Uploaded byDr. Rajesh Bendre
Interpreting Abnormal hemoglobin study
The document provides a detailed overview of abnormal hemoglobin studies, including the structure and types of hemoglobin, their associated disorders, and laboratory evaluation methods such as solubility tests and electrophoresis. It highlights common hemoglobin abnormalities like thalassemias and hemoglobinopathies, and describes case studies illustrating different conditions and their lab findings. Recommendations for interpreting laboratory results and identifying specific hemoglobin disorders based on various metrics are also included.
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Interpreting Abnormal hemoglobin study
- 1. Interpretation of AbnormalInterpretationof Abnormal Hemoglobin studyHemoglobin study Dr Rajesh V BendreDr Rajesh V Bendre MD(path, DNB(path),DPBMD(path, DNB(path),DPB MumbaiMumbai
- 2. Normal Hemoglobin StructureNormalHemoglobin Structure Hemoglobin A is aHemoglobin A is a tetramer composed of 4tetramer composed of 4 subunits:subunits: – 22αα and 2and 2ββ Each subunit has aEach subunit has a porphyrin ring whichporphyrin ring which holds an iron molecule.holds an iron molecule. – This is the bindingThis is the binding site of oxygensite of oxygen Hemoglobin tetramer
- 3. Other Hemoglobins innormalOther Hemoglobins in normal adultsadults HemoglobiHemoglobi nn StructureStructure %% AA αα22 ββ22 92%92% AA22 αα22 δδ22 2.5%2.5% AA1C1C αα22 ((ββ-N-glucose)-N-glucose) 3%3% FF αα22 γγ22 <1%<1% Gower-1Gower-1 ζζ22 εε22 0*0* Gower-2Gower-2 αα22 εε22 0*0* PortlandPortland ζζ22 γγ22 0*0* * Indicates early embryonic form not seen in adults
- 4. Other Hemoglobins innormalOther Hemoglobins in normal adultsadults HbAHbA22:: – Decreased in iron deficiency, alpha-thalassemiaDecreased in iron deficiency, alpha-thalassemia – Elevated in megaloblastic anemia, hyperthyroidism,Elevated in megaloblastic anemia, hyperthyroidism, Beta-thalessemiaBeta-thalessemia HbF:HbF: – Elevated in HPFH, Sickle cell anemia (preferentialElevated in HPFH, Sickle cell anemia (preferential survival of RBCs because HgF inhibits sickling), Betasurvival of RBCs because HgF inhibits sickling), Beta thalessemia majorthalessemia major – Normal levels in Beta-thalassemia minorNormal levels in Beta-thalassemia minor – Normal or mildly elevated in congenital hemolyticNormal or mildly elevated in congenital hemolytic anemiaanemia – Marked elevation in juvenile CML (up to 70%)Marked elevation in juvenile CML (up to 70%)
- 5. Hemoglobin AbnormalitiesHemoglobin Abnormalities There are 3 main categories of inheritedThere are 3 main categories of inherited Hemoglobin abnormalities:Hemoglobin abnormalities: – Structural or qualitative: The amino acid sequenceThe amino acid sequence is altered because of incorrect DNA codeis altered because of incorrect DNA code (Hemoglobinopathy).(Hemoglobinopathy). – Quantitative: Production of one or more globin chainsProduction of one or more globin chains is reduced or absent (Thalassemia).is reduced or absent (Thalassemia). – Hereditary persistence of Fetal HemoglobinHereditary persistence of Fetal Hemoglobin (HPFH):(HPFH): Complete or partial failure ofComplete or partial failure of γγ globin to switchglobin to switch toto ββ globin.globin.
- 6. Abnormal HemoglobinAbnormal Hemoglobin Reasons to suspect a hemoglobinReasons to suspect a hemoglobin disorder:disorder: – Patient presents with suspicious history orPatient presents with suspicious history or physical examphysical exam – Laboratory tests: Microcytic hypochromicLaboratory tests: Microcytic hypochromic RBCs, hemolytic anemiaRBCs, hemolytic anemia – Screening test abnormality (primarily inScreening test abnormality (primarily in neonates)neonates)
- 7. Laboratory Methods toevaluateLaboratory Methods to evaluate HemoglobinHemoglobin Red cell morphologies:Red cell morphologies: – HbS: Sickle cellsHbS: Sickle cells – HbC: Target cells, crystals after splenectomyHbC: Target cells, crystals after splenectomy – Thalassemias: Microcystosis, target cells, basophilicThalassemias: Microcystosis, target cells, basophilic stipplingstippling
- 8. Laboratory Methods toevaluateLaboratory Methods to evaluate HemoglobinHemoglobin Solubility test :Solubility test : – Test to identify HbS. HbSTest to identify HbS. HbS is relatively insolubleis relatively insoluble compared to othercompared to other Hemoglobins.Hemoglobins. – Add reducing agentAdd reducing agent – HbS will precipitate formingHbS will precipitate forming and opaque solutionand opaque solution compared with the clearcompared with the clear pink solution seen in HbSpink solution seen in HbS is not present.is not present.
