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Letters to the Editor |
Paracelsus Medical University, and Landeskliniken Salzburg, Department of Clinical Chemistry, Salzburg, Austria
aAddress correspondence to this author at: Department of Clinical Chemistry, Paracelsus Medical University and Landeskliniken Salzburg, Müllner Hauptstraße 48, 5020 Salzburg, Austria. Fax 43-662-4482-885; e-mail: j.unger{at}salk.at.
There is no agreement as to the optimal sample type to be used for measurements of free hemoglobin (fHb) in plasma. (Because fHb is increased during clotting, serum is not recommended.) Several publications have recommended the use of EDTA as an anticoagulant (2), but another author reported 20-fold higher fHb values in EDTA-plasma than in Li-heparinate plasma and therefore recommended Li-heparinate plasma for such analyses (1). Because Li-heparinate and EDTA plasma are often used for emergency and routine measurements, we explored this issue and searched for a practicable, inexpensive, and rapid method for fHb measurements.
Blood from 49 patients was collected into sample tubes (Monovette, Sarstedt) containing EDTA (1.6 g/L of whole blood) or Li-heparinate (16 kIE/L of whole blood). Samples were collected from patients who required laboratory analyses. Patients gave informed consent, and the study received institutional review board approval. The P-module of the Modular® (Roche), which has the method of Glick et al. (3) preinstalled to determine serum/plasma indices, was used to measure the hemolysis index (HI). For comparison with the HI, we used the 2-wavelength method of Golf et al. (4) to measure fHb and not the more commonly used method of Harboe (5), because measurements at multiple wavelengths are not available for users on the Modular. Briefly, fHb was determined by using the difference of absorbances at 540 and 600nm. A hemoglobin solution, prepared as described by Fairbanks et al. (2), was used as a calibrator at 1250 mg/L. The method detection limit was approximately 50 mg/L as determined by analysis of replicate measurements (n = 10) of calibrator dilutions and the 0 calibrator. At concentrations of 90, 300, 1250 and 9500 mg/L, intraassay imprecision values (CVs) for fHb were 9%, 5.6%, 1.3%, and 0.5%, respectively, and for HI were 15%, 3.6%, 0.6%, and 0.3%. Interassay CVs, determined by replicate measurements during 10 days at 90 and 1250 mg/L fHb concentrations, were 9.4% and 3.5% for fHb and 12.4% and 0.6% for H-index measurements, respectively. The values for fHb in our patients ranged from <50 to 794 mg/L in EDTA plasma and from <50 to 1120 mg/L in Li-heparinate plasma. The corresponding values for H-index ranged from <50 to 760 (EDTA plasma) and <50 to 1070 mg/L (Li-heparinate plasma), respectively. In univariate ANOVA, no significant differences in mean (SD) fHb values were observed between specimens collected in EDTA [232 (179) mg/L] or heparin [254 (226) mg/L]. Similarly, mean (SD) HI did not differ between the 2 materials [153 (173) for EDTA and 173 (225) for Li-heparinate). Correlation coefficients, R, between fHb and H-index were 0.939 in EDTA plasma and 0.967 in Li-heparinate plasma. Difference plots (fHB – HI) for both plasma collections showed no correlation of difference with the mean of fHB and HI. Correlations between fHb and HI in EDTA plasma were substantiated in 200 additional samples (R = 0.990). In these samples, values for fHb and HI ranged from <50 to 6300 mg/L and <50 to 7030 mg/L, respectively. In samples with fHb values <900 mg/L, the correlation remained high (0.860). We therefore could not confirm with these sample collection tubes the statement that fHb may be 20-fold higher in EDTA plasma than in heparin plasma (1).
An effect of EDTA became apparent when we determined fHb and HI in blood from healthy volunteers collected with increasing EDTA concentrations. At EDTA concentrations 3 times the usual, both fHb and HI were >50% higher than values in EDTA plasma collected under standard conditions. In contrast, similar experiments with Li-heparinate plasma showed no dependence of fHb or HI on Li-heparinate concentrations in the range of 16–48 kIE/L whole blood. Thus EDTA influences fHb and HI in a concentration-dependent manner, but the effect can be disregarded when collection tubes are correctly filled.
We conclude that fHb and HI can be measured in blood collected either with heparin or EDTA provided that EDTA tubes are adequately filled. Because of the excellent agreement of values for fHb measurements and HI in the P-module of the Modular, HI may be sufficient in many clinical settings.
Acknowledgments
Grant support/funding: This work was supported by a grant from the Land Salzburg.
Financial disclosures: None declared.
References
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