revised January 9, 2001
Author: Peter A. Lane M.D.
Director, Colorado Sickle Cell Treatment and Research Ctr.
University of Colorado Health Sciences Ctr.

Newborn Screening for Hemoglobin Disorders

The demonstration in 1986 that prophylactic penicillin markedly reduces the incidence of pneumococcal sepsis (1) provided a powerful incentive for the widespread implementation of neonatal screening for sickle cell disease (2). Subsequent experience demonstrated that neonatal screening, when linked to timely diagnostic testing, parental education, and comprehensive care, markedly reduces morbidity and mortality from sickle cell disease in infancy and early childhood (3-5). This chapter provides an overview of the current status of neonatal screening for sickle cell disease in the United States and reviews issues related to the follow-up of neonatal screening results and diagnostic testing. Neonatal screening results indicative of non-sickle hemoglobinopathies, hemoglobinopathy carriers, alpha-thalassemias, and unidentified hemoglobin variants are also briefly discussed.


Forty-one states and the District of Columbia provide universal screening for sickle cell disease (6). Three states target screening to infants of high-risk ethnic groups. The primary purpose of screening is to identify infants with sickle cell disease, the most prevalent disorder included in neonatal screening panels (7). Screening also identifies infants with other hemoglobinopathies, hemoglobinopathy carriers, and in some states, infants with alpha-thalassemia syndromes. The majority of screening programs use isoelectric focusing (IEF) of an eluate from the dried blood spots also used to screen for hypothyroidism, phenylketonuria, and other disorders. A few programs use high performance liquid chromatography (HPLC) or cellulose acetate electrophoresis as the initial screening method. Most programs retest abnormal screening specimens using a second complimentary electrophoretic technique, HPLC, immunologic tests, or DNA-based assays (8-9).

The sensitivity and specificity of current screening methodology is excellent (6), and 99% of U.S. infants at highest risk for sickle cell disease are born in states with universal screening (7). However, neonatal screening systems are not foolproof. A few infants, even in states with universal screening, may not be screened. Other infants with sickle cell disease may not be detected because of mislabeled specimens, clerical errors, extreme prematurity, blood transfusion prior to screening, or the inability to locate affected infants after discharge from the nursery (4, 8, 10-14). All infants, including those born at home, must be screened and the initial screening test should always be obtained prior to any blood transfusion, regardless of age. The information requested on screening forms should be accurately and completely recorded to facilitate interpretation of results and follow-up in the event the result is positive. Normal neonatal screening test results cannot be presumed; the result must be documented for each infant. Finally, infants from high-risk ethnic groups born in states without routine neonatal screening or for whom neonatal screening results cannot be documented should be screened by hemoglobin electrophoresis, IEF, or HPLC prior to 2 months of age. Such infants include those of African, Mediterranean, Middle Eastern, (East) Indian, Caribbean, and South and Central American descent.

Hemoglobins (Hb) identified by neonatal screening are generally reported by level of expression as determined by the particular analytical procedure. Because more fetal hemoglobin (Hb F) than normal adult hemoglobin (Hb A) is present at birth, normal infants show Hb FA. Infants with hemoglobinopathies also show a predominance of Hb F at birth. Those with sickle cell disease syndromes show Hb S in absence of Hb A (FS), Hb S with another hemoglobin variant (e.g. FSC, FSDPunjab) or a quantity of Hb S greater than Hb A (FSA). Hundreds of other Hb variants are also identified. Many abnormal hemoglobins produce few or no clinical consequences while some such variants as Hb E can produce significant anemia. Hb E/beta-thalassemia often produces a severe, transfusion-dependent anemia. Many screening programs also detect and report Hb Bart's, indicative of alpha-thalassemia.


As shown in Table 1, a number of different neonatal screening results may indicate sickle cell disease syndromes (8, 15, 16). These include FS, FSC, FSA, and occasionally other phenotypes such as FSDPunjab. Hemoglobin FS in infancy reflects a variety of genotypes with a wide range of clinical severity. Most infants with FS screening results have homozygous SS, but other genotypes including sickle beta-0-thalassemia, sickle beta-(+)-thalassemia, sickle delta/beta-thalassemia, and sickle HPFH are possible. The co-inheritance of alpha-thalassemia may complicate differentiation of genotypes in some infants (17). Infants with positive screening tests should have confirmatory testing of a second blood sample prior to 2 months of age allowing prompt implementation of education, prophylactic penicillin, and comprehensive care (6, 8). In many states, where primary screening programs using hemoglobin electrophoresis (cellulose acetate and citrate agar) confirmation is done by IEF, HPLC, and/or DNA-based methods. Solubility tests are inappropriate, in part because high levels of fetal hemoglobin give false-negative results.

Table 1. Sickle Hemoglobinopathies: Neonatal Screening and Diagnostic Test Results.



