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Neonatal Anemia: Recognizing Thalassemia and Hemoglobin Variants
James H. Nichols, PhD, DABCC, FACBProfessor of Pathology, Microbiology, and Immunology
Medical Director, Clinical ChemistryAssociate Medical Director of Clinical Operations
Vanderbilt University School of MedicineNashville, TN 37232‐5310
Objectives
• Describe hemoglobin genetics• Interpret hemoglobin chromatograms and IEF• Recognize common hemoglobin variants
Case
• 4 mo male, African American, abnormal newborn screen, seen for follow‐up testing
• Newborn screen shows hemoglobin FS at birth
Audience Poll
• What do these results indicate?A. Normal profileB. Abnormal amounts of hemoglobin FC. Sickle cell diseaseD. Sickle cell trait
Chromosomal Organization of Globin Genes
Hemoglobin Concentration Structure
Hb A ~90% 2 2
Hb F ~1.0% 2 2
Hb A2 ~2.5% 2 2
Hb A1 ~7.0% Mixture of post-translational
variants of Hb A
Normal Hemoglobins in Adults
Reasons for Requesting Hemoglobin Variant Analysis
• Follow‐up to abnormal newborn screen• Adoption• Prenatal screening – patients of ethnic origin• Anemia of unknown origin in ethnic patient• Athletic exam for competitive sports
Hemoglobinopathies
1. Structural – substitution, addition or deletion of one or more AAs in the globin chain i.e HbS, HbC, HbE, HbD, HbO, etc…
2. Thalassemia‐ quantitative defect in globin chain production i.e. alpha and Beta Thalassemia
3. Combination of 1 and 2
4. Asymptomatic disorders – i.e. Hereditary persistence of fetal Hemoglobin
Sickle Cell Disease
• Disease diagnosis based upon presence of a specific variant gene, the sickle gene – Mutation = Glu6Val substitution in the β-globin protein
• One bs gene = Sickle trait• 2 variant genes (bs or other) = disease • Others include Hb C, D, O and b-Thal
Symptoms of Sickle Sell Disease:
• Pallor• Pain Crises• Jaundice• Hand‐foot syndrome• Eye problems• Stroke• Acute chest syndrome• Weakness, general
• Delayed growth & puberty
• Priapism• Infections• Gallstones• Sores (ulcers) on the legs (chronic)
• Spleen dysfunction
Severity Depends on Genotype (i.e. S/C = mild, vs S/S and S/OArab = severe)
Laboratory Diagnosis of Sickle Cell Disease
• Sickle Chex (Dex) – Qualitative Solubility Kit ‐red cells lysed, hemoglobin released – reduced HbS insoluble – cloudy/turbid suspension‐detects homo and heterozygous HbS
• HPLC – BioRad Variant II ‐thal program
• Electrophoretic Separation Technique– Isoelectric focusing (IEF)– Citrate Agar Electrophoresis (Acid)– Cellulose Acetate Electrophoresis (Alkaline)
• DNA Sequencing of ‐globin gene
High Performance Liquid Chromatography (HPLC)
Peak name
Retention time, min
F window 0.98–1.20
A0 window 1.90–3.10
A2 window 3.30–3.90
D window 3.90–4.30
S window 4.30–4.70
C window 4.90–5.30
BioRad Variant II-Thal Short Program
Hb A1c Hb EHb D
Treatment of Sickle Cell Disease
Hydroxyurea
• Increases Hb F levels in RBCs• Decreases neutrophil counts
– 4‐12 weeks after initiation– SS neutrophils have enhanced binding to
fibronectin and are more prone to activation– Modest neutropenia may be beneficial
• Increases the water content of RBCs• Alters the adhesion of RBCs to the
endothelium• Increases the flexibility of sickled cells
Case• 1 day male, Hispanic/Latino. Normal newborn screen. Here for follow‐up testing.
