Scand. J . din. Lab. Invest. 43, 171-173, 1983.

Predictive value of a new osmotic test in the screening of heterozygous p thalassemias

P I E R 0 A N I C H I N I , G I O V A N N A R E Y N A U D O , & G I U L I O P A C I N I

Laboratorio di Analisi Chimico Cliniche dell'ospedale di Camerata, U.S.L. 10/G, Firenze, Italy

Anichini, P., Reynaudo, G . & Pacini, G. Predictive value of a new osmotic test in the screening of heterozygous p thalassemias. Scand. J . din. Lab. Invest. 43, 171-173, 1983.

A new osmotic test (time for 50% haemolysis in standard solution) has been applied in patients affected with heterozygous p thalassemia in a population consisting of 19 thalassemic patients, 15 sideropenic patients, and 52 controls. The same population was examined for heterozygous p thalassemia using elec- tronic measurement of the erythrocyte indices. Sensitivity, specificity, predictive value and efficiency of the two tests were calculated. Statistical analysis did not show any significant differences in sensitivity and specificity between the two methods. The authors conclude that the osmotic test is sensitive, reliable and rapid; it seems to be a valid substitute for electronic haematology analysers in countries where they are not available.

Key- words: osmotic fragility; screening test for thalassemia; thalassemia

Piero Anichini, M.D., Laboratorio di Analisi Chimico Cliniche dell'ospedale di Camerata, via della Piazzola 68, 501 00 Firenze, Italy

The prevention of homozygous p thalassemias is based on genetic counseling, which in turn depends on the possibility of performing a re- liable screening of heterozygous thalassemia. The determination of erythrocyte indices by elec- tronic haematology analysers is generally used for this purpose [6]. However, these devices are expensive and are not available in every country. Resistance to haemolysis by hypotonic saline solutions is increased in heterozygous /3 thalas- semias, but presently available tests based on this phenomenon do not satisfy the criteria of rapidity and reliability required for screening. Nonetheless, a new osmotic test which seems to be quick, specific and reliable has recently been

0036-55 13/83/040&0171 $02.00 @ 1983 Medisinsk Fysiologisk Forenings Forlag

proposed [3]. In our paper, we compare this new test with the electronic measurement of erythro- cyte indices from the point of view of predictive value.

M A T E R I A L S A N D M E T H O D S

The study was carried out on 19 patients (seven males, 12 females, 7-87 years) affected with heterozygous p thalassemia, and on 15 patients (three males, 12 females, 17-89years) with iron deficiency due to various causes. Fifty-two con- trol subjects (25 males, 27 females, 14-69years) were also examined; they did not show any clini- cal or laboratory evidence of haematological disorders. No one in the control or patient group showed jaundice.

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172 P. Anichini, G. Reynaudo & G. Pacini

For every control or patient, erythrocyte indices, free serum iron, total and latent iron binding capacity and ‘osmotic index’ [3] were determined.

Haematologic parameters were evaluated on blood collected with K3 EDTA on the electronic haematology analyser Clay A d a m Ultra Logic 800 (Becton-Dickinson, Parsippany, NJ, USA), following the manufacturer’s instructions. Every determination was run in duplicate.

Free serum iron, total and latent iron binding capacity were evaluated by ferrozine method [ l ] (Dow Diagnostics, Indianapolis, IN, USA).

To determine osmotic index, 10 ml of venous blood was placed in a plastic plain tube contain- ing 1 drop (about 20 pl) of heparin 5000 IUlml (Novo Industri AJS, Copenhagen, Denmark). The blood was centrifuged for 10 min at 2000 rpm and plasma was decanted. The test was car- ried out on the erythrocyte concentrate following the instructions of Flatz & Flatz [3]. 0.010 ml of erythrocyte concentrate was suspended in 10 ml of distilled water and the absorbance at 620 nm was recorded. The same amount of erythrocyte concentrate was suspended in 10 ml of a solution containing Na,HPO, 8.0 mmoV1, NaH,PO, 1.8971 mmoV1, NaCl48.1 mmoV1,glycerine 180 mmoV1. Haemolysis was followed at 620 nm (temperature 37”C), beginning exactly 15 sec after the erythrocyte transfer. The time in seconds elapsed until 50% of the erythrocytes were haemolysed was the ‘osmotic index’. The absorbance corresponding to 50 % erythrocyte haemolysis was quickly calculated as follows:

Absorbance at 50 % haemolysis =

(Initial absorbance - absorbance in water)/:!

To avoid falsely low results due to contamina- tion of first cell suspension with any water re- maining in the spectrophotometer aspiration system and cuvette, the first suspension was used for priming, without recording results. After priming, three more samples were examined and results were averaged. Haemolysis recording was stopped 4 min after starting the test. Every determination was done within 2 h after blood sampling.

Haemoglobin electrophoresis was performed on blood samples showing MCV <70 f andlor osmotic index 2 120 sec. Electrophoretic sep- aration was carried out on cellulose acetate (Labometrics s.r.l., Milano, Italy), following the

manufacturer’s instructions. In the same sam- ples, foetal haemoglobin was measured using the method of Nierhaus & Betke [5].

Diagnosis of heterozygous p thalassemia and sideropenic anaemia were made according to usual criteria [8].

R E S U L T S

The osmotic index in the controls ranged from 26to95sec(meank SD =47.17? 16.75).The osmotic index in thalassemic patients was in every case > 4 min, whereas in sideropenic patients it ranged from 120 sec to more than 4 min. Since all thalassemic patients samples showed an osmotic index > 4 min, it was decided to consider this value as a positive result. MCV ranged from 80 f to 96 fin controls (mean 2 SD = 88.04 k 3.31), from 56 f to 69 fin thalassemic patients (mean ? SD = 62.42 ? 3.36), and from 51 f to 82 f in sideropenic patients (mean & SD = 66.07 k 9.41). MCV <70 f was considered a positive result in the screening. Distribution of positive and negative results is shown in Table I.

