Determination of hemoglobin is one of the most commonly

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1 ORIGINAL ARTICLE Multiple-Site Analytic Evaluation of a New Portable Analyzer, HemoCue Hb 201+, for Point-of-Care Testing Sten-Erik Bäck, PhD,* Carl G. M. Magnusson, PhD, Lena K. Norlund, MD, PhD, Henning H. von Schenck, MD, PhD, Monica E. Menschik, BS, and P. E. Stellan Lindberg, MS Abstract: The analytic performance of the HemoCue Hb 201+ system was validated to major automatic cell counters in the marketplace and to the previous HemoCue B-Hemoglobin system. In addition, the performance of all instruments was compared against the ICSH reference method. Between 119 and 138 samples were analyzed at 4 different hospitals. In all, 497 patient samples were tested. Imprecision calculated from duplicate samples for all systems ranged from 0.5 to 1.1%. The correlation for all systems against the reference method was better than 0.99 overall, with biases ranging from 0.28 to 0.21 g/dl. Correlations between the new Hb 201+ and ADVIA 120 (Bayer Diagnostics), GEN-S (Beckman Coulter), Sysmex XE 2100, CellDyn 4000 (Abbott), and the HemoCue B-Hemoglobin system showed all r > The HemoCue Hb 201+ had a mean bias of 0.10 g/dl from the reference method and between 0.06 to 0.43 g/dl against the other instruments. Imprecision calculated from duplicate samples on the HemoCue Hb 201+ system was 0.75%. The authors conclude that all hemoglobin methods tested including the new HemoCue Hb 201+ system showed excellent correlation and very small bias to the international reference method. The Hb 201+ system yields results that agreed well with all tested systems, with an accuracy and precision similar to the cell counters. Key Words: automatic cell counters, HemoCue Hb 201+, ICSH reference method, hemoglobin methods (Point of Care 2004;3:60 65) Determination of hemoglobin is one of the most commonly performed analyses in point-of-care (POC) testing situations and the first choice in diagnosing anemia. Accuracy and From the *Department of Clinical Chemistry, Central Hospital, Kristianstad; the Department of Clinical Chemistry, Hospital of Engelholm; the Department of Clinical Chemistry, University Hospital, Lund; the Department of Clinical Chemistry, County Hospital, Halmstad; and HemoCue AB, Department of Research and Development, Engelholm, Sweden. Reagents and laboratory tests supported by HemoCue AB. No other grants were received. Reprints: P. E. Stellan Lindberg, HemoCue AB, Department of Research and Development, Box 1204, SE Engelholm, Sweden ( stellan. lindberg@hemocue.se). Copyright 2004 by Lippincott Williams & Wilkins precision are essential features of hemoglobin determinations because many decisions are based on fixed cutoff levels (eg, diagnosis of anemia, acceptance for blood donation, indications of bleeding, and doping status). The International Committee for Standardization in Hematology (ICSH) method 1,2 has been established as the international reference method for hemoglobin determination. Nevertheless, the use of this method in routine analysis has disadvantages. The reagent is toxic, there are needs for precise pipetting, and the method is susceptible to turbidity. 3 In hospital laboratories, large automated cell counters are most often used to determine blood hemoglobin levels. However, the need to provide accurate, reliable small instrumentation to the POC testing environment is becoming increasingly important. The dominating POC system in the market is the HemoCue B-Hemoglobin system. 3 It is based on an optical measuring cuvette of small volume (10 µl) and a short light path (approximately 0.14 mm) with a dry reagent deposited in the cavity of the cuvette. The cuvette is used as a sampling device and the blood sample is drawn into the cuvette cavity by capillary action. A new portable hemoglobin instrument, the HemoCue Hb 201+ system, was recently introduced. The cuvette and the reagents are similar to those in the HemoCue B-Hemoglobin system. The new instrument is of smaller size (dimensions, mm; weight, 350 g batteries included) and is battery powered. The reaction in the HemoCue cuvette is a modified azide methemoglobin reaction first described by Vanzetti et al. 4 First, the erythrocyte membranes are disintegrated by sodium deoxycholate, followed by the conversion of the hemoglobin iron from the ferrous to the ferric state by nitrite to form methemoglobin, which then combines with azide to form azide methemoglobin. The HemoCue Hb 201+ system and HemoCue B-Hemoglobin system are both factory calibrated with human blood against the ICSH method (discussed later) according to Kwant et al. 5 There is no need for any further calibration by the user. The hemoglobin calibration process for the HemoCue systems is also verified by comparison of results from human blood samples distributed to several reference laboratories over the world. 60 Point of Care Volume 3, Number 2, June 2004

