Clin PatholThe thalassaemia syndromes are inherited disorders of haemoglobin (Hb) production, characterised by a reduction in globin chain synthesis leading to an imbalance ofthe glo-bin

Clin Pathol 1994;47:289-295

Leaders~~~~~~~~~~~~~. '.. ..:.

Guidelines for investigation of the a and /Bthalassaemia traits

The Thalassaemia Working Party of the BCSH General Haematology Task Force

These Guidelines should beread in conjunction with theGuidelines for Fetaldiagnosis of Globin GeneDisordersThe Members of theWorking Party were:B BainM BhavnaniM Brozovic (chair)JM OldA D StephensThe members of the BCSHGeneral Haematology TaskForce were:B BainM Brozovic (Chair)DW DawsonJM EnglandRM RowanA D StephensS M LewisN K ShintonM F MurphyJ K Wood

Correspondence to:Dr A D Stephens,Department ofHaematology, StBartholomew's Hospital,London EClA 7BC

Accepted for publication7 September 1993

IntroductionThe thalassaemia syndromes are inheriteddisorders of haemoglobin (Hb) production,characterised by a reduction in globin chainsynthesis leading to an imbalance of the glo-bin chains. There are many different DNAmutations leading to these disorders (seeguidelines for the fetal diagnosis of the globinchain disorders') but it is helpful to subdividethem into two groups: a' and,B° in which no

globin is synthesised and a+ and ,(+ in whichsome, but a reduced amount, of globin is syn-thesised. Although most patients have eithera or fi thalassaemia these may interact witheach other (afi thalassaemia), with thalas-saemia (Qfl thalassaemia), and with Hb vari-ants such as Hb S, Hb E, Hb O-b, and HbLepore. Although most cases of hereditarypersistence of fetal Hb (HPFH) have no clini-cal or genetic implications a few are associ-ated with a fi thalassaemia-like phenotype andcan occasionally interact with a fi thalas-saemia trait to produce a fi thalassaemia inter-media phenotype. Cases with thalassaemicfeatures may be genetically and pheno-typically related to 5fl thalassaemia.

Although the carrier states for these condi-tions are clinically silent, the homozygote anddouble heterozygote states may have majorclinical implications and it is therefore essen-

tial to be able to detect and identify the car-

rier states shown in the table. Knowledge of

the carrier state can also be useful in explain-ing hypochromic, microcytic red cell indices.The thalassaemia disorders are common in

people of Mediterranean, Middle Eastern,African, Pakistani, Indian, and South EastAsian ancestry, but occur sporadically in allpopulations. In our increasingly multiculturalsociety it is becoming important to considerthe diagnosis of a thalassaemia trait regardlessof the apparent ethnic origin. The universalavailability of electronic cell counters pro-

vides the haematologist with a simple screen-

ing procedure that can be used to selectpatients for further investigation.The purposes of these guidelines are to:(a) advise on which people should be

tested and on how the diagnosis should beestablished.

(b) Document the routine proceduresavailable in most laboratories which are nec-

essary to make the diagnosis. DNA analysis iscovered in separate guidelines.'

(c) Provide useful addresses for coun-

selling, haemoglobinopathy cards, etc.

Selection of patients to be investigatedInvestigation is usually carried out for geneticpurposes-that is, to identify those at risk ofproducing offspring with a clinically signifi-cant form of thalassaemia (homozygous or

doubly heterozygous) such as fi thalassaemia

Characteristics of thalassaemia

UsualHb H

Phenotype Genotype MCV MCH Hb A2 bodies

a Thalassaemia trait -a/aa a+/a N N Na Thalassaemia trait -,a/-a or l--/aa a+/a' or a°/a 4 4 N or 4 +Hb H disease (mild) --/a- a'/a+ 4 4 N or 4 +++Hb H disease (severe) --/aaT aY/aT 4 4 N or 4 +++Hb Barts hydrops ---- a'/a4