Approximate % of US patients
Neonatal screening results 1
Hb separation by 2 months of age 1


Serial CBC, reticulocytes

Hematologic studies by 2 years



dot blot

Hb A23


Hb F


Hb F


Hemolysis and anemia by

6-12 months

N or I 4
beta s
Mild or no anemia by 2 years
N or D
NA 5
NA 6
beta s beta c
S beta-thal
FSA or FS 7
Mild or no anemia by 2 years
N or D
NA 6
beta A beta s
S betao-thal
Hemolysis and anemia by

6-12 months

D 4
beta A beta s
S delta/beta--thal
Mild anemia by 2 years
beta s
No hemolysis or anemia
N or D
beta s

Hb = hemoglobin, thal = thalassemia, N = normal, I = increased, D= decreased

Table shows typical results ˇ exceptions occur. Some rare genotypes (eg. SDPunjab, SO Arab, SC Harlem, S Lepore, SE) not included

  1. Hemoglobins reported in order of quantity (e.g. FSA = F>S>A)
  2. Normal MCV: >70 at 6-12 months, > 72 at 1-2 years
  3. Hb A2 results vary somewhat depending on laboratory methodology
  4. Hb SS with co-existent alpha-thalassemia may show decreased MCV and Hb A2 >3.6%; however neonatal screening results from such infants usually show Hb BartÝs.
  5. NA = not applicable ˇ quantity of Hb A2 can not be measured by hemoglobin electrophoresis or column chromatography in presence of Hb C.
  6. NA = not applicable ˇ test not indicated.
  7. Quantity of Hb A at birth sometimes insufficient for detection.
Hemolytic anemia and clinical signs and symptoms of sickle cell disease are rare before 2 months of age and develop variably thereafter as Hb F levels decline. Thus for infants with an FS phenotype, serial CBC and reticulocyte counts may not clarify the diagnosis during infancy. Parental testing and/or DNA analysis may be helpful in selected cases (8). In all cases, infants with Hb FS should begin penicillin prophylactics by 2 months of age, and parents should be educated about the importance of urgent medical evaluation and treatment for febrile illness and for signs and symptoms of splenic sequestration (6, 8).


As shown in Table 2, neonatal screening identifies some infants with non-sickle hemoglobinopathies (8, 18-23). Some of these conditions may be severe and require chronic blood transfusions. Infants who express solely Hb F could be normal infants who do not yet show Hb A because of prematurity. They could, on the other hand, have beta-thalassemia major or another thalassemic syndrome. Premature infants without Hb A need repeat testing to identify those with sickle cell disease and other hemoglobinopathies such as homozygous beta-thalassemia, a severe transfusion dependent disorder. Infants with FE require family studies, DNA analysis, or repeated hematologic evaluation during the first 1-2 years of life to differentiate homozygous Hb E, which is asymptomatic, from Hb E beta-0-thalassemia, which is variably severe (19-22). Importantly, most infants with beta-thalassemia syndromes (i.e. beta-thalassemia minor and beta-thalassemia intermedia) are not identified by neonatal screening.

Table 2. Non-sickle Hemoglobinopathies Identified by Neonatal Screening
Screening Results
Possible Condition
Clinical Manifestations
F only Homozygous betao-thalassemia  Premature Infant Severe thalassemia Repeat screening necessary
FE EE E betao -thalassemia Microcytosis with mild or no anemia Mild to severe anemia
FC CC C betao -thalassemia Mild microcytic hemolytic anemia Mild microcytic hemolytic anemia
FCA C beta +-thalassemia Mild microcytic anemia



The red cells of newborns with alpha-thalassemia contain hemoglobin Bart's, a tetramer of gamma globin. Many, but not all neonatal screening programs detect and report Hb Bart's (8, 18, 24, 25). As shown in Table 3, infants with Hb Bart's at birth may be silent carriers or have two-gene deletion alpha-thalassemia, Hb H disease (three-gene deletion alpha thalassemia), or Hb H Constant Spring disease. Silent carriers, the largest group with Hb Bart's at birth, have a normal CBC. Persons with two-gene deletion alpha-thalassemia generally show a decreased MCV with mild or no anemia. Newborns with >10% hemoglobin Bart's by IEF or >30% hemoglobin Bart's by HPLC and/or who develop more severe anemia may have Hb H disease or Hb H Constant Spring disease (26). More extensive diagnostic testing and consultation with a pediatric hematologist is generally needed for accurate diagnosis and appropriate treatment. The identification of Hb Bart's in Asian infants can have important genetic implications for couples who may be at risk for pregnancies complicated by hydrops fetalis (27).