- 9. Laboratory Methods toevaluateLaboratory Methods to evaluate HemoglobinHemoglobin
- 10. Laboratory Methods toevaluateLaboratory Methods to evaluate HemoglobinHemoglobin Electrophoresis:Electrophoresis: – Alkaline (Cellulose Acetate) pH 8.6:Alkaline (Cellulose Acetate) pH 8.6: All Hemoglobin molecules have a negative charge, and migrate towards the anodeAll Hemoglobin molecules have a negative charge, and migrate towards the anode proportional to their net negative charge.proportional to their net negative charge. – Amino acid substitutions in hemoglobin variants alter net charge and mobility.Amino acid substitutions in hemoglobin variants alter net charge and mobility. – Acid (Citrate agar) pH 6.2:Acid (Citrate agar) pH 6.2: Hemoglobin molecules separate based on charge differencesHemoglobin molecules separate based on charge differences andand their ability totheir ability to combine with the agar.combine with the agar. – Used to differentiate Hemoglobin variants that migrate together on theUsed to differentiate Hemoglobin variants that migrate together on the cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).
- 11. Laboratory Methods toevaluateLaboratory Methods to evaluate HemoglobinHemoglobin High-Performance LiquidHigh-Performance Liquid Chromatography (HPLC):Chromatography (HPLC): – Weak cation exchange column.Weak cation exchange column. The ionic strength of the elutingThe ionic strength of the eluting solution is gradually increasedsolution is gradually increased and causes the variousand causes the various Hemoglobin molecules to have aHemoglobin molecules to have a particular retention time.particular retention time. Amino acid substitutions willAmino acid substitutions will alter the retention timealter the retention time relative to HbA.relative to HbA. There is some analogyThere is some analogy between retention time andbetween retention time and pattern on alkalinepattern on alkaline electrophoresis.electrophoresis.
- 12. Laboratory Methods toevaluateLaboratory Methods to evaluate HemoglobinHemoglobin
- 13. Most common HemoglobinMostcommon Hemoglobin abnormalitiesabnormalities ThalassemiasThalassemias – AlphaAlpha – BetaBeta HemoglobinopathiesHemoglobinopathies – HbS trait; diseaseHbS trait; disease – HbC trait; diseaseHbC trait; disease – HbEHbE – Hereditary Persistence of Hemoglobin FHereditary Persistence of Hemoglobin F (HPHF)(HPHF)
- 14. Approach for ReportingApproachfor Reporting Reporting Protocol for Abnormal Hemoglobin study by HPLC (Beta2 variant Programme-Biorad)- Although a cut off of >4.0% of HbA2 is recommended for identifying b- thalassemia carriers, each laboratory needs to establish their own normal ranges. Also some cases of silent b-thalassemia can have HbA2 between 3.6 to 3.9% Blood transfusion within 3 months will have dilutional effect & false low HbA2 & Hb F Coexisting Iron deficiency tends to decrease Hb A2 & cause microcytosis Coexisting Folate & B12 deficiency tend to falsely elevate Hb A2 levels Some hemoglobins co-elute in the Hb A2 window like Hb E, Hb D Iran, Hb G Copenhagen, Hb Lepore and Hb Osu Christianbourg making it impossible to quantitate HbA2 in the presence of these hemoglobins
