1215 Bioanalysis (2014) 6(9), 1215–1226 ISSN 1757-6180 Research Article part of Valentina Appierto ‡,1 , Maurizio Callari ‡,1 , Elena Cavadini 1 , Daniele Morelli 2 , Maria Grazia Daidone* ,1 & Paola Tiberio 1 1 Unit of Biomarkers, Department of Experimental Oncology & Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, 20133 Milan, Italy 2 Unit of Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133 Milan, Italy *Author for correspondence: Tel.: +02 2390 2238 Fax: +02 2390 2764 mariagrazia.daidone@istitutotumori.mi.it ‡ Authors contributed equally A lipemia-independent NanoDrop ® -based score to identify hemolysis in plasma and serum samples 10.4155/BIO.13.344 © 2014 Future Science Ltd Background: The identification and management of hemolyzed samples are crucial issues in the development of new blood-based biomarkers. Results: Using experiments of controlled hemolysis and lipemia and two plasma series from cancer patients, we developed and validated a lipemia-independent hemolysis score (HS). HS resulted strictly associated with the amount of lysed erythrocytes and with serum index measurement (reference method), highly reproducible, and able to identify as hemolyzed plasma/serum samples containing ≥6.1 mg/dl of free hemoglobin. Conclusion: We developed a simple, robust, sensitive, cost-effective, spectrophotometrically-based system to identify hemolyzed plasma/serum specimens. The procedure requires only 2 µl of sample, thus representing a useful tool for research studies and an essential preanalytical quality control for an optimal biobanking of liquid biopsies. Background Hemolysis is the breakdown or disruption of the integrity of erythrocytes (red blood cells [RBC]) and induces the consequent release of hemoglobin and all the other intra- cellular components into surrounding fluid (e.g., plasma/serum). In vivo hemolysis may be caused by more than 50 different medical conditions (e.g., immune hemolytic anemia, genetic disorders, and chemotherapic treat- ments), does not depend on the technique of the healthcare provider and is thus virtu- ally unavoidable [1] . On the contrary, in vitro hemolysis may be due to suboptimal blood collection and improper sample processing or storage [2] . Hemolysis estimation is a very critical issue for clinical laboratories as it strongly influ- ences and jeopardizes the accuracy and reli- ability of several medical test results (typically producing overestimation of iron, alanine aminotransferase, aspartate aminotransfer- ase, lactate dehydrogenase values and under- estimation of albumin, glucose, sodium lev- els) [3,4] . It is well documented that the mere visual estimation of hemolysis (pink or red coloration) may under-estimate the real sever- ity of hemolysis in the samples and does not allow the detection of samples with low hemo- lysis levels [2] . In fact, endogenous interfering substances can markedly impair the capabil- ity to visually detect hemolysis, an example of which is the underestimation of hemolysis in the presence of elevated concentration of bilirubin [5] . To overcome the intrinsic limi- tations of the procedure, different methods have been developed, and most of them are based on the specific absorption spectrum of hemoglobin (in particular, on oxyhemoglo- bin’s absorbance peak at 414, 541 or 576 nm) [2] . Currently, diagnostic laboratories are equipped with automated instrumentation based on semiquantitative spectrophotomet- ric measurements, that can automatically test for hemolysis, together with other interfer- ences including icterus and lipemia, and that can grade interfering substances into several categories (serum indices) [4,6] . However, an increasing number of ex vivo studies is attempting to identify new diag- nostic, prognostic or predictive circulating biomarkers in plasma or serum samples, as they could be measured in a noninva- sive way and, thus, represent liquid biopsies For reprint orders, please contact reprints@future-science.com