Hema 2 Lec Module 12: RBC MEMBRANCE DISORDERS & RBC ENZYME DEFICIENCIES

peripheral membrane proteins

Lattice network is formed by _________ and certain proteins which also anchor the cytoskeleton to the membrane and the integral proteins.

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

When erythrocytes lyse or burst while inside the blood vessel.

EXTRAVASCULAR HEMOLYSIS

Lysis occurs in the reticuloendothelial organs like the spleen, and with the help of macrophages and complement proteins.

hemoglobinuria / hemosiderinuria

Both types of hemolysis showcase similar laboratory findings. One thing that does differentiate them is the presence of ___________ or __________ , which is seen in intravascular types of hemolysis.

False. they are MORE likely

True or False: Extravascular hemolytic anemias are less likely to present with splenomegaly.

both

State whether it is Intravascular Hemolysis, Extravascular Hemolysis, both, or none: Reticulocyte count increased

both

State whether it is Intravascular Hemolysis, Extravascular Hemolysis, both, or none: Lactate dehydrogenase increased

Intravascular Hemolysis

State whether it is Intravascular Hemolysis, Extravascular Hemolysis, both, or none: Indirect bilirubin increased

Extravascular Hemolysis

State whether it is Intravascular Hemolysis, Extravascular Hemolysis, both, or none: Indirect bilirubin increased/normal

both

State whether it is Intravascular Hemolysis, Extravascular Hemolysis, both, or none: Haptoglobin decreased

Extravascular Hemolysis

State whether it is Intravascular Hemolysis, Extravascular Hemolysis, both, or none: Urinary hemosiderin absent

Intravascular Hemolysis

State whether it is Intravascular Hemolysis, Extravascular Hemolysis, both, or none: Urinary hemosiderin present

hereditary

Most defects in the RBC membrane that can cause hemolytic anemia are _________ however, acquired defects also exist.

False. It CAN be classified

True or False: Hereditary membrane defects cannot be classified by morphologic features and also according to those that affect membrane structure and those that affect membrane transport.

HEREDITARY SPHEROCYTOSIS

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

True

True of False: HEREDITARY SPHEROCYTOSIS is characterized by spherocytic red cells, splenomegaly, and familial occurrence (in 75% of families, it is inherited in autosomal dominant manner, although in 15%-30% of cases, neither parent is affected - autosomal recessive)

ANK1

- which codes for ankyrin-1

- most cases in the US and Europe; only 5% 10% of cases in Japan

SPTA1

- which codes for ɑ-spectrin

- fewer than 5% of cases -> rare but associated with a severe form of HS

SPTB

- which codes for β-spectrin (15%-30%)

EPB42

- which codes for protein 4.2

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

transmembrane

Mutations of genes for _______ proteins can also occur, including the SLC4A1 gene which codes for band 3 (20%-35% of cases)

False. NO mutation is identifed

True or False: In approximately 10% of patients, mutation is identified.

"vertical defect"

The defect in HS is referred to as a __________ because the association between the lipid bilayer and the red cell cytoskeleton is faulty.

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.

poorly deformable spherocytes

Small portions of the membrane form vesicles, and the vesicles are gradually released resulting in the gradual loss of surface area and the eventual formation of _________ which are easily destroyed in the spleen (extravascular hemolysis).

True

True or False: As spherocytes attempt to move through the narrow sinusoids and fenestrations of the endothelial cells lining splenic sinusoids, they acquire further membrane loss or become trapped in the sinusoids, and are rapidly removed by splenic macrophages

sodium and potassium

In HS, the erythrocytes are also abnormally permeable to cations, especially _____ and _____ which is likely due to disruption of the integrity of the protein cytoskeleton but the exact mechanism is not clear.

False. it is INCREASED

True or False: No defect in energy metabolism, in fact, it is decreased (compensate for membrane defect) and can give rise to overactive Na+-K+ ATPase pumps which cause a reduction in intracellular cations and water diffusing out of the cell, leading to cellular dehydration.

anemia, jaundice, and splenomegaly

Three (3) key clinical manifestations

Silent carriers

are clinically asymptomatic with normal laboratory findings.

bilirubinemia

Often a chronic extravascular hemolytic process (hemolytic anemia), with indirect __________.

splenectomy

Hemolysis varies in severity and is corrected by ______ (surgical removal of the spleen) but the spherocytosis remains.