The osmotic test had a sensitivity of 100% and a specificity of 79.1% in screening for thalas- semic patients. The predictive value and effi- ciency respectively were 57.6 and 83.7%. Elec- tronic evaluation of erythrocyte indices showed a sensitivity of 100% and a specificity of 86.1 %; the predictive value and the efficiency were 67.6 and 87.2 % respectively.

Statistical analysis was performed by the t test for correlated data according to Galen & Gam- bin0 [4]. The osmotic test and electronic MCV measurement had the same sensitivity, whereas no statistically significant differences in specific- ity were found between the two screening tests (0.20>P> 0.15).

TABLE I. Distribution of positive and negative results of the two tests in controls, sideropenic and thalas- semic patients

Electronic Screening test Osmotic test measurement

Limit value <4 min > 4 min >70 f <70 f Controls (52) 52 0 52 0 Sideropenic (1 5) 1 14 4 11 Thalassemic (19) 0 19 0 19

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A new test in thalassernia 173

D I S C U S S I O N

In our experience, the mean values of the osmo- tic index in normal, thalassemic and sideropenic subjects were different from those reported by Flatz & Flatz [3]. The mean value in control subjectsis 47.17 ? 16.75 sec, whereasinFlatz & Flatz series it was 50 +- 12 sec. Patients affected with iron deficiency show an osmotic index > 120 sec in every case, whereas 10 of 23 cases showed such pattern in Flatz & Flatz series. All thalassemic patients had an osmotic index > 4 min, in our series; in the other study, an osmotic index of more than 120 sec was found. This may arise from differences in the population studied (Italian instead of German) or from slight differ- ences in the method. We want to point out that centrifugation is a critical step, since it may in- crease osmotic fragility of erythrocytes; in our study, it was done at 2000 rpm for 10 min. We were not able to find out any information about time and speed of centrifugation in the Flatz & Flatz paper. It is also critical to p e r f o m the test within a few hours after sampling; in our laboratory, it was done within 2 h.

The new osmotic test shows a n excellent sen- sitivity and a good specificity. No statistically significant differences in sensitivity and specific- ity were found between the two methods. The predictive value is limited by false positives occurring with sideropenic patients; however, this is a cause of false positives with haematology analysers, too. We did not use the test in jaun- diced patients, since false positives may occur when it is applied to patients affected with cholestasis. This is not a cause of false positives when using an electronic haematology analyser. It must be considered, however, that in prelimi- nary assays we noticed osmotic indices >4 min in patients suffering from cholestatic jaundice with total serum bilirubin >70 pmoV1, and consistent values of direct bilirubin. We think that clinical examination and/or plasma observation are suf- ficient to identify such patients, in whom the test may give false positives.

Since specificity, predictive value and effi- ciency are strictly dependent on the prevalence of heterozygous /3 thalassemia and iron deficiency in a population, the values we have reported are not to be considered as absolute; they should be corrected according to the prevalence of such diseases in the country where the test has to be used [4].

From our results, the osmotic test appears to be almost as reliable as electronic size deter- minations in screening for heterozygous /3 thalassemias. Obviously, electronic haema- tology counters supply much more information than the osmotic test does. Moreover, a dis- criminatory function has been developed that can distinguish iron deficiency from thalassemia trait with erythrocytes indices alone [2]; also a rapid screening test based on electronic size dis- tribution curves has been described, to dis- tinguish thalassemic trait from sideropenic anaemias [7]. However, these tests are not widely used; their results need to be confirmed by determining serum iron, total and latent iron binding capacity, 4 and foetal haemoglobin. So, they d o not give a definitive diagnosis but just a provisional one. In our opinion, in screening of heterozygous /3 thalassemias the osmotic test we used may validly substitute for haematology analysers, in countries where they are not avail- able .

A C K N O W L E D G M E N T S

We are indebted to Mrs Carla Lander for techni- cal assistance.

R E F E R E N C E S 1 Ceriotti, F. & Ceriotti, G. Improved direct specific

determination of serum iron and total iron binding capacity. Clin. Chem. 26 (Z), 327, 1980.

2 England, J.M. & Fraser, P.M. Differentiation of iron deficiency from thalassemia trait by routine blood count. Lancer i, 449, 1973.

3 Flatz, S.D. & Flatz, G. Population screening for p thalassaemia. Lancet ii, 495, 1980.

4 Galen, R.S. & Gambino, S.R. OItre il Concerto di Normalita: il Vafore Preditfivo e YEfficienza delle Diagnosi Mediche. Padova, Piccin editore, pp.

5 Nierhaus, K. & Betke, K. Eine vereinfachte Mod- ifikation der sauren Elution fur die cytologische Dartstellung von fetalem Hamoglobin. Klin. Wschr. 46,47, 1968.

6 Pearson, H.A., OBrien, R.T. & McIntosh. S. Screening for thalassemia trait by electronic measurement of mean corpuscular volume. N. Engl. J . Med. 288,351, 1973.

7 Torlontano. G. , Tata, A. & Camagna, A. A rapid screening test for thalassemic trait. Acra Hemarol. 48, 234, 1972.

8 Wintrobe, M.M. Clinical Hemarology. Phila- delphia, Lea and Febiger, pp. 626633,1974.

lo-19,131-133,1980,

Received 27 September 1982 Accepted 3 December 1982

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