads to alpha chain excess…no Hgb A is produced

– Rigid RBCs with Heinz bodies destroyed in bone marrow and blood (ineffective erythropoiesis)

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Stippled NRBC

NRBC

Target cell

Wright’s stained blood smear

HJB

Beta Thal Major (Homozygous)• Clinical findings• Lab findings

– Severe anemia, target cells, nucleated red cells– RBC inclusions– No hemoglobin A; compensatory Hgb F

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Pap bodiesNRBC

Transfused RBC

Target cell

Hypercellular Bone Marrow (10x)

Blood smear

Howell-Jolly body

Target cells

Blood smear Transfused

RBC

Beta Thal Major (Homozygous)

• Treatment– Transfusion– Splenectomy– Iron chelation

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Wright’s stained blood smear

Stippled RBC

Target cell

Beta Thal Minor (Heterozygous)

• One abnormal beta gene– Slight decreased rate of

beta chain production– Blood picture can look

similar to iron deficiency• Lab findings

– Mild anemia, target cells, no nRBCs, stippled RBCs

– No Heinz bodies– Normal iron tests– Compensates with

Hgb A2

Ovalocytes

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Alpha Thal Major/Homozygous

• Deletion of all 4 alpha genes results in complete absence of alpha chain production– No normal hemoglobin types made

• Known as Barts Hydrops Fetalis– Die of hypoxia….Bart’s Hgb

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Target cells

Wright’s stain blood smear

Heinz bodies Excess beta chains Supravital

stain

Alpha Thal Intermedia = Hgb H Disease

• Three alpha genes deleted– Moderate decrease in alpha chains leads to beta

chain excess…unstable Hgb H– Moderate anemia

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Alpha Thal Minor (Heterozygous)• One or two alpha genes deleted (group)

– Slight decrease in alpha chain production – Mild or no anemia, few target cells– Essentially normal electrophoresis; many undiagnosed

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Beta Thalassemias

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Alpha Thalassemias

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+

HGB Synthesis Defects

Differential Diagnosis of Microcytic Anemia

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2. Macrocytic Normocytic Anemias

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Macrocytic Normocytic Anemias

Characteristics ??????

Wright’s stained blood smear

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A. MEGALOBLASTIC ANEMIA

• Vitamin B12 deficiency• Folate deficiency• Abnormal metabolism of folate and vit B12

B. Non megaloblastic anemia

• Liver disease• Alcoholism• Post splenoctomy• Neonatal macrocytosis• Stress erythropoiesis

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A. Megaloblastic Anemia • Macrocytosis due to a deficiency of vitamin B12 or folic acid

that causes impaired nuclear maturation – Vitamin B12 & folate are DNA coenzymes necessary for

DNA synthesis and normal nuclear maturation– Results in megaloblastic maturation…nucleus lags

behind the cytoplasm and leads unbalanced growth called maturation asynchrony

• Both deficiencies cause enlarged fragile cells– Many cells die in the marrow (ineffective)– Show a similar blood picture and clinical findings

• Only vitamin B12 deficiency causes neurological symptoms…required for myelin synthesis

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RBC maturation in microcytic anemias…IDA

Normoblastic RBC maturation normocytic red cells

Megaloblastic RBC maturation macrocytic red cells

Megaloblastic Anemia

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Lab Findings of Megaloblastic Anemia

Mild to severe anemia, – Increased MCV & MCH, normal MCHC– Low RBC, HGB, WBC and PLT counts (fragile

cells) due to ineffective hematopoiesis.– Low reticulocyte count – Macrocytic ovalocytes and teardrops; – Marked anisocytosis and poikilocytosis – Schistocytes/microcytes - due to RBC breakage

upon leaving the BM– Erythroid hyperplasia - low M:E ratio (1:1) – Iron stores increased.