(a thalassaemia major)/1 Thalassaemia trait #f/fl or fl+/fl T -

5,f Thalassaemia trait bfl/fl N or 4 -

f, Thalassaemia trait (normal Hb A2) f+I/fl N -

Hb Lepore trait Hb Lepore/fl N -

f, Thalassaemia intermedia Heterogeneous 4 4 1, N or T -,B Thalassaemia major fl0/fl0,flo+, fl+/fl+ 4 4 1, N or T -

For a thalassaemia: There are two pairs of allelic structural genes that code for the a globin chains and "classical" a thalassaemiadeletion (a-) but non-deletional forms (aar) also occur that may vary in severity and more than 30 genotypes have been charac-terised. The above is a classification of a thalassaemia that indicates two ways of expressing the genotype.For fl thalassaemia: There is only one pair of allelic genes that code for the /1 globin chains but there are more than 100 DNAsubtypes of fi thalassaemia, most of which are non-deletional.N = normal.

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A simpleflow chartfordiagnostic tests

*Refer to Estimation ofHb A2, point 10.

major, Hb Barts hydrops, or Hb Sfl thalas-saemia; or for clinical purposes, to establishthe cause of microcytosis. The methods ofselecting patients for investigation depend onwhich of these circumstances applies and onwhether or not the patient is a pregnantwoman.

RED CELL INDICES AND THALASSAEMIA TRAITSMost people with thalassaemia traits have areduced mean corpuscular volume (MCV)(microcytosis) and a reduced mean corpuscu-lar Hb (MCH) in the presence of a normal ornear normal mean corpuscular Hb concentra-tion (MCHC). The Hb concentration is usu-ally normal or only slightly reduced and thered cell count is often raised. Although manypeople with the a+ thalassaemia trait havenormal red cell indices,2 (WHO unpublishedreport HDP/WG/HA 87.5. Alpha thalas-saemia) with information available at present,it is highly likely that those with the a' thalas-saemia trait will have an MCH below 26 pg.Those with the rare ,B thalassaemia trait(silent f, thalassaemia trait) may also havenormal red cell indices.

Haematologists rely heavily on their auto-mated blood cell counters to select whichpatients require further investigation for theseconditions. Careful attention must thereforebe paid to the calibration of the cell counterand its performance should be satisfactoryboth by local assessment and in the NationalExternal Quality Assessment Scheme. Eachlaboratory should establish its own referencerange for the haemoglobin concentration andthe red cell indices. Selection of patients forinvestigation may then be based on the MCHor the MCV or on various formulae. It is stillnecessary, however, to undertake further test-ing to distinguish microcytosis due to a tha-lassaemia trait from that associated with iron

deficiency, the anaemia of chronic disorders,or sideroblastic anaemia and to identify whichtype of thalassaemia trait a person has inher-ited.

USE OF FORMULAE BASED ON RED CELLINDICESVarious formulae can be used34 to determinewhether the blood count is more suggestive ofa thalassaemia trait or iron deficiency butthese formulae are not applicable to children,pregnant women, or polycythaemic patientswith iron deficiency and will only predict thecorrect diagnosis in 80%-90% of patients.5The following formula may be used in whicheach of the variables included makes a contri-bution, and the overall function is better thanany single measurement:MCV (fl) -RBC ( x 1012/1) -5 x Hb (g/dl) -KWhere K is a constant factor dependent onthe instrument calibration and is adjusted byeach laboratory so that a negative result withthis formula suggests a diagnosis of thalas-saemia trait3 whereas a positive result suggestsiron deficiency.

THALASSAEMIA TRAIT WITHOUT MICROCYTOSISThe red cell indices may also be affected byconcomitant B12 or folate deficiency or liverabnormalities and in these situations peoplewith a thalassaemia trait may have normal redcell indices. Those with certain /J thalas-saemia mutations may also have normal redcell indices.'

