Hematology unit 2.2 chp 17 and 18

vertical interaction?

interactions between skeletal lattice and its attachment to integral protein and lipids

role of vertical interactions

stabilize the lipid bilayer membrane

vertical interaction defects

selective loss of portions of the lipid bilayer; net loss of cell membrane; decrease in surface-area-to-volume ratio; formation of spherocytes; hemolysis of RBCs

horizontal interaction

provides mechanical stability; spectrin heterodimer head-to-head association; forms tetramers and skeletal protein interactions

horizontal interaction defects

disruption of the skeletal lattice; membrane destabilization; cell fragmentation; formation of poikilocytes

causes of vertical defect

ankyrin, band 3, protein 4.2, a-spectrin, b-spectrin

causes of horizontal defect

protein 4. 1R; glycophorin C, a-spcrtin, b-spectrin, actin

lipid compositions abnormalities

defection that affect the composition of the membrane lipid bilayer

what types of RBC morphologies show lipid composition abnormalities?

acanthocytes or stomatocytes

normal RBC membrane lipid composition amount

equal amounts of free cholesterol and phospholipid

what happens if there is excess free plasma cholesterol?

accumulates in the outer bilayer, expansion of out face as compared to the inner

-form acanthocytes

hereditary spherocytosis

RBCs deficient through abnormally permeable to monovalent cation and problems with deformability and permeability

Hemolysis of hereditary spherocytosis

mild to moderate hemolysis

hereditary spherocytosis defects in spectrin and ankyrin

weakening of vertical connections; uncoupling between inner membrane skeleton and outer lipid bilayer; shedding of lipid bilayer, forms microvesicles

hereditary spherocytosis membrane loss

decrease in surface-area-to-volume-ratio; changes shaped from discoyte to spherocyte; increase in cytoplasmic viscosity; reduced cellular flexibility

hereditary spherocytosis other membrane abnormalities

total lipids in membrane are decrease; abnormally permeable to Na+, compensatory increase in catio pump

destruction of hereditary spherocytosis cells in spleen

trapped in splenic cords (lacks flexibility), hypoglycemia, acidic, hypoxic environment

hereditary spherocytosis clinical symptoms

jaundice, splenomegaly, bile stones, aplastic crisis associated with viral infection

how hereditary spherocytosis have variety in clinical severity?

~25% compensated hemolytic disease; or can be homozygous for HS, and most HS patient exhibit partial compensated HA but appear asymptomatic

RBC lab eval of HS

Hb levels normal or decrease, reticulocyte >8%, RBC smear: spherocytes not alway prominent, young RBCs are normal, polychromasia

MCV 77-87

MCH normal

MCHC >36

other markers of hemolysis by HS

increase in serum bili, decrease haptoglobin, increase in LD, increase in urine and fecal urobilinogen

osmotic fragility

measures RBC’s resistance to osmotic stress

-screening test for HS

methodology of osmotic fragility

RBC incubated in varying conc. of NaCL. spherocytes can’t expand as normal RBCs,

osmotic fragility requirements

spherocytes must constitute >1-2% and incubation at 37 C for 24 hrs

autohemolysis test

can differentiate congenital, nonspherocytic hemolytic anemia,

autohemolysis test methodology

blood incubated at 37 C; measures degree of spontaneous hemolysis, depends on membrane integrity

HS= 5-25% at 24 hours

antihuman globulin test

used in differentiating HS from immune hemolytic anemia; detects antibodies or complement bound RBCs in vivo

-DAT negative in HS, positive in immune HA

Hereditary elllipotocytosis (HE)

autosomal dominant inheritance; elliptocytes are prominent peripheral blood findings; heterogenous in degree of hemolysis and clinical severity

classification of hereditary ellipotcytosis

based on RBC morphology; can be common HE, spherocytic HE, or stomatocytic HE

spherocytic HE

hemolytic ovalocytosis

stomatocytic HE

melanesian ovalocytosis, southeast Asian ovalocytosis, autosomal recessive inheritance

erythrocyte shape of common HE

biconcave elliptocytes, variable hemolysis

erythrocyte shape of spherocytic HE

spherocytes and fat elliptocytes, present hemolysis

erythrocyte shape of southeast asian ovalocytosis

roundish elliptocytes that are also stomatocytic, mild or absent hemoysis

how is HE’s shape abnormal?

defect in one of the skeletal proteins, elliptocytic shape acquired in circulation, RBCs subjected to shear stress as circulate and acquire elliptical shape

what is the lifespan of HE?

normal lifespan

HE elliptocytosis with membrane fragementation

decrease in cell surface area, reduced cell deformability, RBC life span severely shortened

what is the effect of HE having abnormally permeable to cations?

requires increase of ATP to run cation pump to maintain osmotic equilibrium

SAO variant of HE

abnormal band 3 protein, rigid RBCs

what is the variant of common HE in black infants?

