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Hematology Pathophysiology: Hemoglobinopathies (Sarnaik + Pathoma) NORMAL HEMOGLOBIN: Structure: Tetrameric protein with 4 peptide chains o HbA= 2 alpha + 2 beta chains o Molecule held together by interactions between the chains Each Hb chain has a heme moiety (carries O2) o Heme bound to globin chains by covalent bonds o Iron in Fe2+ form (changes to Fe3+ form results in inability to carry O2) Alpha1 beta2 interface is an important region for the unique O2 carrying capacity of Hb o Abnormalities in this region will alter O2 affinity Areas where chains are in contact with eachother OR the heme molecules are functionally important o Have been highly conserved throughout evolution Summary: o Primary structure (sequence of amino acids) o Secondary structure (folding into helix) o Tertiary structure (binding of heme into heme pocket of helix) o Quaternary structure (binding of four globin chains into a tetramer) Function: Function is oxygen transport and delivery to tissues Relationship between O2 saturation and O2 tension is a sigmoid (S-shaped) curve o Allows tissues to extract ~25% of O2 brought to it O2 tension in venous system is 40mm (PO2 in venous system=40) O2 saturation after unloading to tissues is 75% o If the relationship were not this way, the Hb would only be able to unload O2 if the PO2 were to fall very low Important Values: o P50: oxygen tension (PO2) at which Hb is 50% saturated (measure of the AFFINITY of Hb for O2) Normal value is 26torr (under physiologic conditions) Lower P50= higher affinity (LEFT-SHIFT) Higher P50= lower affinity (RIGHT SHIFT) Lower pH Higher pCO2 (Bohr effect) Higher temperature Higher 2,3-BPDG levels Genetics: Synthesis of Hb control by genes that are switched on and off at certain stages of human life o Results in different globin chain synthesis at different times Alpha chain genes located on cs 16 (4 genes total) o Alpha synthesized at high levels throughout prenatal and post natal periods Beta chain and beta-like chain genes (delta, gamma) located on cs 11 (2 genes each) o Beta chains begin to be synthesized ~9th week of gestation and continue to rise after birth HbA detected around this time o Gamma chains synthesized at high level in prenatal period and decline after birth HbF declines but persists until ~9 months of age (switch to beta is complete) Therefore, B abnormalities do not present at birth Small amount of HbF present in adults (<1%) Detected by exposing RBCs to acid (HbF resistant to acid) o Delta chains made at low level both before birth and after HbA2 normally present at 1-3% of total Hb Diagnostically elevated in beta thalassemias* Types of Hb: HbA: alpha2beta2 (96%) HbA2: alpha2delta2 (3%) HbF: alpha2gamma2 (1%) ABNORMAL HEMOGLOBIN SYNTHESIS: Production of STRUCTURALLY Abnormal Globin Chains: HbS, HbC etc. Recall that these are INTRINSIC RBC defects that result in hemolytic anemia (normocytic anemia) Production of Structurally Normal but DECREASED Amounts of Globin Chains: thalassemias Recall that these result in DECREASED Hb and MICROCYTIC anemia Failure to Switch Globin Chain Synthesis: hereditary persistence of fetal Hb THALASSEMIAS: Definition: a genetic decrease in globin chain synthesis Genetics: caused by mutations in globin gene clusters Various defects Deletional (usually alpha thalassemias) and non-deletional mutations (usually beta thalassemias) Alpha Thalassemias: Basics: most commonly DELETIONAL defects, although other mutations have been described Types: o Single Gene Deletion: silent carrier state o Two Gene Deletion: mild microcytic, hypochromic anemia (similar to iron deficiency) o Three Gene Deletion (HbH Disease): moderate microcytic, hypochromic anemia Also hepatosplenomegaly due to extramedullary hematopoiesis HbH results from tetramers of beta globin chains that form since they cannot combine with alpha chain Hb Barts can be detected in first few weeks of life with 3 gene deletions as well o Four Gene Deletion (Hydrops Fetalis): incompatible with life, resulting in intrauterine death or hydrops fetalis HbBarts due to tetramers of gamma chains Diagnosis: o CBC: microcytosis and hypochromia with anemia (2 and 3 gene mutations) o Demonstration of decreased alpha chains: globin chain synthesis measure in reticulcytes Expensive and difficult test to perform o Abnormal Hb Tetramers: demonstrated by cresyl blue stain and Hb electrophoresis (migrate faster