RBC membrane disorders and RBC enzyme deficiency

TRUE

TRUE OR FALSE: It is vital that the cytoskeleton and its relationship to the integral proteins are functional to ensure that the red cell is not prematurely removed from the circulation.

Intravascular hemolysis

A type of hemolysis where erythrocytes lyse or burst while inside the blood vessel

Extravascular hemolysis

A type of hemolysis where lysis occurs in the reticuloendothelial organs like the spleen, and with the help of macrophages and complement proteins

presence of hemoglobinuria/hemosiderinuria

What differentiates intravascular hemolysis from extravascular hemolysis?

splenomegaly

What differentiates extravascular hemolysis from intravascular hemolysis?

Hereditary spherocytosis

• Characterized by spherocytic red cells, splenomegaly, and familial occurrence

• Affects 1 in 5000 people; occurs predominantly in those of Northern European ancestry.

• ANK1

• SPTA1

• SPTB

• EPB42

In hereditary spherocytosis, mutations can occur in several genes that encode for cytoskeletal proteins including?

ANK1

• codes for ankyrin-1

• (most cases in the US and Europe; only 5% 10% of cases in Japan)

SPTA1

• codes for ɑ-spectrin

• (fewer than 5% of cases are rare but associated with a severe form of HS)

SPTB

codes for β-spectrin

EPB42

• codes for protein 4.2

• (45%-50% of cases in Japan, more commonly seen in Asia)

SLC4A1 gene

In hereditary spherocytosis, mutations of genes for transmembrane proteins can also occur, including the?

Vertical defect

• The referred defect in hereditary spherocytosis

• The association between the lipid bilayer and the red cell cytoskeleton is faulty.

TRUE

TRUE OR FALSE: RBCs in HS are characterized by an unstable lipid bilayer which facilitates the release of lipids from the membrane, resulting in loss of surface area and the formation of poorly deformable spherocytes.

• anemia

• jaundice

• splenomegaly

Three key clinical manifestations of hereditary spherocytosis:

TRUE

TRUE OR FALSE: In hereditary spherocytosis, hemolysis varies in severity and is corrected by splenectomy (surgical removal of the spleen) but the spherocytosis remains.

• hemolytic crises

• aplastic crises

• megaloblastic crises

Complications that could occur in patients with hereditary spherocytosis

Mild hereditary spherocytosis

• (about 20-30% HS cases)

• May be asymptomatic because an increase in erythropoiesis compensates for the RBC loss or hemolysis.

• Laboratory findings: Normal hemoglobin, slightly increased bilirubin, slight reticulocytosis, few spherocytes

Moderate hereditary spherocytosis

• (about 60%, more common phenotype)

• Incompletely compensated hemolytic anemia, causing low Hgb levels.

• Laboratory findings: Low-Normal hemoglobin, increased bilirubin (>2 mg/dl), 6-10% reticulocytosis, more spherocytes

Severe hereditary spherocytosis

• (about 3-5%, rare)

• Most patients have a well compensated hemolytic anemia (rarely symptomatic) but complications may occur (can occur in hemolytic anemias in general)

• Laboratory findings: Low hemoglobin (<g mg/dl), increased bilirubin (>3 mg/dl), >10% reticulocytosis, more spherocytes

Hemolytic crises

Often arises after viral infections

Aplastic crises

• Dramatic decrease in Hgb level and reticulocyte count

Megaloblastic crises

• Commonly occurs in pregnant HS patients

• When folic acid deficiency develops because of increased folate utilization to support the chronic erythroid hyperplasia when the bone marrow is trying to replace all the destroyed RBCs

Spherocytes on PBS

• Hallmark of hereditary spherocytosis

Spherocytes

are small, round, dense RBCs lacking a central area of pallor but they are NOT specific for HS

>4%

What is the cut off of the total red blood cells ((hyperchromic, spherocytic cells) has been proposed to screen for hereditary spherocytosis?

• normal-appearing RBCs

• polychromasia

• varying degrees of anisocytosis and poikilocytosis

Other peripheral blood findings in hereditary spherocytosis includes?

