Hemoglobinopathies and thalassemia's Flashcards
How do you screen for Hb S: Sickle Solubility test
screen for hgb S
-based on principle that hgb s is less soluble than hb A when it gets deoxygenated and precipitates causing turbidity
-blood is added to buffer salt solution with a reducing agent like (NA dithionite, Na hydrosulfite - reduces ferric iron so it cant bind o2 = deO2) and detergent lysing agent like Saponin (hgb gets released from RBC by dissolving membrane lipids)
-DeO2 Hgb S polymerizes in solution creating turbidity. Solutions WITHOUT Hgb S, remain clear
-look at solution against lined paper
What gives you A FALSE NEG when screening for Hb S: Sickle Solubility test
-Infants <6 months:
-mutant beta chains did not replace normal gamma chains. Thus low levels of Hgb S present, means no polymerization occurs
-low hematocrit
What gives you A FALSE POS when screening for Hb S: Sickle Solubility test
-hyperlipidemia
-Hemoglobinopathies (ex. Hgb C Harlem)
-Has 2 substitutions – one is the one seen in Hgb S (valine replacement)
too much blood added
-hgb like harlem which have two aa substitutions so the HbS and then one other one.
Screening Tests for Hb S: Sickling Test
what is usually pos or neg
-Induces sickling on a glass slide
-one drop of whole blood mixed with 2 drops of Na Metabisulphite (reducing agent) on slide
-seal under cover slip to prevent O2 from entering - inducing deO2 hgb S- polymerization
Hb C Harlem is negative when using this test however newborns that contain < 10% Hb S are weakly positive and easily missed (mostly Hb F)
What are some Screening Tests for Sickle Cell Anemia
Sickling and Solubility tests are used for screening
Detects presence of Hgb S but does not distinguish (AS) and disease SS
Alkaline Hemoglobin Electrophoresis:
What is it used of for
-confirms hemoglobinopathies
-separates hgb molecules in electric field based on differences in total molecular charge
-charged molecules move through pH 8.4 (alkaline) producing mobility patterns to detect variant and normal hgb (hgb assumes a neg charge and moves toward the anode (positive)
-some hgbs have the same charge so they move with similar mobility patterns so you need to run in acidic environment to ID the hgb present
Alkaline Electrophoresis vs acidic
Hgb molecules have negative charge and migrate toward the positive pole (anode) in the gel, others will take on positive charge (go to cathode) - like Hb S migrates with D and G on alkaline eletrophoresis but separates
l from D and G on aide.
-likewise C migrates with E and O on alkaline but separates on acid
Alkaline must be done first and is enough to diagnose SS, AS and Thalassemia but acidic is needed for confimation
What is HPLC
-Confirmed Hgb variants with electrophoresis best used for B thalassemia because it can quantify A, A2, and F
-Molecular methods are best for determining definitively
Process:
Patient’s RBC lysate is injected into a cation exchange column.
Both normal and variant Hgbs will bind to column
Elution buffer is injected and forms a gradient of varying ionic strength
Various Hgb types will be differentially eluted from the column at their own retention time
A detect will measure the absorbance of the fraction at 415 nm
The area under the peak is used to quantify Hgb fraction – reported as % of total Hgb
Thalassemia
Quantitative Hemoglobin Mutations
Reduced production of globin chain
-mutations in Alpha or beta chain are most sign because they are the major type of hemoglobin
-Disrupts regular alpha-beta ratio, resulting in premature destruction of RBCs
-divided into alpha and beta thalassemia
-thalassemia belt coincides with Malarial prevalence
possibility because a reduction in parasite invasion and growth in thalassemia cells
Reduced attachment of infected thalassemia cells to endothelial cells and greater binding to anti-malarial antibodies to infected cells
-Therefore, increased phagocytosis and immune clearance of parasites
process of normal HGB
-switch from embryonic to fetal Hb occurs between 6 and 10 weeks of gestation
-fetal to adult Hb occurs at approximately the time of birth.
New born has Hb A (20-30%), Hb F (65-90), hbA2 (<1)
Adult Hb A (95-100), hb F 0-2, hb a 0-3.5
Normal Individual Genotype:
one cluster of B globin genes are inherited on chromosome 11
normal B chain synthesis = BB
2 copies of a-globin gene are inherited on chromosome 16 aa/aa
clinical manifestations of thalassemia result from:
-Reduced or absent transcription of messenger RNA
-mRNA processing Errors because of mutations that add or remove splice sites resulting in nog globin chains or altered chain production
-Translation errors due to mutations that change codon reading frame - add a stop codon, substitute an incorrect on
Unequal production of the α- or β-globin chains causing an imbalance in alpha beta ratio > Reduced RBC precursors ** more important
PBF:
-Hypo/micro RBCs
Mechanisms in β - Thalassemia
unpaired excessive chains
-Unpaired, excess alpha chains precipitate and form inclusion bodies
-Inclusion bodies cause oxidative stress and damage to cellular membranes.
