Hemolytic anemias

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  • Sickle Cell Anemia (Hemoglobinopathies)By: Jessica Hebert, Rob McElroy, Beth McQuinn, Yuway Tsing, Elias Kass, Bill Walter, Amelia Kramer

  • What are hemolytic anemias?Decreased life-span of erythrocytesRemoval of RBCs may be:Intravascular (within the blood)Extravascular (within the spleen)Associated with:Increased erythropoiesisIncreased hemoglobin catabolism byproducts

  • Possibilities?Many types of hemolytic anemias:G6PD-DeficiencyHemoglobinopathiesSickle Cell DiseaseHereditary SpherocytosisThalassemiasImmunohemolysisOthers

  • http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=sickle+cell+prevalence+AND+hstat%5Bbook%5D+AND+340265%5Buid%5D&rid=hstat6.section.17004Background:Prevalence in Populations

  • Clinical Presentation: Signs and Symptomshttp://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/sca.shtml

  • Background

  • There are a range of Treatments per extremes or per symptomsClinical Presentation

  • Sickle Cell AnemiaA hereditary genetic disorder that results in the production of abnormal hemoglobins

  • In Sickle Cell, the Beta chain is affected by a point mutationSource: Robbins

  • One small point mutation leads to a major problem when HbS is deoxygenated Deoxyhemoglobin A (HbA)http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/hbb.shtmlDeoxyhemoglobin S (HbS)

  • A prolonged environment of low oxygen leads to aggregation and polymerization of hemoglobins into long chains of rod-like fibers, causing the RBC to form the shape of a crescent or sickle.http://www.humanillnesses.com/original/images/hdc_0001_0003_0_img0235.jpgDonut-shapedSoft and malleableAble to pass through small spacesLifespan ~120 daysSickle-shapedHard and rod-likeGets stuck in narrow spacesLifespan ~20 days

  • Sickling of red blood cells leads to:Vaso-occlusive complications

    Severe anemia

    Chronic hyperbilirubinemia

  • Vaso-occlusive complicationsOccur in areas with: Prolonged, low oxygen tension

    Decreased pH

    Inflammation

    Low blood flowSource: Robbins

  • Severe Anemia and Chronic Hyperbilirubinemia Young RBCs with HbS can alternate between the sickled and normal formation.

    However, sickling causes oxidative damage to the RBCs cell membrane (and to the cells around it).

    Eventually, RBCs become irreversibly sickled.

  • Severe Anemia and Chronic HyperbilirubinemiaIrreversibly sickled cells end up in the spleen.

    Hemolysis occurs at the splenic cords.

    Decreased RBCs and increased bilirubin

    Infarction and fibrosis causes autosplenectomy.

    Source: Robbins

  • G6PD-Deficiency Distribution

  • G6PD-Deficiency Distribution12% of males of African descent (A type)22% of Southeast Asian males (Mahedian type)50% of Kurdish males (Mediterranean type)Also, a significant prevalence in males of Italian, Greek, Lebanese, Iraqi, and Sephardic heritage (Mediterranean type)

  • G6PD-DeficiencyPresenting SymptomsFatigue or muscle weaknessJaundice or pallorDyspneaDark urinePain (especially abdominal)Splenomegaly

  • Glucose 6-Phosphate

  • G6PD-Deficiency PathologyGenetic abnormalities (of which there are several varieties) lead to deficient or impaired enzyme functionHigh levels of oxidants (from foods, drugs, exercise, infections) may damage or denature proteins within the erythrocyteRBCs may then be subject to intravascular or extravascular hemolysis

  • G6PD Crisis Precipitation - BeansBroad beans (favism)Fava beans, bell beans, fever beans, hava beans, pigeon beans, horse beans, tick beans, silkworm beans, English dwarf beans

  • G6PD Crisis Precipitation - Oxidizing DrugsAntimalarials (primaquine, quinine, quinidine)NSAIDs (aspirin, ibuprofen)Antibiotics (sulfonamides)NitrofurantoinTCM herbs (rhizoma coptidis, margarita, calculus bovis)

  • Other oxidantsHenna primarily infants and pregnant womenNaphthlene (moth balls) regular exposure

  • Early Lab Tests CBCRBC LowHct LowHb LowMCV Normal to HighMCHC Low to NormalMCH Low to NormalSoinitial CBC is only your starting point

  • Early Lab Tests UrinalysisHemoglobinElevated post-crisisBilirubinElevated post-crisisUrobilinogenElevated post-crisisMay also see hematuria, proteinuria, LDHThese account for dark color of urine following a crisis, but are also non-specific for any given type of hemolytic anemia

