Optimized Treatment of Heparinized Blood Fractions to Make Them Suitable for Analysis

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.

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.

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.