-Apoptosis occurs
Mechanisms in β - Thalassemia
Iron Overload
Children (Iron causes growth retardation and absence of sexual maturity),
Adults (Cardiomyopathy, Fibrosis, Cirrhosis, Exocrine dysfunction)
-Mostly due to transfusion required by beta thalassemia major
Consistently elevated EPO = erythropoietin
-Promotes erythroferrone from erythroblasts which suppresses hepcidin
-Low hepcidin levels allows more iron absorption by the intestines
-in ineffective erythropoiesis , this hyperabsorption of iron is not needed because iron is recycled back into plasma
Mechanisms in β - Thalassemia
Ineffective Erythropoiesis
ineffective erythropoiesis and increased destruction
-bone marrow cannot release sufficiently viable cells into circulation
-cells that have been released have inclusions so they are quickly destroyed by splenic macrophages (EXTRAvas)
Mechanisms in β - Thalassemia
Ineffective Erythropoiesis
enlargened spleen and liver
Worsens anemia and cause neutropenia and thrombocytopenia
The released Hgb leads to increased bilirubin and jaundice
when do symptoms of thalassemia occur
-asypm during fetal life and then symps start showing up between 6-24 months
-decreasing of alpha chains causes increase of gamma chains in fetus/newborn and then B chains after birth
-delta and beta chains can form a tetramer
-these tetramers precpitate in mature RBCs forming inclusion bodies which are removed by splenic macrophages = anemia
α – Thalassemia – Pathological Hemoglobins
Unlike β thal, α thal affects fetus & newborns
- a decrease in alpha causes excess γ chains form Hb Bart’s - γ4. Stable and dont precipitate but form tetramers. After 6 months they will switch to Beta and a decrease in alpha chains will now be an increase in Beta chains
Very high oxygen affinity
Poor/no release of oxygen to tissue
excess β chains form Hb H - β4
beginning few weeks after birth and throughout life
Vulnerable to oxidation
Precipitates out forming many bodies of denatured Hgb
β-thalassemia is divided into four categories:
-β-thalassemia silent carrier (heterozygous state) – no anemia, no clinical symptoms
-β-thalassemia minor (heterozygous state – mild hemolytic anemia, no clinical symptoms
-β-thalassemia major (homozygous or compound heterozygous state) – severe anemia, severe clinical symptoms, needs transfusion
-β-thalassemia intermedia –mild/moderate anemia, moderate clinical symptoms, doesn’t need transfusion
Beta(silent)Beta – Silent carrier state
what does it result in
nearly normal alpha, beta chain ratios AND no hematologic abnormalities
β-thalassemia minor trait what does it result in
Micro/hypo; target cells, elliptocytes, teardrop cells and basophilic stippling
-low mcv and mchc
when 1 beta globin gene is affected by a mutation which decreases or abolishes its expression AND the other gene is normal
-mild, asymptomatic anemia
-Hepatomegaly, splenomegaly
β Thalassemia Major
characterized by severe anemia needing regular transfusions
-diagnosed between 6months - 2 years old (after gamma to beta switch)
HAP: reduced or absent
LD: markedly elevated
NO Hb A or decreased Hb A
marked erythroid hyperplasia
low retics occurs when erythroid precursors undergo apoptosis due to ineffective erythropoiesis
β Thalassemia Major
PBS
marked hypo/micro - because of decreased hgb production
poly - ineffective but increased EPO production
Poik - increased dectruction in BM and Spleen Targets, tears, ellip, frags and all inclusions
β Thalassemia Major Treatment
Transfusion & Iron chelation
-major therapeutic option
-use donor cells that are max 10 days old
-regular transfusions are used to suppress anemia and marked erythropoiesis.
-if there is a decrease in erythropoiesis there is decreased iron absorption in the intestines
-children in this treatment will not get hepatosplenomegaly
-however , chronic transfusions can lead to iron overload because there is no way for iron to leave the body so the iron in transfused RBCs accumulate in organs outside the bone marrow such as the heart liver and pancreas
β Thalassemia Major Treatment
Hematopoietic stem cell transplant (HSCT)
Only curative therapy. Teens with HLA donor match and no iron overload have good outcomes
allogenic cells (cord blood unit)
β Thalassemia Major Treatment Hydroxyurea (Hb F induction agents)
Switches on the gamma gene to produce more gamma chains. Gamma chains will combine with excess alpha chains to form Hgb F. Thus partially correcting alpha:beta ratio imbalance
β Thalassemia Intermedia
-symptoms fall between b-thalassemia minor and b-thalassemia major, but without the need for regular transfusion therapy to
maintain the hemoglobin level and quality of life
-Patient may develop iron overload 🡪 must monitor for iron overload after age 10 even without regular transfusions - ineffective erythropoiesis which suppresses hepcidin production by the liver caused increased iron absorption by the intestines
chelation therapy
Patients have higher risk of thrombosis due to iron overload and precipitation of RBC chains
What is B^0
b^+
b^s
B^0 - no B chain production
b^+ - decreased chain production
b^s -mildly decreased
always look at family history
