The Hematopoietic & Lymphoid System

Dr. Maha Shomaf

Red Cells Disorders

• Types :

• 1- increase production

• 2- decrease production

Anemia

• Reduction in oxygen transporting capacity of blood mainly due to reduction of the total red cell mass to below normal amounts .

• Causes of anemia :

• 1- excessive bleeding

• 2- increased RBCs destruction

• 3- decreased RBCs production

Anemia due to blood loss

• 1- acute : trauma

• 2- chronic : GIT lesions & GYN problems

Anemia due to increased destruction (hemolytic anemia)

• 1- intrinsic (intracorpuscular)

abnormalities).

a- hereditary

membrane abnormalities

enzyme deficiency

disorder of Hg synthesis

• b- acquired

membrane defect (PNH)

• 2- Extrinsic (extracorpuscular

abnormalities)

a- antibodies mediated

b- mechanical trauma to RBCs e.g

-microangiopathic hemolytic

anemias: TTP, DIC

-intravascular  Infections: malaria

Anemia due to impaired production

• 1-disturbance of proliferation & differentiation of stem cells

aplastic anemia, pure red cell aplasia

• 2-disturbance of proliferation & maturation of erythroblasts

a-defective DNA synthesis: megaloblastic anemias

b-defective Hg synthesis   Deficient heme synthesis: iron deficiency anemia   Deficient globin synthesis: thalassemias:      Anemia of renal failure

c-unknown or multiple mechanisms

• Anemias also can be classified depending on the morphology of the RBCs into :

• 1-normocytic

• 2-microcytic

• 3-macrocytic

• Or degree of hemoglobinization into :• 1-Normochromic• 2-Hypochromic• Or RBC shape :• 1-spherocytosis• 2-Ovalocytosis• 3-stomatocytosis• 4-elliptocytosis

• Results • 1-Erythroid hyperplasia

• 2-Extramedullary hematopoiesis (liver,spleen ,LNs)

• 3-Reticulocytosis

• RBCs indices are measured by: 1-MCV: the average volume/RBC femtoliters(cubic microns) 2-MCH: the content(mass) of Hg /RBC picogram 3-MCHC:the average concentration of Hg in a given vol. of packed RBCs grams/dl

4- RDW: red cell distribution width

the coefficient of variation of RBC volume

5- HCT(hematocrit): the percentage of RBCs in a known

volume of blood

Clinical features of anemia

• 1-pallor• 2-fatique • 3-lassitude• 4-hyperbilirubinemia & jaundice• 5-GB stones• 6-secondary hemochromatosis• 7-growth retardation• 8-skeletal abnormalities• 9-cachexia

Anemia of blood loss

Hemmorhage

• If acute it can lead to hypovolemic shock

• Hemodilution begins at once & full effect within 2-3 days

• The anemia is normocytic normochromic

• Recovery is inhanced by ↑ erythropoietin level

• The marrow response is marked by reticulocytosis

• Chronic blood loss is associated with iron deficiency leading to chronic anemia of under production

Anemia of diminished

erythropoiesis

Iron deficiency anemia

• Anemia affects 25-50% of the population of developing countries &10% of population of developed countries

• IDA is the most common cause of nutritional deficiency anemia

• Total body iron is 2gms in women

6gms in men

• Distribution :

1- 80% in Hg

2- 20% in myoglobin & iron-containing enzymes (catalase &cytochromes)

• Iron stores :-Hemosiderin & Fe-binding ferritin contain 10-20% of total body iron-Stored Fe is mainly found in liver ,spleen & BM-Transferrin is 33% saturated with iron

normally-serum iron in men is 120 µg/dL in women 100 µg/dL- (TIBC) total iron binding capacity of serum is

300 -350 µg/dL

• There is no regulated pathway for iron excretion

• 1-2 mg/d are lost by shedding of mucosal & skin epithelial cells

• Iron balance is largely regulated by regulating the absorption of dietary iron

• Normal daily western diet contains 10-20mg of Fe mostly heme containing animal products & the remainder as inorganic Fe in vegetables

• About 20% of heme Fe is absorbable

• Only 1-2% of non-heme Fe is absorbable

• The average western diet contains sufficient Fe to balance the daily loss loss

Iron absorption

• Iron is absorbed in duodenum• Nonheme iron is reduced by ferric reductase

then transported by the divalent metal transporter (DMT 1) into the cytoplasm

• Iron is transferred to transferrin in the plasma through the action of 2 proteins:

• 1-ferroportin• 2-hephaestin

• Both DMT 1& ferroportin are widely distributed in the body

• Only a fraction of the iron that enters the body is deliverd to transferrin by the action of ferroportin

• The remainder is bound to ferritin & lost through the exfoliation of mucosal cells

• There is balance between the amount of iron deliverd to transfferin & the iron loss

• The balance is regulated by hepcidin synthesized by the liver in an iron- dependent fashion

• Hepcidin causes degradation of ferroportin

& thus when hepcidin level is high less iron is transferred to transferrin

• When hepcidin levels are low iron transport is enhanced as in IDA & ineffective erythropoiesis

Causes of iron deficiency anemia

• 1-low dietary intake• 2-malabsorption • 3-increased demands not met by increased

uptake• 4-chronic blood loss MENORRHAGIA

PEPTIC ULCERSTOMACH CANCERULCERATIVE COLITISINTESTINAL CANCERHAEMORRHOIDS

Clinical manifestation

• Mostly asymtomatic• Weakness• Pallor• Glossitis , stomatitis• Dysphagea • Atrophic gastritis• Dry skin• Hair loss

• Thinning & spooning of finger nails

(koilonychia)

• Pica : the compunction to eat non-foodstuffs such as dirt or clay

• Stages of IDA

• Iron depletion – Stage One

• Iron deficient erthyropoiesis – Stage Two

• Iron deficiency anemia – Stage Three

Stage 1 (prelatent)

• Iron storage is exhausted - indicated by decrease in serum ferritin levels

• Hb (N), MCV (N), iron absorption (), transferrin saturation (N), serum ferritin (), marrow iron ()

• No anemia – RBC morphology is normal

• May have elevated RDW

Stage 2 ( Latent )

• Insufficient iron to insert into protoporphyrin ring to form heme

• Protoporphyrin accumulates in cell and complexes with zinc to form ZPP

• Hb (N), MCV (N), serum ferritin (), transferrin saturation (), TIBC (),

• marrow iron (absent)