- 15. HPLC assignedwindows & hemoglobin variants may require capillary electrophoresis for correct categorization During interpreting capillary electrophoresis- HbA2 always elutes at 240, all other hemoglobins to be interpreted with reference to Hb A2. Approach for ReportingApproach for Reporting P1 (0.63 – 0.85) F (0.98 – 1.20) P2 (1.24 -1.40) P3 (1.40 – 1.90) A0 (1.40- 1.90) A2 (1.90 -3.90) D (3.90- 4.30) S (4.30- 4.70) Unknown (4.70- 4.90) C (4.90 – 5.30) No Hb variant. However, part elution of tetramers Hb barts, Hb H in alpha thalassemia, Hb F1 (acetylated form) in neonates, high bilirubin HbF > 10% which is not age appropriate Can have other Hb variants requiring DNA studies HbA1C elutes here, however >7% needs to rule out diabetic status Denotes the status of sample integrity, >10 % can have other Hb variants like Hb J- alpha chain variants Hb A elutes here. Co- eluting Hb variants are Hb Koln (peak in initial upward slope + secondary peak at 4.90), Hb twin peaks (hump on downward slope) Hb A2> 11% can have other Hb variants like HB E, Hb Lepore (hump on downward slope), Hb D Iran, Hb G Honolulu Hb D Punjab, Hb Korle-bu, Hb G philadelphia Hb S, Hb Q thailand Hb hasharon, Hb O arab Hb C, Hb G sriraj, Hb E saskatoon
- 16. Hb A2 Levels Hb FLevels (always correlate with Age) Microcytic hypochromic anaemia with or without NRBC Remark Always correlation with Age, H/o transfusion within past 3 months & Patient Ethnic Origin is necessary 3.5 – 3.8 3.6 – 7.5 6 – 8 3.6 – 11 Upto 3 Less than 3 1 – 5 2 – 10 20 – 50 10 – 90 80-100 1 - 3 Present(+) Present(+) Present (++) Present (+++) Present (+++) Present (++) B-thalassemia carrier (advised DNA studies) B- thalassemia trait B-thalassemia intermedia or Delta Beta thalassemia trait B-thalassemia Syndrome(?major / ?intermedia) (advised family studies ) Delta Beta thalassemia disease(advised family studies & DNA studies) Alpha thalassemia trait/Hb H disease (advised family studies & DNA studies) Approach for ReportingApproach for Reporting
- 17. Hb A2 Levels Hb F Levels Typeof anaemia Remark 3.5- 3.8 Upto 3.2 1 – 2 20 - 60 Normocytic normochromic Advised DNA studies to rule out Silent/ carrier BTT Hereditary persistence of Hb F (HPFH- Deletional / Non-Deletional variants)) 3.5 – 4 2 -10 Macrocytic Advised Vitamin B12 & Folate studies & repeat HPLC after treatment of hematinics to rule out BTT 3.6 – 7.5 2-10 Microcytic (RDW- >20) BTT & advised Iron studies • In India- common hemoglobinopathies seen with geographic distribution (Gujarat, Rajasthan, Maharashtra- Betathalassemia, sickle cell)(Pujab,Haryana-Hb D) (west Bengal,North East-Hb E). Hb C is extremely rare. Approach for ReportingApproach for Reporting
- 18. Case 1Case 1 47 year old female47 year old female presents with apresents with a history of peptic ulcerhistory of peptic ulcer disease, H. Pylori andisease, H. Pylori an anemia.anemia. Labs:Labs: Hgb: 10.2Hgb: 10.2 Hct: 30.9Hct: 30.9 MCV: 96.4MCV: 96.4 B12: 338B12: 338 Iron: 122Iron: 122 Ferritin: 304.5Ferritin: 304.5 IBC: 226IBC: 226
- 19. Case 1Case 1 HbF:1.3% HbA2: 4.1%Sickle solubility test POSITIVE
- 20. Case 1Case 1 Hemoglobin S/C disease:Hemoglobin S/C disease: – Second most commonSecond most common hemoglobin variant inhemoglobin variant in Africans; 1 in 1000 births ofAfricans; 1 in 1000 births of African AmericansAfrican Americans – Relatively benign condition;Relatively benign condition; Milder disease than SickleMilder disease than Sickle cell disease. Patients havecell disease. Patients have normal growth andnormal growth and developmentdevelopment – Do not see the classic sickleDo not see the classic sickle cellscells – Peripheral smear revealsPeripheral smear reveals anisocytosis, target