Hemolysis Flashcards
Describe the red cell membrane
- Allows 4x increase in hemoglobin concentration
- Protects hemoglobin from oxidation
- Allows modulation of oxygen affinity by 2,3-BPG
Composition
Lipids
• Asymmetric bilayer: choline phospholipids in outer half; aminophospholipids in inner half
• Also contains cholesterol and glycoplipids
Membrane proteins
Integral proteins
• Receptor and antigen-bearing proteins (glycophorin A)
• Transport proteins (Na/K ATPase)
• Anion transport protein (for Cl- and HCO3-)
• Channel proteins (glucose diffusion)
• Glycolipid anchored proteins (protect from lysis)
Structural proteins
• On inner surface of membrane
• Include: spectrin, actin, ankyrin, some enzymes
• Spectrin-actin network = supports and stabilizes lipid bilayer
o Maintains shape and flexibility
Define hemolysis and list the three mechanisms by which it may occur.
Definition: shortened RBC survival (<100 days)
3 mechanisms:
Loss of membrane deformability:
• Viscoelastic properties of membrane (dependent on spectrin-actin cytoskeleton)
• Surface-to-volume ratio of cell
• Mean corpuscular hemoglobin concentration of RBC
• Increased hemoglobin → less flexible
• Physical state of hemoglobin in the cell
• Less flexible with polymerized/precipitated hemoglobin
• Ex: sickle cell anemia, G6PD deficiency, thalassemia
Phagocytosis of antibody- and/or complement-coated RBC by macrophages
• Occurs in liver and spleen
• Extravascular hemolysis
Disruption of membrane integrity
• Intravascular hemolysis
Occurs when:
• Hemolytic complement (C5-C9) fixed to membrane (ex: ABO mismatched transfusion reactions) → forms hole → bursts RBC
• Oxidized membrane sulfhydryl groups (ex: G6PD deficiency)
• Parasite invasion (ex: malaria)
• Fibrin cuts RBC (ex: microangiopathic hemolytic anemia; abnormal heart valves)
Describe normal RBC breakdown
Progressive decrease in enzyme activity as RBC ages
• Decreased ATP → Ca2+ concentration rises
• Fragmentation and loss of membrane → spherocytosis
• Decreased deformability
• Entrapment by spleen and liver macrophages
As RBC ages, membrane sialoglycoproteins are altered
• IgG autoantibody attaches to altered proteins
• With enough coating → macrophages able to recognize and phagocytose
After macrophage engulfment:
• Hemoglobin broken into globin, iron and protoporphyrin
• Globin = degraded, returned to amino acid pool
• Iron = conserved
Protoporphyrin converted to bilirubin
o Diffuses out of macrophage
o Complexes with albumin = “indirect” or “unconjugated” bilirubin
o Transported to liver
o Conjugated with glucuronide (via glucuronyl transferase) = “direct” or “conjugated” bilirubin
o Excreted in bile
o Overall: produces carbon monoxide (released in lungs)
Be able to distinguish extravascular and intravascular hemolysis using laboratory tests, and to use this information to help determine the cause of hemolysis.
Extravascular hemolysis
o RBC’s processed by spleen and liver
o Iron is conserved
o If liver’s capacity to conjugate increased bilirubin exceeded → increased serum levels of unconjugated (indirect) bilirubin
Intravascular hemolysis
Haptoglobin = plasma protein that binds free hemoglobin
• Forms complex → cleared by hepatocytes
• With intravascular hemolysis = depleted = levels with be low/absent
Remaining intravascular hemoglobin: Some = oxidized to methemoglobin • Excreted in urine Rest = filtered in urine Some reabsorption by tubular cells: o Globin degraded to amino acids o Protoporphyrin converted to bilirubin Most iron = stays in tubular cell (ferritin or hemosiderin) o Lost when cell exfoliated into urine Indicators of intravascular hemolysis: o Hemosiderinuria o Free hemoglobin in plasma and/or urine
Describe the role of the spleen in hemolysis.