2 Point of Care Volume 3, Number 2, June 2004 HemoCue Hb 201+ TABLE 1. Overview of the Participating Laboratories Site Location Instrument No. of Patients A Central Hospital of Kristianstad Beckman Coulter GEN-S 138 B Hospital of Engelholm Bayer ADVIA C University Hospital of Lund Sysmex XE D County Hospital of Halmstad Abbott CellDyn This study was performed mainly to evaluate the performance of the HemoCue Hb 201+ hemoglobin analyzer against the international reference method for hemoglobin testing according to the ICSH, 1,2 and to compare with results obtained from common clinical laboratory instruments. MATERIALS AND METHODS The study was performed in collaboration with 4 clinical chemistry laboratories in Sweden: Central Hospital of Kristianstad (A), Hospital of Engelholm (B), University Hospital of Lund (C), and County Hospital of Halmstad (D). The study was performed in August to October Between 119 to 138 patient samples selected to cover the relevant measuring range were chosen from specimens submitted for routine analysis in each hospital. The samples were drawn in K 2 -EDTA (Becton Dickinson) and were first analyzed by the routine method in duplicate, followed by duplicate measurements using the HemoCue B-Hemoglobin system and the new HemoCue Hb 201+ system (HemoCue AB, Engelholm, Sweden). In all, 497 patient samples were analyzed and then transported to HemoCue AB in Engelholm, Sweden, for ICSH measurements. All studies were performed according to the NCCLS EP9-A guideline. 6 The following routine instruments were used: GEN-S (Beckman Coulter, Pasadena, CA), ADVIA 120 (Bayer Diagnostics, Tarrytown, NY), Sysmex XE 2100 (Sysmex Corporation, Kobe, Japan), and CellDyn 4000 (Abbott Laboratories, Abbott Park, IL; Table 1). All instruments were run, calibrated, and controlled according to recommendations from the manufacturers. Recalibration of the instruments was not performed during the length of the study. All hospital laboratories are accredited according to EN The ICSH method was initially performed according to recommendations 1,2 and then again with a slight modification of the procedure using a second blanking wavelength at 700 nm instead of filtration. All measurements were performed on a Cary 300 spectrophotometer (Varian Inc., Palo Alto, CA). The method was calibrated using a commercial cyanmethemoglobin standard from Euro-Trol (CNMetHb batch B518a). In addition, at site B (Table 1), to prove our modification of the reference method all samples with an absorbance greater than at 750 nm were filtered and reanalyzed. The results of the filtrated samples were compared with the ICSH method using only one wavelength (540 nm), and with our modified method, which uses a second blanking wavelength at 700 nm. RESULTS The susceptibility of the ICSH reference method to turbidity in the absence of filtration or other means for removing the interference effect is evident from Figure 1A. The correlation using a single wavelength on the ICSH method demonstrated ICSH (Abs 540 nm ) = ICSH Filtered , r = 0.986, and s yx = A much better correlation was obtained between nonfiltered samples processed with the double-wavelength method and filtered samples assayed with the original procedure: ICSH (Abs nm ) = ICSH Filtered , with r = and s yx = 0.12 (Fig. 1B). The results of the new HemoCue Hb 201+ system, as well as those from all hematologic systems evaluated, compared with the modified ICSH method are illustrated in Figure 2. The 2-wavelength method showed a scatter (s yx ) for all systems in the range of 0.13 to 0.21 g/dl. Only minor biases from FIGURE 1. ICSH methods with and without correction of background for samples with turbidity The straight line in A and B represents the regression equation y = x. (A) The regression equation for the comparison for ICSH(Abs 540 nm ) against ICSH Filtered is Y = 0.99x ; r = 0.986; s yx = (B) The regression equation for the comparison for ICSH (Abs nm ) against ICSH Filtered is Y = 1.01x ; r = 0.998; s yx = Lippincott Williams & Wilkins 61

3 Bäck et al Point of Care Volume 3, Number 2, June 2004 FIGURE 2. Comparison of hemoglobin measurements by different instruments and ICSH(Abs nm ). (A) Beckman Coulter GEN-S. (B) Bayer ADVIA 120. (C) Sysmex XE (D) Abbott CellDyn (E) HemoCue B-Hemoglobin. (F) HemoCue Hb All views plotted against ICSH(Abs nm ) Lippincott Williams & Wilkins

4 Point of Care Volume 3, Number 2, June 2004 HemoCue Hb 201+ FIGURE 3. Comparison of hemoglobin measurements by Hb 201+ and different instruments. (A) Hb 201+ plotted against the Beckman Coulter GEN-S. (B) Against Bayer ADVIA 120. (C) Against Sysmex XE (D) Against Abbott Cell-Dyn (E) A summary plot against all cell counters. (F) Against HemoCue B-Hemoglobin Lippincott Williams & Wilkins 63