INVESTIGATION FOR GENETIC PURPOSESThis can be carried out before conception orduring pregnancy. In either circumstance it isessential that most cases of the fi thalassaemiatrait be detected ("silent" fl thalassaemia traitcannot be detected from red cell indices) and

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Guidelines for investigation of the a and fi thalassaemia traits

that the a thalassaemia trait be detected inthose ethnic groups where the - -faa (or a')genotype occurs and where the condition HbBarts hydrops fetalis is therefore possible.Patients may be selected for further testing onthe basis of either the MCH or the MCV.Further testing may be carried out on allthose who fall below the lower limit of thereference range for the selected variable.Alternatively, a laboratory may investigate alarge population of subjects with a suspectedthalassaemia trait to establish a satisfactorylower cut off point, thus reducing unneces-sary testing.

If the patient is not pregnant, formulaesuch as that given may be applied to deter-mine whether iron deficiency or a thalas-saemia trait is more likely. A provisionaldiagnosis of iron deficiency should be con-firmed and treatment given. If the red cellindices do not return to normal after correc-tion of the iron deficiency investigation for athalassaemia trait is then indicated. When apatient is already pregnant such formulaebecome invalid and either the MCH or theMCV should be used to select patients forfurther investigation. If the woman is preg-nant it may be necessary to test for thalas-saemia even though she is known to be irondeficient. This is because both thalassaemiaand iron deficiency can occur in the samepatient and undue delay may occur if thalas-saemia testing is not undertaken until the irondeficiency is corrected; this is especially likelyif the woman is not of northern Europeanextraction. If the pregnant woman is thoughtto have the ,6 thalassaemia trait or esfl thalas-saemia trait, or has an interacting Hb variantsuch as Hb S, C, E, 0-b, or Lepore, theputative father should have similar investi-gations.

At present it seems that Hb Barts hydropsis extremely rare in those of ethnic orginother than from South East Asia (see also theassociated guidelines,6 7). If the woman isthought to have the a thalassaemia trait andoriginates from South East Asia she may havethe a' genotype and the precise genetic diag-nosis may therefore be important and theputative father should also be tested for athalassaemia.

INVESTIGATION FOR CLINICAL PURPOSESThis is most commonly undertaken in peoplewith microcytosis or a low MCH in theabsence of iron deficiency or after correctionof iron deficiency. Except in the special cir-cumstances of pregnancy it is not generallyuseful to investigate for a thalassaemia trait inthe presence of iron deficiency and moder-ately severe anaemia (Hb < 8 g/dl).

DIAGNOSIS IN SPECIAL GROUPSInfants less than 1 year oldThe presence of Hb Barts hydrops at birthindicates a thalassaemia trait and if it isnoticeably raised the baby may have Hb Hdisease89 and may need specialist follow up.fi Thalassaemia heterozygotes will developthalassaemic indices and increased concentra-

tions of Hb A, between 3 and 6 months ofage. At present the diagnosis of fi thalas-saemia trait cannot be made reliably in theneonatal period, but if clinically indicated,studies can be carried out when the baby isover 6 months old.

Thalassaemia interactions with structuralvariantsIn families that carry certain structural Hbvariants (for example, Hb S, C, E, O-ab, andsome unstable Hbs), it is necessary to investi-gate for the presence or absence of 16 thalas-saemia genes that may interact with thesestructural variants.

How to establish the diagnosisIn most clinical situations the first step in thediagnosis of the thalassaemia traits and thephenotypically similar syndromes such as HbLepore trait is the measurement of the redcell indices. If the cells are normocytic, thediagnosis can be excluded with sufficientaccuracy for clinical purposes as long as thereis no coexisting disorder such as B 12 or folatedeficiency, or liver disease which may raisethe MCV and MCH into the normal range.Once the possibility of a thalassaemia traithas been identified specific tests are requiredto confirm or exclude the diagnosis of thalas-saemia, and if present, to determine whetherit is a, ,B, or 5fl thalassaemia, Hb E trait, orHb Lepore trait. The table summarises thegeneral classification of the thalassaemia syn-dromes together with the typical haematologi-cal features for those more than 1 year of agewho have the common genotypes discussed inthese guidelines. More precise diagnosesrequire the measurement of globin chain syn-thesis ratios and/or DNA analysis.

