Hemolytic disorders due to Inherited abnormalities in Red cell

cytoskeleton

Guvera VasireddyDepartment of Pathology

OMC

RED CELL CYTOSKELETON The remarkable elasticity and durability of the red cell

are due to the properties of its specialized membrane skeleton

Lies closely apposed to the internal surface of the plasma membrane.

Its chief protein component, spectrin, consists of two polypeptide chains, α and β, which form intertwined (helical) flexible heterodimers.

The “head” regions of spectrin dimers self-associate to form tetramers, while the “tails” associate with actin oligomers

INTERACTIONS BETWEEN VARIOUS CYTOSKELETAL PROTEINS Each actin oligomer binds multiple spectrin

tetramers creating a two-dimensional spectrin-actin skeleton that is connected to the cell membrane by two distinct interactions.

The first, involving the proteins ankyrin and band 4.2, binds spectrin to the transmembrane ion transporter, band 3.

The second, involving protein 4.1, binds the “tail” of spectrin to another transmembrane protein, glycophorin A.

RED CELL CYTOSKELETON

Protein composition of the red blood cell membrane skeleton. The major components of the erythrocyte membrane as separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and revealed by Coomassie blue staining. G3PD, glucose 3-phosphate dehydrogenase.

HEREDITARY INTRINSIC MEMBRANE DEFECTS

Hereditary spherocytosis

Spherocytic elliptocytosis

Hereditary elliptocytosis

Southeast Asian ovalocytosis

Hereditary pyropoikilocytosis

Hereditary stomatocytosis

Hereditary xerocytosis Rh antigen deficiency Hereditary

acanthocytosis Abetalipoproteinemia McLeod Syndrome

(Ke11 antigen deficiency)

Chorea-acanthocytosis syndrome

In(Lu)

ACQUIRED MEMBRANE DEFECTS

Acquired spherocytosis Clostridia

septicemia Thermal burn Hypophosphatemia Zieve’s syndrome Snake, spider, and

insect bites

Acquired acanthocytosis Spur cell anemia Vitamin E deficiency Infantile

pyknocytosis Paroxysmal

nocturnal hemoglobinuria

ERYTHROCYTE MEMBRANE PROTEIN DEFECTS IN INHERITED DISORDERS OF RED CELL SHAPE

Protein Disorder CommentAnkyrin HS Most common cause of typical dominant HSBand 3 HS, SAO,

NIHF, HAc"Pincered" HS spherocytes seen on blood film presplenectomy; SAO results from 9 amino acid deletion

β-Spectrin HS, HE, HPP, NIHF

"Acanthocytic" spherocytes seen on blood film presplenectomy; location of mutation in β-spectrin determines clinical phenotype

α-Spectrin HS, HE, HPP, NIHF

Location of mutation in α-spectrin determines clinical phenotype; α-spectrin mutations most common cause of typical HE

Protein 4.2 HS Primarily found in Japanese patientsProtein 4.1 HE Found in certain European and Arab

populationsGPC HE Concomitant protein 4.1 deficiency is basis of

HE in GPC defects

HEREDITARY SPHEROCYTOSIS (HS) The pathogenic mutations most commonly affect ankyrin, band 3,

spectrin, or band 4.2, the proteins involved in the first of the two tethering interactions.

Most mutations cause shifts in reading frame or introduce premature stop codons, such that the mutated allele fails to produce any protein.

The prevalence of HS is highest in northern Europe, where rates of 1 in 5000 are reported.

An autosomal dominant inheritance pattern is seen in about 75% of cases.

The remaining patients have a more severe form of the disease that is usually caused by the inheritance of two different defects (a state known as compound heterozygosity).

PATHOGENESIS Young HS red cells are normal in shape.

Deficiency of membrane skeleton reduces the stability of the lipid bilayer, leading to the loss of membrane fragments as red cells age in the circulation.

The loss of membrane relative to cytoplasm “forces” the cells to assume the smallest possible diameter for a given volume, namely, a sphere.

