4Normal Hemoglobin Structure FePorphyrin ringO2 binding siteThe oxygen atom binds to the Fe atomperpendicular to the porphyrin ring
5Hemoglobin FunctionThe function of the Hemoglobin molecule is to pick up oxygen in the lung and deliver it to the tissues utilizing none of the oxygen along the way.
6Hemoglobin FunctionThe normal hemoglobin molecule is well suited for its functionAllows for O2 to be picked up at high O2 tension in the lung and delivered to the tissues at low O2 tension.The oxygen binding is cooperative:As each O2 binds to hemoglobin, the molecule undergoes a conformational change increasing the O2 affinity for the remaining subunits.This creates the sigmoidal oxygen dissociation curve
7Normal Hemoglobin Function The hemoglobin dissociation curve
8Normal Hemoglobin Function Many variables influence the dissociation curve:pH:An increase in pH (dec. CO2) shifts the curve to the left (increased O2) affinityA decrease in pH (inc. CO2) shifts the curve to the right (decreased O2 ) affinityTemperature:Increased temp with increased metabolic demands causes decreased O2 affinity (right shift) and increased O2 delivery2,3 DPG:Lowers O2 affinity by preferentially binding to Beta chain of deoxyhemoglobin, stabilizing it and reduces the intracellular pHAs hemoglobin concentration decreases, 2,3 DPG increases, allowing more O2 to be unloaded
9Other Hemoglobins in normal adults Structure%Aα2 β292%A2α2 δ22.5%A1Cα2 (β-N-glucose)3%Fα2 γ2<1%Gower-1ζ2 ε20*Gower-2α2 ε2Portlandζ2 γ2* Indicates early embryonic form not seen in adults
10Other Hemoglobins in normal adults HbA2:Decreased in iron deficiency, alpha-thalassemiaElevated in megaloblastic anemia, hyperthyroidism, Beta-thalessemiaHbF:Elevated in HPFH, Sickle cell anemia (preferential survival of RBCs because HgF inhibits sickling), Beta thalessemia majorNormal levels in Beta-thalassemia minorNormal or mildly elevated in congenital hemolytic anemiaMarked elevation in juvenile CML (up to 70%)
11Hemoglobin Abnormalities There are 3 main categories of inherited Hemoglobin abnormalities:Structural or qualitative: The amino acid sequence is altered because of incorrect DNA code (Hemoglobinopathy).Quantitative: Production of one or more globin chains is reduced or absent (Thalassemia).Hereditary persistence of Fetal Hemoglobin (HPFH): Complete or partial failure of γ globin to switch to β globin.
12Abnormal Hemoglobin Reasons to suspect a hemoglobin disorder: Patient presents with suspicious history or physical examLaboratory tests: Microcytic hypochromic RBCs, hemolytic anemiaScreening test abnormality (primarily in neonates)
13Laboratory Methods to evaluate Hemoglobin Red cell morphologies:HbS: Sickle cells
17Laboratory Methods to evaluate Hemoglobin Red cell morphologies:HbS: Sickle cellsHbC: Target cells, crystals after splenectomyThalassemias: Microcystosis, target cells, basophilic stippling
18Alpha Thalassemia with basophilic stippling The basophilic stippling is due to precipitated globins that formed in excess (I.e. HbH, Beta tetramer)
19Laboratory Methods to evaluate Hemoglobin Electrophoresis:Alkaline (Cellulose Acetate) pH 8.6:All Hemoglobin molecules have a negative charge, and migrate towards the anode proportional to their net negative charge.Amino acid substitutions in hemoglobin variants alter net charge and mobility.Acid (Citrate agar) pH 6.2:Hemoglobin molecules separate based on charge differences and their ability to combine with the agar.Used to differentiate Hemoglobin variants that migrate together on the cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).
21Laboratory Methods to evaluate Hemoglobin High-Performance Liquid Chromatography (HPLC):Weak cation exchange column. The ionic strength of the eluting solution is gradually increased and causes the various Hemoglobin molecules to have a particular retention time.Amino acid substitutions will alter the retention time relative to HbA.There is some analogy between retention time and pattern on alkaline electrophoresis.
23Laboratory Methods to evaluate Hemoglobin Solubility test (Sickledex):Test to identify HbS. HbS is relatively insoluble compared to other Hemoglobins.Add reducing agentHbS will precipitate forming and opaque solution compared with the clear pink solution seen in HbS is not present.
