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Icsh recommendations for the measurement of haemoglobin f

International Journal of Laboratory Hematology
The Official journal of the International Society for Laboratory Hematology I N T E R NATI O N A L J O U R NA L O F L A B OR ATO RY H E M ATO LO G Y ICSH recommendations for the measurement of Haemoglobin F A. D. STEPHENS*, M. ANGASTINIOTIS†, E. BAYSAL‡, V. CHAN§, B. DAVIS–, S. FUCHAROEN**,P. C. GIORDANO††, J. D. HOYER‡‡, A. MOSCA§§, B. WILD–– ON BEHALF OF THE INTERNATIONALCOUNCIL FOR THE STANDARDISATION OF HAEMATOLOGY (ICSH) College London Hospitals, London, UK†Thalassaemia International Federation Measurement of the Haemoglobin F in red cell haemolysates is important in the diagnosis of db thalassaemia, hereditary persis- tence of fetal haemoglobin (HPFH) and in the diagnosis and man- ticsCentre,AlWasiHospital,Dubai,UAE§Department of Medicine, The agement of sickle cell disease. The distribution of Hb F in red cells is useful in the diagnosis of HPFH and in the assessment of feto- maternal haemorrhage. The methods of quantifying Hb F are described together with pitfalls in undertaking these laboratory tests with particular emphasis on automated high-performance liquid chromatography and capillary electrophoresis.
Mahidol University, Thailand††Hemoglobinopathies ReferenceLaboratory, Human and ClinicalGenetics Department, LeidenUniversity Medical Center, Leiden, TheNetherlands‡‡Division of Hematopathology, TheMayo Clinic College of Medicine,Rochester, MN, USA§§Dipartimento di Scienze e TecnologieBiomediche, Universita` degli Studi diMilano, Italy––UK NEQAS(H), Watford, UK Correspondence:Dr A. D. Stephens, Department ofHaematology, University College LondonHospitals, 250 Euston Road, LondonNW1 2PJ, UK.
Tel.: 020 344 79638;Fax: 020 344 79911;E-mail: adrian.stephens@mac.com Received 13 April 2011; accepted forpublication 13 June 2011 KeywordsHaemoglobin F, high-performanceliquid chromatography, capillaryelectrophoresis, recommendedmethods, haemoglobinopathy,thalassaemia Ó 2011 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
assessment of the proportion of red cells of fetal originis clinically useful in assessing the amount of FMH that It is now 30 years since the first ICSH recommenda- has occurred and the medication to be given. Assessing tions were published (International Committee for the proportion of cells that contain a high proportion of Standardization in Haematology, 1979) concerning the fetal haemoglobin (often called F cells) is also useful in quantification of Haemoglobin F (Hb F); and during the diagnosis of ‘classical’ deletional HPFH.
this time, there have been several new analytical devel-opments in the field, and therefore the ICSH Boardconsider that the original recommendations should be revised. Hb F is a haemoglobin tetramer composed of In 1866, it was noted that fetal haemoglobin is more two a and two c globin chains (a2c2). It is a normal resistant to alkali than adult haemoglobin (von Kor- haemoglobin and is the major haemoglobin present in ber), but it was not until 1951 (Singer, Chernoff & the fetus but is gradually replaced after birth by Hb A Singer, 1951) that this property was utilized to measure (a2b2) as the c chains are replaced by b chains. The the amount of fetal haemoglobin present in red cell measurement of Hb F in red cells is clinically useful in haemolysates (the one-minute alkali denaturation the diagnosis of db thalassaemia because the amount of technique). This technique was refined by in 1959 Hb F is raised in this condition. Hb F is also slightly (Betke, Marti & Schlicht, 1959) when they published raised (1–5%) in pregnancy (Pembrey, Weatherall & their two-minute alkali denaturation technique and Clegg, 1973), in hereditary persistence of fetal haemo- this was further modified in 1972 (Pembrey, Mcwade & globin (HPFH) and sometimes in b thalassaemia trait. It Weatherall, 1972). This two-minute alkali denaturation is occasionally raised in other situations (Table 1), but technique has been considered to give accurate and apart from sickle cell disease and feto-maternal haem- clinically useful results in the 0–40% range of Hb F but orrhage (FMH), there is little clinical utility in measur- to underestimate the amount of Hb F at higher levels; ing the Hb F in these other situations (Mosca et al., although in clinical situations, this is not a problem.