• No anemia, no hypochromia, but slight microcytosis

Stage 3

• All laboratory tests for iron status become abnormal

• Most significant finding is microcytic,hypochromic anemia

Diagnosis

• Microcytic hypochromic anemia• ↓ MCV & MCHC• ↓ serum ferritin & iron levels• Low transferrin saturation• ↑ TIBC• ↑ platelets• BM cellularity is only mildly increased

although erythropoietin is increased• Extramedullary hematopoiesis is uncommon

Adult reference ranges

malefemaleunits

Hg 13.6-17.212.0-15.0g/dl

Hct 39-4933-43%

RBC count4.3-5.93.5-5.0106/mm3

reticulocye0.5-1.50.5-1.5%

MCV76-10076-100fl

MCH27-3327-33pg

MCHC33-3733-37g/dl

RDW11.5-14.511.5-14.5-

Normal blood smear

Hypochromic microcytic anemia

Hypochromic microcytic anemia

Anemia of chronic disease

• The most common type of anemia in hospitalized patients

• It is related to inflammation-induced sequestration of iron within the cells of reticuloendothelial system

• Causes :1-chronic microbial infection as- osteomyelitis- bacterial endocarditis- lung abscess2-chronic immune disorder as RA3-neoplasms as HD , lung & breast

carcinoma

Lab findings

• Normocytic normochromic anemia

• Or microcytic hypochromic anemia

• ↑ iron stores

• ↑ serum ferritin

• ↓ TIBC

Etiology

High circulating hepcidin level ↓ inhibition of ferroportin ↓ blockage ofmononuclear iron transfer erythroid Phagocytes → → cells

• Inflammatory cytokines

1- → enhance hepcidin synthesis by the liver

2- → inhibit the compensatory increase in erythropoietin levels which is

inadequate for the degree of anemia

Megaloblastic anemia

• Megaloblastic anemia is macrocytic anemia which results from inhibition of DNA synthesis in red blood cells

• This is often due to deficiency of vitamin B12 and/or folic acid.

Vitamin B12

• It is synthesized in nature by microorganisms.• It is a water soluble vitamin.• It is play a key role in the normal functioning of the brain

and the formation of blood. • It is one of the eight B vitamins. • It is normally involved in DNA synthesis and regulation,

fatty acid synthesis and energy production. • A common synthetic form of the vitamin is

cyanocobalamin which does not occur in nature but is used in many pharmaceuticals, supplements and as food additive.

• Peptic digestion releases dietary vit B12 • Vit B12 binds to salivary B12-binding

proteins called cobalophilins (R binders)• R-B12 complexes transported to

doudenum • Vit B12 is released through the action of

pancreatic proteases• Vit B12 attaches to the intinsic factor(IF)

secreted from parietal cells in the stomach

• IF-B12 complex is attached to specific surface receptor for IF in the distal ileum

where it is absorbed

• The absorbed vit B12 is bound to transcobalamins which is synthesized in liver, macrophages and ileum.

• Vit B12 is delivered to liver & other cells

Folic acid (Folate)

• Folate is widely present in nearly all types of food

• It is readily dystroyed by 10-15 min of cooking

• Best sources are fresh uncooked vegetables & fruits

• Food folates are in polyglutamate form & must be digested into monoglutamate for absorption

• It is absorbed in the proximal of small intestine

• It is transported in the blood as monoglutamate methyl THF

• Within the cells it is metabolized into several derivatives most importantly from DHF into THF by the enz DHF reductase

Function of folate

• 1-Folate is necessary for the production and maintenance of new cells.This is especially important during periods of rapid cell division and growth such as infancy and pregnancy.

• 2-Folate is needed to synthesize DNA bases (most notably thymine, but also purine bases) needed for DNA replication since THF acts as acceptor & donor of one-carbon units .

• Thus folate deficiency hinders DNA synthesis and cell division, affecting mostly bone marrow and cancer cells.

• Deficiency of folate in pregnant women has been implicated in neural tube defects.

Vitamin B12 and Folate Nutritional aspectVitamin B12 and Folate Nutritional aspect

Vitamin B12FolateDaily take7 – 10 ug200 – 250 ug

Main foodAnimal product onlyLiver, green leafs

CookingresistantEasily destroyed

Adult daily requirement1 – 2 ug150 ug

Body stores2-3 mg (sufficient for 5-20 yrs)

10 - 12 mg (sufficient for 4 months)

Absorption sitesTerminal ileumDuodenum, jejunum

MechanismsBinding to IFConversion to MTH

Limts2 – 3 ug / day50-80% of dietary content

Endohepatic circulation5 - 10 ug / dally90 ug / day

Transport in plasmaMost bond to TCI, TCIIWeakly bound to albumin

Causes of Causes of Megaloblastic AnemiasMegaloblastic Anemias

1. Vitamin B12 deficiency.2. Folate deficiency.3. Abnormalities of V B12 or folate

metabolism, transcobalamin deficiency, nitrous oxide, anti-folate drugs.

4. Other defects of DNA synthesis:- Congenital enzyme deficiencies, e.g. orotic

aciduria.- Acquired, e.g. alcohol, therapy with

hydroxyurea

Causes of vit B12 deficiency

1- long-standing malabsorption2- Pernicious anemia due to atrophic gastritis autoimmune helicobacter pylori3- Gastrectomy4- Resection of ileum5- Ileal diseases as Crohn,s disease Tropical sprue Wipple disease6- Gastric atrophy & achlorhydria 7- Chronic pancreatitis

Causes of Folate DeficiencyCauses of Folate Deficiency

1-Alcoholism

2-Deficient intake

3-Increased needs: pregnancy,hemolytic anemia

4-Malabsorptive disease as celiac disease

5-Intestinal resection

6-Druges as anticonvulsants (phenytoin ,methotrexate)

pathogenesis

• Impairment of DNA synthesis• Delay in nuclear maturation & cell division• Synthesis of RNA & cytoplasmic elements

proceeds at a normal rate in contrast to nucleoli→→ nuclear-cytoplasmic asynchrony

• Ineffective erythropoiesis : defective megaloblasts undergo apoptosis in the marrow with delay in maturation resulting in decrease in the number of cells reaching the circulation

• Pancytopenia

anemia

granulocytopenia

thrombocytopenia

Symptoms of megaloblastic Symptoms of megaloblastic anemiaanemia

1. Weak muscles 2. Numbness or tingling in hands and feet 3. Nausea 4. Decreased appetite 5. Weight loss 6. Irritability 7. Lack of energy or tiring easily (fatigue) 8. Diarrhea 9. Increased heart rate (tachycardia) 10. stomatitis . 11. Glossitis12. jaundice

Results

1. Megaloblastic anemia.2. Macrocytosis of epithelial cell surface.3. Sterility.4. Reversible melanin skin pigmentation

(rarely).5. Decrease osteoblast activity.6. Neuropathy (vit B12 only).7. Neural tube defect in fetus ( folate only).