24Most common Hemoglobin abnormalities ThalassemiasAlphaBetaHemoglobinopathiesHbS trait; diseaseHbC trait; diseaseHbEHereditary Persistence of Hemoglobin F (HPHF)
25Case 147 year old female presents with a history of peptic ulcer disease, H. Pylori an anemia.Labs:Hgb: 10.2Hct: 30.9MCV: 96.4B12: 338Iron: 122Ferritin: 304.5IBC: 226
26Case 1HbF: 1.3%HbA2: 4.1%Sickledex test POSITIVE
28Case 1 Hemoglobin S/C disease: Second most common hemoglobin variant in Africans; 1 in 1000 births of African AmericansRelatively benign condition; Milder disease than Sickle cell disease. Patients have normal growth and developmentDo not see the classic sickle cellsPeripheral smear reveals anisocytosis, target cells, poikilocytosis, polychromasia
29Case 1 Hemoglobin S/C disease: Most patients have moderate splenomegaly with many having autosplenectomy, usually older age than with Sickle cell diseaseMay have veno-occlusive disease, but less common and less severe than in sickle cell diseaseMay have aseptic necrosis of bone with osteomyelitis~50% HbS: 50% HbC; rarely is HbF >2%
30Case 2A 45 year old German man who is asymptomatic is seen for microcytosis.Peripheral smear shows microcytosis, hypochromia, target cells, basophilic stippling, polychromasiaLabs:Hgb: 11.8Hct: 37.5MCV: 65.9Iron: 119Ferritin: 506IBC: 275Fe Sat: 43%
33Case 2 Beta Thalassemia Minor: The thalassemia seen most commonly is caucasians (primarily Mediterranean descent)Beta thalassemia minor is loss of one of two genes for Beta globin on chromosome 11Patients generally asymptomaticMay have mild microcytic anemia (MCV: 60-70; Hgb: 10-13) with a normal or slightly increased RBC countThe peripheral smear will show target cells and basophilic stipplingSee increased HbA2 in the range of 5-9% with normal HbFThalassemia found most commonly in caucasiansSee mild microcytosis
34Case 2 Beta Thalassemia Minor: Primary indication is a slightly elevated HbA2 detected by HPLC (usually around 4-7%, up to 10%) typically without elevation of HbFDiagnosis may be obscured in concomitant iron deficiency present because Beta-thalassemia causes an increase in HbA2 while iron deficiency causes a decrease in HbA2. Both create a microcytosis.May see a anemia that partially responds to iron therapyAlways want to look at iron studies when interpreting hemoglobin electrophoresis; usually wait to diagnose until nutritional deficiencies have first been corrected.
35Case 2 Beta Thalassemia Major: Homozygous double gene deletion with no Beta globin productionPresents with lethal anemia, jaundice, splenomegaly, growth retardation, bone malformations, deathSevere hypochromic, microcytic anemia with very bizarre cellsHbA2 is not increasedHgF is at nearly 100%Abundant intra-erythrocyte precipitation of alpha monomers that are insoluble
36Case 347 year old African American female presents to the ER with drug intoxication and marked anemia. She is unable to provide any adequate history to the clinicians.Labs:Hgb: 5.9Hct: 17.8MCV: 97.1RDW: 20.9Iron: 83Ferritin: 394.3IBC: 144Fe Sat: 58%
37Case 3 HbF: 1.0%; HbA: 38.7%; HbA2: 4.4%; HbS: 56.1% Sickledex is POSITIVE; Peripheral smear with 2+ sickle cells
39Case 3 Sickle cell anemia: In sickle cell trait, usually see HbS concentrations of 35 to 45% of total Hemoglobin because the HbS has a slower rate of synthesis than HbAIf HbS is less than 33%, start thinking about S-alpha-thalassemiaIf HbS is greater than 50%, worry about S-Beta-thalassemia or Sickle cell disease with transfusion
40Case 3 Sickle cell anemia: This patient was transfused with two units of RBCs before the HPLC was performed.It is important to know the appropriate ratios of HbS: HbA expected. If the patient does not fit, always look at the transfusion history.If concerned about overlying Beta-thalassemia, repeat HPLC after four months of most recent transfusion
45Case 4 Hemoglobin C trait: Hemoglobin C trait (Heterozygotes) are clinically and hematologically wellModerate target cells seen on peripheral smearHbA and HbC in a 60:40 ratio on HPLC2% of African Americans have HbC traitHomozygotes have mild hemolytic disease, cholelithiasis and occasional aplastic crisis.See reduced MCV with increased MCHCIntracellular HbC crystals, block-like structures may be seen and are pathognomonic of HbC.