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

james.h.nichols@vanderbilt.edu

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

SickleDex = Positive

HbF = 33.8%HbA = <1%HbA2 = 2.7%HbS = 62.5%

A

NB

SF

C

Audience Poll

• What do these results indicate?A. Normal profileB. Abnormal amounts of hemoglobin FC. Sickle cell diseaseD. Sickle cell trait

Hemoglobin Tetramer

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

Globin Chain Expression

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

Isoelectric Focusing(IEF)

H/Barts AFS C

A D

EA

NB

+

Application

-

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

Hydroxyurea Therapy

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

Alpha Thalassemia

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.