• mean cell hemoglobin concentration (MCHC)

• red cell distribution width (RDW)

What CBC parameter is increased in hereditary spherocytosis?

FALSE

TRUE OR FALSE: Bone marrow analysis is required for the diagnosis of HS

Osmotic fragility test

• has been used as a supporting test.

• The principle stems from the observation that RBCs that are more spherical (spherocytes), with a decreased surface/volume ratio have a limited capacity to expand in hypotonic solutions and lyse at a higher concentration of NaCl than do normal biconcave RBCs.

Increased

Spherocytes have increased or decreased OFT?

TRUE

TRUE OR FALSE: Increased osmotic fragility is characteristic but it is not specific for HS; it also lacks sensitivity.

TRUE

TRUE OR FALSE: In HS, after splenectomy, osmotic fragility improves but the spherocytes remain.

Fresh heparinized plasma

Specimen used in OFT

Sterile, defibrinated blood

Specimen used in autohemolysis

Incubated osmotic fragility test

• A variation of OFT

• Entails incubating the blood at 37°C for 24 hours before performing the OFT allows HS cells to become more spherical

• Often needed to detect mild cases of HS

Autohemolysis test

• Another test that supports the diagnosis of HS

• Glucose is added and provides the ATP needed to drive cation pumps to help maintain the osmotic balance in the RBCs.

Eosin-5’-maleimide (EMA) Binding Test

• More sensitive and specific than OFT (> 90%)

• Contains fluorescent dyes that binds to transmembrane proteins band 3, Rh, RhAg, and CD47 in the RBC membrane; its fluorescence can be detected by flow cytometry.

TRUE

TRUE OR FALSE: Specimens from HS patients show a lower fluorescence intensity than RBCs from normal controls and from patients with spherocytes due to immune-mediated hemolysis.

Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis (SDSPAGE)

Can be used to identify membrane protein deficiencies

Osmotic Gradient Ektacytometry

Can determine variation in membrane surface area and cell water content

Hypertonic Cryohemolysis Test

Cells from HS patients are particularly sensitive to cooling at 0°C in hypertonic solutions (greater than 20% hemolysis versus 3% to 15% hemolysis in normal cells)

no treatment

Treatment for mild hereditary spherocytosis

Splenectomy

results in longer RBC survival in the circulation but it does not get rid of the spherocytes; the anemia is usually corrected but leukocytosis and thrombocytosis can occur.

lifelong risk of sepsis and even death secondary to encapsulated bacteria.

Major drawback of splenectomy

regular transfusions

Treatment for severe cases of hereditary spherocytosis

Hereditary elliptocytosis

• Mainly an autosomal dominant condition.

• Affects approximately 3-5 per 10,000 people (US population), because majority are symptomatic the actual prevalence is not known.

• It is more common in Africa and in Mediterranean regions, where there is a high prevalence of malaria.

Horizontal defect

• All cases of HE are associated with defects in proteins that disrupt the horizontal or lateral interactions in the protein cytoskeleton

• SPTA1

• SPTB

• EPB41

The HE phenotype can result from various mutations in at least 3 genes:

SPTA1

• codes for ɑ-spectrin

• accounts for 65% of cases of hereditary elliptocytosis

SPTB

• codes for β-spectrin

• accounts for 30% of cases of hereditary elliptocytosis

EPB41

• codes for protein 4.1

• accounts for 5% of cases of hereditary elliptocytosis

• mutations result in weakened spectrin-actin protein 4.1 junctional complexes

TRUE

TRUE OR FALSE: In hereditary elliptocytosis, the spectrin mutations result in impaired association of spectrin dimers into spectrin tetramers and oligomers.

1. Common HE (including hereditary pyropoikilocytosis [HPP])

2. Spherocytic HE

3. Southeast Asian Ovalocytosis (Hereditary Ovalocytosis)

On the basis of RBC morphology, hereditary elliptocytosis can be divided into 3 groups:

Common hereditary elliptocytosis

• Most affected people (~90%) are nonanemic and only 10% to 20% exhibit mild hemolysis.