than HbA) Hb Barts: gamma chain tetramers (prenatal or infancy) HbH: beta chain tetramers (in adults) o Routine Hb Electrophoresis: important to note that while this is useful for detecting tetramers in 3 and 4 gene deletions, it is not useful for 1 and 2 gene deletions Why? All Hb types are decreased proportionately o Restriction Mapping: reserved for prenatal diagnosis Blood Smear: o Microcytic, hypochromic RBCs o Target cells o Heinz bodies (beta chain tetramers- HbH) Beta Thalassemias: Types: o Beta Thalassemia Trait/Thalassemia Minor (B/B0): one normal beta gene (not a disease) HbA slightly decreased (90%) HbA2 diagnostically elevated (>4%) HbF may be slightly elevated (2-5%) o Beta Thalassemia Intermedia (B+/B+ or B+/B0): miler disease because beta globin synthesis is NOT completely suppressed o Thalassemia Major (B0/B0): completely suppressed beta globin synthesis HbA VERY low (<20%) HbA2 diagnostically elevated (>4%) HbF increased (70-80%; age-related) Pathophysiology: o Decreased beta chain synthesis leads to excess of alpha chains Some of these used up by beta-like chains to form HbF and HbA o Free alpha chains form tetramers (very insoluble) Accumulate/precipitate in RBC and cause problems resulting in cell death - - - Accumulation in cytoplasm decreased RBC deformability Accumulation in membrane increased K+ flux Accumulation in nucleus G1 cell cycle arrest Overall result of above changes: RBC lifespan very short (extravascular hemolysis due to removal by spleen) RBCs may be destroyed in bone marrow ineffective erythropoeisis o Lack of beta chains results in microcytic, hypochromic anemia Attempts to increase red cell mass (expansion of marrow cavity, extramedullary hematopoiesis) Increased iron absorption Clinical Features of Beta Thalassemia Major (B0/B0): o Severe anemia at 4-6 months of age (switch to adult Hb and beta chains occurs) Hemolytic anemia (removal of alpha tetramers by spleen) + microcytic, hypochromic anemia (decreased Hb production) o CBC: Microcytic, hypochromic anemia Evidence of compensation for hemolytic anemia (polychromasia and nucleated RBCs) Extravascular hemolysis due to removal of damaged RBCs by spleen (damage caused by alpha chain tetramers) Increased RDW and reticulocytes o Evidence of extramedullay hematopoiesis and marrow expansion: Hepatosplenomegaly Typical facies (bossing, chipmunk cheeks) Thinning of bony cortex on X-ray Osteoporosis (due to hypoparathyroidism) Hair on end appearance of skull bones Management of Beta Thalassemia Major: o Chronic Transfusions: required to avoid growth failure and other consequences of severe anemia Results in development of secondary hemochromatosis (iron overload) Chelation therapy required due to iron overload: Desferoxamine (daily infusions via battery operated pumps) Desferasirox (oral qd; hepatic and renal issues) Desferiprone (oral qd; agranulocytosis and arthralgias) Iron overload causes organ dysfunction: Liver enzyme elevation Pancreas diabetes Pituitary gland growth failure and delayed puberty Heart arrhythmias and cardiomyopathy Increased risk of infection with Yersinia due to iron toxicity and chelation therapy o Splenectomy: performed if there is an increased transfusion requirement from hypersplenism (ie. trapping transfused RBCs in the spleen) Increased risk of infections due to pneumococcus, hemophilus and meningococcus (encapsulated organisms) o Folic Acid Supplementation: due to increased requirements o Bone Marrow Transplant: when chronic transfusions and chelation are not possible Note that there is NO treatment for beta thalassemia minor: o DO NOT TREAT WITH IRON danger of iron overload STRUCTURAL HEMOGLOBINOPATHIES: Basics: Cause: result from point mutation in alpha or beta globin gene, which produces an amino acid insertion in the polypeptide chain of the Hb molecule functional abnormality of Hb Possible Results of Mutation: o No physical abnormality and no clinical problem o Increased tendency to aggregate (HbS, HbC) o Instability of Hb molecule (hemolytic anemia) o Altered O2 affinity (increased or decreased) o Decreased O2 carrying capacity (HbM) Sickling Disorders: Types: o SS (Sickle Cell Anemia): homozygous for mutant beta chain HbS No normal beta alleles No HbA o SC: double heterozygosity with HbS and HbC (mutant beta chains) No normal beta alleles No HbA o Sickle-B Thalassemia: double heterozygosity for HbS and B-thalassemia One meta gene directs synthesis of HbS One B gene