Specific tests are an essential step in thediagnosis of thalassaemia trait and include thefollowing: (a) electrophoresis of Hb on cellu-lose acetate membrane at alkaline pH; (b)measurement of Hb A2; (c) assessment of HbF; (d) looking for Hb H inclusion bodies; (e)assessment of iron status.

Additional tests are sometimes requiredsuch as isoelectric focusing, globin chain syn-thesis, or DNA analysis.

ELECTROPHORESIS OF HBElectrophoresis of Hb at alkaline pH on cellu-lose acetate membrane should be carried outon all samples tested for thalassaemia becausethis procedure will detect the Hb variants, HbLepore, Hb S, Hb C, Hb E, and Hb OQb,together with Hb H, Hb Barts, and most HbA, variants, all of which have medical impli-cations when associated with a thalassaemiatrait. The technique used is based on thatdescribed by Schneider et alP0 and given indetail by the International Committee forStandardization in Haematology (ICSH).9

(1) Tetrasodium EDTA (1 g/l in 100 mg/lpotassium cyanide) should be used as thehaemolysing reagent.5 The haemolysate isliable to oxidation and should be used withinseven days.

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(2) Strong detergents such as Triton X-100 should not be used with adult specimensas they are likely to denature Hb H and if thishappens Hb H disease may be missed; how-ever, they do not seem to harm Hb Barts andso can be used for neonatal specimens.

(3) If a variant Hb is detected the sampleshould also be examined by Hb electrophore-sis on citrate agar at acid pH9 or by a similartechnique on commercial precast agar oragarose gels. Some workers find commercialgels easier to use than gels prepared in housebut they are expensive and some agarose gelsdo not give as much information as a well runcitrate agar gel.

(4) It is important to detect the presence ofHb A2 variants during the Hb electrophoreticrun as it is the sum of the "normal" Hb A2and the Hb A2 variant that must be assessedin the diagnosis of the ,6 thalassaemia trait(see later). If the presence of an Hb A2 variantis missed it may mean that any coexistent,Bthalassaemia trait is also missed.

ESTIMATION OF HB A2A raised Hb A2 is the hallmark of the classic 16thalassaemia trait. It should be measuredwhenever a thalassaemia trait is suspected.Details of techniques are to be found inICSH recommended methods11 but thefollowing points should be observed:

(1) Preparation and storage of haemo-lysate: the haemolysate for Hb A2 and Hb Fdeterminations should be prepared by wash-ing packed red cells with three volumes ofisotonic saline (0 15 M). Mix the washed,packed red cells with an equal volume of dis-tilled water and a half volume of carbon tetra-chloride and mix well for 10 minutes.Centrifuge and remove the supernatant Hbsolution, check the Hb concentration andadjust to 10 ± 2 g/dl. The Hb solution maythen be stored in a stoppered tube at 4°C forup to two weeks.

(2) Both electrophoresis and elution fromcellulose acetate and microcolumn chro-matography are recommended by ICSH" butthe precision and accuracy of automatedscanning densitometry are inadequate for HbA2 estimations (see (8)). Routine techniquescannot be used for the determination of HbA2 in the presence of Hb C, Hb E, and Hb 0(and some other Hbs) because of their similarmobility to Hb A2 on cellulose acetate andthese microcolumns. Family studies and/orDNA analysis may be helpful in these situa-tions.

(3) When preparing TRIS buffers it isessential to use a pH electrode that is knownto be suitable for use with TRIS becauseTRIS interferes with the function of someelectrodes. If an electrode can be calibratedwith two standard buffers after immersion inTRIS buffer for at least 15 minutes it may beconsidered suitable for use with TRIS.