SickleDex = Negative
HbF = 77.4%HbA = 22.6%HbS = <2%
Audience Poll
• What is the most appropriate interpretation of these results?A. Hereditary persistence of FB. Normal profileC. Sickle cell diseaseD. Beta thalassemia
Hemoglobin F in Health and Disease
• Normal– adult <2%– newborn ~80%– 10‐week‐old ~50%– 6‐month‐old ~2%– pregnancy 3 ‐ 15%
• Anemias– Aplastic 5 ‐ 25%– Iron def. 2 ‐ 8%
• Malignancies– Leukemias 2 ‐ 20%
• Hb Disorders– Hb S <20%– Unstable Hb <10%– ‐thal <30%– ‐thal 30 ‐ 95%– S/‐thal 10 ‐ 30%– ‐thal 25 – 35%– ‐thal <1%
• Hereditary Persistence– HPHF 10 ‐ 100%
Hereditary Persistence of F (HPFH)
• Molecular studies have identified two groups of disorders where expression of the globin gene of Hb F persists at high levels in adult erythroid cells with normal RBC indices and morphology
• Form of ‐thalassemia
Pancellular forms• clearly increased Hb F in heterozygotes (15 – 35%)• usually due to major deletions of the globin gene cluster,
including the gene silencers• Evenly distributed among RBCs
Heterocellular forms• modest elevations in Hb F (1 - 4 %) distributed in an uneven
fashion among the F cells• molecular lesions include promoter mutations in the globin
genes and mutations distant from the globin cluster, including a determinant on chromosome 6q in some families
• HbF expression is not evenly distributed among RBCs
HPFHType % Hb F GA Hb F Cellular DistributionAfrican GA
HeterozygousHomozygous
13 - 31100
2:3variable
pancellularpancellular
African G
Heterozygous 15 - 20 Gonly pancellularSwiss
Heterozygous 1 - 3 variable heterocellularNormal neonate 50 - 80 3:1 heterocellular
Normal adult <2 2:3 heterocellular
Case• 23 mo female, African American. Abnormal newborn screen showing HbFS (84% F, 16% S)
SickleDex = Positive
HbF = 27.3%HbA = <1%HbA2 = 3.2%HbS = 68.5%
NB
AFSC
Audience Poll
• What is the most appropriate interpretation of these results?A. Normal profileB. Hereditary persistence of HbFC. Sickle cell diseaseD. Sickle cell disease with hereditary persistence HbF
Case• 33 mo male, unknown ethnicity with anemia (9.7 Hgb),
microcytosis (small cells), anisocytosis (unequal size). Mentzer’s index = 13.4 (indeterminate)
SickleDex = Negative
HbF = 9.6%HbA = 85.5%HbA2 = 4.9%
NB
AFSC
Audience Poll
• What is the most appropriate interpretation for these results?A. Normal profileB. Sickle cell diseaseC. Suggestive of beta thalassemiaD. Hereditary persistence of HbF
Mentzer’s Index• If CBC shows microcytic anemia, ratio of MCV/RBC can distinguish iron deficiency from thalassemia.
• < 13 beta thalassemia more likely (thalassemia is a disorder of globin synthesis, normal amount of cells, but cells produced are smaller and more fragile, RBC normal, but MCV down, so ratio is low)
• >13 iron deficiency more likely (in iron deficiency bone marrow can’t produce as many cells and cells are small, both MCV and RBC down)
• Not reliable, iron deficiency and beta thalassemia can coexist, and ferritin more reliable measure of iron deficiency
Beta Thalassemia• A group of genetic disorders resulting in dimished (or absent
0 ‐chain synthesis
• β‐thalassemia is commonly associated with decreased Hb A and increased HbA2 in heterozygotes
• Heterozygous thal is asymptomatic
• Homozygous thal is a severe disorder associated with transfusion dependent hemolytic anemia
• Homozygous + thal is a heterogenous disorder with severity depending on mutation and % of HbA (the more HbA, the less severe the disease)
Expected Hgb chain distributions in β‐thalassemia
HbA HbA2 HbF
0) present 4 – 8% 0 – 5%β0/ β0 none 1‐6% >94%β+/ β+ present 2.4‐8.7% 20‐90%β0/ β+ present 0.6‐3.4% >75%δβ0/ δβ0 none 0% 70‐92%
Case• 13 y/o female, Asian, 9 yrs status post bone marrow
transplant. On chronic transfusions for anemia, premature deliver at 28 wks to prevent hydrops (in utero transfusions)
SickleDex = NegativeHb= 10.0 (low)Ferritin = 962 on exjade for chronic transfusions
HbF = <1%%HbA = 67.3%HbA2 = 2.8%HbH = 28.9%
AFSC
Audience Poll
• What is the most appropriate interpretation of these results? A. Normal profileB. Suggestive of beta thalassemiaC. Alpha thalassemiaD. Sickle cell trait
Alpha Thalassemia
• Our patient has alpha thalassemia major.• A group of genetic disorders associated with defective ‐chain synthesis
• Difficult to diagnose after neonatal period• Characteristic HPLC profile – HbBarts (4 chains – most common in neonate) and HbH (4 ‐chains).