• Non-hypochromic elliptocytes number about 15% in a blood film (normally less than 5% of the RBCs are elliptical).

Fetal hemoglobin

In affected neonates with common hereditary elliptocytosis, worsening of hemolysis has been attributed to the presence of?

Hereditary pyropoikilocytosis (HPP)

• Considered a severe form of common HE

• Autosomal recessive inheritance; occurs primarily in blacks

• Cells fragment at 45-46 degrees Celsius

• It features a severe congenital hemolytic anemia characterized by microcytosis, striking micropoikilocytosis and fragmentation.

Spherocytic hereditary elliptocytosis

• Accounts for 10% of cases of HE.

• Features mild to moderate hemolytic anemia and splenomegaly.

• both elliptocytes and spherocytes are present, along with abnormal osmotic fragility and autohemolysis tests

Southeast Asian Ovalocytosis

• Also known as Hereditary ovalocytosis

• Inheritance pattern is autosomal dominant; the underlying defect is related to a deletion of 27 bases from the band 3 gene (SLC4A1).

• Occurs with high frequencies (20% to 30%) in certain populations of the Far East, particularly Malaysia and regions of the malaria belt of Southeast Asia

• Hemolysis is usually absent or mild

• No treatment is required

Leach phenotype

• Is due to a mutation in the genes that encode for GPC, a protein where Gerbich antigens are expressed at its extracellular domains.

• Heterozygotes have normal RBC morphology while homozygotes have mild elliptocytosis but no anemia.

Gerbich antigens and glycoprotein C

What is lacking in leach phenotype?

TRUE

TRUE OR FALSE: In leach phenotype, the reason for the elliptocyte morphology may be a defect in the interaction between GPC and protein 4.1 in the junctional complex.

Sodium

Cell volume is determined by the intracellular concentration of cations, particularly?

prematurely hemolyzed

When RBCs lose the ability to regulate volume, the cells are?

Stomatocyte

If the total intracellular cation content is increased, water enters the cell and increases the cell volume, forming a?

Dehydrated RBC or a Xerocyte

If the total intracellular cation content is decreased, water leaves the cell which decreases cell volume and produces a?

Hereditary stomatocytosis

• Rare, autosomal dominant disorder

• Has 2 types

Dehydrated Stomatocytosis (DHst) or Heredity Xerocytosis

• More common form of hereditary stomatocytosis

• Due to mutations in the PIEZO1 gene

• The potassium leak is not balanced by an increase in sodium, thus the intracellular cation content is reduced and water then is lost from the cell.

• Phenotype varies from asymptomatic to severe hemolytic anemia but generally it is mild to moderate.

• Most patients do not require treatment.

PIEZO1 gene

• This protein combines with other proteins to form a core in the RBC membrane to mediate cation transport.

• Mutations of this protein results in an increase in ion channel activity, and thus an increase in cation transport.

• The cation permeability gets disrupted (abnormal).

TRUE

TRUE OR FALSE: In hereditary stomatocytosis, splenectomy does not improve the anemia and is contraindicated. Doctors should not perform splenectomy on these kinds of patients.

MCHC

What CBC parameter is increased in hereditary stomatocytosis?

Overhydrated stomatocytosis (OHst)

• Very rare; a defect in cation permeability is still present

• The RBC membrane is excessively permeable to sodium and potassium in this case.

• There is an influx of sodium into the cell that exceeds the loss of potassium which leads to a net increase in the intracellular cation concentration

• Mutation in the RHAG gene (According to studies)

• Blood films show 5% to 50% stomatocytes.

1. Markedly increased MCV (110-150 fL)

2. Decreased MCHC

3. Increased osmotic fragility

What is the characteristic result of CBC parameter in patients with overhydrated stomatocytosis?

Acquired stomatocytosis

There are cases wherein stomatocytes are in peripheral blood films and does not necessarily mean that the patient has hereditary stomatocytosis.