Laboratory findingsLaboratory findings

Blood count

- Decreased RBC count- Low hemoglobin - ↑MCV (> 110fL) (m.b Normal with associated Fe def.)- MCHC is normal

- Pancytopenia- The reticulocyte count m.b low for the degree

of anemia

Blood film

-Macrocytic red cells

-Hypersegmented neutrophils (4or5 lobes)

-Platelets m.b large & misshapen

Megaloblastic anemiablood film(macrocytes &

hypersegmented neutrophils)

Normal neutrophils

Megaloblastic anemiablood film

Diagnostic features of pernicious anemia

• Low serum vit B12 levels• Normal or ↑ folate levels• Serum antibodies to parietal cells or IF• Moderate to severe megaloplastic

anemia• Leukopenia & hypersegmented

neutrophils• Reticulocytic response (within2-3days)

to parenteral vitB12

Aplastic anemia

• Suppression of multipotent stem cells leading to marrow failure & pancytopenia

• Characterized by :

I. Peripheral blood Pancytopenia.II. Reticulocytopenia.III. Bone marrow hypocellularity.IV. Depletion of hematopoitic stem cells.

• Can be moderate, severe or very severe.

• Paul Ehrlich introduced the concept of aplastic anemia in 1888,who described a patient with severe anemia and neutropenia exhibiting a yellow hypocellular marrow, However, it was not until 1904 that Anatole Chauffard named this disorder aplastic anemia.

Classification of Aplastic anemia

• 1- acquired a-idiopathic b-secondary

• 2- congenital (inherited)1. Fanconi anemia 2. Dyskeratosis congenita 3. Schwachman – Diamond syndrome.

Idiopathic aplastic anemia

• The cause of the bone marrow failure in idiopathic aplastic anemia is unknown.

• It is accounts for approximately 70-80% of aplastic anemia cases.

Secondary aplastic anemia

Secondary aplastic anemia is associated with:

I. Exposure to Ionizing radiation, exposure to radiation suppresses the

bone marrow in a predictable dose-dependant manner.

– Depending on the dose and exposure time, the bone marrow generally recovers after withdrawal of the agent.

II. Certain drugs:

– Aplastic anemia may occur secondary to idiosyncratic adverse reactions to certain drugs such as chloramphenicol and gold compounds .

– In idiosyncratic reactions the bone marrow failure is unpredictable and unrelated to dose, and the bone marrow doesn’t usually recover when the agent is withdrawn.

– Aplastic anemia due to idiosyncratic reactions is a rare event and is likely due to a combination of genetic and environmental factors in susceptible individuals.

– Also the antimetabolite drugs (e.g. methotrexate) and mitotic inhibitors (e.g. daunorubicin) may cause only temporary aplasia.

III. Infectious agent:

– Acquired Aplastic anemia appears occasionally as a complications from certain infections like the viral infection.

– Viruses that have been linked to the development of aplastic anemia include Epstein-Barr, cytomegalovirus, parvovirus B-19 and HIV.

– also Acquired Aplastic anemia is present in up to 2% of patients with acute viral hepatitis .

IV. Pregnancy:

Very rare, may happen during pregnancy and resolve with delivery or with spontaneous or induced abortion.

V. Autoimmune disorders: An autoimmune disorder in

which the immune system attacks healthy cells as stem cells in the bone marrow.

VI. as complication in paroxysmal nocturnal hemoglobinuria(PNH) .

pathogenesis

• Aplastic anemia occurs due to either:

I. Reduction in the number of haemopoietic stem cells due to inherited abnormality or due to an immune reaction against them.

II. A primary fault in the bone marrow microenvironment ( rare cause).

• The mechanism of acquired aplastic anemia involves the severe depletion of hematopoietic stem and progenitor cells from the bone marrow by a direct or indirect mechanism.

I. Direct mechanism: Cytotoxic chemotherapy, radiation, chemical,

or virus damage the DNA of the stem and progenitor cells cause apoptosis of these cells.

II. In the indirect method:

• Exposure to certain drugs or chemicals in susceptible individuals result in an autoimmune T cell (CD8) attack that destroys the stem and progenitor cells.

• These cells produce inhibitory cytokines, such as

gamma-interferon and tumor necrosis factor, which can suppress stem and progenitor cell growth by affecting the mitotic cycle and cell killing by inducing Fas-mediated apoptosis.

• In addition, these cytokines induce nitric oxide production by marrow cells, which contributes to immune-mediated cytotoxicity and the elimination of hematopoietic stem cells.

Clinical presentation

• The clinical presentation of patients with acquired aplastic anemia includes symptoms related to the decrease in bone-marrow production of hematopoietic cells:

I. Anemia with pallor, headache, palpitations, dyspnea, fatigue, or foot swelling.

II. Thrombocytopenia, leading to increased risk of hemorrhage, bleeding gum , bruising, Nosebleeds and petechiae.

III. Leukopenia, leading to increased risk of bacterial or fungal infection particulary mouth and pharyngeal ulcerations.

• Hepatosplenomegaly and lymphadenopathy do not occur; their presence suggests an underlying leukemia.

Diagnosis• CBC:

– Pancytopenia is typical, although initially only one or two cell lines may be decreased.

– The Hb is usually less than 10 g\dl.– Normal MCH and MCHC, – The MCV is normal or increased ( Anemia is

normochromic, normocytic or macrocytic). – Absolute granulocytopenia (neutrophil count is

decreased).– Monocytopenia is usually present.– Absolute lymphocyte count may be normal or

decreased.– Thrombocytopenia is always present.

• Reticulocyte Count:– low reticulocyte levels.