is completely suppressed (SB0-thal) OR partially suppressed (SB+-thal) SB0-thal may be as severe as SS HBA2 is elevated o SO Arab or SD: double heterozygosity for HbS and HbO/D Potentially as severe as SS o SE: heterozygosity for HbS and HbE HbE causes hypochromic thalassemic phenotype and decreases severity o HbS with Hereditary Persistence of Fetal Hb: failure to switch from gamma to beta chain synthesis Patients have high HbF and very mild clinical symptoms due to its protective effects o Sickle Cell Trait: benign carrier state of HbS (10% incidence in Blacks) Vast majority of people have no symptoms Incurs resistance to malaria Can cause hematuria and loss of urine concentration capacity May also cause intravascular sickling with strenuous exercise at high altitudes OR flying at high altitudes in an unpressurized aircraft Inheritance: o All sickling disorders are autosomal CODOMINANT Since being heterozygous carries discernible clinical findings (although minor) If both parents carry one abnormal gene, then each pregnancy has a 1 in 4 risk If one parent has the disease and the other one abnormal gene, then each pregnancy has a 1 in 2 risk Pathophysiology of Sickling: o HbS: point mutation of AG leads to replacement of a glutamic acid with a valine HbS is extremely insoluble when deoxygenated Therefore, low O2 conditions, acidosis and dehydration result in sickling Polymers form in the RBC that causes the shape change to sickled form Shape change results in: Increased rigidity Loss of deformability Increased adhesiveness to endothelial cells RBC membrane damage All changes adversely affect flow properties of red cells through microvasculature vasoocclusion Results in stasis, low pO2 in tissues, high acidity (accumulation of metabolites), increased viscosity and FURTHER vaso-occlusion PMNs, platelets and the coagulation cascade also play a role in this process Hemolytic anemia results (normocytic) due to membrane damage, which decreases RBC lifespan to 15 days instead of 120 days Clinical Features: o In general, sickled cells lead to: Vaso-occlusion (pain, ACS, stroke, priapism) Chronic organ damage (spleen, liver, kidney, lung) Shortened RBC survival (chronic anemia, aplastic crisis, sepsis, CHF) o Hemolytic Anemia (Extravascular): Pallor Jaundice Increased fatigue Gallstones - - Poor growth Aplastic crisis following viral infections (can result in life-threatening decrease in Hb) o Vascular Obstruction: Cause: intravascular sickling Result: episodic musculoskeletal pain (can be frequent and severe) Although termed “pain crises”, they are usually uncomplicated and NOT lifethreatening Typical Areas of Obstruction: Bone pain/infarction: very common at all ages o Dactylitis: swelling of the hands and feet due to bone infarction (early childhood symptom) o Osteonecrosis: of spine or femoral heads (often seen in adults; cause of chronic pain) Spleen: o Splenic Sequestration Crisis: sudden pooling of blood in the spleen with hypovolemic shock (life threatening and recurrent syndrome in kids) o Loss of Splenic Function (Autosplenectomy): due to vaso-occlusion and fibrosis Lifelong risk of severe bacterial infection (encapsulated) Howell Jolly bodies on blood smear Increased risk of Salmonella osteomyelitis Strokes: uncommon but highly recurrent (requires chronic transfusions to prevent) Acute Chest Syndrome: pulmonary infarction and/or pneumonia are common causes (indistinguishable from one another) Kidneys: o Loss of urine concentration capacity due to sickling in vessels around loop of Henle (large volumes of dilute urine may deter patient from taking in fluids to prevent dehydration) o Hematuria (medullary infarcts) o Glomerular nephropathy o Disease is EXTREMELY variable in severity: Factors affecting disease severity include: Presence of genetic markers (ie. beta gene haplotype) Co-inheritance of alpha-thalassemia (beneficial) Amount and distribution of HbF (higher levels are beneficial and prevent sickling) Diagnosis: o Clinical history and findings o Demonstrate the presence of HbS Solubility test (saponin lyses RBCs and releases Hb into solution of Na-diathionite, which deoxygenates Hb; solution will become turbid if HbS present) Hb electrophoresis (presence of HbS and absence of HbA) HbS moves slower than HbA Modern technique is HPLC o Demonstrate the presence of hemolytic anemia Low Hb Normocytic, normochromic RBCs High reticulocyte counts High bilirubin Low haptoglobin o Morphologic