• Spleen = hypoxic, acidotic and hypoglycemic
o RBC less able to generate ATP → swells = spherocytic
• Even mildly deformed RBCs = trapped and removed by splenic macrophages
• Spleen is the body’s “fine filter”
Be able to use laboratory test results and information from the blood smear and bone marrow biopsy to determine whether hemolysis is present.
Marrow response to hemolysis:
Within 2 days = erythroid hyperplasia
Within 1 week = brisk reticulocytosis
5x (or greater) increase in RBC production
• Due to greater iron availability (extravascular hemolysis)
• Compensates for hemolysis
Expanded marrow space
• In axial skeleton, long bones, skull
Extramedullary hematopoiesis in chronic severe hemolysis
• In liver and spleen (because produced RBCs in utero)
Diagnosis of hemolysis
Peripheral smear:
• Shift cells, spherocytes, elliptocytes, spur cells, target cells, sickled cells, schistocytes
Reticulocytosis (corrected absolute retics>150,000)
Products of RBC destruction:
• LDH (intravascular > extravascular)
• Bilirubin (unconjugated)
• Low haptoglobin (intravascular > extravascular)
Hemoglobinuria, hemoglobinemia (acute intravascular)
Urine hemosiderin (intravascular)
Describe the complications of hemolysis.
Anemia
o If RBC lifespan < 20 days
Aplastic crisis
o Associated with Parvovirus B19 infection
o Self-limited lasting about 1 week
o Requires transfusions until RBC production resumes
Folate deficiency
o Worsening anemia, decreased reticulocyte counts, hypersegmented neutrophils and macrocytes, megaloblastic changes in marrow
Skeletal abnormalities
o In children → “chipmunk” appearance (forehead bossing, broad cheek bones, protruding maxilla)
o On skull x-ray = “hair on end” appearance
Kernicterus (newborns)
o From unconjugated bilirubin → CNS = affects basal ganglia → seizures, mental impairment
Gallstones
o Black, bilirubin-containing stones
Iron overload
o Due to conservation of iron in extravascular hemolysis, increased absorption, and transfusions
o Treat with Fe chelator
Vascular/endothelial injury (intravascular hemolysis)
o From free hemoglobin and RBC membrane fragments:
• Activate clotting cascade → DIC
• Activate complement (C3a, C5a) → release of vasoactive amines → renal vasoconstriction, shock, death
Be able to diagnose hereditary spherocytosis using clinical and laboratory information, and to explain its pathophysiology.
Autosomal dominant
o Most prevalent in Northern Europeans
o Associated with ankyrin gene on chromosome 8
Abnormal ankyrin → spectrin deficiency
Progressive loss of membrane → spherocyte formation
• High MCHC; low surface: volume ratio → less deformable RBC
Makes cells leaky = permeable to Na+
• Excess Na+ enters → increased Na/K ATPase activity
• Requires more glucose for energy
When enters spleen = low glucose → rapid decrease in ATP
• Na+ and water enter RBC → swelling
• Phagocytosed by macrophages
Spleen = main site of RBC destruction so splenectomy is curative
• Problem: more susceptible to encapsulated bacteria
• Try to wait until 5 years old
• Give pneumococcal vaccine
Clinical findings:
o Mild anemia, splenomegaly, jaundice
o Many = asymptomatic
o Increased risk of bilirubin gallstones and cholecystitis
o May get aplastic crises and folic acid deficiency
Lab findings:
o Spherocytes ~ 20% of cells
o Reticulocytosis
o Elevated serum indirect bilirubin
Diagnosis:
o Osmotic fragility test = measures surface to volume ratio of cells
o Determines how much water cells can accommodate before bursting
• Spherocytes can accumulate less water than biconcave disc
• Begin to lyse around 0.65%; normal cells around 0.5% saline
Microangiopathic hemolytic anemia
Membrane disorder
o Fibrin strands or platelet clots strung across vessels or severe endothelial damage
o Cause RBCs to fragment
Can be seen in Trombotic Thrombocytopenic Purpura (TTP)
o Life-threatening
o Platelet thrombi occlude microvasculature
o Result: renal failure, fluctuating neurologic signs (seizures, paresthesias, coma), fever, thrombocytopenia, microangiopathic hemolytic anemia
o In children = Hemolytic uremic syndrome (HUS)
• Similar to TTP but neurologic disease is rare, usually not fatal, often follows viral upper respiratory infection or Shigella or E. coli gastroenteritis
Found in other conditions:
o DIC, vasculitis, malignant HT, disseminated carcinoma, pregnancy complications (pre-eclampsia, abruption placenta); march hemoglobinuria with running
Lab findings: o Schistocytes (RBC fragments) o Intravascular = hemosiderinuria, negative iron balance
Describe the biochemical pathways by which the red cell generates energy and maintains membrane integrity.