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Macrocytic OvalocytesBlood NRBC Blood

Howell-Jolly body

Teardrop

Schistocyte

Stippled RBC & Cabot Ring

Giant PlateletPap bodies Hypersegmented Neutrophil >5

lobes

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Vitamin B12 (Cobalamin) Deficiency

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Vitamin B12 deficiencyOccur as a result of one of the following

conditions1. Nutritional Coballamin deficiency

• Strict vegetarianism

2. Abnormal intragasteric events ( i.e. inadequate proteolysis of food Coballamin)• atrophic gastritis.

3. Loss or atrophy of gastric mucosa ( deficient IF)• total or partial gasterectomy

– May develop B12 and iron deficiency with macro and micro red cells…a dual (dimorphic) RBC population

• pernicious anemia

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Cont…..4. Abnormal events in the small bowel lumen

– Inadequate pancreatic protease– Competing agents like fish tape worms (D.latum)– Disorders of ileac mucosa– Diminished or absent of IF – Coballamin receptor– Drug effects– Metabolic disorders ( coballamin is not used by

cells)

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Folate (Folic acid) Deficiency: • Deficient intake. • Increased needs: pregnancy,

infant, rapid cellular proliferation, and cirrhosis

• Malabsorption (congenital and drug-induced)

• Inherited DNA Synthesis Disorders: Deficient thiamine and factors (e.g. enzymes) responsible for folate metabolism.

• Toxins and Drugs:

Two RBC populations Dimorphism

Macrocytic RBCs

Microcytic RBCs

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1. Pernicious Anemia

• it is defined as anemia resulting from defective secretion of IF associated with autoimmune attack on the gastric mucosa leading to atrophy of the stomach or Abs that block IF action.

• Abs block the site of IF where vit B12 binds.

• The diagnosis is confirmed by low serum B12 level and typically abnormal results of schilling test

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Schilling test

• Used to diagnose pernicious anemia and determine if IF is available.

• If absorbed a portion of oral dose of vit B12 (not

used by the body) ---- excreted in urine

• If not absorbed (malabsorption)…….. Not detected in urine but pass out in feces

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Stage I. To diagnose malabsorption• give the patient a radio active cobalt labeled vit B12 orally

and • Non labeled vit B12 intramuscularly to saturate liver

• If IF is present in the stomach (no other disease) vit B12 is absorbed and labeled vit B12 detected in urine

• If absorption is impaired labeled vit B12 not detected in urine instead in stool

• Diagnose malabsorption if it is not appear in urine by proceeding stage II.

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Stage II. Differentiate cause of malabsorption

• Oral dose labeled vit B12 + IF

• Appear in urine…… pernicious anemia

(give IF)

• If not appear in urine it is other cause of malabsorption

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• Performed to determine if the patient suffer from mal absorption of the IF – B12 complex secondary to small intestinal bacterial overgrowth.

• If this is the condition then tetracycline should normalize vit B12 absorption.

Stage III

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Polychromatophilic RBCs Wright’s stain

NRBC

B. Non-Megaloblastic Anemia

• Macrocytosis that is NOT due to vitamin B12 or folate deficiency

• Accelerated erythropoiesis– Regenerating marrow or marked reticulocyte

response following recent blood loss

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Stomatocytes, Alcoholic Stomatocytes, Alcoholic EchinocytesAcanthocytes

Target cells

Non-Megaloblastic Anemia• Liver disease and alcoholism

– Complex & multiple problems– Degree of anemia varies, round macrocytes– Target cells/acanthocytes - due to abnormal lipid metabolism.– Echinocytes are also commonly found on the smear in liver disease.

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Blood smear

Differential Diagnosis of Macrocytic Anemia

• Megaloblastic and non-Megaloblastic

– Perform B12 and folate levels

– Specific morphology

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3. Normocytic Normochromic Anemia

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3. Normocytic Normochromic Anemia

• It includes• Aplastic anemia

due to BM failure

• Blood loss anemia

• Hemolytic anemia

Is a condition in which the size & Hgb content of RBCs is normal but the number of RBCs is decreased.

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A. Aplastic Anemia

– Condition of blood pancytopenia caused by bone marrow failure…decreased production of all cell lines and replacement of marrow with fat.

– Due to damaged stem cells, damaged bone marrow environment or suppression

– No extramedullary hematopoiesis

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Types of aplastic anemia

–Primary/idiopathic = 50%

–Secondary/acquired….chemicals, drugs, infections, radiation = 50%

–Congenital….Fanconi’s • Aplasia plus dwarfism, skeletal

abnormalities, mental retardation, abnormal skin pigmentation.

107Bone marrow, decreased # Bone marrow, decreased #

precursor cellsprecursor cells

10X10X

Normal RBCs No Platelets

Blood

Lab diagnosis of Aplastic Anemia

• Normochromic –Normocytic RBC (normal MCV & MCH)• Low reticulocyte count & Hgb • Pancytopenia • No abnormal cells • Hypoplasia Bone marrow•Normal Serum iron, vitamin B12 and folate levels

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B. Hemolytic anemia

• Result from an increase in the rate of pre mature red cell destruction.