(4) Improved results may be obtained withmicrocolumn chromatography when a 10 mMrather than a 50 mM TRIS buffer is used.'2

(5) If a glycine "developer" is used the pHmust be adjusted very slowly as the molarity

of the developer is an important factor in theelution of the Hb fractions and if the devel-oper is made too acidic it must be discardedand not made alkaline again because thiswould increase the total ionic concentrationand therefore alter the elution pattern.

(6) Commercial microcolumn kits for theestimation of Hb A2 are available. It is impor-tant to follow the manufacturer's instructionscarefully regarding the preparation, equilibra-tion (especially temperature), and the use ofthe columns.

(7) Automated high performance liquidchromatography can save time but is expen-sive in both capital and revenue costs. If used,it is important to examine the peak shape,baseline, and resolution and to ensure thatthey are satisfactory before using the com-puted peak areas.

(8) Reference materials have been devel-oped by the ICSH for use in Hb A2 measure-ment and will be available from the NationalInstitute of Biological Standards andControls (NIBSC).

(9) If an Hb A2 variant is present it isessential to measure both the normal Hb A2and the variant Hb A2 because it is the totalHb A2 which indicates whether or not a per-son has the 16 thalassaemia trait. If an Hb A2variant is present, electrophoresis and elutionof the Hb A2 and the Hb A2 variant bands isusually the best technique to use for measure-ment.

(10) Interpretation: values below 3-3% areusually normal and over 3-7% usually indi-cate the 16 thalassaemia trait. Some overlap inHb A2 values between normal and 16 thalas-saemia trait has been reported'2 and valuesbetween 3-3 and 3-7% need to be interpretedwith care. Knowledge of the iron state of theindividual patient is important as iron defi-ciency sufficient to cause moderately severeanaemia (Hb < 8 g/dl) can reduce the Hb A2level and lead to people with the 16 thalas-saemia trait being classified as not having thiscondition."-'5 Normal Hb A2 concentrationscan be found in the ,B1 thalassaemia trait andin the Hb Lepore trait (table) although boththese conditions have the same clinical signif-icance as the 16 thalassaemia trait. Raised lev-els have been described in the presence ofsome unstable Hb 16 globin variants and insome cases of pernicious anaemia. A valueabove 7% suggests either an analytical erroror the presence of another Hb that is not HbA2.

IfHb A2 concentrations in the "borderline"area (3 3%-3-7%) in the absence of Hb S(see below), the estimation should berepeated on another blood sample and theiron state assessed. If the patient is iron defi-cient the iron deficiency should be correctedbefore the analysis is repeated. If repeatanalysis again gives a borderline result globinchain synthesis or DNA analysis should beconsidered if the diagnosis of the 16 thalas-saemia trait, or its exclusion, would alter theclinical management of the patient or thegenetic advice given to a couple. An exampleof such a situation would be if a pregnant

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Guidelines for investigation of the a and/ thalassaemia traits

woman had the /1 thalassaemia trait and herpartner had borderline results.

If a person has both a sickle cell trait andthe a thalassaemia trait the Hb A2 may beslightly raised (3 5%-4 0%) but this does notof itself indicate a coexistent ,B thalassaemiatrait. The simplest way of obtaining a correctdiagnosis in this situation is to quantify theHb S. In the uncomplicated sickle cell trait(Hb A + Hb S) the Hb S comprises less than40% of the total Hb. If the a thalassaemiatrait is also present the Hb S will be lower16whereas the combination of sickle cell traitand ,B thalassaemia trait results in an Hb Sconcentration of more than 60% and usually80%-90%.