Life span of the affected red cells is decreased on average to 10 to 20 days from the normal 120 days

The left panel shows the normal organization of the major red cell membrane skeletal proteins. Various mutations involving α-spectrin, β-spectrin, ankyrin, band 4.2, or band 3 that weaken the interactions between these proteins cause red cells to lose membrane fragments. To accommodate the resultant change in the ratio of surface area to volume these cells adopt a spherical shape. Spherocytic cells are less deformable than normal ones and therefore become trapped in the splenic cords, where they are phagocytosed by macrophages. GP, glycophorin.

Role of the red cell membrane skeleton in hereditary spherocytosis.

PATHO

PHYSIO

LOG

Y OF H

EREDITARY SPH

EROCYTO

SIS.

MEMBRANE DEFECTS THAT LEAD TO HS

CLINICAL PRESENTATION Presents at any age.

Highly variable from asymptomatic to severely anaemic, but usually there are few symptoms.

Well-compensated haemolysis;

Features of haemolytic anaemia: splenomegaly, gallstones, mild jaundice may be present.

Occasional aplastic crises occur, e.g. with parvovirus B19 infection.

LABORATORY DIAGNOSIS

BLOOD FILM Erythrocyte morphology in HS is variable.

Typical HS patients have blood films with easily identifiable spherocytes lacking central pallor

May present with only a few spherocytes on the film or with numerous small, dense spherocytes and bizarre erythrocyte morphology with anisocytosis and poikilocytosis.

Rarely, spherostomatocytes are seen.

Specific morphologic findings have been identified in patients with certain membrane protein defects, such as pincered erythrocytes (band 3) or spherocytic acanthocytes (-spectrin).

Peripheral blood film of spherocytic hemolysis.Spherocytes are round, are slightly smaller than normal red blood cells, and lack central pallor.Note the nucleated red blood cells and polychromatophilic cells.

Note the anisocytosis and several dark-appearing spherocytes with no central pallor. Howell-Jolly bodies (small dark nuclear remnants) are also present in red cells of this asplenic patient.

PERIPHERAL SMEAR IN HS

Click icon to add pictureMarrow smear from a patient with hemolytic anemia. The marrow reveals greatly increased numbers of maturing erythroid progenitors (normoblasts).

OSMOTIC FRAGILITY Osmotic fragility is tested by adding increasingly hypotonic

concentrations of saline solution to red cells.

Spherocytes, which already are at maximum volume for surface area, burst at higher than normal saline concentrations.

Some HS individuals have a normal osmotic fragility on freshly drawn red blood cells, with the osmotic fragility curve approximating the number of spherocytes.

After incubation at 37°C for 24 hours, HS red cells lose membrane surface area more readily than normal because their membranes are leaky and unstable.

Incubation accentuates the defect in HS erythrocytes and brings out the defect in osmotic fragility, making incubated osmotic fragility the standard test in diagnosing HS.

A. Histograms of the distribution of (top) MCV and (bottom) MCHC in red cells of a patient with HS before splenectomy. Vertical lines mark the normal limits of the distributions. B. Osmotic fragility testing. The shaded area is the normal range. Results representative of both typical and severe spherocytosis are shown.A "tail," representing very fragile erythrocytes that have been conditioned by the spleen, is common in many HS patients prior to splenectomy.

OSMOTIC FRAGILITY

HS Trait or Carrier

Mild Spherocytosis

Moderate Spherocytosis

Moderately Severe Spherocytosis*

Severe Spherocytosis

Fresh blood

Normal Normal or slightly increased

Distinctly increased

Distinctly increased

Distinctly increased

Incubated blood

Slightly increased

Distinctly increased

Distinctly increased

Distinctly increased

Markedly increased

Laboratory Findings

HS Trait or Carrier

Mild Spherocytosis

Moderate Spherocytosis

Moderately Severe Spherocytosis*

Severe Spherocytosis

Hemoglobin (g/dL)

Normal 11–15 8–12 6–8 <6

Reticulocytes (%)

1–2 3–8 ± 8 ≥10 ≥10

Bilirubin (mg/dL)

