A Brief Overview of Hemoglobin Electrophoresis

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Ordinary Hemoglobin Structure. Hemoglobin A will be a tetramer made out of 4 subunits: 2a and 2

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A Brief Overview of Hemoglobin Electrophoresis Sarah Walter, M.D.

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Normal Hemoglobin Structure Hemoglobin A will be a tetramer made out of 4 subunits: 2 α and 2 β Each subunit has a porphyrin ring which holds an iron particle. This is the coupling site of oxygen

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Normal Hemoglobin Structure Hemoglobin tetramer

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Normal Hemoglobin Structure O Fe Porphyrin ring O 2 restricting site The oxygen particle ties to the Fe iota opposite to the porphyrin ring

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Hemoglobin Function The capacity of the Hemoglobin atom is to get oxygen in the lung and convey it to the tissues using none of the oxygen en route.

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Hemoglobin Function The ordinary hemoglobin particle is appropriate for its capacity Allows for O 2 to be grabbed at high O 2 strain in the lung and conveyed to the tissues at low O 2 pressure. The oxygen restricting is agreeable: As every O 2 ties to hemoglobin, the atom experiences a conformational change expanding the O 2 fondness for the rest of the subunits. This makes the sigmoidal oxygen separation bend

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Normal Hemoglobin Function The hemoglobin separation bend

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Normal Hemoglobin Function Many factors impact the separation bend: pH: An expansion in pH (dec. CO 2 ) shifts the bend to one side (expanded O 2 ) partiality An abatement in pH (inc. CO 2 ) shifts the bend to one side (diminished O 2 ) partiality Temperature: Increased temp with expanded metabolic requests causes diminished O 2 liking (right move) and expanded O 2 conveyance 2,3 DPG: Lowers O 2 proclivity by specially official to Beta chain of deoxyhemoglobin, settling it and lessens the intracellular pH As hemoglobin fixation diminishes, 2,3 DPG increments, permitting more O 2 to be emptied

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Other Hemoglobins in ordinary grown-ups * Indicates early embryonic frame not found in grown-ups

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Other Hemoglobins in typical grown-ups HbA 2 : Decreased in iron inadequacy, alpha-thalassemia Elevated in megaloblastic pallor, hyperthyroidism, Beta-thalessemia HbF: Elevated in HPFH, Sickle cell paleness (particular survival of RBCs in light of the fact that HgF represses sickling), Beta thalessemia significant Normal levels in Beta-thalassemia minor Normal or somewhat lifted in intrinsic hemolytic frailty Marked rise in adolescent CML (up to 70%)

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Hemoglobin Abnormalities There are 3 primary classes of acquired Hemoglobin variations from the norm: Structural or subjective: The amino corrosive arrangement is adjusted in view of mistaken DNA code (Hemoglobinopathy). Quantitative: Production of at least one globin chains is lessened or missing (Thalassemia). Inherited constancy of Fetal Hemoglobin (HPFH): Complete or halfway disappointment of γ globin to change to β globin.

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Abnormal Hemoglobin Reasons to speculate a hemoglobin issue: Patient presents with suspicious history or physical exam Laboratory tests: Microcytic hypochromic RBCs, hemolytic frailty Screening test variation from the norm (essentially in neonates)

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Laboratory Methods to assess Hemoglobin Red cell morphologies: HbS: Sickle cells

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Sickle cells on fringe spread

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Laboratory Methods to assess Hemoglobin Red cell morphologies: HbS: Sickle cells HbC: Target cells, precious stones after splenectomy

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HbC gems with Target cells

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Laboratory Methods to assess Hemoglobin Red cell morphologies: HbS: Sickle cells HbC: Target cells, gems after splenectomy Thalassemias: Microcystosis, target cells, basophilic stippling

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Alpha Thalassemia with basophilic stippling

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Laboratory Methods to assess Hemoglobin Electrophoresis: Alkaline (Cellulose Acetate) pH 8.6: All Hemoglobin particles have a negative charge, and relocate towards the anode corresponding to their net negative charge. Amino corrosive substitutions in hemoglobin variations change net charge and versatility. Corrosive (Citrate agar) pH 6.2: Hemoglobin particles isolate in view of charge contrasts and their capacity to join with the agar. Used to separate Hemoglobin variations that relocate together on the cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).

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Hemoglobin Electrophoresis Patterns

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Laboratory Methods to assess Hemoglobin High-Performance Liquid Chromatography (HPLC): Weak cation trade segment. The ionic quality of the eluting arrangement is bit by bit expanded and makes the different Hemoglobin atoms have a specific maintenance time. Amino corrosive substitutions will modify the maintenance time in respect to HbA. There is some similarity between maintenance time and example on basic electrophoresis.

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Normal HPLC design

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Laboratory Methods to assess Hemoglobin Solubility test (Sickledex): Test to distinguish HbS. HbS is generally insoluble contrasted with different Hemoglobins. Include decreasing operator HbS will encourage shaping and misty arrangement contrasted and the unmistakable pink arrangement found in HbS is not present.