moderately severe anemia and jaundice, peripheral blood blood has budding and fragile bizarre poik,

clinical presentation of spherocytic HE

rare with presence of hemolysis, minimal changes in RBC morph: both spherocytes and elliptocytes

clinical presentation of southeast asian variant of HE

stomatocytic HE; mild or absent hemolysis, increase in RBC cation permeability, expression of blood group antigens is muted, HE cells are resistant to malaria parasites

what does the blood smear look like in HE?

microelliptocytes, bizarre poik, schistocytes, spherocytes; reticulocyte is high

HE osmotic fragility and autohemolysis tests

unincubated and incubated abnormally increase, no need to perform

hereditary pyropoikilocytosis

Rare, autosomal recessive disorder, severe subtype of HE,

when is HPP prominent?

Primarily in blacks; Infancy or early childhood have Severe hemolytic anemia and Extreme poikilocytosis

HPP thermal stability

fragment at 45-46 C, disintegrate when incubated at 37 C > 6hrs

HPP clinical presentation

present at birth: hemolytic anemia, hyperbilirubinemia, serologic studies for HDB are neg

therapy for HPP

splenectomy

HPP peripheral smear

Striking poikilocytes→ Budding, fragments, microspherocytes, elliptocytes, triangulocytes, bizarre forms

-decrease MCV

Osmotic fragility of HPP

Abnormal especially after incubation, increase fragmentation on Thermal sensitivity test

Hereditary Stomatocytosis Syndromes rare autosomal dominant

Hemolytic anemias, Abnormalities in cation permeability in RBC membrane

Hereditary Stomatocytosis Syndromes types of syndromes

Overhydrated hereditary stomatocytosis (OHS) and Dehydrated hereditary stomatocytosis (DHS)

Overhydrated hereditary stomatocytosis (OHS)

Abnormally permeable to Na+ and K+; Intracellular concentration of cations increase; Water enters the cells, stomatocytes

Dehydrated hereditary stomatocytosis (DHS)

Net loss of K+; Exceeds passive Na+ influx and net Na+ gain; Cell dehydrates appears

lab evaluation of HSS

anemia: mild to moderate Hb; increase in bili, moderate reticulocytosis

OHS lab evaluation

MCHC of stomatocytes↓; MCV ↑; 10–50% stomatocytes; ↑ osmotic fragility and autohemolysis

DHS lab evaulation

target cells, RBCs with Hb puddled at periphery, slight increase in MCV; increase in MCHC, decrease in osmotic fragility

therapy of hereditary stomatocytosis syndromes

splenectomy contraindicated

Acanthocytosis

Abnormalities of lipid membrane; Acquired or hereditary; Liver disease; Abetalipoproteinemia

how does the concentration of plasma lipid increase in acanthocytosis

RBCs acquire excess lipids; Expand RBC membrane → target cells, leptocytes, acanthocytes

Abetalipoproteinemia

Hereditary acanthocytosis; Rare autosomal recessive disorder, Absence of serum β-lipoprotein; Defective processing and secretion of apolipoprotein B

-increase in cholesterol/phospholipid ratio

what do acanthocytes have in abetalipoproteinemia?

decrease lecithin, increase sphingomyelin, decrease in membrane fluidity

what is hereditary acanthocytosis characterizied by?

Steatorrhea; Retinitis pigmentosa; Neurological abnormalities

what do acanthocytes exhibit in hereditary acanthocytosis

Normal permeability, glucose, osmotic fragility; increase in autohemolysis at 48 hrs; Minimal hemolysis, little or no anemia; Normal to slight increase in reticulocyte count

Paroxysmal Nocturnal Hemoglobinuria (PNH)

Rare acquired disorder of RBC membrane; RBC abnormally sensitive to lysis by complement'; Intermittent bouts of intravascular hemolysis is classic pattern; Nocturnal hemoglobinuria

Acquired stem cell somatic mutation of PNH

Abnormal clone of differentiated hematopoietic cells; RBCs, platelets, neutrophils; Bind abnormally large amounts of complement; Abnormally sensitive to complement lysis

GPI-anchoring deficiency

Somatic mutation of the PIG-A gene; Mutated progenitor cell has a proliferative advantage; Develops after damage to BM or idiopathic

PNH clinical presentation

occurs most often in adults; acute intravascular hemolysis; hemoglobinuria often associated with sleep, venous thrombosis is a common cause of death; infection wiht leukopenia present

disease manifestations of PNH

hyperhemolysis, venous thrombosis, BM hypoplasia

lab evaluation of PNH

severe anemia; Hb 8-10; RBCs are normocytic or macrocytic, and microcytic if IDA develops, hemosiderinuria,