• Compensated hemolytic disease

• Uncompensated hemolytic disease

• It leads to– Erythropoietic hyperplasia – BM produces red cells 6 to 8X the normal rate– Marked reticulocytosis

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Hemolytic anemia

• Two main mechanisms for RBC destruction in HA–Intravascular hemolysis: in the

circulation

–Extravascular hemolysis: in RE system (reticuloendothelial system)

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Extravascular hemolysis

Aged RBC 120 day

Abnormal RBC

During destruction RBC releases Hgb

Hgb

Exstravascularly removed by Macrophage (RES) in BM, liver and spleen

Iron reabsorbed

Globin

Amino acid

Protein synthesis

Protoporphyrin

Unconjugated bilirubin liver (glucuronic acid)

conjugated bilirubin gut reabsorbed &

Excreted as urobilin & urobilinogen

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Extravascular hemolysis

• Lab features –Increased RBC break down

• Serum bilirubin increase

• Stool urobilinogen increase

• Blood urobilinogen increase

• Urine urobilinogen increase

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Intravascular hemolysis • Red cells are destroyed in blood vessels and Hgb is released into

the circulation:

Free Hgb

Saturates plasma haptoglobin

Excess free Hgb is filtered by the glomerules (kidney)(if rate of hemolysis saturates renal reabsorption capacity)

Free Hgb enters urine

Fe is released in bladder tubule

Renal tubule loaded with hemosiderin

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Intravascular hemolysis

• Lab features– Hemoglobinemia and hemoglobinuria– Hemosiderin uria– Reduced/absent serum haptoglobin

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1. Hereditary hemolytic anemia• This is a congenital hemolytic anemia. some of

which present at birth and other later in life, while still others may remain silent unless a physiological stress is super imposed

• Result of intrinsic red cell defects – Membrane defect (Hereditary Shperocytosis,

Elliptocytosis and sickle cell anemia)

– Metabolic defect : G6PDH and PK defic

– Hgb chain defect (hemoglobinopatheis) : sickle cell anemia

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Spherocytes

A. Hemolytic Anemias due to Membrane Defects

• Most common is Hereditary Spherocytosis (HS)– Membrane defect is

decreased spectrin and increased permeability of membrane to sodium ions

• Lab findings– Anemia varies– Few to many

spherocytes on smear, high MCHC

– Increased OF test

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H Ovalocytosis

Normocytic ovalocytes

H Ovalocytosis/Elliptocytosis• Membrane defect is

polarization of cholesterol or hemoglobin at ends and increased sodium permeability

• Over 25% ovalocytes– Most asymptomatic– Mild anemia in 10-

15%

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H Stomatocytosis

Hereditary Stomatocytosis• Membrane defect is

abnormal permeability to sodium and potassium

• Caused by edema • 20-30% stomatocytes

on blood smear– Mild to severe

hemolytic anemia

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H Acanthocytosis = Abetalipoproteinemia

H Acanthocytosis

• Defect is increased membrane cholesterol due to abnormal plasma lipids

• Numerous acanthocytes on smear– Mild anemia – Also known as abetalipoproteinemia

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Hypotonic

Osmotic Fragility Test (OF)• Most commonly used to diagnose Hereditary

Spherocytosis– Red cells are placed in hypotonic solutions

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Osmotic Fragility Test (OF)

Decreased Surface: Volume Ratio “Easy

to Lyse”

Increased Surface: Volume Ratio “Hard to Lyse”

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B. Defect red cell metabolism (Enzyme defect)

• G6PD deficiency – G6PD is the only source of NADPH in red cell – NADPH is reduced for the production of reduced

Glutathione. – Hgb and RBC membrane are usually protected from

oxidant stress by reduced glutathione (GSH)– In G6PD deficiency NADPH and GSH synthesis is

impaired, rendering the red cells vulnerable to oxidant stress.

– Most individuals with G6PD are asymptomatic except during oxidant stress resulting from drugs or other causes.