HB FThe Hb F is raised (by about 1%-3%) in onethird to half of the people with the ,B thalas-saemia trait. A raised Hb F (5%-20%) in thepresence of hypochromic, microcytc, red cellindices, however, is the hallmark of the 5flthalassaemia trait and it should be measuredwhenever Hb F is detected on Hb elec-trophoresis on cellulose acetate membrane. Itmay also be raised in other conditions such asin pregnancy and in the myeloproliferativedisorders.

(1) Hb F should be measured by the twominute alkali denaturation techniquedescribed by Betke et a17 and in detail byPembrey et al.8 Attention to detail isextremely important when undertaking thisassay. In particular, the concentration of thehaemolysate (10 ± 2 g/dl), the temperature ofthe alkali denaturation mixture (20 ±2°C),and the time that the denaturation is allowedto proceed (two minutes) must all be care-fully controlled. If the Hb F is above 50% amore accurate result may be obtained by thetechnique of Jonxis and Visser'9 but theincreased accuracy will rarely, if ever, alterthe clinical management of the patient.

(2) Reference materials developed byICSH for use in Hb F measurement will beavailable from NIBSC.

(3) If the Hb F is 5%-20%, the patientmay have cfl thalassaemia or HPFH.Pronounced microcytosis favours the first. Ifthe Hb F is more than 20% it is likely that thepatient has HPFH and in this condition theKleihauer cytochemical test for Hb F willshow a pancellular distribution. If the patientis pregnant the partner should be tested andif his tests are abnormal a definitive diagnosisby DNA analysis should be considered.

HB H INCLUSIONS(1) Hb H inclusions are due to the oxidationand precipitation of Hb H within the red celland they can be generated in vitro by theredox action of certain dyes. They are foundin some forms of a thalassaemia. Unfor-tunately there is batch to batch variation withthe dye and any new batch should be checkedwith a known positive sample.

(2) Blood (preferably anticoagulated withEDTA and used within 24 hours of venesec-tion) is incubated with 1% brilliant cresyl

blue or new methylene blue at room tempera-ture (18%-25°C) for four hours; a normalcontrol should always be included (CSRHatton and DR Higgs, personal communica-tion). Blood films are made and examined.Hb H inclusions form a "golf ball" appear-ance inside the cells.

(3) In people with the a thalassaemia traitthere are usually a few red cells (1/1000 to1/10 000) that contain typical inclusions(table)20 but in Hb H disease they are foundin 30%-100% of the red cells. The absence ofHb H inclusions does not, however, excludean a thalassaemia trait.

ISOELECTRIC FOCUSINGAs a general rule the separation of Hbs onisoelectric focusing is similar to that obtainedby electrophoresis on cellulose acetate mem-brane but the bands are sharper and the reso-lution of some Hbs is better. Isoelectricfocusing is not usually indicated in districtservice laboratories but can be very useful inreference centres and also in regional labora-tories where large numbers of samples areprocessed. This is a useful technique to dif-ferentiate Hb Barts from "fast" variants suchas Hb J and Hb N in anticoagulated umbilicalcord blood samples. It is also useful in labora-tories undertaking neonatal screening ondried blood spots from Guthrie cards becauseit reduces the interference from met Hbwhich usually prevents adequate resolution ofHb bands when using electrophoresis toanalyse Hb eluted from such dried bloodspots.

GLOBIN CHAIN SYNTHESISThis technique is valuable in detecting thepresence of the a or fi thalassaemia trait incouples considering prenatal diagnosis whenthe usual tests give equivocal results; it maysometimes be also useful in fetal diagnosis(see associated guidelines'). The technique istime consuming and involves culturing theblood in the presence of tritiated leucine andtherefore facilities for the use and disposal ofradioactive material must be available. Forthese reasons this test is usually only under-taken in reference centres. If it is undertaken,it is essential that adequate numbers ofnormal and abnormal controls are analysedregularly.

DNA ANALYSISDNA can be analysed from white blood cells,or obtained from amniocytes or chorionictissue obtained by transabdominal or trans-vaginal sampling methods. These techniquesare usually undertaken in specialist referralcentres, and the laboratory techniques arediscussed in the associated guidelines.'

