Use of the ADVIA 120 for differentiating extracellular and intracellular hemoglobin

Background: The use of cell-free hemoglobin (Hgb) solutions, such as Oxyglobin (Biopure Corp, Cambridge, MA, USA), as a blood substitute for the treatment of acute anemias is increasing in veterinary medicine. These solutions interfere with colorimetric tests, which do not discriminate between cellular Hgb (Hgb_cell) from the patient and extracellular Hgb (Hgb _delta) from the Oxyglobin, and therefore make the monitoring of anemia, based on Hgb concentration, difficult. The ADVIA 120 hematology analyzer (Bayer Diagnostics, Tarrytown, NY, USA) evaluates Hgb by 2 methods, a standard cyanmethemoglobin colorimetric method and flow cytometry, and therefore might provide the means to differentiate extracellular and intracellular Hgb. Objective: The objective of this study was to determine the accuracy and precision of the ADVIA 120 in differentiating extracellular from intracellular Hgb. Methods: Anticoagulated whole blood samples from 10 healthy dogs were analyzed in triplicate on the ADVIA 120. Hgb_delta concentration was determined by adding Oxyglobin (13 g/dL) to the whole blood samples at dilutions of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32. Hgb_cell and Hgb _total values were calculated and compared with actual values by linear regression. Analyses were done in triplicate and repeated 9 consecutive times to evaluate intra-assay precision of Hgb_total and Hgb _cell determinations. Results: Correlation between Hgb values obtained by colorimetric (Hb_total) and flow cytometric (Hgb_cell) methods on whole blood samples was high (R² = .99; n = 10) with a slope of 0.96 and intercept of 0. Correlation between actual and predicted Hgb_cell values also was high (R² = .99), with a small positive bias (0.289 ± 0.185; n = 60). Intra-assay precisions were high, with most coefficients of variation <2%. Conclusion: The ADVIA 120 is capable of differentiating Hgb_cell from Hgb_delta. The flow cytometric method is accurate and precise when compared with the cyanmethemoglobin method. A small bias between the results is unlikely to be clinically significant but may affect the ability of the ADVIA to differentiate small quantities (<0.3 g/dL) of Hgb_delta.