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B. Hemolytic Anemias due to Enzyme Defects

• Inherited enzyme deficiencies that lead to premature RBC death

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PK Deficiency

Echinocytes

Hemolytic Anemias due to Enzyme Defects

• PK deficiency– ↓ATP impairs cation pump– Severe hemolytic anemia– Echinocytes

• G-6-PD Deficiency– Unable to protect Hgb due to

decreased NADPH – No clinical problems unless

exposed to oxidants– Exposure to oxidants induce

Heinz body formation and RBC destruction Normal RBCs if no

exposure to oxidant

G-6-PD Deficiency

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G-6-PD deficiency after exposure to oxidant

Heinz bodies - denatured Hgb Supravital stain

G-6-PD deficiency Hemolytic episode

Damaged RBCs Wright’s stain

G-6-PD Deficiency• Blood findings after oxidant exposure:

– Mod to severe anemia– Schistocytes, spherocytes due to pitting out of Heinz

bodies by spleen• Enzyme assay

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Target cells/Codocytes

C. Normocytic anemias due to hemoglobinopathies

• Inherited hemoglobin defect with production of structurally abnormal globin chains;– All have target cells

• Beta chain amino acid substitution = variant Hgb – Hgb S = valine substituted for glutamic acid @ 6th of ß – Hgb C = lysine substituted for glutamic acid @ 6th of ß

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HGB S Disease (Hgb SS)

Sickle cell

Target cell

Hemoglobin S Disorders

– Two sickle cell genes inherited (both beta chains are abnormal)

– Symptomatic after 6 months of age

• Lab findings– Severe anemia – Targets, sickle cells– NRBCs, inclusions– No Hgb A, >80% Hgb S,

↑ F

A. Hemoglobin S disease/Sickle cell anemia/Hgb SS

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Target cells only NO Sickle cells

HGB S Trait (Hgb SA)

Hemoglobin S Disorders

– One sickle cell gene inherited

• Lab findings– Asymptomatic, targets only– No anemia or sickle cells– ~60% Hgb A, ~40% Hgb S

• Potential problems if hypoxic

B. Hemoglobin S trait/Sickle cell trait/Hgb SA

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C crystals

HGB C Disease (Hgb CC)

Target cell

Hemoglobin C Disorders

• Lab findings– Mild anemia – Many target cells – Intracellular C crystals– No Hgb A, >90% Hgb C– Decreased OF

A. Hemoglobin C disease/Hgb CCTwo C genes inherited (both β chains are abnormal)C crystals polymerize differently and look like blocky Hgb packed rods in the red cells....intracellular.

129

HGB C Trait (Hgb CA)

Target cells only NO C crystals

Hemoglobin C Disorders

B. Hemoglobin C trait/Hgb CA– One C gene inherited

• Lab findings– Asymptomatic, no anemia– Targets, no C crystals– ~60% Hgb A, ~40% Hgb C– Normal Hgb A2 and F

130

SC Crystals

Target cells

HGB SC Disease (Hgb S & Hgb C)

Hemoglobin SC Disease

• Lab findings– Intermediate in severity

between Hgb SS & SA – Several target cells – Many SC crystals– No Hgb A, ~50% Hgb

S, ~50% Hgb C, ↑ F

Hemoglobin SC disease/Hgb SC• One sickle gene and one C gene inherited• Double heterozygote‑ inherit sickle gene (S) from one parent and C gene from other parent; • Both β chains are abnormal

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2. Acquired hemolytic anemia

• A variety of acquired conditions result in shortened survival of previously normal red cells. These include immune mediated destruction, red cell fragmentation disorders, acquired membrane defects, spleen effects

• Result of extrinsic causes– Immune HA; warm HIHA, cold AIHA– Drug associated– Infection associated

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Spherocytes & polychromasia

Blood

Warm Autoimmune HA (WAIHA)• Altered immune response causes production of an

IgG warm autoantibody against ‘self’ RBC antigens– Antibody/complement attaches to RBC antigen…

partially phagocytosed (loss of membrane) spherocytes

• Cause: Primary (idiopathic) or secondary to disease

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Ingestion of coated RBC

RBC

Electron Microscopy

Blood

Monocyte with ingested RBC

RBC

Warm Autoimmune HA (WAIHA)

• Lab findings– Mod to severe anemia, spherocytes, high MCHC– Erythrophagocytosis – Looks similar to H spherocytosis but positive DAT– Increased OF, bilirubin– Erythroid hyperplasia

134

50x

RBC Agglutination

100x

Cold Autoimmune HA (CAIHA)• Altered immune response causes production of an IgM

cold autoantibody against ‘self’ RBC antigens– Antibody/C3 attaches to RBC antigen agglutination