2009). The clinical effects of sickle cell disease can be This method was selected by the ICSH as the recom- reduced by using drugs such as hydroxyurea (hydroxy- mended method for clinical use (International Com- carbamide) which raise the Hb F level and measuring mittee for Standardization in Haematology, 1979), and the Hb F level can then be clinically useful in helping in the same article, a method was described for prepar- ing a standard for Hb F. The method for producing a HbF standard was further modified by Wild (1986) to pro- Table 1. Conditions in which Hb F is raised duce the 1st WHO International Reference Material forHb F (World Health Organization Expert Committee on Biological Standardization, 1994) for use with the two- minute alkali denaturation method. This is a stabilized, freeze-dried cyanmethaemoglobin preparation pre- pared from blood obtained from the father of a patient with beta thalassaemia major and is held at National Institute of Biological Standards and Controls (NIBSC) Hereditary persistence of fetal haemoglobinSickle cell anaemia ± treatment with In 1958, column chromatography of haemolysates from human blood showed that fetal haemoglobin could be separated from adult haemoglobin and that a small proportion of the fetal haemoglobin separated from the main Hb F peak (Allen, Schroeder & Balog, 1958), and it was later shown that this small peak was acetylated Hb F (Schroeder et al., 1962). Becausethe acetylated Hb F eluted before the main peak, it was called FI and the main peak FII, but the main Ó 2011 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
peak was later renamed Fo when the main peak of may mislead analysts into believing that the accuracy adult haemoglobin was renamed Ao. It was later is also good. It is important that the sample loading is shown that these two types of fetal haemoglobin within the manufacturer’s recommended range as not could also be separated by isoelectric focusing (Basset only must the column not be overloaded, but it is et al., 1978). Automated, dedicated high-performance essential that the optical absorbance is kept within the liquid chromatography (HPLC) was introduced in the linear range because if too much sample is applied late 1980s to simplify and speed up screening of any large peaks may be underestimated leading to a maternal blood samples for Hb A2, Hb F and common relative overestimation of small peaks such as the Hb haemoglobin variants (such as Hb S, C, D, E and O).
F. It is equally important that the baseline used for Recently, automated capillary zone electrophoresis the integration is correct as only those parts of the (CZE) has been introduced as an alternative to auto- peak that are above the baseline will be quantified, mated HPLC for screening blood samples for Hb A2, and this may lead to parts of a peak being excluded Hb F and the common variant haemoglobins (Van from the integration if the baseline is incorrectly Delft et al., 2009). The c-chains of Hb F may contain assigned. As a general rule, with the equipment used either glycine or alanine at position 136 (cG and cA) for quantifying Hb F, the baseline should be straight because of the presence of two c genes (cG and cA) and close to the horizontal. Ideally, the baseline coding for almost identical products, but these two should be obtained by running a blank sample twice forms do not separate on HPLC or CZE, but at the (so that there is no haemoglobin present) at the present time, there is no clinical advantage in measur- beginning of each batch and recording the chromato- ing the cG & cA chains separately. A radial immuno- gram produced with the second blank (to avoid any diffusion technique has also been developed and is carry over), and this should be used as the baseline commercially available. With the huge increase in for that batch of samples. If a haemoglobin peak (such throughput of Hb A2 and Hb F measurements in as Hb H) elutes with the void volume and if as occurs hospital laboratories because of both the mandatory with some systems, the integration excludes the ‘void and the voluntary screening programmes, automated peak’; then, in the presence of Hb H, the total amount HPLC or CZE have become the main tool to quantify of haemoglobin will be underestimated leading to an Hb F and Hb A2 in most laboratories in Europe and overestimation of the Hb F peak. Accurate quantifica- North America and is being increasingly used in Asia tion of a peak also requires that that peak is com- and Africa, and capillary isoelectric focusing (cIEF) is pletely separated from neighbouring peaks, and this is also being introduced. However, dedicated instru- especially important for small peaks such as Hb F. For ments are available from different companies, and all these reasons, it is extremely important that the these approach the analytical issues in different ways.
chromatogram, or electropherogram, is inspected care- When it is necessary to assess the proportion of red fully before the results are authorized. Because Hb F is cells that are of fetal origin either a cytochemical tech- reported as a ratio of Hb F to the total haemoglobin, as nique such as the Kleihauer method (Kleihauer, long as there is an appropriate baseline associated with Braun & Betke, 1957; Kleihauer, 1974) or the good peak integration, area calibrators should not be Shepard modification of it (Shepard, Weatherall & necessary as they are a poor substitute for inadequate Conley, 1962) or flow cytometry can be used (Nelson chromatography or peak quantification. However, et al., 1998; Leers et al., 2007). These techniques can standards and controls remain essential to verify that also be used to study cases of HPFH.