5 Bäck et al Point of Care Volume 3, Number 2, June 2004 the reference method were observed. The ADVIA showed slightly lower results, with an average bias of 0.18 g/dl. The GEN-S showed results on average 0.28 g/dl and the negative bias was more apparent at higher hemoglobin concentrations. The CellDyn gave results nearly equal to the reference method. Results from the HemoCue B-Hemoglobin system were about 0.21 g/dl higher than the ICSH method. Comparisons between the Hb201+ and the 2-wavelength reference method showed y = 1.009x 0.01; r = 0.998; bias = 0.10 g/dl; n = 497. Results between the HemoCue Hb 201+ and the other systems tested are summarized in Figure 3. The 201+ system gave excellent correlation to all methods, with r = to and s yx = 0.14 to There was a slight positive bias to the ADVIA of 0.30 g/dl and to the GEN-S of 0.43 g/dl. The bias was greater with the GEN-S at higher hemoglobin concentrations. The same result was found in the comparison of the GEN-S against the ICSH method. The graph in Figure 3E shows the results of the Hb 201+ compared with a summary of all cell counters. The correlation was excellent: y = 1.014x and the bias was, on average, 0.23 g/dl. Compared with the HemoCue B-Hemoglobin system, the Hb 201+ system gave, on average, a 0.11 g/dl lower result. Duplicate samples were analyzed on all systems according to the NCCLS protocol. 6 The means, standard deviations and coefficients of variation (CV) are shown in Table 2. For the cell counters the CVs varied from 0.51 to 0.72% over the range 4 to 20 g/dl. Hb 201+ presented similar CVs ( %) at all sites, which are slightly better than the HemoCue B-Hemoglobin system. DISCUSSION In this study comparing hemoglobin methods, it was clear that the choice of blood specimens could be a source of error. The effect of sample turbidity from lipids, proteins, and cell stroma often produces falsely elevated results on hemoglobin determinations. The number of patients affected by these errors was higher than expected. Rather than using the original ICSH reference method for hemoglobin requiring filtration of the samples before measurement, we demonstrated that a more simple way to correct for background turbidity is to include a second wavelength at 700 nm when performing the ICSH measurement. For both HemoCue systems a second wavelength, 880 nm, is incorporated to compensate for background turbidity. They are therefore less susceptible to high lipid contents and high levels of leukocytes. The CLIA 88 analytic quality requirement 7 for hemoglobin states that 95% of the samples should be within ±7% compared with results of the reference method. All methods evaluated in this study fulfilled this requirement compared with the 2-wavelength reference method. The accuracy and precision of hemoglobin determinations are extremely important because many decisions are based on fixed cutoff levels (eg, diagnosis of anemia, acceptance for blood donation, indications of bleeding, and doping status). All instruments evaluated in this study gave results that met these criteria of needed performance including the new HemoCue Hb 201+ system. The data from this study show that the Hb 201+ has a low CV, similar to the automated laboratory cell counters, and proven accuracy to the ICSH reference method. The new, smaller design makes it attractive in POC testing, which, together with the battery power, makes it a flexible system to use in ambulances and screening programs. CONCLUSION All examined cell counters and hemoglobin systems demonstrated excellent correlation and very small bias to the ICSH reference method. Both accuracy and precision of all systems were extremely good. The new HemoCue Hb 201+ system provides results that correlate well to the ICSH reference method and to all automated cell counters tested. Based on this study, the performance of the HemoCue 201+ system is equal to that obtained by many common clinical laboratory TABLE 2. Comparative Methods and Hb 201+ Comparative Methods (X) n Comparative Method (X), mean, g/dl Hb 201+ (Y), mean, g/dl Comparative Method (X) Hb 201+ (Y) SD,* g/dl CV, % SD,* g/dl CV, % Beckman Coulter GEN-S Bayer ADVIA Sysmex XE Abbott CellDyn All laboratory instruments ICSH A 540 A HemoCue B-Hemoglobin *Standard deviation (SD) for 2 replicates is calculated according to the following formula: d 2 /2n, where d is the difference between each sample and n is the total number of samples Lippincott Williams & Wilkins

6 Point of Care Volume 3, Number 2, June 2004 HemoCue Hb 201+ methods currently in use. The new, smaller HemoCue device is, therefore, a suitable alternative for use in POC testing. ACKNOWLEDGMENT The authors thank all laboratory staff involved for technical assistance. REFERENCES 1. Reference and Selected Procedures for the Quantitative Determination of Hemoglobin in Blood. 2nd ed., vol. 14, no. 6. Approved standard. NCCLS document no. H15-A2. Wayne, PA: 1987: May Recommendation for reference method for haemoglobinometry in human blood (ICSH standard 1995) and specifications for international haemoglobincyanide standard. J Clin Pathol. 1996;49: Von Schenck H, Falkensson M, Lundberg B. Evaluation of HemoCue, a new device for determining hemoglobin. Clin Chem. 1986;32: Vanzetti G, Nardeschi A. An azide methemoglobin method for hemoglobin determination in blood. J Lab Clin Med. 1966;January: Kwant G, Oeseburg B, Zwart A, et al. Calibration of a practical haemoglobinometer. Clin Lab Haematol. 1987;9: Method Comparison and Bias Estimated Using Patient Samples: Approved Guideline. Vol. 15, no. 17. NCCLS document no. EP9-A. December CLIA 88 Proficiency Testing Criteria Acceptable Analytical Performance. Code of Federal Register 57 (40); ; February Lippincott Williams & Wilkins 65