J Clin Pathol 1995;48:861-864

Comparison of haemoglobin H inclusion bodieswith embryonic 4 globin in screening for ct

thalassaemia

L C Chan, J C C So, D H K Chui

AbstractAims-To compare the haemoglobin (Hb)H inclusion test with immunocytochem-ical detection of embryonic 4 chains inscreening for a thalassaemia.Methods-Blood samples from 115 patientswith relevant clinical history and hypo-chromic microcytic indexes were screenedusing the HbH inclusion test and the Vari-ant Hemoglobin Testing System (BioRad,Hercules, CA, USA).Results-The HbH inclusion test waspositive in 61 of 115 cases, three of whomhad HbH disease confirmed by electro-phoresis. The remaining 58 had a thal-assaemia 1. All three HbH cases and 56of 58 cases of a thalassaemia 1 expressedembryonic 4 chains, giving a specificity of96-7%. Fifty four of115 cases had a negativeHbH inclusion test, of whom 50 had Pthalassaemia trait and three had iron de-ficiency. No diagnosis was reached for theremaining patient.Conclusion-The immunocytochemicaltest is as sensitive as the HbH inclusiontest in screening for a thalassaemia. Thepresence of 4 chains is highly specific for athalassaemia 1 incorporating the (--/SEA)deletion. The specificity and simplicity ofthe immunocytochemical test make it thetest of choice in screening for a thal-assaemia.(JT Clin Pathol 1995;48:861-864)

Keywords: HbH inclusion test, 4 chains, a thalassaemia.

Haematology Section,Department ofPathology,Queen Mary HospitalCompound,University of HongKong, Hong KongL C ChanJ C C So

Department ofPathology,McMaster University,Hamilton, Ontario,CanadaD H K Chui

Correspondence to:Dr L C Chan.

Accepted for publication30 March 1995

In South East Asia, where a and thalassaemiaare prevalent, the haemoglobin (Hb) H in-clusion test, based on incubation of eryth-rocytes in brilliant cresyl blue, is usedextensively in many laboratories as a screeningtest for subjects with a thalassaemia. The pres-ence of HbH inclusions is correlated with theaocl-- genotype (including those with deletionsinvolving the complete 4-a gene cluster) aspresent in oa thalassaemia 1 carriers and thea-/-- genotype associated with HbH disease.'Based on positive findings, couples at risk ofproducing fetuses with hydrops fetalis due toHb Barts are identified for genetic counsellingand prenatal diagnosis. Recently, an im-munocytochemical test which detects em-

bryonic 4 chain in erythrocytes was shown to behighly specific and sensitive for a thalassaemia 1carriers of the (--SEA/) deletion'-that is, theoca/-- genotype in which the 4 globin gene

cluster is intact. We have compared the HbHinclusion test with immunocytochemicaldetection of 4 globin chains in screening for athalassaemia.

MethodsBlood samples from 115 patients with relevantclinical history and microcytic (mean cor-puscular volume (MCV) < 80 fl) and hypo-chromic indexes were subjected to the followingstudies: (1) HbH inclusion test; (2) estimationof HbA2 and HbF by the Variant HemoglobinTesting System3 (BioRad, Hercules, California,USA); and (3) electrophoresis of the haem-olysate in cellulose acetate at pH 8-55 usingthe Super Z kit Hb system as appropriate.Using the Variant Hemoglobin Testing System,the normal adult reference ranges for HbA2and HbF in our laboratory are, respectively,2 3-3O0% and 0-0 9% (unpublished data). Thenormal adult reference range for MCV in ourlocal population, based on determination oferythrocyte indexes using the Technicon H*1blood analyzer, is 83-96 fl.4 Serum iron andserum ferritin concentrations were determinedin 40 cases.

HBH INCLUSION TESTAir dried peripheral blood smears were madefrom a mixture of two parts 1% brilliant cresylblue to one part blood following incubation at37°C for exactly 30 minutes. For detection ofinclusions, 1000 to 5000 red blood cells wereexamined under an oil immersion lens whichrequires up to 15 minutes per case.

DETECTION OF EMBRYONIC 4 CHAINSAir dried peripheral blood smears were fixedin a solution containing 90% absolute acetoneand 10% absolute methanol at room tem-perature for five to 10 minutes. They were thenwashed in phosphate buffered saline (PBS)containing 3% bovine serum albumin (BSA);10 ml of a 1 in 20 dilution of murine mono-clonal antihuman 4 globin chain conjugatedwith fluorescein isothiocyanate was then added.The slides were incubated in a humidifiedchamber at room temperature for 30 minutesand were then washed in BSA free PBS, coveredwith glycerol and examined under a fluor-escence microscope (Zeiss). Cord blood andblood from an individual with a normal fullblood count were used as positive and negativecontrols, respectively, for 4 globin expression.