0–1 1–2 ± 2 2–3 ≥3

Spectrin content (% of normal)‡

100 80–100 50–80 40–80§ 20–50

Blood film Normal Mild spherocytosis

Spherocytosis

Spherocytosis

Spherocytosis and poikilocytosis

DIFFERENTIAL DIAGNOSIS OF HS HS must be differentiated from acquired hemolytic anemias that

produce circulating spherocytes and abnormal osmotic fragility. Autoimmune hemolytic anemia (AHA) usually produces

spherocytes. The direct antiglobulin test (direct Coombs test), readily distinguishes most AHA from HS.

On microscopic examination, HS usually produces more uniform spherocytosis than does AHA.

An elevated MCHC may also help differentiate HS from AHA. Other causes of acquired spherocytosis such as transfusion

reactions, AB0 incompatibility, oxidant erythrocyte damage, thermal bums, snake venom, and Clostridia sepsis are distinguished from HS by the clinical setting and lack of chronicity.

Unusual inherited conditions occasionally confused with HS include Rh antigen deficiency, hereditary stomatocytosis, unstable hemoglobins, and the oxidant hemolysis of Wilson’s disease.

Peripheral blood film of microspherocytes seen in Clostridium perfringens sepsis.Although regular spherocytes are usually smaller than normocytic red blood cells, microspherocytes are even smaller than that. This finding is usually seen in critically ill, septic patients with severe C. perfringens infection.

MANAGEMENT Patients with HS may require red cell transfusions for a plastic crisis,

chronic folate administration to ward off megaloblastic crisis, or cholecystectomy for biliary lithiasis.

The most significant therapeutic decision, however, centers on the issue of splenectomy.

Splenectomy does not eliminate the spherocytic defect but dramatically improves the rate of hemolysis in HS patients.

In very mild cases (older age, normal hemoglobin, minimal hemolysis, and no complications), there is no need for splenectomy.

In more severely affected individuals (young age, moderate anemia, active hemolysis, and complications), splenectomy is clearly indicated and beneficial.

Newer surgical techniques such as laparoscopic splenectomy and partial splenectomy are beginning to impact on the treatment of HS, but there are insufficient data to ascertain whether the indication for splenectomy has changed.

Vaccination against , pneumoniae prior to splenectomy is strongly recommended.

COMPLICATIONS

NEONATAL PERIOD AND IMMUNO COMPROMISED PATIENTS Parvovirus B19 selectively infects

erythropoietic progenitor cells and inhibits their growth.

Parvovirus infections frequently are associated with mild neutropenia, thrombocytopenia, or pancytopenia.

Parvovirus infection presents with fever, chills, lethargy, vomiting, diarrhea, myalgia, and a maculopapular rash on the face (slapped cheek syndrome), trunk, and extremities.

APLASTIC CRISIS During the aplastic phase, hematocrit level and reticulocyte

count fall, marrow erythroblasts disappear, and, as the plasma iron turnover decreases, plasma iron level increases.

Giant pronormoblasts, a hallmark of the cytopathic effects of parvovirus B19, often appear in the marrow. As production of new red cells declines, the remaining cells age, and microspherocytosis and osmotic fragility increase.

Return of marrow function is heralded by a fall in serum iron concentration and emergence of granulocytes, platelets, and, finally, reticulocytes.

Aplastic crises usually last 10 to 14 days (about half the life span of typical HS red cells), the hemoglobin value usually falls to about half its usual level before recovery occurs.

HEMOLYTIC CRISIS Hemolytic crises usually are associated with viral

illnesses and typically occur in childhood.

They generally are mild but during severe hemolytic crises, marked jaundice, anemia, lethargy, abdominal pain, and tender splenomegaly occur.

Hospitalization and erythrocyte transfusion may be required.

The most common etiologic agent in these cases is parvovirus B19, which causes erythema infectiosum.

MEGALOBLASTIC CRISIS Megaloblastic crisis occurs in HS patients

with increased folate demands,

Occurs in pregnant patients, growing children, or patients recovering from an aplastic crisis.