the equipment is working satisfactorily. Although forHPLC and capillary isoelectric focusing it is the peakareas that are related to the amount of haemoglobin present, it should be noted that in CZE the peak areas With automated systems such as HPLC, CZE and cIEF, should not be used for quantification because of the the accuracy of peak quantification is dependent on different migration velocities through the detector, both the peak resolution and the method of peak inte- instead the ‘spatial area’ (integrated area divided by gration. Poor peak quantification will result in poor the migration time) should be used (Huang, Coleman accuracy but may still result in good precision that & Zare, 1989; Hempe & Craver, 1999).
Ó 2011 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
S A M P L E C O L L E C T I O N A N D S T O R A G E cut-off in adults, this is not a problem. Nevertheless,CVs of the order of 2–3% can be obtained at higher Any anticoagulant can be used although it is common Hb F concentrations (Papadea & Cate, 1996; Paleari to use K2EDTA which is the anticoagulant used for et al., 2005). Accuracy is harder to establish and moni- the analysis of blood counts. Ideally, the analysis tor but comparison with National or Regional quality should be undertaken as soon as possible after vene- assessment schemes can be helpful. A WHO reference section, but storage of a sealed whole blood sample at material is available, but it was only analysed by the 4 °C for 2–3 weeks is acceptable as there will be mini- two-minute alkali denaturation technique.
mal oxidation at 4 °C during that time (Tietz, 1990).
Where there are high ambient temperatures, such asin tropical areas, suitable means of transport must be H I G H - P E R F O R M A N C E L I Q U I D used to prevent deterioration because considerable denaturation of haemoglobin occurs if a sample is Several companies now produce dedicated HPLC sys- kept at 50 °C for more than 1 h. Sample stability after tems (Van Delft et al., 2009). These are all similar in freezing at )20 °C or at )80 °C is not well docu- that they all utilize a weak cation-exchange column in mented, although some manufacturers claim an over- their instruments. A sample of RBC lysate is injected all stability of 1 month at )20 °C and 3 months at into the system; haemoglobin molecules will attach to )80 °C. When freezing samples, it is essential that the the column as they are charged molecules in the buffer blood samples are frozen as quickly as possible system used. An eluting solution is then injected into because slow freezing of proteins promotes denatur- the system; the composition of this solution changes by ation; one satisfactory way is to freeze drops of whole increasing the ionic strength. The haemoglobin frac- blood or haemolysate in liquid nitrogen. Thawing and tions (normal plus any haemoglobin variant) will elute re-freezing of blood samples should be discouraged. A off the column when the ionic strength of the eluting partially coagulated blood sample should not be solution is greater than the retention to the column.
accepted for Hb F quantification by automated equip- Different haemoglobin variants will have different ment as it may contaminate or block the tubing.
overall charges because of the amino acid substitutionthat is present. Thus, the time at which the haemoglo- bin molecule elutes off the column (retention time) ischaracteristic and reproducible, but not unique, for To be clinically useful, the results of the Hb F quantifi- each haemoglobin variant. As the haemoglobin frac- cation have to be expressed as a percentage of the total tions elute off the column, they pass through a detec- haemoglobin (which will usually include Hb A, Hb F, tion system which utilizes absorbance readings at 415 Hb A2 and any glycated, acetylated or aged adducts).
and 690 nm. The percentage of each haemoglobin frac-tion (Hb A, Hb F, Hb A2, and any variant) is calculatedby summing the area under the curve of each peak of the chromatogram. Glycated haemoglobins, such as Hb As stated earlier, both precision and accuracy are AIc, and methaemoglobin elute from the column as extremely important because a raised level of Hb F separate peaks distinct from and before Hb A as does may be indicative of the diagnosis of HPFH or delta the ‘aged peak’ AId. Fast variants, such as Hb H or Hb beta thalassaemia trait. The precision obtainable in the Barts, may not be quantified as they may elute off the manual techniques is ±0.1% in the final answer that is column before the instrument begins to integrate equivalent to an SD of 0.05% (leading to a CV of 5% in many systems designed for adult samples, and this in the normal range and better when raised as often will affect the quantification of Hb F. However, they occurs in thalassaemia). A similar precision should be are usually quantified in systems designed for neonatal obtainable in modern automated HPLC and capillary samples that start integrating sooner.