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inter- CHVR 5V1 Na2 va1

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a2 al 01

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-SEA deletion

1=5'-GCGATCTGGGCTCTGTGTTCT-3'2=5'-GTTCCCTGAGCCCCGACACG -3'3=5'-ACTGCAGCCTTGAACTCCTG -3'

Figure 1 Alpha globin gene loci and primers for PCR analysis.

DNA EXTRACTION AND POLYMERASE CHAINREACTIONDNA was extracted from peripheral bloodusing a commercial kit (Intragene PurificationMatrix; BioRad). To detect the (--/SEA) de-letion, three primers were synthesised usingdetails of the sequences provided by Chang etal5 (fig 1). Primers 1 and 2 amplify a 314nucleotide band if normal or if a thalassaemia2 is present, whereas a thalassaemia 1 with the(--/SEA) genotype will yield a 195 nucleotideband on amplification with primers 1 and 3.DNA (250 ng) was amplified using the poly-merase chain reaction (PCR) with primers ata final concentration of 2 mtM for 35 cycles on

Figure 2 Immunofluorescent erythrocytes stained with anti-C chain antibody in a case ofax thalassaemia 1. (Magnification x 2000.)

an automated themocycler under the followingconditions: denaturation at 94°C for 30 sec-onds, annealing at 58°C for 45 seconds andextension at 72°C for one minute, except forthe first cycle when denaturation took place at95°C for 90 seconds and at 94°C for 30 sec-onds. After amplification, DNA was elec-trophoresed on a 2% agarose gel at 100 Vfor one hour and visualised using ethidiumbromide.

STATISTICAL ANALYSISThe sensitivity and specificity of the im-munocytochemical test for the diagnosis of axthalassaemia were calculated as described bySimel.6

ResultsThe HbH inclusion test was positive in 61 ofthe 115 cases studied. Three of these 61patients had HbH disease based on the pres-ence of multiple inclusions and a HbH bandon electrophoresis. In the remaining 58 thepattern ofHbH inclusions was compatible witha diagnosis of a thalassaemia 1. All three cases

of HbH disease expressed embryonic 4 chainsas did 56 of the 58 cases of a thalassaemia 1.Therefore, the sensitivity of the immuno-cytochemical test compared with conventionalmethods for the diagnosis of a thalassaemiawas 96-7% (59/59+2). Figure 2 shows thefluorescence pattern of erythrocytes, revealingvarying staining intensities in a patient with1athalassaemia 1.Of the two patients (cases 47 and 71) with

HbH inclusions, but without embryonic 4chains, one had iron deficiency anaemia whilstthe other had an unexplained hypochromicmicrocytic anaemia with no family history ofthalassaemia. As expected, the (-/SEA) de-letion was not detected on DNA analysis ofthe a globin locus by PCR in both of thesecases. By contrast, an amplified band of 195base pairs, confirming the presence of the (--/SEA) deletion, was present in all seven casesof a thalassaemia 1-that is, both HbH and 4chain positive. The results of the DNA analysisare presented in fig 3.

Fifty four cases had a negative HbH inclusiontest. Fifty had thalassaemia trait based onraised A2 and F concentrations and three were

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Screening methods for a thalassaemia

MCase 1

ACase 47

A BBCase 71

A B

404-367-

314-242-

195- 190-

Figure 3 An ethidium bromide stained agarose gel ofDNA amplified from cases 1 (HbH positive, 4 chain positive),47 and 71. Lane A, primers 1 and 2; lane B, primers 1 and 3; M, DNA markers.

1 15 cases (MCV < 80 fl)

HbH positive(n = 61)

HbH disease a Thalassaemiapattern (n = 3) (n = 58)(confirmed by

electrophoresis)-all 4 chain positive

4 positive(n = 56)d

HbH negative(n = 54)

P Thalassaemia Iron deficiency (n = 3)b(n = 50)a Unknown (case 74)c

4 negative(n = 2)e-iron deficiency (case 71)-unexplained (case 47)

Figure 4 Flow chart of results of the HbH inclusion test and C globin chain expression. aFive offive cases 4 chainnegative; btwo of two cases C chain negative; 'C chain positive; seven of seven cases (CISEA) positive; '(-ISEA)negative.

iron deficient. The 4 chain was expressed inthe patient (case 74) whose iron status wasunknown and whose HbH inclusion test wasnegative, but not in the five cases of ,B thal-assaemia and two cases of iron deficiency ex-amined. As no DNA was available to resolvethe oa globin status of case 74, the specificityofthe immunocytochemical test compared withconventional methods (after excluding case 74)for the diagnosis of oa thalassaemia was 100%(7/0 + 7). A summary of the data is shown infig 4.