cells,anisocytosis, target cells, poikilocytosis,poikilocytosis, polychromasiapolychromasia Hemoglobin S/C disease:Hemoglobin S/C disease: – Most patients have moderateMost patients have moderate splenomegaly with manysplenomegaly with many having autosplenectomy,having autosplenectomy, usually older age than withusually older age than with Sickle cell diseaseSickle cell disease – May have veno-occlusiveMay have veno-occlusive disease, but less commondisease, but less common and less severe than in sickleand less severe than in sickle cell diseasecell disease – May have aseptic necrosis ofMay have aseptic necrosis of bone with osteomyelitisbone with osteomyelitis – ~50% HbS: 50% HbC; rarely~50% HbS: 50% HbC; rarely is HbF >2%is HbF >2%
- 21. Case 2Case 2 A 45 year old GermanA 45 year old German man who isman who is asymptomatic is seenasymptomatic is seen for microcytosis.for microcytosis. Peripheral smearPeripheral smear shows microcytosis,shows microcytosis, hypochromia, targethypochromia, target cells, basophiliccells, basophilic stippling,stippling, polychromasiapolychromasia Labs:Labs: Hgb: 11.8Hgb: 11.8 Hct: 37.5Hct: 37.5 MCV: 65.9MCV: 65.9 Iron: 119Iron: 119 Ferritin: 506Ferritin: 506 IBC: 275IBC: 275 Fe Sat: 43%Fe Sat: 43%
- 22. Case 2Case 2 HbF:1.6%* HbA2: 5.1%
- 23. Case 2Case 2 Beta Thalassemia Minor:Beta Thalassemia Minor: – The thalassemia seen mostThe thalassemia seen most commonly is caucasians (primarilycommonly is caucasians (primarily Mediterranean descent)Mediterranean descent) – Beta thalassemia minor is loss ofBeta thalassemia minor is loss of one of two genes for Beta globin onone of two genes for Beta globin on chromosome 11chromosome 11 – Patients generally asymptomaticPatients generally asymptomatic – May have mild microcytic anemiaMay have mild microcytic anemia (MCV: 60-70; Hgb: 10-13) with a(MCV: 60-70; Hgb: 10-13) with a normal or slightly increased RBCnormal or slightly increased RBC countcount – The peripheral smear will showThe peripheral smear will show target cells and basophilic stipplingtarget cells and basophilic stippling – See increased HbASee increased HbA22 in the range ofin the range of 5-9% with normal HbF5-9% with normal HbF – Thalassemia found most commonlyThalassemia found most commonly in caucasiansin caucasians – See mild microcytosisSee mild microcytosis Beta Thalassemia Minor:Beta Thalassemia Minor: Primary indication is a slightlyPrimary indication is a slightly elevated HbAelevated HbA22 detected by HPLCdetected by HPLC (usually around 4-7%, up to 10%)(usually around 4-7%, up to 10%) typically without elevation of HbFtypically without elevation of HbF Diagnosis may be obscured inDiagnosis may be obscured in concomitant iron deficiency presentconcomitant iron deficiency present because Beta-thalassemia causes anbecause Beta-thalassemia causes an increase in HbAincrease in HbA22 while iron deficiencywhile iron deficiency causes a decrease in HbAcauses a decrease in HbA22. Both. Both create a microcytosis.create a microcytosis. – May see a anemia that partiallyMay see a anemia that partially responds to iron therapyresponds to iron therapy – Always want to look at ironAlways want to look at iron studies when interpretingstudies when interpreting hemoglobin electrophoresis;hemoglobin electrophoresis; usually wait to diagnose untilusually wait to diagnose until nutritional deficiencies have firstnutritional deficiencies have first been corrected.been corrected.