• Peripheral blood film show:

– low RBCs density.– Neutrophils, monocytes and platelets are

decreased.– RBCs are normocytic or macrocytic.– Toxic granulation may be observed in the

neutrophils, – RBCs and platelets are normal in

appearance.– Blasts and other immature cells are

characteristically absent.

Pancytopenia

Bone marrow– Bone marrow aspirate and biopsy specimens have

prominent fat cells(which comprises over 75% of the marrow).

– Trephine biopsy are required for an accurate quantitative assessment of the marrow cellularity, and severe hypocellularity is a characteristic feature.

– Erythroid, granulocytic, and megakaryocytic cells are severely reduced or absent.

– The main cells present in the bone marrow are lymphoid cells.

– Increased nonhematopoietic elements, such as plasma cells and mast cells are present.

Normal vs hypocellular bone marrow

• Chemistry :

– Serum iron level and transferrin saturation are increased reflecting the decreased use of iron for erythropoiesis.

– Plasma ferritin is increased due to condensation of iron in storage sites.

– Liver function tests may be abnormal if the pancytopenia was preceded by hepatitis.

Hemolytic anemia

• Normal red blood cells life span is 120 days

• As RBCs become older membrane changes occur and the cells are phagocytosed

• Hemolytic anemias are associated with accelerated destruction of RBCs

- Types :1-extravascular • 80%-90% of destruction is within macrophages of the liver and spleen• RBCs membrane and Hb become separated

heme + globin Hg→

2 -intravascularly.

• when RBC destruction occur within the vascular spaces• free Hg released to blood plasma and bound to haptoglobin• which can not be excreted by renal glomeruli• mostly taken up by the liver & BM

1-Mismatched blood transfusion2-G6PD deficiency with oxidant stress

3-Red cell fragmentation syndromes4-Some Autoimmune HA

5-Some drug- & infection-HA6-March hemoglobinuria

7-Paroxysmal nocturnal hemoglobinuria8-Unstable hemoglobin

RBCs disorders that include: 1-Basic membrane structure

2-Enzymes

3-Heamoglobin(Hb) molecules

4.Erythrocyte nucleotide metabolism (rare)

Causes of exravasculr hemolysis

Red blood cell disordersStructural

membrane defectEnzyme defectHg defect

Hereditary spherocytosis

G6PD defeciencyHb C disorder

AcanthocytosisGlutathione reductase deficiency

Hb S-C disorder

Hereditary elliptocytosis

Hexokinase deficiency

Hb S-S disorder

Hereditary stomatocytosis

Pyruvate kinase deficiency

Thalassemia

Hereditary xerocytosis

Rh null

diease

Results of intravascular hemolysis

• 1-hemoglobinemia

• 2-hemoglobinuria

• 3-hemosiderinuria

• 4-unconjugated hyperbilirubinemia

(jaundice)

• 5-acuter tubular necrosis

• 6-absent haptaglobin from plasma

Results of extravascular hemolysis

• 1- No hemoglobinemia• 2- No hemoglobinuria• 3- Jaundice• 4- Pigment stones (chronic hemolysis)• 5- Decreased haptaglobin level in the plasma 6- Splenomegaly 7- Secondary hemochromatosis

HEREDITARY SPHEROCYTOSIS

• Inherited intrinsic defect in the red cell membrane rendering cells spheroidal

• Transmitted as:

autosomal dominant 75%

autosomal recessive 25%

pathogenesis

• HS the primary abnormality resides in one of a group of proteins that form a meshlike supportive skeleton on the intracellular face of the red cell membrane

• The major protein in this skeleton is Spectrin a long, flexible heterodimer that is linked to the

membrane at two points: 1- through ankyrin & band 4.2 to the intrinsic membrane protein band 3 2- through band 4.1 to the intrinsic membrane protein glycophorin.

The horizontal spectrin-spectrin and vertical spectrin-intrinsic membrane protein interactions serve to

1-stabilize the membrane

2-responsible for the normal shape, strength, and flexibility of the red cell.

• common pathogenic feature of all HS mutations is weakening the vertical interactions between the membrane skeleton and the intrinsic

membrane proteins• The mutations most frequently involve :1- ankyrin2- band 33- spectrin

but mutations in other components of the skeleton have also been described.

Four abnormalities in red cell membrane

proteins have been identified:(1) spectrin deficiency alone

(2) combined spectrin and ankyrin deficiency

(3) band 3 deficiency

(4) protein 4.2 defects • Spectrin deficiency is the most common

defect

• In all types of HS the red cells have reduced membrane stability and consequently loose membrane fragments after their release into the periphery, while retaining most of their volume.

• As a result, the ratio of surface area to volume of HS cells decreases until the cells become spherical, at which point no further membrane loss is possible

-The spleen plays a major role in the destruction of spherocytes.

-Red cells must undergo extreme degrees of deformation to leave the cords of Billroth and enter the splenic sinusoids.

-The discoid shape of normal red cells allows considerable latitude for changes in cell shape.

-In HS because of spheroidal shape and limited deformability, spherocytes are sequestered in the splenic cords and eventually destroyed by macrophages.

Lab findings

• Hg is low

• Reticulocytosis > 8%

• ↓ MCV

• Normal MCH

• ↑ MCHC

• ↑ osmotic fragility test

Lab findings

• Blood Smears

• red cells lack the central zone of pallor because of their spheroidal shape

• Spherocytosis, though distinctive, is not diagnostic

HS –anisocytosis & Howell-Jolly bodies

Sphere shape allow a much smaller surface area to volume ratio than biconcave shape

Scanning electron micrograph of a normal biconcave erythrocyte

Scanning electron micrograph of a spherocyte. The spherocyte is smaller, round, and lacks concavities

• Splenomegaly is greater and more common in HS than in any other form of hemolytic anemia.

• The splenic weight is usually between 500 and 1000 gm and can be even greater.

• The enlargement results from marked congestion of the cords of Billroth and increased numbers of mononuclear phagocytes.

• Phagocytosed red cells are frequently seen within macrophages lining the sinusoids and, in particular, within the cords.

• In long-standing cases there is prominent systemic hemosiderosis.

• The other general features of hemolytic anemias earlier are also present

cholelithiasis occurs in 40% to 50% of HS patients

• Spherocytes are round and fragile and do not change shape to pass through certain organs as easily as normal red blood cells.