• Clinical symptoms range from mild microcytosis – severe hemolytic anemia depending on the number of mutations
Alpha Thalassemia
Alpha Thalassemia (AA)-Thal silent (-/) ~ 28%-Thal-1 mutation (--/) is virtually
non-existentConsequently, incidence of (--/- and
--/--) is extremely rare in this group
α α / α α Normal
- - / α α Homozygous
- α /- α Homozygous
- - / α - Hgb H disease
- - / - - Hydrops Fetalis
Alpha Thal Intermedia
Alpha Thal Major
- α / α α Heterozygous
Thalassemia minor1
Thalassemia minor2
Silent Carrier
Summary• Hemoglobin analysis is utilized to confirm hereditary causes of
anemia• Neonatal testing is generally not definitive before 1 – 2 years
of age• Hemoglobin variant analysis in chemistry can provide the
opportunity for combined interpretations with heme/path.• Always consider hemoglobin interpretation in light of the
patient’s ethnicity, symptoms (anemia), and recent transfusion history
Case• 38 y/o male, African American, ED visit for pain crisis with past Hx sickle cell disease
HbF = <1%HbA = 96.2%HbA2 = 2.8%
Audience Poll
• What is the most appropriate diagnosis for these results?A. Normal profileB. Sickle cell diseaseC. Beta thalassemiaD. Hereditary persistence of HbF
Answer
• Specimen shows a normal profile for the patient’s age.
• Inconsistent with claimed Hx of sickle cell disease, unknown Hx of recent transfusion
• Follow‐up with state prescription registry indicated patient’s name on registry for doctor shopping of prescription opiates!
• ED visit with claims of pain crises to seek prescription opiates
Case• 26 mo female, unknown ethnicity, chronic anemia (10.4 Hgb), Ferritin 15 (low), Mentzer’s index = 17.3
SickleDex = Negative
HbF = 3.0%HbA = 95.4%HbA2 = 1.6%
AFSC
Audience Poll
• What is the most appropriate interpretation of these results?A. Normal profileB. Beta thalassemiaC. Low HbA2D. Sickle cell trait
Answer
• This patient has a low HbA2.• Low HbA2 can be seen in anemias, including iron‐deficiency anemia
• This patient has low ferritin, indicative of iron deficiency.
• Note Mentzer’s Index = 17.3 is >13 and could support iron deficiency
Case• 12 y/o female, ethnicity unknown, Hx anemia (currently 12.1
range 12‐16). Mentzer’s index = 13.7 (indeterminate), ferritin = 87 (norm), iron = 50 (norm)
SickleDex = Negative
HbF = 1.2%HbA = 70.6%HbA2 = 28.2% A
FSC
NB
Audience Poll
• What is the most appropriate interpretation for these results?A. Normal profileB. Hemoglobin E traitC. Beta thalassemiaD. Elevated hemoglobin A2 with iron‐deficiency
anemia
Answer• Hemoglobin A2 is elevated. Elevations in hemoglobin A2 are
indicative of beta thalassemia, however beta thalassemia is never >10% HbA2.
• This patient has 28.2% HbA2, and indicates another hemoglobin variant comigrating on HPLC. Hemoglobin E migrates in HbA2 region on HPLC.
• Since patient has significant HbA, this is Hb E trait and not HbE disease.
• Hemoglobin E is most prevalent of Southeast Asian, Thai, Laos, Cambodian descent. Most people have no symptoms or mild anemia.
• As with other unstable hemoglobins, hemoglobin E trait will not show 50:50 ratio to HbA, but rather a 60:40 or even 70:30 split as seen in this patient.