  • Early Lab Tests - SmearOften readily seen with initial CBC, or easily ordered alongsideIf anemias are suspected, probably best to run a smear in the early stagesG6PD-Deficiency may show:Heinz bodiesDenatured Hb bound to cell membraneMay damage cell membrane, leading to intravascular hemolysisBite cellsSpleen removes portion of RBC that had Heinz body, preventing intravascular hemolysisThese arent typically present immediately following an acute hemolytic crisis (problem cells have been destroyed)

  • Other Non-Specific Lab TestsReticulocyte countMay be elevated following acute crisis (6-8 weeks)May be normal in non-crisis situationsMay be reduced in aplastic crisis (could go to zero)Should be considered in conjunction with smear, other dataSerum bilirubinPresent in the unconjugated (prehepatic) formElevated after an acute hemolytic crisisSerum haptoglobinBinds to Hb in the blood for removal by spleen (preventing Hb from excretion in the urine, which could lead to renal failure)Decreased (depleted) after an acute hemolytic crisis

  • Screening for G6PDCells from G6PD-deficient subjects cant convert the oxidized substrate to a reduced stateDifferent tests use different substratesSubstrates can either be the enzymes natural substrate (NADP) or substrates from secondary reactions

  • Fluorescent Screening TestG6PD generates NADPHNADPH fluoresces under long-wave UV lightNo G6PD? No fluorescence.

  • Fluorescent Screening TestShortcomings:False Negative: young red blood cells have more G6PD activity. During an acute hemolytic episode, reticulocytosis results in a bigger population of young red blood cells, so even in G6PD Deficiency, the fluorescence may appear normal.False Positive: if the patient is anemic, very little fluorescence may be seen because there are very few red blood cells.

  • Methemoglobin Reduction TestSodium nitrite converts Hb (hemoglobin) to Hi (methemoglobin)Adding methylene blue should stimulate the pentose phosphate pathway, reducing methemoglobinIn G6PD Deficiency, methemoglobin persists

  • Methemoglobin Reduction TestNormal blood clear red colorDeficient blood brown colorHeterozygotes intermediate color

  • Methemoglobin Reduction Test- Takes longer than fluorescence+ Extremely inexpensive+ Requires only a water bath

    Test should be complemented by cytochemical analysis

  • Cytochemical AnalysisOnly way to reliably detect deficiency in heterozygous womenMore reliable than assayGood complement for other tests

  • Cytochemical AnalysisTreat red cells to convert their oxyhemoglobin (HbO2) to methemoglobin (Hi)In the presence of G6PD, Hi converts back to HbO2Add MTT, which will be reduced by HbO2 to an insoluble formIn G6PD Deficiency:Hi persistsMTT is not reducedErgo, cells are not stained

  • Cytochemical Analysis

  • Cytochemical AnalysisInterpretation:Normal G6PD activity: majority stainedHemizygous: majority unstainedHeterozygotes:Most show mosaicism (40-60% unstained)Some have only 2-3% unstained (fortunate lyonization)

  • G6PD AssayTracks activity of enzyme by following rate of production of NADPH over time NADPH has peak of UV light absorption at 340nm

    Note potentially misleading results in following table

  • G6PD AssayValues are examples only (Lewis 10e, p226)

    Male

    Gd+ (normal)

    Gd-

    (entirely deficient)

    Female

    Gd+/Gd+ (normal)

    Gd-/Gd-

    (entirely deficient)

    Gd+/Gd- (somewhat deficient)

    In health

    7-10

  • G6PD AssayInterpretation:Red cells are likely to lyse from G6PD deficiency if they have less than ~20% of normal enzyme activityConfounding factors:With reticulocytosis, the relatively large population of reticulocytes yields a higher enzyme activity, covering up deficiencyTherefore, a low-normal enzyme activity assay during reticulocytosis points towards G6PD deficiency (without deficiency, values would be very high during reticulocytosis) Re-evaluate after reticulocytosis slows

  • Choosing Appropriate TestsMore than one test should be usedAre they in the middle of a hemolytic crisis?Anemia + Reticulocytosis = Misleading resultsFor females, cytochemical analysis should be usedNormal and deficient control blood should always be used for comparisonSheep are naturally deficient, bah!

    In sickle cell trait, about half the Hemoglobin in the RBC is sickle and will exhibit sickling when under severe conditions of low oxygenation.Who is affected by sickle cell disease?Sickle cell disease is estimated to affect more than 50,000 Americans and has been identified in persons from several different racial backgrounds. The estimated prevalence of the common sickle cell disease variants in African-American live births is approximately 1 in 375 for sickle cell disease, 1 in 835 for Hb SC disease. While sickle cell disease is most commonly found in persons of African ancestry, it also affects persons of Mediterranean, Caribbean, South and Central American, Arabian, and East Indian ancestry

    Is Sickle Cell only in African Americans?Sickle cell is in many nationalities including African Americans, Arabs, Greeks, Italians, Latin Americans and people from India. All races should be screen