QUALITY CONTROLAs these procedures are all used to make, orexclude, the diagnosis of an inherited condi-tion, it is essential to ensure that adequatequality control is undertaken for the determi-nation of the red cell indices and all the othertests described in these guidelines. In house

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protocols should clearly elucidate all aspectsof the procedures to be undertaken. In partic-ular quantitative diagnostic analyses such asHb A, and Hb F should be undertaken insuch a way that duplicates agree to within0-2% of each other (SD < 005%). All labora-tories should check their reference rangeswith each method they use to ensure thattheir results are accurate as well as precise.The Hb concentration of the haemolysateused for the Hb A, and Hb F determinationsshould be recorded, as should the tempera-ture of the reagents for the Hb F.Laboratories should participate in the appro-priate National and Regional QualityAssessment schemes.

Interpretation of resultsAlthough a definitive diagnosis of one of thethalassaemia traits requires detailed DNAanalysis (see associated guidelines', a diagno-sis that is accurate for most clinical purposesexcept for fetal diagnosis can usually beobtained by assessing the results of a group oflaboratory tests. The presence of one of thesetraits or one of the phenotypically similarvariant haemoglobins such as Hb Lepore, HbConstant Spring, or Hb E trait may havebeen suspected because of the presence of redcells with reduced MCH and MCV or themeasurement of these indices may be used asthe first of a series of investigations.

f, Thalassaemia trait is typically associatedwith a raised Hb A, but the Hb A, is notraised in the 5fl thalassaemia trait and it maynot be raised if there is coexistent severe irondeficiency anaemia. Hb Lepore, which is phe-notypically similar to the fi thalassaemia trait,can be detected by Hb electrophoresis.

a Thalassaemia is not associated with aconsistent marker such as the raised Hb A, inthe fi thalassaemia trait but Hb H bodies maysometimes be detected. The diagnosis of athalassaemia trait must usually be made bythe exclusion of iron deficiency and otherforms of thalassaemia in a person who hashypochromic, microcytic red cells. HbConstant Spring (and other variant Hbs withglobin chain elongation that are phenotypi-cally similar to a thalassaemia) may bedetected by Hb electrophoresis, but may beconfused with an Hb A, variant or a carbonicanhydrase variant. Hb A, variants can be dif-ferentiated from carbonic anhydrase variantsby haem staining with dyes such as o-diani-sidine.9The typical haematological features of the

common thalassaemia traits are given nextand in the table. The subtypes of a thalas-saemia are classified according to the numberof deleted genes that cause each type as genedeletion is the cause of classical a thalas-saemia. Non-deletional forms also occur,however, and are clinically important in thatthey can cause a severe form of Hb H disease.

a Thalassaemia trait (one gene deletion)can often be detected at birth by the presenceofHb Barts. This condition is often haemato-logically silent in later life although it may be

associated with some of the changes found inthe variety of a thalassaemia trait caused bytwo gene deletions (see below).

a Thalassaemia trait (two gene deletion) ischaracterised by red cells with a reducedMCH (usually less than 26 pg) and MCV inthe absence of iron deficiency and no evi-dence of fi or Bfl thalassaemia trait (a normalor reduced Hb A,-less than 3 5%). Hb Hbodies may be detected, but their absencedoes not exclude a thalassaemia. Hb elec-trophoresis will be normal unless a variantHb is also present.Hb H disease (three gene deletion) is char-

acterised by anaemia and by red cells with areduced MCH and MCV in the absence ofiron deficiency and with no evidence of fi or6fl thalassaemia trait (a normal or reduced HbA2-less than 3 5%). Hb H bodies are foundin 30%-100% of the red cells. Electro-phoresis of Hb shows the presence of anabnormal band anodal to Hb A comprisingabout 1%-40% of the total Hb.

f, Thalassaemia trait is characterised by redcells with a reduced MCH and MCV in theabsence of iron deficiency. The Hb A2 israised (3 5-7 0%) and the Hb electrophoresisis otherwise normal unless a variant Hb isalso present.

a Thalassaemia trait combined with ,Bthalassaemia trait is characterised by red cellswith a normal or reduced MCH and MCV inthe absence of iron deficiency. The Hb A2 isusually raised and Hb electrophoresis isotherwise normal unless a variant Hb is alsopresent.