electrophoretic equipment and is sufficient for diag- nostic purposes. Some HPLC systems are not able to sufficient accuracy for clinical purposes as long as the detect Hb F below 1% but because 1% is the normal Hb F peak is adequately separated from the other Ó 2011 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
peaks and from any variant haemoglobins present.
sures the absorbance at 415 nm. An electropherogram Because the FI peak is only a very small proportion of (similar to a chromatogram) is thus generated; the per- the total Hb F, it is only apparent on neonatal centage of each haemoglobin fraction (Hb A, Hb F, Hb samples, or adult samples with fairly large amounts of A2, plus any haemoglobin variants) is calculated (but Hb F. If an alpha globin variant is present that see the paragraph aforementioned on peak integration separates from Hb A, then there will be a Hb F variant because ‘spatial areas’ have to be used for quantifica- that will often separate from normal Hb F but it may tion because of the different velocities of peaks past not separate from the other haemoglobin adducts the detector). For haemoglobinopathy, work CZE has present and then the total Hb F will not be adequately the same advantages as HPLC over manual methods quantified. Hb F variants may also be due to muta- but some equipment and reagents have an advantage tions in the c globin chain, and again this may result over HPLC in that haemoglobin adducts such as glycat- in a separate peak and incorrect quantification. Some ed XIa,b,c and the ageing glutathione adduct, XId, do b-chain variants and/or their adducts will not separate not separate from the main haemoglobin peaks, and from Hb F, and this will also lead to incorrect quanti- this makes interpretation easier than with HPLC.
fication. In the absence of a haemoglobin variant, theHb F separates from Hb A, its glycated adducts and C A P I L L A R Y I S O E L E C T R I C F O C U S S I N G the Hb A2, and therefore its quantification should besatisfactory. The presence of any haemoglobin variant In cIEF, the electro-osmotic flow is minimized by coat- eluting before Hb Ao on HPLC will frequently lead to ing the inside of the capillary with a solution to neu- inaccurate quantification. In these situations, a differ- tralize the silanols. The capillary is filled with a ent technique such as the two-minute alkali denatur- mixture of ampholytes and haemolysate. After applica- ation method will give a clinically useful result.
tion of the voltage for a period of time, the haemoglo-bins become focussed at their pI value. They are thenmobilized and pass through the detector operating at C A P I L L A R Y Z O N E E L E C T R O P H O R E S I S 415 nm (Hempe & Craver, 1999), and an electrophero- This method utilizes a thin capillary made of fused gram (similar to a chromatogram) is thus generated; silica with an outer coating of polyimide and usually the percentage of each haemoglobin fraction (Hb A, Hb with an inner diameter of 50 or 75 lm. Because the F, Hb A2, plus any haemoglobin variants) is calculated.
capillary has a very large surface to volume ratio, it isexcellent at dissipating the heat generated by the C Y T O C H E M I C A L T E C H N I Q U E applied voltage. Thus, very large voltages (10–30 kV)can be used and because of these high voltages, the For semi-quantitative estimation of the distribution of fetal run times are significantly shortened and the resolu- Hb in the red cell population. A blood smear is prepared tion increased. The inner surface of the capillary tube on glass slide, fixed in 80% ethanol and then immersed has a negative charge because of the bare silica. When for 5 min at 37 °C in citrate-phosphate buffer pH 3.3 an electric field is applied, the buffer solution within (citric acid 0.075 M and disodium phosphate 0.05 M). The the capillary generates an electro-endosmotic flow elution time and pH of the buffer have to be carefully con- (EOF) that moves towards the cathode. A sample of trolled (Shepard, Weatherall & Conley, 1962; Kleihauer, haemolysate is injected into the system, and the 1974). In patients with deletional HPFH, all RBCs show electric current applied causes separation of individual almost uniform retention of haemoglobin (pancellular haemoglobins because of differences in overall charges.
distribution), whereas in b thalassaemia trait with Haemoglobin variants, if present, separate because of a increased Hb F, and nondeletional HPFH, a hetero- charge difference resulting from the amino acid substi- cellular distribution of haemoglobin occurs.
tution. However, regardless of the overall charge ofeach haemoglobin fraction, the EOF is still stronger than any attraction to either pole, and all haemoglobinfractions will move towards the cathode. All the hae- Flow cytometric assay can be used to assess the moglobin molecules move past a detector which mea- number and distribution of ‘F cells’ in the blood.
