Optimized Treatment of Heparinized Blood Fractions to Make Them Suitable for Analysis.
BACKGROUND
It has been known for decades that many cytokines, such as IL-2, IL-6, and IL-12, bind to heparin. Even though some enzyme-linked immunosorbent assays (ELISA) use antibody-recognizing epitopes not affected by this binding, ELISA manufacturers often warn that heparinized plasma or serum fractions containing more than 3 IU (international units)/mL of heparin should not be used in assays so as to prevent heparin interference in the reaction. In addition, enzyme-based nucleic acid amplifications from heparinized samples have been shown defective by several research groups. The aim of this study was to determine optimal degradation and/or removal of heparin from heparinized blood samples to best turn them into fractions for appropriate ELISA and RT-PCR analysis.


RESULTS
A colorimetric reporter assay based on the metachromatic effect of the binding of heparin to toluidine blue was shown to be a low-cost effective method to discriminate assay compatible blood fractions with heparin levels below 3 IU/mL. Heparin removal from human blood fractions was best achieved by treatment with either Bacteroides Heparinase II or the less expensive Heparinase I at a final concentration of 0.1 U/μL and incubations at 30°C for a period between 30 min and 4 h, or by adsorption to Ecteola slurries at a concentration of 20 mg/mL for 20 min at room temperature (RT). The fact that both enzymatic and resin-based optimized treatments allowed for replication of the readings obtained with heparin-free equivalent fractions in both ELISA and RT-PCR assays indicates they should be appropriate for quantitative studies such as expression profiling at both the protein and nucleic acid level.


CONCLUSIONS
The cost-effective protocols developed in this study could make heparinized, otherwise unusable, blood-derived collections suitable for analysis by ELISA and RT-PCR amplifications, among other analyses, enhancing the possibilities for studying valuable bio-banked heparinized human samples.
1 Facultad de Medicina, Universidad Católica de Valencia "San Vicente Mártir" , Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain .
María Teresa
MT
Sánchez-Fito
1 Facultad de Medicina, Universidad Católica de Valencia "San Vicente Mártir" , Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain .|2 Instituto Valenciano de Patología (IVP), Universidad Católica de Valencia "San Vicente Mártir" , Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain .
Elisa
E
Oltra
D001774:Blood Chemical Analysis / Q000379:methods*; D001800:Blood Specimen Collection / Q000379:methods*; D002482:Cellulose / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D015925:Cryopreservation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005615:Freezing; D006493:Heparin / Q000737:chemistry*; D019763:Heparin Lyase / Q000737:chemistry; D006801:Humans; D018664:Interleukin-12 / Q000097:blood; D007376:Interleukin-2 / Q000097:blood; D015850:Interleukin-6 / Q000097:blood; D010949:Plasma / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction; D014048:Tolonium Chloride / Q000737:chemistry
D007376:Interleukin-2; D015850:Interleukin-6; D014048:Tolonium Chloride; D018664:Interleukin-12; D002482:Cellulose; D006493:Heparin; D011133:Polysaccharide-Lyases; C114663:heparinase II; D019763:Heparin Lyase