(lysis by complement or macrophage)• Primary (idiopathic) or secondary to disease

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Cold Autoimmune HA (CAIHA)

• Lab findings– Agglutination of red cells in

extremities....ears, toes, nose tissue damage gangre

– Severity varies with seasons….avoid the cold

– IgM antibodies cause RBC agglutination

– Reticulocytosis

– Positive Direct Antiglobulin Test (detects complement)

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Hemolytic Transfusion Reaction

• Incompatible blood transfusion

– Recipient has antibodies to antigens on the donor red cells received

– Donor cells are destroyed

• ABO worst

– Intravascular hemolysis that is complement-induced lysis…immediate

– Can be life-threatening

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Hemolytic Disease of the Newborn • Caused by maternal IgG antibodies directed against

baby RBC antigens– Antibodies cross placenta and destroy fetal red

cells• HDN due to Rh incompatibility

– Rh negative mother forms Rh antibody after exposure– HDN due to Rh

• Sever anemia• Many nucleated red cells

• HDN due to ABO incompatibility – Mother’s ABO blood type is O; baby is type A or B– HDN due to ABO

• Mild, no anemia• Spherocytosis

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Schistocytes

Fibrin Strands

RBC

RBC fragmentation on fibrin strands

Hemolytic Anemias due to Trauma• Fragmentation syndromes…most common finding on smear

are schistocytes; anemia varies• Types of trauma

– Mechanical…prosthetic heart valves/cardiac abnormalities – Microangiopathic (MAHA)…small vessels

(DIC.........bleeding) – March hemoglobinuria…forceful contact…. Schistocytes

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Normocytic/normochromic Hemolytic Anemias due to Trauma –

Fragmentation Syndromes

A.When RBCs are exposed to excessive trauma within the cardiovascular system, they may undergo fragmentation and lysis.Schistocytes are the most common finding on the smear; RBC destruction tests are abnormal. Severe trauma causes intravascular hemolysis.

B.Three types of trauma: 1. Mechanical trauma

A. Prosthetic heart valves or cardiac abnormalities fragment red cells. B. Mod to severe anemia with schistocytes and polychromasia.

140

Normocytic/normochromic Hemolytic Anemias due to Trauma –

2. Microangiopathic hemolytic anemia (MAHA) – trauma occurs in small vessels

A. Disseminated Intravascular Coagulation (DIC) is a widespread clotting disorder initiated by conditions such as OB obstetrical) complications or sepsis. In DIC, clotting factors and platelets form fibrin fibrin deposited in the microvessels fragment red cells. See anemia with schistocytes on smear, decreased platelets and depletion coagulation factors leads to severe bleeding; can be fatal..

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2. Microangiopathic hemolytic anemia (MAHA) cont’d……..

B. Hemolytic uremic syndrome (HUS) - most often occurs in children following GI infection (E. coli); noted for renal failure fibrin damages kidney; hemolytic anemia with echinocytes and schistocytes, decreased platelets; often requires dialysis; can be fatal

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Normocytic/normochromic Hemolytic Anemias due to Trauma –

3. March Hemoglobinuria A. Transient, occurs after forceful contact of

body with hard surfaces....joggers, soldiers after long march, bongo drum players.

B. Hemoglobinuria; schistocytes may be present on smear.

143Schistocytes & Spherocytes

Hemolytic Anemias due to Infectious Agents, and Thermal Burns

• Anemia varies, with severe hemolysis – Schistocytes and spherocytes on blood smear– Parasitize RBC, elaborate lytic toxins or cause direct

damage to red cell membrane• Malaria fever• Closteridal infections..release toxins

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Normocytic/normochromic Hemolytic Anemia due to Infectious Agents

A. Malarial infection

1. Damage to membrane occurs when parasite is pitted out of red cell by splenic macrophages or the entire RBC is removed; chills and fever as red cells rupture.

2. P. falciparum causes severe hemolysis called Blackwater fever.

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Normocytic/normochromic Hemolytic Anemia due to Infectious Agents

B. Clostridial infections - Clostridia elaborate toxins which damage RBC membrane causing severe intravascular hemolysis.

Both malaria and clostridial infections are characterized by schistocytes and spherocytes on the blood smear; other infectious organisms can cause hemolysis, e.g. Toxoplasma, Bartonella, Babesia, Ehrlichia.

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END OF ANEMIA