Ó 2011 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
Adult F cells are erythrocytes where the haemoglobin commercially with associated standards covering the is represented by a mixture of Hb A and Hb F (Wood et al., 1975). All individuals have some F cellsestimated at an average of 2.7% of cells in normal I N T E R P R E T A T I O N O F R A I S E D L E V E L S adults by an earlier nonflow cytometric fluorescei- nated antibody technique (Davis et al., 1998). Flowcytometric techniques suggest that this level may be At birth, the average level of Hb F is around 80% but even higher in normal individuals (Chen, Bigelow & depends on the age of gestation. After birth, the Hb F Davis, 2000). F cells can be more prevalent in anaemia steadily decreases to reach adult levels (<1%) during and in some haemoglobinopathies. The enumeration of the second year of life. In thalassaemia trait levels of ‘light pink’-staining F cells vs. ‘deep red’-staining fetal up to 10% may be recorded depending on the muta- cells by the traditional Kleihauer slide test may be tion and the haplotype. In db thalassaemia trait levels difficult (CLSI, 2001). Quantification of F cells may be from 5% to 20% are found. In HPFH heterozygotes, helpful in the management of patients with sickle cell Hb F levels from 2.5% in the ‘Swiss’ type and to up to disease treated with hydroxyurea and also when 30% in the deletional forms are found. In homozygous monitoring patients with myelodysplasia. However, deletional HPFH and in homozygous nontransfused bo measurement of the total Hb F in a haemolysate by thalassaemia major, the Hb F level will be 100%. Ery- HPLC or CZE is usually easier and clinically just as use- thropoetic stress and malignancies may induce variable ful. Commercial assays have been developed for under- As is the case with all clinical assays, the use of internal quality control is important. Controls cannotonly be used to assess the quality of Kleihauer staining It is important to be able to quantify Hb F for the but are also used to better define the region of analysis diagnosis of db thalassaemia and HPFH and in sickle positive for fetal cells in flow cytometry. Controls can cell disease at diagnosis, and whilst monitoring the be made locally in the laboratory, using artificial mix- effect of hydroxycarbamide (hydroxyurea) in sickle tures of fresh cord blood and adult blood samples, or a cell disease or b thalassaemia major or intermedia.
commercial preassayed product can be used. Quality The accuracy and precision of Hb F quantification in performance of the FMH or fetal red cell detection blood samples with Hb F levels in, or very close to, assays are described in the CLSI H52-A document the normal range is often poor, but this is rarely a (CLSI, 2001). This lists potential problems with the diagnostic problem. Hb F levels of 2–3% calculated in Kleihauer and flow cytometry methods.
HPLC areas crowded with glycated fractions should becritically examined for overestimation. Assessment ofthe cellular distribution is useful in the diagnosis of T W O - M I N U T E A L K A L I D E N A T U R A T I O N HPFH and when investigating suspected FMH. Meth- ods used in clinical laboratories first involve separating If a manual technique is required, the two-minute the Hb F from Hb A and Hb A2 and from any alkali denaturation method (Betke, Marti & Schlicht, other haemoglobin present and then quantifying the 1959) as modified by Pembrey, Mcwade and Weather- different fractions and calculating the proportions of all (1972) is still a useful way of assessing the Hb F the different haemoglobins because it is the propor- concentration in clinical situations but has the disad- tion, not the absolute amount, of Hb F that is clini- vantage that it is time consuming and requires experi- cally important. Historical methods of analysis. The two-minute alkali denaturation technique (Pembrey,Mcwade & Weatherall, 1972; International Committeefor Standardization in Haematology, 1979) which R A D I A L I M M U N O D I F F U S I O N T E C H N I Q U E were discussed in a previous publication can produce Haemoglobin F can be quantified by radial immuno- results that are satisfactory for most clinical purposes diffusion. Plates containing anti-Hb F are available but are time consuming and need experience to Ó 2011 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
obtain good results. Automated HPLC with dedicated cially available capillaries and buffers are becoming available and can also produce satisfactory results if increasingly being introduced and can give satisfactory no haemoglobin variant is present, but considerable results if no haemoglobin variant is present, but experience is necessary to interpret the electro- considerable experience is necessary to interpret the pherogram. At present, the only internationally recog- chromatograms. If any haemoglobin variants are nized standard for Hb F is the WHO 1st Reference present, the analysis may lead to misleading results. It Material (85/616) that is available from NIBSC, is essential that the reagents are optimized for Hb F Blanche Lane, South Mimms, Potters Bar, Hertford- rather than Hb AIc and that the baseline is appropri- shire EN6 3QG, UK (http://www.nibsc.ac.uk), but it was only analysed by the two-minute alkali denatur- automated capillary isoelectric focusing with commer- fetal haemoglobin reference preparations and denaturation. Journal of Clinical Pathology fetal haemoglobin determination by the alkali Allen D.W., Schroeder W.A. & Balog J. (1958) denaturation method. British Journal of Hae- Pembrey M.E., Weatherall D.J. & Clegg J.B.