sickling on blood smears Sickling is NOT seen with sickle cell trait Management: o Symptomatic and supportive care of pain episodes (analgesics, local heat packs, adequate hydration, acid-base balance, avoid hypoxia and exposure to cold) o Treatment of febrile episodes EARLY and AGGRESSIVELY with Abx (can die of fever if mismanaged) o Blood transfusions in some scenarios: Prevention of strokes in children Treatment of pneumonia if respiratory distress is present Severe anemia (Hb <5; usually with aplastic crisis or splenic sequestration) Before general anesthesia In problem pregnancies o Early diagnosis by screening of newborns (allows for prevention of high childhood mortality) Anticipatory guidance for care-givers Routine prophylactic penicillin Pneumococcal vaccine o Psychosocial support Pain attacks can be barrier to independent living and self-determination o Non-directive genetic counseling for couples at risk Offer prenatal diagnosis by CVS Choice of therapeutic abortions for involved pregnancies o Newer therapies: Hyroxyurea to stimulate HbF production (protective against sickling) Bone marrow or cord blood stem cell transplant Used in patients with bad outcome (ie. stroke in children) o Future directions: Anti-sickling agents (ie. membrane active drugs) Gene therapy Rarer Structural Hemoglobinopathies: Unstable Hemoglobins: o Cause: usually occur near the HEME POCKET, resulting in Hb that is unstable (results in tendency for heme to separate from globin chain with small amounts of oxidative stress) Denature Hb precipitates in the red cell and forms Heinz bodies (cells sequester to the spleen and extravascular hemolysis and hemolytic anemia result o Diagnosis: Demonstration of hemolytic anemia Detection of Heinz bodies by supravital staining Heart precipitation test (exposure to 50 degree temperature produces a precipitate) Hb electrophoresis not always helpful since Hb rapidly denatures o Management: Avoid oxidant drugs Transfusions as needed Splenectomy if anemia is severe Hemoglobins with High O2 Affinity: o Hb Bethesda (Prototype): amino acid substitution near the alpha1-beta2 interface, resulting in tight binding of O2 Release of O2 to tissue is SLOW, resulting in inefficient tissue oxygenation End result is increased Hb production due to high EPO levels o Diagnosis: High red cell mass High arterial O2 saturation (>90%) A markedly LEFT shifted O2 dissociation curve Presence of familial erythrocytosis (polycythemia) Exclusion of other causes of polycythemia (Polycythemia Vera, cyanotic heart disease) Hemoglobins with Low O2 Affinity: o Hb Kansas (Prototype): amino acid substitution near the alpha1-beta2 interface, resulting in low O2 affinity Hb picks up O2 poorly from the lungs resulting in high deoxyHb levels (cyanosis) Clinically detectable cyanosis require >5gms/dl of deoxygenated Hb (found in patients with chronic cyanosis) o Diagnosis: Right shifted O2 dissociation curve Normal Hb level and RBC mass o Management: No specific management necessary or effective Cyanosis is relatively well tolerated if strenuous activities are avoided - - Hemoglobin M: o Hb M Boston (Prototype): amino acid substitution close to the heme pocket, near the site of the Fe molecule Mutant Hb loses ability to keep Fe in ferrous state (constantly in ferric state- metHb) MetHb is unable to carry O2 resulting in chronic cyanosis o Diagnosis: History of cyanosis since birth, with normal O2 saturation Brown discoloration of freshly drawn blood (does not change with aeration) Spectrophotometry confirms presence of metHb Electrophoresis demonstrates abnormal Hb o Management: None required Amount of HbM is not sufficient to cause physiological derangements Hemoglobins C/D/E: o Basics: structural variants synthesized at a lower rate than normal beta chains and comprise less than half the total Hb in heterozygotes o AC: heterozygous for HbC Mild target cells No anemia o CC: homozygous for HbC (glutamic acid lysine at position 6 on beta chain) Mild hemolytic anemia (extravascular hemolysis) Significant RBC changes (target cells, HbC crystals, microspherocytes) o AE: heterozygous for HbE (HbE common in Far East) Mild thalassemic phenotype Mild micrcytosis and hypochromasia o EE: homozygous for HbE Moderate thalassemic phenotype Significant hypochromasia, microcytosis and mild anemia o E-B0 Thalassemia: heterozygous for both traits Results in transfusion dependent thalassemic phenotype (major issue)*