True

True of False: Most patients have a well-compensated hemolytic anemia and are rarely symptomatic although complications may occur: hemolytic, aplastic, and megaloblastic crises

cholelithiasis

Half of patients, even those with mild disease also experience _______ (bilirubin gallbladder stones) due to the chronic hemolysis.

MILD HS

- about 20-30% HS cases

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

MODERATE HS

- about 60%, more common phenotype

- Incompletely compensated hemolytic anemia, causing low Hgb levels.

SEVERE HS

- 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)

HEMOLYTIC CRISES

Often arises after viral infections

APLASTIC CRISES

Dramatic decrease in Hgb level and reticulocyte count

parvovirus B19

Usually occurs when a patient is concomitantly infected with _________, which suppresses erythropoiesis, and the bone marrow is unable to compensate for the hemolytic anemia (complication).

MEGALOBLASTIC CRISES

Commonly occurs in pregnant HS patients

MEGALOBLASTIC CRISES

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

Mild HS

Determine whether it is Mild, Moderate, or Severe HS:

- Normal Hgb

- Slightly increased bilirubin

- Slight reticulocytosis

- Few spherocytes

Moderate HS

Determine whether it is Mild, Moderate, or Severe HS:

- Low Hgb

- Increased bilirubin (>2 mg/dL)

- 6%-10% reticulocytosis

- More spherocytes

Severe HS

Determine whether it is Mild, Moderate, or Severe HS:

- Low Hgb (often <6 mg/dL)

- Increased bilirubin (>3 mg/dL)

- >10% reticulocytosis

- More spherocytes

Spherocytes

Hallmark of HS: _____ on peripheral blood films

False. they are NOT specific for HS

True or False: Spherocytes are small, round, dense RBCs lacking a central area of pallor and are specific for HS

>4%

Cutoff of ____% of the total RBCs (hyperchromic, spherocytic cells) has been proposed to screen for HS

poikilocytosis

Other peripheral blood findings include normal-appearing RBCs, polychromasia (as expected in hemolytic anemias), and varying degrees of anisocytosis and _________.

True

True or False: Because of the spherocytosis, there is an increase in mean cell hemoglobin concentration (MCHC), as well as an increase in the red cell distribution width (RDW).

False. it is NOT required

True or False: Bone marrow analysis is required for the diagnosis of HS.

OFT (Osmotic Fragility Test)

_______ has been used as a supporting test.

NaCl

Uresh, heparinized blood is mixed with ____ solutions ranging from 0.85% to 0.0%.

True

True or False: In 0.85% NaCl, the amount of water entering the red blood cell is equivalent to the water leaving the cell. At this solution, the intracellular and the extracellular osmolarity is the same and there is equilibrium.

hypotonic solutions

RBCs are suspended in a series of tubes containing increasingly _________ of NaCl, varying from 0.9% to 0.0%, incubated at room temperature for 30 minutes then centrifuged.

spectrophotometric

Percent hemolysis in the supernatant solutions is measured by _________ methods (absorbance of the supernatant at 540 nm) and an osmotic fragility curve is drawn.

equilibrium

In each tube, water enters and leaves the RBCs until ______ between the intracellular and extracellular compartments is achieved

hypotonic

In a _____ solution -> more water will enter the cell to dilute the intracellular contents until equilibrium is reached. As this phenomenon occurs, the cells will swell (cell swelling).

True

True or False: As the RBCs are subject to increasingly hypotonic solutions, swelling intensifies (more water will keep entering the RBCs) until the cell bursts (lysis).

decreased

The principle stems from the observation that RBCs that are more spherical (spherocytes), with a _____ 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.

Spherocytes

are thus said to have increased osmotic fragility.

False: Normal biconcave RBCs show initial hemolysis at 0.45% NaCl

True or False: Normal biconcave RBCs show initial hemolysis at 0.54% NaCl and complete hemolysis generally occurs between 0.35% and 0.30% NaCl.

True

True or False: Cells that are hypochromic and flatter have a greater capacity to expand in hypotonic solutions, lyse at lower concentrations of NaCl than normal cells - cells with increased surface/volume ratio are osmotic resistant. These cells are seen in iron deficiency, thalassemia, liver disease, and reticulocytosis.

spherocytes

In HS, _____ have decreased surface/volume ratio thus they have a lesser capacity to expand and swell in hypotonic solutions.