Overview: glycolytic pathway and pentose phosphate pathway:
Glycolytic pathway:
Glucose generates ATP, 2,3-BPG, NADPH
ATP: Powers Na/K pump to maintain RBC membrane
2,3-BPG
• Produced via the Rapaport-Leubering shunt
• 1,3-DPG forms 2,3-BPG
• Role in regulating Oxygen dissociation curve
• No net ATP synthesis
NADPH
• Reduces methemoglobin (Fe3+) back to hemoglobin (Fe2+)
• Because methemoglobin can’t bind and deliver oxygen
• Donates electron via methemoglobin reductase
Pentose phosphate pathway
Function: produce NADPH to protect from oxygen radical damage
Glucose-6-phosphate → (via glucose-6-phosphate dehydrogenase) 6-phosphogluconate
• Reduces NADP to NADPH
• NADPH = electron donor to reduce oxidized glutathione (GSSG to GSH)
• GSH = able to prevent oxidant denaturation
Be able to diagnose glucose 6-phosphatase deficiency based on clinical and laboratory data, and to describe the genetics and pathophysiology of this condition.
X linked inheritance
o Can get manifestations in females if random inactivation is skewed toward defective gene
Higher incidence in Africa, Mediterranean basin, and Middle East
o May offer some malaria protection
Many different variants:
G6PD B = normal wild type
• Half life of 60 days
G6PD A = normally functioning variant found in 20-40% of African descendants
G6PD A- = oxidant-stress induced
• Mild severity
• Half-life of 13 days
• When stressed = older cells hemolyze; younger cells and reticulocytes survive
• More common in African-Americans
G6PH Mediterranean = oxidant stress induced
• Severe form
• Half-life = hours
• With stress → severe intravascular hemolysis, anemia, hemoglobinuria, jaundice
Drug-induced hemolysis:
o In patients with G6PD A- or G6PD Mediterranean
• Sulfamethoxizole
• Nitrofurantoin (antibiotic for UTI’s)
• Primaquine (treat malaria or pneumocystis pneumonia)
• Dapsone (treat leprosy and some skin diseases)
• Doxorubricin (cancer chemotherapy)
Lab findings:
o Precipitated globin chains = Heinz bodies
Diagnosis:
o Able to measure G6PH activity
Describe the effects of pH, CO2, and 2,3 DPG on the oxygen affinity of hemoglobin, and the physiologic significance of each effect.
• Normal P50 ~27 mmHg
Right shift = higher P50; reduced oxygen affinity (more oxygen released)
o Decreased pH
o CO2
o Increased temperature
o 2,3-DPG (decreases hemoglobin-oxygen affinity)
• Stimulated by anemia or hypoxia (lung disease, heart failure, high altitude)
• Does not bind gamma chains of fetal hemoglobin = produces left shift = allows fetal blood to bind more oxygen in placenta
Describe the effects of carbon monoxide on hemoglobin function and oxygen delivery.
Carbon monoxide + hemoglobin → carboxyhemoglobin
o Greater exposure from smoking or incompletely burned carbon or hydrocarbon fuels
Signs:
o Bright red color to blood
o Bright, cherry red skin and mucous membranes
Physiology:
o Higher affinity of hemoglobin for CO than oxygen → Displaces oxygen from hemoglobin → Disrupts oxygen transport
CO shifts dissociation curve to left:
• Lowers release of oxygen to tissues
• Result: better able to tolerate reduction in blood oxygen capacity due to anemia than CO