• Because spherocytes cannot change their shape easily, they stay in the spleen longer than normal red blood cells, and the membrane surrounding the cell becomes damaged.

• After circulating through the spleen many times, the cell eventually becomes so damaged that it is destroyed by the spleen.

OSMOTIC FRAGILITY TEST

• Osmotic fragility is determined by measuring the degree of hemolysis in hypotonic saline solution

• With the unincubated test, red cell osmotic fragility is considered to be increased if hemolysis occurs in a sodium chloride concentration >0.5%.

• Normal RBCs begin to hemolyze around 0.5% of NaCl conc.& completed at 0.3%

• Increased osmotic fragility is characteristically associated with hereditary spherocytosis(0.65-0.40%)

OSMOTIC FRAGILITY TEST

1-Normal osmotic fragility is seen at top

2-Abnormal lysis of RBCs in mildly hypotonic solutions is seen at lower row (increased osmotic fragility as in HS).

Sickle cell anemia

• The prototypical (and most prevalent) hemoglobinopathy is caused by a mutation in the β-globin chain gene that creates sickle hemoglobin (HbS).

- HbS, like 90% of other abnormal hemoglobins, results from a single amino acid substitution in the globin chain.

- Normal hemoglobins are tetramers composed of two pairs of similar chains α and β

- On average, the normal adult red cell contains 96% HbA (α2β2) 3% HbA2 (α2δ2) 1% HbF fetal Hb (α2γ2).

- Substitution of valine for glutamic acid at the sixth position of the β-chain produces HbS.

- In homozygotes all HbA is replaced by HbS, whereas in heterozygotes only about half is replaced.

Epidemiology

• 8% of American blacks are heterozygous for HbS.

• In parts of Africa where malaria is endemic the gene frequency approaches 30%, as a result of a small but significant protective effect of HbS against Plasmodium falciparum malaria.

• In the United States sickle cell anemia affects approximately 1/600 blacks

Pathogenesis

• Upon deoxygenation, HbS molecules undergo polymerization, a process also referred to as gelation or crystallization.

• These polymers distort the red cell, which assumes an elongated crescentic, or sickle, shape

• Sickling of red cells is initially reversible upon reoxygenation

• membrane damage occurs with each episode of sickling, and eventually the cells accumulate calcium, lose potassium and water, and become irreversibly sickled

A.Peripheral blood smear from a patient with sickle cell anemia.showing sickle cells, anisocytosis, poikilocytosis, & target cells.

B, Higher magnification shows an irreversibly sickled cell

The variables that influence sickling of red cells in vivo.

• The three most important factors are as follows: • 1-The presence of hemoglobins other than HbS. -In heterozygotes approximately 40% of Hb is HbS; the remainder is HbA, which interacts only weakly with deoxygenated HbS. -The presence of HbA slows the rate of polymerization greatly -as a result the red cells of heterozygotes have little tendency to sickle in vivo.

- The presence of HbC(substitution of lysine substitution of lysine for glutamic acid at the 6for glutamic acid at the 6 thth amino acid of the amino acid of the ββ globin chain)globin chain)

- The carrier rate for HbC in American blacks is about 2.3%

- about 1/1250 newborns are double heterozygotes because they have inherited HbS from one parent and HbC from the other.

• HbC has a greater tendency to aggregate with HbS than does HbA

• those with HbS and HbC (called HbSC disease) are symptomatic.

• HbF interacts more weakly with HbS, and therefore newborns with sickle cell anemia do not manifest the disease until they are 5 to 6 months old, when the HbF falls to adult levels

• 2- The concentration of HbS in the cell .• The tendency for deoxygenated HbS to form the

insoluble polymers that create sickle cells is strongly dependent on the concentration of HbS.

• Thus, 1- red cell dehydration, which increases the Hb

concentration enhances sickling and can trigger occlusion of small blood vessels.

2- the coexistence of α-thalassemia reduces the Hb concentration and therefore the severity of sickling.

3- The relatively low concentration of HbS also contributes to the lack of sickling in heterozygotes with sickle cell

• 3-The length of time that red cells are exposed to low oxygen tension

-sickling is confined to microvascular beds where blood flow is sluggish as in the spleen and the bone marrow

- In other vascular beds important pathogenic roles are played by two factors:

1-inflammation 2-increased red cell adhesion

- blood flow in inflamed tissues is slowed →

1-adhesion of leukocytes and red cells to activated endothelium 2-exudation of fluid through leaky vessels.

→prolongs the red cell transit times →sickling

- Sickle red cells also have a greater tendency than normal red cells to adhere to endothelial cells, apparently because membrane damage makes them sticky.

• Two major consequences stem from the sickling of red cells:

• 1- repeated episodes of deoxygenation → membrane damage and dehydration of red cells, which become rigid and irreversibly sickled→ chronic extravascular hemolytic anemia.

• Overall, the mean life span of red cells in sickle cell anemia patients averages only 20 days (one-sixth of normal).

• 2- the sickling of red cells → widespread microvascular obstructions → ischemic tissue damage and pain crises.

• Vaso-occlusion does not correlate with the number of irreversibly sickled cells and therefore appears to result from factors, such as infection, inflammation, dehydration, and acidosis, that trigger the sickling of reversibly sickled cells.

• 3-in children there is moderate splenomegaly due to red pulp congestion• 4-autosplenectomy, is complete by

adulthood. The chronic splenic erythrostasis results in

progressive hypoxic tissue damage, which eventually reduces the spleen to non functional fibrous tissue.

• 5-Vascular congestion, thrombosis, and infarction

• can affect any organ, including bones, liver, kidney, retina, brain, lung, and skin.

• The bone marrow is particularly prone to ischemia, because of its relatively sluggish blood flow and high rate of metabolism.

• Priapism, penile fibrosis and eventual erectile dysfunction.

Complications

• 1-acute chest syndrome • can be triggered by pulmonary infections or fat

emboli from necrotic marrow that secondarily involve the lung.

• The blood flow in the inflamed, ischemic lung becomes sluggish and "spleenlike," leading to sickling within hypoxemic pulmonary beds.

• This exacerbates the underlying pulmonary dysfunction, creating a vicious cycle of worsening pulmonary and systemic hypoxemia, sickling, and vaso-occlusion.

• 2-Central nervous system stroke

• which sometimes occurs in the setting of the acute chest syndrome.