Observations on the chromatographic hetero- (1973) Maternal synthesis of Haemoglobin F Kleihauer E. (1974) Determination of fetal in pregnancy. Lancet 1, 1350–1355.
hemoglobin: a study of the effects of crystalli- zation and chromatography on the heteroge- Detection of Hemoglobinopathies (eds R.M.
neity and isoleucine content. Journal of the Schmidt, T.H.J. Huisman & H. Lehmann), acetyl-containing hemoglobin. Biochimica et American Chemical Society 80, 1628–1634.
20–22. CRC Press, Cleveland, Ohio.
Basset P., Beuzard Y., Garel M.C. & Rosa J.
Kleihauer E., Braun H. & Betke K. (1957). cited Shepard M., Weatherall D. & Conley C. (1962) (1978) Isoelectric focusing of human hemo- Semi-quantitative estimation of the distribu- globin: its application to screening, to the von Korber E. (1866). cited by Singer K, Cher- tion of fetal hemoglobin in red cell popula- characterization of 70 variants, and to the tions. Bulletin of the Johns Hopkins Hospital study of modified fractions of normal hemo- Leers M.P., Pelikan H.M., Salemans T.H., Giord- ano P.C. & Scharnhorst V. (2007) Discrimi- Singer K., Chernoff A.I. & Singer L. (1951) Betke K., Marti H.R. & Schlicht L. (1959) Esti- Studies on abnormal haemoglobins: 1. Their mation of small percentage of fetal haemoglo- Chen J.C., Bigelow N. & Davis B.H. (2000) Pro- of alkali denaturation. Blood 6, 413–428.
posed flow cytometric reference method for Reproductive Biology 134, 127–129.
Tietz N., Finley P.R. & Pruden E.L. (1990) Clini- the determination of erythroid F-cell counts.
Mosca A., Paleari R., Leone D. & Ivaldi G.
cal Guide to Laboratory Tests. 2nd edn W.B.
(2009) The relevance of hemoglobin F mea- surement in the diagnosis of thalassemias and Van Delft P., Lenters E., Bakker-Verweij M., De Korte M., Baylan U., Harteveld C.L. & Giord- ano P.C. (2009) Evaluating five dedicated Davis B.H., Olsen S., Bigelow N.C. & Chen J.C.
Nelson M., Zarkos K., Popp H. & Gibson J. (1998) (1998) Detection of fetal red cells in fetoma- A flow-cytometric equivalent of the Kleihauer diagnostics in multi-ethnic populations. Inter- ternal hemorrhage using a fetal hemoglobin national Journal of Laboratory Hematology Paleari R., Cannata M., Leto F., Maggio A., Demartis F.R., Desogus M.F., Galanello R. & Wild B.J. (1986). The development of reference Hempe J.M. & Craver R.D. (1999) Laboratory Mosca A. (2005) Analytical evaluation of the materials for Hb A2 and Hb F. PhD, Brunel.
diagnosis of structural hemoglobinopathies Wood W.G., Stamatoyannopoulos G., Lim G. & and Thalssemias by capillary isoelectric focus- for hemoglobin A2 and F determination.
Nute P.E. (1975) F-cells in the adult: normal ing. In: Clinical Applications of Capillary Clinical Biochemistry 38, 159–165.
values and levels in individuals with heredi- Electrophoresis (ed. S.M. Palfrey), 81–98.
Papadea C. & Cate J.C.T. (1996) Identification tary and acquired elevations of Hb F. Blood and quantification of hemoglobins A, F, S, Huang X., Coleman W.F. & Zare R.N. (1989) and C by automated chromatography. Clini- World Health Organization Expert Committee Analysis of factors causing peak broadening on Biological Standardization (1994). Biologi- in capillary zone electrophoresis. Journal of Pembrey M.E., Mcwade P. & Weatherall D.J.
(1972) Reliable routine estimation of small WHO Technical Report Series – 848. World International Committee for Standardization in Ó 2011 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.

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