Stomatocytes induced by drying artifacts

Most common acquired stomatocytosis

TRUE

TRUE OR FALSE: True stomatocytosis: stomatocytes should be seen in all areas of the blood film (not just in one area or not just a few)

1. Mutation in spectrin that disrupts spectrin heterodimer self association

2. Partial spectrin deficiency that results in a decreased spectrin/band 3 ratio

Hereditary pyropoikilocytosis is associated with 2 abnormalities;

Glucose-6-phosphage dehydrogenase (G6PD)

is an enzyme that catalyzes the first step in a series of reactions that detoxify hydrogen peroxide formed from oxygen radicals.

6-phosphogluconate

Glucose 6-phosphate dehydrogenase (G6PD) generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) by converting glucose 6-phosphate into?

reduced glutathione (GSH) and NADP

Reduced NADPH that is formed is used by the enzyme glutathione reductase to reduce oxidized glutathione (GSSG) to?

Glutathione peroxidase

will use the reduced glutathione generated in the previous step to detoxify hydrogen peroxide and make it water.

G6PD gene

is located on the X chromosome and encodes for the G6PD enzyme which assembles into a dimer and tetramer in its functional configuration.

TRUE

TRUE OR FALSE: 85% of the mutations in G6PD deficiency are single missense mutations (specifically amino acid substitutions).

G6PD deficiency

• is the most common RBC enzyme defect, with a prevalence of 5% of the global population (approximately 400M people worldwide; it is common in the Philippines).

• The prevalence varies by geographic location: highest prevalence in areas where malaria is endemic (e.g. sub-Saharan Africa, Middle East, Asia), it is also common in the Philippines

G6PD-B

The normal variant of G6PD

G6PD Mediterranean, G6PD-Madrid

G6PD variant that are associated with severely deficient activity

G6PD-A

G6PD variant that is associated with mild to moderately deficient activity

TRUE

TRUE OR FALSE: Older RBCs are preferentially destroyed or hemolyzed because reticulocytes and young RBCs have 5 times more G6PD activity.

TRUE

TRUE OR FALSE: Enzyme activity of G6PD decreases as the cells age.

Heinz bodies

In G6PD deficiency, hemoglobin is particularly affected by oxidation, resulting in conversion to methemoglobin, which is also not as soluble thus hemoglobin precipitates as?

Oxidative stress

can induce an acute hemolytic episode; triggers include certain oxidizing drugs

Hemolysis secondary to drug exposure

is the classic manifestation of G6PD deficiency

infection

the most common cause of hemolysis in individuals with G6PD deficiency

Favism

• Rare, severe hemolytic episode that occurs in some G6PD-deficient individuals after ingesting fava beans.

• Can manifest as sudden onset of acute intravascular hemolysis within hours of ingestion.

• Only a small percentage of G6PD-deficient patients manifest this, most have the G6PD Mediterranean variant.

normocytic, normochromic erythrocytes, varying degrees of anisocytosis, poikilocytosis, spherocytosis, and schistocytosis

Peripheral smear of a G6PD deficienct shows?

TRUE

TRUE OR FALSE: G6PD deficient patient have negative DAT

Quantitative spectrophotometric assays

• Gold standard to determine G6PD activity

• It is able to make a definitive diagnosis, and assess the severity of the deficiency.

• Based on direct measurement of NADPH generated.

• Cutoff points are usually set at less than 20% of normal activity.

• The rate of NADPH formation is proportional to G6PD activity and is measured as an increase in absorbance at 340 nm.

Qualitative tests

• serve as rapid screening tools.

• Results merely tell if a specimen is “G6PD-deficient” or “normal”.

Fluorescent spot test

• Based on the principle that NADPH generated is fluorescent, while NADP in the reagent is not.

• Specimens with normal G6PD activity will show moderate to strong fluorescent spots under UV light while those with decreased or no activity do not fluoresce or show only weak fluorescence.

Dye-reduction assay

• Employ the same G6PD enzymatic reaction as the fluorescent spot test but with an additional step in which the NADPH produced reduces a dye, giving a visualized color change.

• Has an advantage of not requiring a UV light, but is more expensive.