Immunophenotyping detects what in PNH

CD55, CD59, CD14 or type I, type II, type III

Fluorescent–labeled inactive toxin aerolysin (flaer) test for PNH

Toxins binds directly to GPI anchor and Direct measurement of GPI-deficient cells

Inherited RBC enzyme deficiency

Compromise integrity of cell membrane or Hb and Hemolysis

Two most common enzyme defects

1Glucose-6-phosphate dehydrogenase (G6PD) and Pyruvate kinase (PK)

Glucose-6-phosphate dehydrogenase (G6PD)

Affects hexose monophosphate shunt and found more frequently

Pyruvate kinase (PK)

Affects glycolytic pathway (Embden-Meyerhof) and Second most common

Hexose Monophosphate Shunt job of catabolizing glucose

Maintains adequate levels of reduced glutathione (GSH) by conversion of NADPH into NADP and NADP is reduced back to NADPH by G6PD

Hexose monophosphate shunt job of GSH

Protects RBC from oxidant damage; Maintains HB in the reduced functional state; Preserves vital cellular enzymes

Enzyme deficiencies in HMP shunt

Oxidation of hemoglobin; Formation of Heinz bodies; Spleen removes Heinz bodies; Extravascular hemolysis

Glycolytic Pathway

Maintains adequate levels of ATP, needed for Active cation transport across the cell membrane, Maintains membrane deformability, and Maintains RBCs’ biconcave shape

glycolytic pathway deficiencies in enzymes

Decreased ATP and impaired cation pumping and increase in osmotic fragility, hemolysis

Rapoport-Luebering shunt

Provides RBC with 2,3-bisphosphoglycerate (2,3-BPG) and Stimulated during hypoxia to facilitate O₂ delivery to tissues

-released by O2 to tissues

diagnosis of RBC enzyme deficiencies

Diagnostic workup suggested by: Absence of a detectable abnormal Hb, A negative direct antiglobulin test, Lack of spherocytes and Normal erythrocyte fragility test

G6PD facts?

• Primarily Mediterranean area, Africa, and China

• Sex-linked recessive inheritance—fully expressed in males and females with homozygous inheritance

• Varying levels of severity

• Generally asymptomatic except when challenged with oxidizing chemical, drug, or severe infection

G6PD deficiency pathophysiology

Generation of NADPH impaired and Generation of GSH impaired → cellular oxidants accumulate → Hb has decreases solubility → precipitate to form Heinz bodies

what happens in G6PD deficiency when Heinz bodies attach to the RBC membrane?

Cause increased cation permeability, osmotic fragility, cell rigidity; Removal by splenic macrophages producing “bite” cells and blister cells; progressive membrane loss

Oxidant stress in G6PD

Oxidizes membrane lipids and proteins and membrane damage causing RBCs to be removed by spleen or cells can hemolyze in circulation

Reticulocytes have 5× higher ___ than oldest circulating RBCs

enzyme activity

what happens in G6PD when there is severe oxidant stress?

Overwhelm the system → Hemolysis is generally self-limited and Older stressed cells are hemolyzed →Replaced by younger cells with higher GSH levels

Female heterozygotes with G6PD deficiency

One normal and One G6PD deficient population of cells; random inactivation of one X chromosome in each cell

G6PD deficiency clinical presentation for acute, acquired hemolytic anemia

Most have no clinical symptoms, no anemia; Hemolytic episodes occur after Infectious illness or Exposure to certain drugs

what does the hemolysis depend on in G6PD deficiency clinical presentation for acute, acquired hemolytic anemia?

degree of oxidant stress, G6PD variant, sex of patient

clinical presentation of drug induced G6PD deficiency clinical presentation

Acute intravascular hemolysis; 1–3 days after exposure; 3–4 g/d L drop in Hb; Abdominal and lower back pain; Dark or black urine

G6PD deficiency ingestion of fava beans

Sudden severe hemolytic episode (favism); usually affect children 2-5 years old

G6PD deficiency presentation of symptoms

Malaise, severe lethargy, nausea, vomiting, abdominal pain, chills, tremor, fever; Hemoglobinuria a few hrs after ingestion; Jaundice

Hereditary (chronic) nonspherocytic HA

Associated with G6PD variants; Low in vitro activity or are markedly unstable; chronic hemolysis, hemolysis is usually compensated, increased reticulocytosis

Neonatal hyperbilirubinemia

Some neonates with G6PD deficiency that have severe hyperbilirubinemia,

G6PD deficiency laboratory eval immediately following hemolytic episode

Peripheral blood has Polychromasia, occasional spherocytes, small hypochromic cells, RBC fragments, bite cells, Blister cells, Reticulocytosis; increase leukocytes,

increase in unconjugated bili and LD; decrease haptoglobin

definitive diagnosis of G6PD

Requires demonstration of a ↓ in erythrocytic G6PD activity and perform assay 2-3 months after hemolytic episode