This complication is preventable with appropriate folate supplementation.

GALLBLADDER DISEASE Formation of bilirubinate gallstones, the most

frequently reported complication in up to half of HS patients.

Coinheritance of Gilbert syndrome uridine diphosphoglucuronate glucuronosyltransferase gene polymorphism markedly increases the risk of gallstone formation.

Most gallstones occur in adolescents, children, and young adults.

Routine management should include interval ultrasonography to detect gallstones because many patients with cholelithiasis and HS are asymptomatic.

OTHER COMPLICATIONS Dermatologic manifestations of HS, including skin ulceration, gouty

tophi, and chronic leg dermatitis, are uncommon. Findings attributable to extramedullary hematopoiesis have been

described in some HS patients. Thrombosis has been reported in several HS patients, usually

postsplenectomy. Iron overload has been described in untransfused HS patients both with

coinherited hemochromatosis and in patients without HFE (hemochromatosis gene) mutations.

Several HS kindred have been reported with neuromuscular abnormalities including cardiomyopathy, slowly progressive spinocerebellar degenerative disease, spinal cord dysfunction, and movement disorders.

The observation that erythrocyte ankyrin and -spectrin are also expressed in muscle, brain, and spinal cord raises the possibility that these HS patients suffer from defects of one of these proteins.

Heterozygous defects of band 3 have been described in patients with inherited distal renal tubular acidosis and normal erythrocytes.

THERAPY AND PROGNOSIS Splenectomy alleviates the anemia in the overwhelming majority of

patients.

Postsplenectomy, spherocytosis and altered osmotic fragility persist, but the "tail" of the osmotic fragility curve, created by conditioning of a subpopulation of spherocytes by the spleen, disappears.

Erythrocyte life span nearly normalizes, and reticulocyte counts fall to normal or near-normal levels.

Changes typical of the postsplenectomy state, including Howell-Jolly bodies, target cells, siderocytes, and acanthocytes, become evident on the blood film.

Postsplenectomy, patients with the most severe forms of HS still suffer from shortened erythrocyte survival and hemolysis, but their clinical improvement is striking.

HEREDITARY ELLIPTOCYTOSIS, PYROPOIKILOCYTOSIS, AND RELATED DISORDERS

HEREDITARY ELLIPTOCYTOSIS SYNDROMES Disorders characterized by elliptical red cells on the

peripheral blood smear

Most are clinically silent and are discovered incidentally when a blood smear is reviewed..

The clinical expression of hemolytic HE ranges from a moderate hemolytic anemia to severe, near-fatal or fatal hemolytic anemia.

These disorders have identified abnormalities of various erythrocyte membrane proteins, including - and -spectrin, protein 4.1, and GPC.

The majority of defects occur in spectrin, the principal structural protein of the erythrocyte membrane skeleton

VARIANTS OF HE Hereditary pyropoikilocytosis (HPP) is a severe hemolytic

anemia, with red cell fragments, poikilocytes, and microspherocytes seen on peripheral blood smear.

Once regarded as a separate condition, HPP is now recognized to be a variant of the HE disorders.

Spherocytic HE is a rare condition in which both ovalocytes and spherocytes are present on the blood smear.

Southeast Asian ovalocytosis (SAO), also known as stomatocytic elliptocytosis, is an HE variant prevalent in the malaria-infested belt of Southeast Asia and the South Pacific.

Characterized by rigid spoon-shaped cells that have either a longitudinal slit or a transverse ridge.

A: Micropoikilocytes and elliptocytes in a neonate with transient poikilocytosis and an α-spectrin gene mutation. B: Same child at 7 months of age, now exhibiting morphology of common hereditary elliptocytosis.

C: Compound heterozygous hereditary elliptocytosis due to two α-spectrin self-association–site structural mutations. Note distorted red cell shapes, elliptocytes, and fragments. D: Hereditary pyropoikilocytosis. Red cell abnormalities are similar to those in (A) and (C) with prominent budding and fragmentation.