Table 3. Alpha Thalassemia Syndromes Identified by Neonatal Screening
Screening Results
Possible Condition
Clinical Manifestations
FA+BartÝs Alpha-thalassemia silent carrier Alpha-thalassemia minor

Hb H disease

Hb H Constant Spring

Normal CBC

Microcytosis with mild or no anemia

Mild to moderately severe microcytic hemolytic anemia

Moderately severe hemolytic anemia

FAS+BartÝs FAC+BartÝs



Alpha-thalassemia with  structural Hb variant Clinical manifestations, if any, depend on the structural variant (e.g. Hb E) and severity of alpha thalassemia

Hb = hemoglobin


Approximately 50 infants who are carriers of hemoglobin variants (i.e. hemoglobin traits) are identified for every one with sickle cell disease. Initial confirmation by the screening laboratory using complimentary methodology can usually confirm the carrier state accurately. Some programs recommend testing of a second specimen from the infant for confirmation, in part to exclude the possibility of a mislabeled specimen.

Table 4. Hemoglobinopathy Carriers Identified By Neonatal Screening
Screening Results
Possible Condition
Clinical Manifestations
FAS Sickle cell trait Normal CBC Generally asymptomatic
FAC Hb C carrier No anemia Asymptomatic
FAE Hb E carrier Asymptomatic  Normal or slightly low MCV without anemia
FA Other Other Hb variant carrier Depends on variant. Most have no clinical or hematologic manifestations

Hb = hemoglobin

Carriers are generally asymptomatic (Table 4) and thus identification is of no immediate benefit to the infant. However, parents can benefit from knowing the child's carrier status, in part because the information may influence their decision on having more children. Therefore, parents of infants who are determined to be carriers through neonatal screening should be offered education and testing for themselves and their family (2, 4, 6, 8). Such testing may raise concerns about mistaken paternity and should not be performed without prior discussion with the mother. Testing of potential carriers requires a CBC and hemoglobin separation by hemoglobin electrophoresis, isoelectric focusing, and/or HPLC. Accurate quantitation of Hb A2 requires column chromatography or HPLC analysis. Assessment of HbA2 is needed to accurately identify patients with beta-thalassemia. Assay of Hb F by alkali denaturation, radial immune diffusion, or HPLC is also needed if the MCV is borderline or decreased.


Many of the more than 600 known hemoglobin variants are detected by current neonatal screening methods. Many are rare. Most are not identifiable by neonatal screening or clinical laboratories. Each year 10,000 to 15,000 infants with unidentified hemoglobin variants are detected by US neonatal screening programs (28, 29). The definitive identification of these variants is accomplished for fewer than 500 of these infants, due in part to limited reference laboratory capacity. Most infants are heterozygotes, and most will have no clinical or hematologic manifestations. However some variants, particularly unstable hemoglobins or those with altered oxygen affinity, can produce clinical manifestations even in heterozygotes. Other variants have no clinical consequences in heterozygous or homozygous individuals. Some, however, cause sickle cell disease when co-inherited with Hb S and thus have potential clinical and genetic implications (15).

The follow-up of these infants is problematic. The clinical uncertainty may cause frustration and anxiety for parents and health care providers. No national consensus exists to guide neonatal screening programs and clinicians in the follow-up of infants with unidentified hemoglobin variants. The following approaches may be considered.

If the infant is a heterozygote (i.e. the quantity of Hb A is equal to or greater than the quantity of the unidentified hemoglobin), the infant is well without anemia or neonatal jaundice, and the family history is negative for anemia or hemolysis, then no further hematologic evaluation may be necessary. Alternatively, some experts recommend repeating the hemoglobinopathy screen and/or obtaining a CBC, reticulocyte count, and peripheral smear at 9-12 months of age. Fetal hemoglobin (Hb Bart's) variants disappear by 1 year of age. The absence of anemia or hemolysis is reassuring for infants with hemoglobin variants that persist (alpha or beta-globin variants).

For some families, hemoglobin electrophoresis, IEF, or HPLC and/or CBC, blood smear, and reticulocyte counts on parents may be appropriate alternatives. With the exception of Hb Bart's variants, one of the parents is expected to show the same variant as the infant. The absence of any clinical history or laboratory evidence of anemia or hemolysis in the parent reassure the physician and family that the unidentified variant is unlikely to affect the infant's health adversely. In addition, this approach potentially identifies families with other hemoglobin variants (e.g. Hb S) for whom the definitive identification of the unidentified variant might have potential genetic implications.

Definitive hemoglobin identification can be performed by protein sequencing, DNA analysis and/or HPLC combined with electrospray mass spectrometry in a specialized reference laboratory (30). Such testing is indicated for infants with clinical or laboratory evidence of hemolysis or abnormal oxygen affinity and for infants without Hb A (homozygotes or compound heterozygotes), especially if the unidentified variant is inherited with Hb S (15, 31). Identification of the hemoglobin variant to clarify genetic risks should also be considered in families where another hemoglobin abnormality (e.g. Hb S) is present.


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