DiscussionThe immunocytochemical detection of em-bryonic 4 chains is as sensitive as the HbHinclusion test in screening for a thalassaemia.

Embryonic 4 chains were present in 56 (96 5%)of 58 cases of oa thalassaemia 1 based on apositive HbH inclusion test. Of the two cases(47 and 74) which did not express the 4 chain,DNA analysis confirmed that the (--/SEA) de-letion was absent as expected. It is possiblethat, in these two cases, the 4 genes were deletedtogether with the oa gene loci, as is the case inadult carriers of deletions such as (--Fill),(-- Thai/), and (--HW/) in South East Asia.78Case 47 had a negative discriminant func-tion as calculated using red blood cell indexes9which is in keeping with the diagnosis of a thal-assaemia. Other possibilities which mayaccount for negative 4 chain expressioninclude the -al- oa, -a/aCsot and ----/C4aax'genotypes, or a "false positive" HbH inclusiontest.

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We have also shown that the immuno-cytochemical test is highly specific for the diag-nosis ofa thalassaemia 1. However, the 4 globinchain was also detected in a single case (case74) for whom the HbH inclusion test wasnegative. As the HbF and A2 concentrationswere normal and no variant haemoglobins weredetected, the absence ofHbH inclusions cannotbe because of the presence of f thalassaemiatrait or HbE, both of which are known todecrease significantly the number of HbH in-clusions.' As no cells were stored for DNAanalysis, we cannot confirm whether the (-SEA/) deletion was present using PCR.We have shown that the immunocyto-

chemical detection of embryonic 4 chains is apractical alternative to the HbH inclusion testin the diagnosis of oc thalassaemia. As stainingis positive in almost all erythrocytes, only smallnumbers of red cells need to be evaluatedcompared with the laborious screening of sev-eral fields for the detection ofHbH inclusions.Detection of 4 chains using immunofluor-escence can be carried out in batches and theresults can be rapidly scored and assigned asdefinitively positive or definitively negative.

It must be emphasised that screening forembryonic 4 chains, like all laboratory tests,will need to be interpreted together with otherrelevant clinical and laboratory data. In a fewpatients with 4-oc thalassaemia the immuno-

cytochemical test will be negative but the HbHinclusion test will be positive. Therefore, werecommend that screening for oc thalassaemiabe undertaken using 4 chain detection; negativecases must be confirmed using appropriate mo-lecular analysis.

We thank F Lee, Amy Chan and JY Chan for excellent technicalassistance and J Kwok for preparing the manuscript. Serumiron and ferritin concentrations were kindly provided by Mr DRobinson, Clinical Biochemistry Unit, Queen Mary Hospital,Hong Kong.

1 Skogerboe KJ, West SF, Smith CS, Terashita ST, LeCroneCN, Detter JC, et al. Screening for a-thalassaemia. Cor-relation of hemoglobin H inclusion bodies with DNA-determined genotype. Arch Pathol Lab Med 1992;116:1012-18.

2 Tang W, Luo H-Y, Eng B, Waye S, Chui HK. Im-munocytochemical test to detect adult carriers of the (--SEA/) deletional oa-thalassaemia. Lancet 1993;342:1145-7.

3 Tan GB, Aw TC, Dunstan TA, Lee SH. Evaluation of highperformance liquid chromatography for routine estimationof haemoglobins A2 and F. J Clin Pathol 1993;46:852-6.

4 Robertson EP, Pollock A, Yau KS, Chan LC. Use of Tech-nicon H* 1 technology in routine thalassaemia screening.Med Lab Sci 1992;49:259-64.

5 Chang J-G, Lee L-S, Lin C-P, Chen P-H. Rapid diagnosisof a-thalassaemia 1 of Southeast Asia type and hydropsfetalis by polymerase chain reaction. Blood 199 1;78:853-4.

6 Simel DL. Playing the odds. Lancet 1985;i:329-30.7 Tang W, Luo H-Y, Albitar M. Human embryonic 4 globin

chain expression in deletional a-thalassaemias. Blood 1992;80:517-22.

8 Chui DHK, Wong SC, Chung S-W, Patterson M, BhargauaS, Poon M-C. Embryonic 1-globin chains in adults: amarker for a-thalassaemia due to a 17.5kb deletion. N EnglJ Med 1986;314:76-9.

9 England JM, Fraser PM. Discrimination between iron de-ficiency and heterozygous-thalassaemia syndromes indifferential diagnosis of microcytosis. Lancet 1979;i: 145-8.

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