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Clinical Chemistry 44, No. 3, 1998


their glyHb content: Abbott Control L, M, and H (Abbott Diagnostici; Abb L, Abb M, and Abb H, codes 1A86–10, lot no. 13207M100); DCA 2000 normal and abnormal controls (Bayer Diagnostics; Bay N, code 5072, lot no. 0068; Bay A, code 5073, lot no. 0056); GlycoHb control I and II (Menarini diagnostics; Gly I, code U8312, lot no. 6031; Gly II, code U8312, lot no. 6031); Lyphocheck (Bio-Rad Labs.; Lyp I, lot no. 33531; Lyp II, lot no. 33532); Precinorm and Precipath (Boehringer Mannheim; Pre N, code 1488422, lot no. 187994; Pre P, code 1488449, lot no. 187995); and Roche HbA1c control N and P (Roche Diagnostics; Roc N, code 0755672, lot no. T1937; Roc P, code 0755680, lot no. T1937). We also analyzed two home-prepared lyophilized materials (Sam 1, Sam 3) tested for homogeneity and stability; one has already been used in an Italian interlaboratory exercise [11]. All the control materials, with the exception of the Abbott, were available in the solid state as lyophilized hemolysates. They were reconstituted with distilled water, or with the appropriate reconstitution fluid (Bayer, Roche) according to the manufacturer’s instructions.

For each method comparison the glyHb concentration was measured simultaneously in 50 samples and in 15 control materials, in five separate studies. In fact, for logistic reasons, 50 blood specimens were analyzed simul- taneously by the Bio-Rad Variant, the Kontron s400, the Merck-Hitachi L-9100, and by the Menarini H8140 meth- ods. Different batches of 50 samples each were then analyzed separately by the other methods (Abbott IMx, Boehringer Tina-quant, Roche Unimate A1c, and Tosoh A1c 2.2). Particular attention was dedicated to sample selection, to study homogeneous specimen groups. As a matter of fact, the 50 sample batches were selected on five different occasions from the routine laboratory, by collect- ing samples from healthy and diabetic subjects to cover approximately the same Hb A1c range. The Hb A1c ranges (Bio-Rad Variant values) analyzed in the five studies were: 5.1–14.5%, 5.2–14.3%, 4.4–12.6%, 4.4–12.8%, and 4.7–13.0%.

analytical methods The following methods for glyHb measurement in blood were used according to the manufacturers’ instructions. Affinity chromatography methods. Abbott IMx test (Abbott Diagnostici).

Hb content, spectral analysis of Hb derivatives, and methemoglobin (MetHb) concentration were measured by standard laboratory techniques [13].

statistical analysis

Method imprecision was evaluated by means of the test of differences of duplicates on 10 normal and 10 diabetic samples. In this test, the mean is equal to the sum of all results divided by their number, whereas SD ( d2/ 2n)1/2, where d2 is the sum of the squares of the differences among the duplicates, and n is the number of


Methods comparison was elaborated with the stan- dardized principal component analysis [14]. In all the comparisons the results obtained by the Bio-Rad Variant method were plotted on the x-axis and the regression equation was calculated from the measurements on blood samples. The results obtained on the control materials were reported as normalized residuals, i.e., as (y y )/Sy x, where y is the results obtained by the y-method (Abbott IMx, Boehringer Tina-quant, Hitachi L-9100, Kon- tron s400, Menarini H8140, Roche Unimate A1c, Tosoh A1c 2.2), y the values calculated from the equations of the

regression line, and Sy x samples.

the residuals calculated on blood

Data correction after calibration was achieved by con- structing, for each method, a calibration curve with the assigned Hb A1c value on the x-axis (i.e., the Bio-Rad Variant value obtained on each control), and the reported y-method value on the ordinate. The original data ob- tained on blood samples with the y-method were then recalculated with the calibration curve and plotted in terms of bias units with respect to the measurements performed with the x-method.

Results The control materials were first analyzed with regard to their composition. Table 1 reports data on total Hb, MetHb, and Hb derivatives content. As can be seen, most of the materials possess a total Hb content similar to that of whole blood, with the tendency of being in the normal- low or anemic range. Their MetHb concentrations were found generally higher than in normal whole blood

(usually MetHb is





1%), from moderate (Gly






I and II, was not

HPLC methods. Bio-Rad Variant (Bio-Rad Labs.); Hitachi system L-9100 (Merck Bracco); Kontron system 400 (Kon- tron Instruments); Menarini H8140 (Menarini diagnos- tics); and Tosoh system A1c 2.2 (Eurogenetics Tosoh).

Immunochemical methods. Boehringer hemoglobin A1c Tina-quant test (Boehringer Mannheim); Roche Unimate A1c (Roche Diagnostics).

Hb electrophoresis was performed on cellulose acetate followed by staining with Ponceau Red and counterstain- ing with tetramethyl benzidine, as described [12]. Total

possible to determine MetHb concentration materials because these controls were found cyanMetHb form.

in the Abbott mainly in the

The composition of the controls was then investigated by cellulose acetate electrophoresis. Fig. 1 reports typical patterns obtained after fixation and staining. The main bands visible under these conditions were those of Hb A, Hb A2, and carbonic anhydrase, but in most of the materials this pattern was altered because of various reasons. In the case of the Abbott L, M, and H materials (Fig. 1, lanes 2–4), the Hb A band was more diffuse than in normal controls, with a cue prominent at the cathodic

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