- 24. Case 2Case 2 Beta Thalassemia Major:Beta Thalassemia Major: – Homozygous double gene deletion with no BetaHomozygous double gene deletion with no Beta globin productionglobin production – Presents with lethal anemia, jaundice, splenomegaly,Presents with lethal anemia, jaundice, splenomegaly, growth retardation, bone malformations, deathgrowth retardation, bone malformations, death – Severe hypochromic, microcytic anemia with verySevere hypochromic, microcytic anemia with very bizarre cellsbizarre cells – HbAHbA22 is not increasedis not increased – HgF is at nearly 100%HgF is at nearly 100% – Abundant intra-erythrocyte precipitation of alphaAbundant intra-erythrocyte precipitation of alpha monomers that are insolublemonomers that are insoluble
- 25. Case 3Case 3 47 year old African47 year old African American femaleAmerican female presents to the ERpresents to the ER with drug intoxicationwith drug intoxication and marked anemia.and marked anemia. She is unable toShe is unable to provide any adequateprovide any adequate history to thehistory to the clinicians.clinicians. Labs:Labs: Hgb: 5.9Hgb: 5.9 Hct: 17.8MCV: 97.1Hct: 17.8MCV: 97.1 RDW: 20.9RDW: 20.9 Iron: 83Iron: 83 Ferritin: 394.3Ferritin: 394.3 IBC: 144IBC: 144 Fe Sat: 58%Fe Sat: 58%
- 26. Case 3Case 3 Sickledexis POSITIVE; Peripheral smear with 2+ sickle cells HbF: 1.0%; HbA: 38.7%; HbA2: 4.4%; HbS: 56.1%
- 27. Case 3Case 3 Sickle cell anemia:Sickle cell anemia: – In sickle cell trait, usuallyIn sickle cell trait, usually see HbS concentrations ofsee HbS concentrations of 35 to 45% of total35 to 45% of total Hemoglobin because theHemoglobin because the HbS has a slower rate ofHbS has a slower rate of synthesis than HbAsynthesis than HbA If HbS is less than 33%,If HbS is less than 33%, start thinking about S-start thinking about S- alpha-thalassemiaalpha-thalassemia If HbS is greater thanIf HbS is greater than 50%, worry about S-50%, worry about S- Beta-thalassemia orBeta-thalassemia or Sickle cell disease withSickle cell disease with transfusiontransfusion Sickle cell anemia:Sickle cell anemia: – This patient was transfusedThis patient was transfused with two units of RBCs beforewith two units of RBCs before the HPLC was performed.the HPLC was performed. – It is important to know theIt is important to know the appropriate ratios of HbS:appropriate ratios of HbS: HbA expected. If the patientHbA expected. If the patient does not fit,does not fit, alwaysalways look at thelook at the transfusion history.transfusion history. If concerned aboutIf concerned about overlying Beta-overlying Beta- thalassemia, repeat HPLCthalassemia, repeat HPLC after four months of mostafter four months of most recent transfusionrecent transfusion
- 28. Case 3Case 3 HbAHbAHbSHbS HbAHbA22 HbFHbF Hb ASHb AS 55-6055-60 40-4540-45 2-32-3 <1<1 Hb SSHb SS 00 90-9590-95 2-32-3 5-105-10 Hb S-Hb S-αα-thal-thal 7575 2525 2-32-3 <1<1 Hb S-Hb S- ββ thal majorthal major 00 90-9590-95 Inc.Inc. 5-105-10 Hb S-Hb S- ββ thal minorthal minor 5-305-30 60-9060-90 Inc.Inc. 5-105-10 Hb S HPFHHb S HPFH 00 70-8070-80 2-32-3 20-3020-30 Hb SCHb SC 00 5050 2-32-3 <1<1 Expected ratios
- 29. Case 4Case 4 31 year old healthy31 year old healthy female, pregnant withfemale, pregnant with moderate target cellsmoderate target cells detected on routinedetected on routine peripheral smearperipheral smear Labs:Labs: Hgb: 15.0Hgb: 15.0 Hct: 42.5Hct: 42.5 MCV: 87.8MCV: 87.8 MCH: 31.0MCH: 31.0 RDW: 12.6RDW: 12.6
- 30. Case 4Case 4 HbF:0.6%; HbA2: 2.9%; HbA: 56.3%
- 31. Case 4Case 4 Hemoglobin C trait:Hemoglobin C trait: – Hemoglobin C trait (Heterozygotes) are clinically andHemoglobin C trait (Heterozygotes) are clinically and hematologically wellhematologically well – Moderate target cells seen on peripheral smearModerate target cells seen on peripheral smear – HbA and HbC in a 60:40 ratio on HPLCHbA and HbC in a 60:40 ratio on HPLC – 2% of African Americans have HbC trait2% of African Americans have HbC trait – Homozygotes have mild hemolytic disease,Homozygotes have mild hemolytic disease, cholelithiasis and occasional aplastic crisis.cholelithiasis and occasional aplastic crisis. See reduced MCV with increased MCHCSee reduced MCV with increased MCHC – Intracellular HbC crystals, block-like structures mayIntracellular HbC crystals, block-like structures may be seen and are pathognomonic of HbC.be seen and are pathognomonic of HbC.
- 33.