False: they tend to lyse at LOWER (less hypotonic) solutions

True or False: spherocytes in HS tend to lyse at HIGHER (more hypotonic) solutions of NaCl and are said to have INCREASED osmotic fragility.

Osmotic fragility

is usually increased in HS but it may be normal in mildly affected patients.

Increased osmotic fragility

is characteristic but it is not specific for HS; it also lacks sensitivity.

True

True or False: The OFT does not differentiate between the spherocytes in hereditary spherocytosis and the spherocytes caused by other conditions (e.g. burns, other immune-mediated hemolytic anemias, other acquired disorders, etc.) It just tells that there are cells in the blood that have decreased surface to volume ratio (it is not a confirmatory/definitive test)

remain

After splenectomy, osmotic fragility improves but the spherocytes ______.

INCUBATED OSMOTIC FRAGILITY TEST

A variation of OFT

37°C for 24 hours

INCUBATED OSMOTIC FRAGILITY TEST Entails incubating the blood at ___°C for ___ hours before performing the OFT allows HS cells to become more spherical

mild

INCUBATED OSMOTIC FRAGILITY TEST is often needed to detect ____ cases of HS (when a low number of spherocytes are seen relative to the total RBC population).

Normal Blood

OFT performed on _______________

- Hemolysis (pinkish to red discoloration of the supernatant fluid) starts at 0.45% NaCl concentration and completes at 0.30% NaCl concentration.

Hereditary Spherocytosis

OFT performed on _______________

- Spherocytes do not have much room to swell up.

- Hemolysis at less hypotonic solution/lyses early

AUTOHEMOLYSIS TEST

Another test that supports the diagnosis of HS

37°C for 48 hours

Sterile, defibrinated blood is incubated at ____°C for ____ hours, then the RBCs undergo a complex series of changes, lose membrane, and become more spherocytic.

False. without added glucose is 0.2% to 2.0%

True or False: Normally, the amount of autohemolysis at 48 hours without added glucose is 0.3% to 2.0% and 0% to 0.9% if with added glucose.

autohemolysis

In HS, _________ is virtually always increased (10%-50%) and with glucose, the lysis is diminished to a variable extent but not to the reference interval.

ATP

If glucose is added, it provides the ____ 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%)

flow cytometry

EMA is a fluorescent dye that binds to transmembrane proteins band 3, Rh, RhAg, and CD47 in the RBC membrane; its fluorescence can be detected by _____________________

False. they show a LOWER fluorescence intensity

True or False: Specimens from HS patients show a higher 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). What test is this?

no treatment

MILD HEREDITARY SPHEROCYTOSIS usually requires ____________

False. its reserved for MODERATE TO SEVERE cases.

True or False: Splenectomy (surgical removal of the spleen) is only reserved for severe cases.

encapsulated bacteria

Major drawback is the lifelong risk of sepsis and even death secondary to _______________

True

True or False: Prior to splenectomy, patients should be vaccinated.

False: DISCOURAGED because of the risks for sepsis and disseminated infection.

True or False: Splenetomy is encouraged in children.

longer RBC survival

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

SEVERE CASES OF HEREDITARY SPHEROCYTOSIS

Usually require regular transfusions to accommodate the anemia

autosomal dominant

HEREDITARY ELLIPTOCYTOSIS IS Mainly an __________________ condition.

HEREDITARY ELLIPTOCYTOSIS

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

HEREDITARY ELLIPTOCYTOSIS

malaria

is more common in Africa and in Mediterranean regions, where there is a high prevalence of _______

"horizontal defect"

All cases of HE are associated with defects in proteins that disrupt the horizontal or lateral interactions in the protein cytoskeleton, known as the _____________________ resulting in weakening of the membrane skeleton.

cell fragmentation

RBCs are biconcave and discoid at first but become elliptical over time after repeated exposure to shear stress in the circulation, eventually __________________ can occur leading to membrane loss and a decreased surface area-to-volume ratio.

SPTA1

The HE phenotype can result from various mutations in at least 3 genes. For this one:

- codes for ɑ-spectrin

- accounts for 65% of cases