• the acute chest syndrome and stroke are the two leading causes of ischemia-related death

• 3-Aplastic crisis

- represents a sudden but usually

temporary cessation of erythropoiesis.

- usually triggered by parvovirus infection of erythroblasts, and, while severe, is self-limited.

• 4-infections. Increased susceptibility to infections by

encapsulatedbacteria, such as pneumococci.

• Causes :1- hyposplenism in adults due to autoinfarction.

2-In children bacterial sequestration and killing by the spleen due to enlargement & congestion caused by trapped sickled red cells is affected & thus even children with enlarged spleens are at risk for fatal

septicemia.

3-Defects in the alternative complement pathway that impair the opsonization of encapsulated bacteria

• For reasons that are not entirely clear, patients with sickle cell disease are particularly predisposed to Salmonella osteomyelitis.

Hg is typically 5 to 8 g/dL. Hg is typically 5 to 8 g/dL. MCV is normal.MCV is normal.Hct 18% to 30% (normal range, 35%-Hct 18% to 30% (normal range, 35%-

45%).45%).Reticulocytosis and hyperbilirubinemia

The blood smear shows target cells The blood smear shows target cells and the characteristic sickled and the characteristic sickled erythrocytes with sharply pointed ends. erythrocytes with sharply pointed ends.

There is a compensatory hyperplasia of erythroid progenitors in the marrow

Sickle solubility test : Sickle solubility test : A mixture of haemoglobin S (Hb S) in a reducing solution A mixture of haemoglobin S (Hb S) in a reducing solution

(such as (such as sodium dithionite) gives a turbid appearance, ) gives a turbid appearance, whereas normal Hb gives a clear solution. whereas normal Hb gives a clear solution.

Haemoglobin electrophoresis : A form of gel electrophoresis on which the

various types of haemoglobin move at varying speed.

• The hypercellular marrow often causes bone resorption and secondary new bone formation, resulting in prominent cheekbones and changes in the skull resembling a "crew-cut" in roentgenograms.

• Extramedullary hematopoiesis can also appear in the spleen and liver.

Thalassemia

Thalassemia

• inherited disorders caused by mutations that decrease the rate of synthesis of α- or β-globin chains.

• As a consequence there is a deficiency of hemoglobin, with additional secondary red cell abnormalities caused by the relative excess of the other unaffected globin chain.

Molecular Pathogenesis

• inherited as autosomal codominant conditions

• HbA, is a tetramer composed of 2 α chains and 2 β chains (2α &2β).

• The α chains are encoded by two α-globin genes on chromosome 11

• The β chains are encoded by a single β-globin gene located on chromosome 16.

• The β-globin mutations associated with β- thalassemia are:

• (1) βo, in which no β-globin chains are produced

• (2) β+, in which there is reduced (but detectable) β-globin synthesis.

• Sequencing of β-thalassemia genes has revealed more than 100 different responsible mutations, the majority of which consist of single-base changes.

• Individuals inheriting one abnormal allele have thalassemia minor or thalassemia trait, which is asymptomatic or mildly symptomatic.

• Most individuals inheriting any two β0 and β+ alleles have β-thalassemia major occasionally, individuals inheriting two β+ alleles have a milder disease termed β-thalassemia intermedia.

• gene deletions rarely underlie β- thalassemias

Types of the mutations in β-thalassemia:

1- mutations in the promoter region that controls the initiation and rate of transcription.

• These lead to reduced globin gene transcription. • some β-globin is synthesized, such alleles are

designated β+.2-Mutations in the coding sequences • associated with more serious consequences. • It leads to the formation of a termination, or "stop" codon,

which interrupts translation of β-globin messenger RNA (mRNA) and completely prevents the synthesis of β-globin. Such alleles are designated β0.

• 3-Mutations that lead to aberrant mRNA processing • are the most common cause of β-

thalassemia.• Most of these affect introns, but some have

been located within exons. • If the mutation alters the normal splice

junctions, splicing does not occur, and all of the mRNA formed is abnormal.

• Unspliced mRNA is degraded within the nucleus, and no β-globin is made.

• However, some mutations affect the introns at locations away from the normal intron-exon splice junction. These mutations create new sites that are substrates for the action of splicing enzymes at abnormal locations-within an intron

• Because normal splice sites remain intact, both normal and abnormal splicing occur,

• Normal β-globin mRNA is decreased but not absent. • depending on their position, splice junction mutations

can create either β0 or β+ alleles.

• Thalassemia major

• Homozygous or compound heterozygous (β0/β0, β0/β+, or β+/β+)

• Severe, requires blood transfusions regularly

• β-thalassemia trait

• β/β+ or β/β0

• Asymptomatic, with mild microcytic anemia, or microcytosis without anemia

the pathogenesis of the anemia in β-thalassemia .

• 1-↓ synthesis of β-globin leads to ↓ HbA formation

- →low MCHC

- hypochromic and microcytic RBCs.

• 2-red cell hemolysis

it results from the unbalanced rates of β-globin and α-globin chain synthesis.

• 3-ineffective erythropoiesis

- intramedullary destruction of erythroid

precursors ( Erythroblasts ) before their maturation into red cells.

-this can ↑ in the absorption of dietary iron → iron overload.

α-Thalassemia

• Most of the α-thalassemias are caused by deletions that remove one or more of the α-globin gene loci.

• The severity of the disease that results from these lesions is directly proportional to the number of α-globin genes that are missing .

• 1-Hydrops fetalis (- -/- - ) Fatal in utero• 2-HbH disease (β4)-Hb Bart (γ4) (- -/- α) Moderately severe anemia• 3-α-thalassemia trait (- -/αα )(Asian) or (-α/-α )(black African) Similar to β-thalassemia trait

• 4-Silent carrier (-α/αα )

Asymptomatic, normal red cells

• the hemolytic anemia and ineffective erythropoiesis tend to be less severe in α-thalassemia than in β-thalassemia because the membrane damage in β-thal is more .

• Unfortunately, both HbH and Hb Bart have an abnormally high affinity for oxygen, which renders them ineffective at delivering oxygen to the tissues.

Clinical manifestations

• thalassemia major manifests itself postnatally as HbF synthesis diminishes.

• Affected children fail to develop normally, and develop growth retardation shortly after birth due to consumption of nutrients in ineffective erythropoiesis.