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Most basic Hemoglobin anomalies Thalassemias Alpha Beta Hemoglobinopathies HbS attribute; malady HbC characteristic; sickness HbE Hereditary Persistence of Hemoglobin F (HPHF)

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47 year old female presents with a past filled with peptic ulcer illness, H. Pylori a frailty. Labs: Hgb: 10.2 Hct: 30.9 MCV: 96.4 B12: 338 Iron: 122 Ferritin: 304.5 IBC: 226 Case 1

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Case 1 HbF: 1.3% HbA 2 : 4.1% Sickledex test POSITIVE

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Case 1

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Case 1 Hemoglobin S/C malady: Second most regular hemoglobin variation in Africans; 1 in 1000 births of African Americans Relatively amiable condition; Milder sickness than Sickle cell infection. Patients have ordinary development and advancement Do not see the great sickle cells Peripheral spread uncovers anisocytosis, target cells, poikilocytosis, polychromasia

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Case 1 Hemoglobin S/C illness: Most patients have direct splenomegaly with many having autosplenectomy, typically more established age than with Sickle cell malady May have veno-occlusive ailment, however less normal and less extreme than in sickle cell infection May have aseptic rot of bone with osteomyelitis ~50% HbS: half HbC; once in a while is HbF >2%

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A 45 year old German man who is asymptomatic is seen for microcytosis. Fringe spread shows microcytosis, hypochromia, target cells, basophilic stippling, polychromasia Labs: Hgb: 11.8 Hct: 37.5 MCV: 65.9 Iron: 119 Ferritin: 506 IBC: 275 Fe Sat: 43% Case 2

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Case 2 HbF: 1.6%* HbA 2 : 5.1%

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Case 2 Cellulose acetic acid derivation gel performed HbS

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Case 2 Beta Thalassemia Minor: The thalassemia seen most ordinarily is caucasians (principally Mediterranean plunge) Beta thalassemia minor is loss of one of two qualities for Beta globin on chromosome 11 Patients by and large asymptomatic May have mellow microcytic sickliness (MCV: 60-70; Hgb: 10-13) with an ordinary or somewhat expanded RBC tally The fringe spread will demonstrate target cells and basophilic stippling See expanded HbA 2 in the scope of 5-9% with ordinary HbF Thalassemia discovered most regularly in caucasians See gentle microcytosis

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Case 2 Beta Thalassemia Minor: Primary sign is a marginally hoisted HbA 2 distinguished by HPLC (as a rule around 4-7%, up to 10%) commonly without height of HbF Diagnosis might be clouded in associative iron lack exhibit since Beta-thalassemia causes an expansion in HbA 2 while press insufficiency causes a lessening in HbA 2 . Both make a microcytosis. May see a frailty that incompletely reacts to iron treatment Always need to take a gander at iron reviews when translating hemoglobin electrophoresis; more often than not hold up to analyze until nutritious lacks have first been revised.

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Case 2 Beta Thalassemia Major: Homozygous twofold quality erasure with no Beta globin generation Presents with deadly frailty, jaundice, splenomegaly, development impediment, bone distortions, passing Severe hypochromic, microcytic iron deficiency with extremely odd cells HbA 2 is not expanded HgF is at about 100% Abundant intra-erythrocyte precipitation of alpha monomers that are insoluble

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47 year old African American female presents to the ER with medication inebriation and checked sickliness. She can't give any sufficient history to the clinicians. Labs: Hgb: 5.9 Hct: 17.8MCV: 97.1 RDW: 20.9 Iron: 83 Ferritin: 394.3 IBC: 144 Fe Sat: 58% Case 3

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Case 3 HbF: 1.0%; HbA: 38.7%; HbA 2 : 4.4%; HbS: 56.1% Sickledex is POSITIVE; Peripheral spread with 2+ sickle cells

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Case 3

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Case 3 Sickle cell frailty: In sickle cell quality, normally observe HbS centralizations of 35 to 45% of aggregate Hemoglobin on the grounds that the HbS has a slower rate of combination than HbA If HbS is under 33%, begin considering S-alpha-thalassemia If HbS is more noteworthy than half, stress over S-Beta-thalassemia or Sickle cell ailment with transfusion

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Case 3 Sickle cell sickliness: This patient was transfused with two units of RBCs before the HPLC was performed. It is critical to know the proper proportions of HbS: HbA anticipated. In the event that the patient does not fit, dependably take a gander at the transfusion history. In the event that worried about overlying Beta-thalassemia, rehash HPLC following four months of latest transfusion

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Case 3 Expected proportions

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31 year old sound female, pregnant with direct target cells identified on routine fringe spread Labs: Hgb: 15.0 Hct: 42.5 MCV: 87.8 MCH: 31.0 RDW: 12.6 Case 4

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Case 4 HbF: 0.6%; HbA 2 : 2.9%; HbA: 56.3%

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Case 4

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Case 4 Hemoglobin C quality: Hemoglobin C characteristic (Heterozygotes) are clinically and hematologically well Moderate target cells seen on fringe spread HbA and HbC in a 60:40 proportion on HPLC 2% of African Americans have HbC attribute Homozygotes have mellow hemolytic sickness, cholelithiasis and infrequent aplastic emergency. See lessened MCV with expanded MCHC Intracellular Hb