5fl Thalassaemia trait is characterised byred cells with a reduced MCH and MCV inthe absence of iron deficiency. The Hb A2 isnormal or slightly reduced and the Hb F israised (5%-20%). Electrophoresis of Hb isotherwise normal unless a variant Hb is alsopresent.Hb E trait is characterised by red cells with

a slightly reduced MCH and MCV in theabsence of iron deficiency. Electrophoresis ofHb shows a variant band (usually 20%-30%)in the position of Hb A2 on cellulose acetatemembrane at alkaline pH which does not sep-arate from Hb A on citrate agar at acid pH.The Hb stability is usually slightly abnormal.Hb Lepore trait is characterised by red

cells with a reduced MCH and MCV in theabsence of iron deficiency and a normal HbA2 (less than 3-5%). Electrophoresis of Hbshows a variant band (7%-15%) in the posi-tion of Hb S on cellulose acetate membraneat alkaline pH which does not separate fromHb A on citrate agar at acid pH.

Note: iron deficiency can occur in conjunc-tion with any of these genotypes and if it ismoderately severe (Hb < 8 g/dl) it mayreduce the Hb A2. For this reason it may benecessary to retest a patient after correctionof any iron deficiency.

ConclusionsNo guidelines can be all inclusive-there willalways be exceptions and cases must be con-

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sidered individually. Patients diagnosed ashaving one of the thalassaemia traits shouldbe counselled and given an explanation of theresults of the tests. A haemoglobinopathycard or other written record of the resultsshould be made available to the patients andtheir doctors. Special counselling and followup arrangements must be established for cou-ples considering prenatal diagnosis.

Usefil addresses(1) Prenatal diagnosis by DNA analysis andother information on the thalassaemias isavailable as a national service from: Dr JohnOld, National Haemoglobinopathy ReferenceService, Institute of Molecular Medicine,John Radcliffe Hospital, Oxford OX3 9DUTelephone 0865-222449 Fax 0865-222500

(2) Haemoglobinopathy cards and leafletcan be obtained from: DSS Leaflets Unit, POBox 21, Stanmore, Middlesex HA7 lAYTelephone 081-952-2311

(3) Multilingual explanatory booklets onsome of these conditions are available from:The UK Thalassaemia Society, 107Nightingale Lane, London N8 7QY.Telephone 081-348-0437

(4) The names and addresses of NHSCounselling Centres can be obtained fromthe UK Thalassaemia Society

1 Guidelines for the fetal diagnosis of globin gene disorders._J Clin Pathol 1994:47:199-204.

2 Weatherall DJ, Clegg JB. The thalassaemia syndromes. 3rdedn. Oxford: Blackwell Sci Publications, 1981.

3 England JM, Frazer PM. Discrimination between iron

deficiency and heterozygous thalassaemia syndrome indifferential diagnosis of microcytosis. Lancet 1979,i:145-8.

4 England JM. Discriminant functions. Blood Cells 1989;15:463-73.

5 d'Onofrio G, Zini G, Ricerca BM, Mancini S, Mango G.Automated measurement of red blood cell microcytosisand hypochromia in iron deficiency and /-thalassaemiatrait. Arch Pathol Lab Med 1992;116:84-9.

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Clin PatholThe thalassaemia syndromes are inherited disorders of haemoglobin (Hb) production, characterised by a reduction in globin chain synthesis leading to an imbalance ofthe glo-bin