• skeletal deformities due to expanded BM spaces.

• prominent splenomegaly, hepatomegaly, and lymphadenopathy due to extramedullary hematopoiesis and the hyperplasia of the mononuclear phagocytes

• They are sustained only by repeated blood transfusions, which improve the anemia and reduce the skeletal deformities associated with excessive erythropoiesis.

• gradually systemic iron overload develops due to :

• 1- repeated blood transfusion.• 2- increased uptake of dietary iron from the gut due to ↓ hepcidin level.

Thal-majorskeletal abnormality

Lab findings

• Peripheral blood smears• Hypochromic microcytic anemia Hb. levels are 1-2 g/dl lower than in normal persons

of the same age and gender.

• ↓MCV, ↓ MCH• Target cells • Poikilocytosis, anisocytosis• Reticulocytosis.• Nucleated red cells (normoblasts)

Target cells

Target cells(arrows) anisocytosis poikilocytosis.

• Bone marrow• The combination of ineffective erythropoiesis

and hemolysis results in a striking hyperplasia of erythroid progenitors, with a shift toward early forms.

• ↑sidroblasts.• The expanded erythropoietic marrow may

completely fill the intramedullary space of the skeleton, invade the bony cortex

Hemoglobin electrophoresis

• ↓ HbA• ↑ HbF• ↑HbA2

Glucose-6-Phosphate Dehydrogenase Deficiency

• The red cell is vulnerable to injury by endogenous and exogenous oxidants normally inactivated by reduced glutathione (GSH).

• Abnormalities affecting the enzymes that are required for GSH production reduce the ability of red cells to protect themselves from oxidative injury and lead to hemolytic anemias.

• The prototype of these anemias is that associated with a deficiency of glucose-6-phosphate dehydrogenase (G6PD).

• One of the most important is the G6PD A- variant, which is carried by approximately 10% of black males in the United States.

• G6PD A- has normal enzymatic activity but a decreased half-life.

• Because red cells lack the capacity for protein synthesis, older G6PD A- red cells become progressively deficient in enzyme activity and more vulnerable to oxidant stress.

• The G6PD gene is on the X chromosome.• More than 400 G6PD variants have been identified, but only a

few are associated with disease.

• Because the G6PD gene is on the X chromosome, all the red cells of affected males are affected & vulnerable to oxidant injury

• in women because of random inactivation of one X chromosome heterozygous females have two distinct populations of red cells, one normal and the other deficient in G6PD activity.

• Most carrier females are asymptomatic, except those with a

very large proportion of deficient red cells (a chance situation known as ( unfavorable lyonization ).

• G6PD deficiency produces no symptoms until the patient is exposed to an environmental factor (most commonly infectious agents or drugs) that results in increased oxidant stress.

• The drugs incriminated include :• 1-antimalarials (e.g., primaquine), • 2-sulfonamides,• 3-nitrofurantoin, • 3-phenacetin, • 4-aspirin (in large doses), • 5-vitamin K derivatives.

• More commonly, episodes of hemolysis are triggered by infections, which induce phagocytes to produce free radicals as part of the normal host response.

• These offending agents produce oxidants such as H2O2 which removed by GSH, which is converted to oxidized glutathione in the process.

• Because regeneration of GSH is impaired in G6PD-deficient cells, hydrogen peroxide is free to "attack" other red cell components, including globin chains, which have sulfhydryl groups that are susceptible to oxidation.

• Oxidized Hb denatures and precipitates, forming intracellular inclusions called Heinz bodies, which can damage the cell membrane sufficiently to cause intravascular hemolysis.

• Other cells that are less severely damaged neverthelss suffer from a loss of deformability, and their cell membranes are further damaged when splenic phagocytes attempt to "pluck out" the Heinz bodies, creating so-called bite cells

• All of these changes predispose the red cells to becoming trapped in the splenic sinusoids and destroyed by the phagocytes (extravascular hemolysis).

• Drug-induced hemolysis is acute and of variable clinical severity.

• Typically, patients develop evidence of hemolysis after a lag period of 2 or 3 days.

• In G6PD A-, the enzyme deficiency is most marked in older red cells, which are thus more susceptible to lysis. Since the marrow compensates by producing new (young) resistant red cells, hemolysis tends to abate even if drug exposure continues.

• In other variants, such as G6PD Mediterranean, found mainly in the Middle East, the enzyme deficiency and the hemolysis that occur upon exposure to oxidants are more severe.

Peripheral blood smear (Heinz bodies) revealed by supravital staining.

"bite cells."

Paroxysmal Nocturnal Hemoglobinuria

• A rare disorder of unknown etiology, • (PNH) is the only form of hemolytic anemia that results

from an acquired membrane defect secondary to a mutation that affects myeloid stem cells.

• The mutant gene, called PIGA, • PICA gene is required for the synthesis of a specific type

of intramembranous glycolipid phosphatidylinositol glycan (PIG), which is membrane-associated protein.

• Without the membrane anchor, these "PIG-tailed" proteins cannot be expressed on the surface of cells.

• The affected proteins include several that limit the spontaneous activation of complement on the surface of cells.

• PIG-deficient precursors give rise to red cells that are inordinately sensitive to the lytic activity of complement.

• The hemolysis is nocturnal because the blood becomes acidic during sleep (because of CO2 retention) and an acid pH may promote hemolysis.

• It is not known why red cell destruction is paroxysmal.

hemoglobinuria

• Several other PIG-tailed proteins are deficient from the membranes of granulocytes and platelets, possibly explaining the striking susceptibility of these patients to infections and intravascular thromboses.

• PIGA is X-linked, and thus normal cells have only a single active PIGA gene, mutation of which is sufficient to give rise to PIG deficiency.

• Because all myeloid lineages are affected in PNH, the responsible mutations must occur in a multipotent stem cell.

• All normal individuals harbor small numbers of PIG-deficient bone marrow cells that have mutations identical to those that cause PNH.

• It is believed that clinically evident PNH occurs only in rare instances in which the PIG-deficient clone has a survival advantage.

• It is hypothesized that in PNH patients, autoreactive T cells specifically recognize PIG-tailed surface antigens on normal bone marrow progenitors.

• Because PIG-deficient stem cells do not express these targets, they escape immune attack and eventually replace the normal marrow elements.

Diagnosis

• Ham’s acidified serum test : -Erythrocytes in PNH are abnormally sensitive

to complement mediated lysis in acidified serum .

