title: Strategy linking several analytical methods of neonatal screening for sickle cell disease.
abstract: BACKGROUND
  The French national programme for the neonatal screening of sickle cell disease (SCD) was set up in 1995. This screening is targeted at newborn infants at risk. Over 5 years, 115,480 newborn infants were tested from 80 maternity departments from the northern part of the Paris area. 250 Patients with SCD were identified--that is, one in 462 newborn infants tested. Carriers for a haemoglobin (Hb) variant are frequent (5.34%). Some uncommon Hb variants were also identified, which gave rise to pitfalls to the testing when associated with HbS: HbKorle-Bu, HbHope, HbBougardirey-Mali, and HbLadésirade (4% of SS-like profiles).
  
  
  OBJECTIVE
  An effective screening strategy was developed to avoid these false positive and false negative responses.
  
  
  METHODS
  Isoelectric focusing (IEF), the method of primary screening, is rapid and inexpensive. Cation exchange high performance liquid chromatography (CE-HPLC), which is automated, fast, and quantitative was selected as a secondary method.
  
  
  RESULTS
  IEF diagnosed normal profiles in 89% of the tested samples from newborn infants. CE-HPLC identified most of the common Hb variants by their retention time and the measure of HbA/HbS ratio, important for the differential diagnosis between an asymptomatic HbS carrier and an HbS/beta+thal compound heterozygote. Furthermore, the high sensitivity of the CE-HPLC detected as little as 0.5% of a Hb variant. This avoided false negatives in samples from premature or transfused newborn infants. All samples with SS-like profiles were confirmed with a second CE-HPLC with another programme. A combination of these three methods confirmed the status of 99.7% of the samples from the tested newborn infants. Some cases required a reverse phase-HPLC method (for gamma-globin or alpha-globin chain variants). Finally, some exceptional samples required confirmation by testing DNA extracted with Güthrie paper for a precise diagnosis.
  
  
  CONCLUSIONS
  This effective strategy combining several methods dramatically reduces the risk of errors. Many families are thus spared unnecessary worrying recalls. The only unavoidable cause of false positives remains the HbS/hereditary fetal Hb (HPFH).
authors.affiliation: Laboratoire de Biochimie Génétique, Hôpital Robert Debré, 48 Boulevard Sérurier, 75935 Paris, France. rolande.ducrocq@rdb.hop-ap-paris.fr
authors.forename: R
authors.initials: R
authors.lastname: Ducrocq
authors.forename: O
authors.initials: O
authors.lastname: Pascaud
authors.forename: A
authors.initials: A
authors.lastname: Bévier
authors.forename: C
authors.initials: C
authors.lastname: Finet
authors.forename: M
authors.initials: M
authors.lastname: Benkerrou
authors.forename: J
authors.initials: J
authors.lastname: Elion
mesh_terms: D000755:Anemia, Sickle Cell / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003951:Diagnostic Errors; D006580:Genetic Carrier Screening; D005820:Genetic Testing / Q000379:methods; D014644:Genetic Variation; D006451:Hemoglobin, Sickle / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007231:Infant, Newborn; D015997:Neonatal Screening / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D010297:Paris; D012680:Sensitivity and Specificity
chemical_list: D006451:Hemoglobin, Sickle; D015345:Oligonucleotide Probes