title: Mass spectrometric analysis of unknown haemoglobin fractions identified by high-performance liquid chromatography in an antenatal screening programme.
abstract: OBJECTIVE
  The majority of haematology laboratories use automated high-performance liquid chromatography (HPLC) as a primary method in the screening of samples for haemoglobin variants and thalassaemia. HPLC detects adducted fractions, such as HbA1C and other peaks of unknown significance. The National Health Service (NHS) Sickle and Thalassaemia Screening Programme does not mandate further assessment of these unknown peaks, but their presence may cause concern or precipitate detailed investigations. Mass spectrometry (MS) is being increasingly used in NHS Laboratories and is an effective method of characterising variant haemoglobins.
  
  
  METHODS
  Antenatal blood samples accepted for NHS Sickle and Thalassaemia Screening Programme from 2005 to 2013 were assessed. Those samples with unidentified peaks on HPLC were further analysed by Liquid chromatography tandem MS (LCMSMS).
  
  
  RESULTS
  58,493 samples were processed. 966 (1.6%) significant haemoglobinopathies were identified. 68 patients (0.11%) were found to have unidentified peaks on HPLC. α chain variants: two were hyperunstable α variants, Hb Adana and Hb H/Constant Spring. The patient with HbH/Constant Spring required transfusion during pregnancy. Other abnormalities include 24 unstable α chain variants of minimal clinical significance and 17 α chain variants of no clinical significance. The frequency of β chain abnormalities was lower, with 10 patients having an insignificant β chain variant, 4 mildly unstable β chain variants (2 of whom had increased oxygen affinity) and 4 with variants associated with increased oxygen affinity.
  
  
  CONCLUSIONS
  The prevalence of unidentified variants on automated HPLC within our population is 0.1%. LCMSMS is an effective technique to assist in the characterisation of unknown haemoglobin variants. α chain variants of limited clinical significance were the most commonly detected abnormality.
authors.affiliation: Haematology Department, Royal Hallamshire Hospital, Sheffield, UK.
authors.forename: M
authors.initials: M
authors.lastname: Griffin
authors.affiliation: Haematology Department, Royal Hallamshire Hospital, Sheffield, UK.
authors.forename: J
authors.initials: J
authors.lastname: Eyre
authors.affiliation: Haematology Department, Victoria Hospital, Kirkcaldy, UK.
authors.forename: S
authors.initials: S
authors.lastname: Dack
authors.affiliation: Haematology Department, Royal Hallamshire Hospital, Sheffield, UK.
authors.forename: J
authors.initials: J
authors.lastname: Wright
mesh_terms: D000755:Anemia, Sickle Cell / Q000097:blood / Q000175:diagnosis* / Q000453:epidemiology / Q000235:genetics; D015415:Biomarkers / Q000097:blood; D002851:Chromatography, High Pressure Liquid; D004739:England / Q000453:epidemiology; D005260:Female; D020022:Genetic Predisposition to Disease; D014644:Genetic Variation; D006454:Hemoglobins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D011237:Predictive Value of Tests; D011247:Pregnancy; D011250:Pregnancy Complications, Hematologic / Q000097:blood / Q000175:diagnosis* / Q000453:epidemiology / Q000235:genetics; D011296:Prenatal Diagnosis / Q000379:methods*; D015995:Prevalence; D021241:Spectrometry, Mass, Electrospray Ionization; D013222:State Medicine; D053719:Tandem Mass Spectrometry; D013789:Thalassemia / Q000097:blood / Q000175:diagnosis* / Q000453:epidemiology / Q000235:genetics
chemical_list: D015415:Biomarkers; D006454:Hemoglobins
keywords: HAEMATOLOGY; HAEMOGLOBINOPATHY; LABORATORY TESTS; MATERNAL-FOETAL