-Several combinations of patient & normal serum & cells are mixed & some are acidified to maximize the hemolytic effect associated with PNH .

Immunohemolytic Anemias

• Antibodies that recognize determinants on red cell membranes cause these uncommon forms of hemolytic anemia.

• The antibodies may arise spontaneously or be induced by exogenous agents such as drugs or chemicals.

• Immunohemolytic anemias are classified based on :

(1) the nature of the antibody

(2) the presence of certain predisposing conditions

• Warm Antibody Type

• Primary (idiopathic) >60%

• Secondary: 25%

1.B-cell lymphoid neoplasms (e.g CLL)

2.autoimmune disorders (e.g SLE)

3.drugs (e.g α-methyldopa, penicillin,

quinidine)

• Cold Antibody Type• Acute: 1.Mycoplasma infection, 2.Infectious mononucleosis • Chronic: 1.idiopathic, 2.B-cell lymphoid neoplasms (e.g

lymphoplasmacytic lymphoma)

Warm Antibody Immunohemolytic Anemias

• These are caused by IgG or, rarely, IgA antibodies that are active at 37°C.

• More than 60% of cases are idiopathic (primary), while another 25% are associated with an underlying disease affecting the immune system (e.g., systemic lupus erythematosus [SLE] or are induced by drugs

• The diagnosis of immunohemolytic anemias depends on the detection of antibodies and/or complement on patient red cells.

• This is done using • 1-The direct Coombs antiglobulin test, It measures the capacity of antibodies raised in animals

against human immunoglobulins or complement to agglutinate red cells from the patient.

• 2-The indirect Coombs test, in which patient serum is tested for the ability to agglutinate defined red cells.

• The hemolysis usually results from the opsonization of red cells by the autoantibodies, which leads to erythrophagocytosis in the spleen and elsewhere..

• Spheroidal cells resembling those seen in hereditary spherocytosis are often found in the peripheral blood smear.

• cell membrane is removed during attempted phagocytosis of Ab-coated cells. This reduces the surface area-to-volume ratio and leads to the formation of spherocytes, which are rapidly destroyed in the spleen

• The clinical severity of immunohemolytic anemias is quite variable. Most patients have chronic mild anemia with moderate splenomegaly and often require no treatment

Cold Antibody Immunohemolytic Anemias

• caused by low-affinity immunoglobulin M (IgM) antibodies

• IgM Abs bind to red cell membranes only at temperatures below 30°C, which are commonly experienced in distal parts of the body (e.g., ears, hands, and toes).

• Although IgM fixes complement well, the later steps of complement fixation occur inefficiently at temperatures below 37°C.

• As a result, most cells with bound IgM pick up some C3b but are not lysed in the periphery. When these cells travel to warmer areas, the weakly bound IgM antibody is released, but the coating of C3b remains

• Because C3b is an opsonin the cells are phagocytosed by the mononuclear phagocyte system, especially Kupffer cells (extravascular hemolysis ).

• Cold agglutinins sometimes develop transiently during recovery from pneumonia caused by Mycoplasma sp. and infectious mononucleosis, producing a mild anemia of little clinical importance.

• A chronic cold agglutinin hemolytic anemia occurs in association with lymphoid neoplasms or as an idiopathic condition.

• In addition to anemia, Raynaud phenomenon often occurs in these patients as a result of the agglutination of red cells in the capillaries of exposed parts of the body

Polycythemia

• an increase in the blood concentration of red cells, which usually correlates with an increase in the hemoglobin concentration.

• Polycythemia may be :• 1-relative, • when there is hemoconcentration caused

by a decrease in plasma volume as in : a.water deprivation, b.prolonged vomiting, c.diarrhea, d.or the excessive use of diuretics

• 2-absolute,

• when there is an increase in the total red cell mass.

A.primary ( PCV )

when the increase in red cell mass results from an autonomous proliferation of the myeloid stem cells,

B.secondary when the red cell progenitors are

proliferating in response to an increase in erythropoietin.

1-Appropriate: lung disease, high-altitude living, cyanotic heart disease  

2-Inappropriate: erythropoietin-secreting tumors (e.g., renal cell carcinoma, hepatoma, cerebellar hemangioblastoma)

• Primary polycythemia (polycythemia vera [PCV]) is a clonal, neoplastic proliferation of myeloid progenitors,

• The disease results from somatic mutation of a single haemopoietic stem cell which gives its progeny a proliferative advantage.

• JAK2 mutation is present in haemopoietic cells in almost 100% of patients.

• Although the increase in red cells is diagnostic findings, in many patients there is also overproduction of granulocytes and platelets.

• PRV has a propensity for clonal evolution into either acute myeloid leukemia (AML) or myelofibrosis with myelolid metaplasia (MMM).

Clinical manifestations

• PRV is a disease of elderly• M=F• Clinical features are the result of hypervicosity ,

hypervolaemia or hypermetabolism:• 1-headache , dyspnoea, blurred vision , night sweats,

pruritis after hot bath can be severe problems.• 2-plethoric appearance :ruddy cyanosis , conjunctival

suffusion and retinal venous enlargement.• 3-splenomegaly in 75% of patients.• 4- hemorrhage ( gastrointestinal, uterine, cerebral).• 5-thrombosis either arterial

(cardiac,Cerebral,peripherial), or venous (deep or superficial leg veins ,portal

Diagnosis

• Major criteria• Total red cell mass : male >35ml/kg

• female>32ml/kg.

• Arterial oxygen saturation >92%.

• Splenomegaly.

• JAK2 mutation.

• Minor criteria• Platelets >400x109.

• White cells> 12x109.

• Increased NAP (neutrophil alkaline phosphatase) score.

• Raised serum vitamin B12 level

Secondary polycythemia

• There is an increase in RBCs production due to• A- compensatory increasing in

erythropoietin secretion: • 1- High altitude.• 2-Pulmonary disease (chronic obstructive

airways ) and alveolar hypoventilation (sleep apnoea)

• 3- Cardiovascular disease especially congenital with cyanosis.

• 4- Heavy cigarettes smoking.

• B- inappropriate Erythrpoietin increase:

• 1- in renal disease

(hydronephrosis, cyst).

• 2- tumors

e.g uterine leiomyoma, hypernephroma , hepatocellular carcinoma