Optimized Procedure for DNA Isolation from Fresh and Cryopreserved Clotted Human Blood Useful in Clinical Molecular Testing

In the routine clinical laboratory, large amounts of uncoagulated blood are collected and the blood clot usually is discarded. In molecular biology, cells from EDTA-anticoagulated or acid-citrate-dextrose-anticoagulated peripheral blood are used as sources of DNA (1)(2)(3). After leukocyte isolation, most procedures utilize enzymatic cell digestion, followed by extraction with hazardous organic solvents (phenol-chloroform) and precipitation with ethanol (4)(5). To minimize the volume of blood collected for laboratory tests, several authors have developed methodologies to isolate DNA from blood clots (4)(5)(6)(7)(8). However, the techniques may be difficult or impractical and may require slicing of the clot with scalpels or other sharp instrument, exposing laboratory personnel directly to contaminated blood (4)(7). Other techniques are time-consuming, using many chaotropic reagents, enzymes, RNA-removal steps, or large volumes of samples and reagents not suitable in the clinical laboratory (4)(5)(6)(7)(8). We have optimized a nonenzymatic, nontoxic procedure for efficient DNA extraction from fresh and cryopreserved clotted blood. Blood samples were obtained from 24 unrelated individuals who had given informed consent. We compared 10 paired samples of EDTA-anticoagulated blood and fresh blood clot and studied 14 samples of cryopreserved clot that had been frozen for 2 years at −20 °C. Blood clots were homogenized with 9 g/L NaCl, using a Potter-MARCONI MA 099 system, for 30 s. The homogenizing Teflon probe was cleaned three times with 700 mL/L ethanol and 9 g/L NaCl between samples to avoid cross-contamination. One milliliter of each homogenized sample was centrifuged at 1200g for 5 min, and the supernatant was removed. Blood cells were lysed with 1 mL of Tris buffer 1 (10 mmol/L Tris-HCl, pH 8.0, 10 mmol/L KCl, 10 mmol/L MgCl2, 2 mmol/L EDTA, pH 8.0, and 25 mL/L Triton X-100). After centrifugation, the pellet was washed twice with Tris buffer 1 and lysed with 220 μL of Tris buffer 2 (10 mmol/L Tris-HCl, pH 8.0, 10 mmol/L KCl, 10 mmol/L MgCl2, 2 mmol/L EDTA, pH 8.0, 0.4 mol/L NaCl, and 10 g/L sodium dodecyl sulfate) and incubated for 15 min at 56 °C. Cellular proteins were removed by precipitation, after addition of 100 μL of 5 mol/L NaCl. DNA was isolated by ethanol precipitation and solubilized in Tris-EDTA buffer (10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA). The DNA concentration was measured by spectrophotometry at 260 nm, and DNA purity was determined by the A260/A280 ratio (3). The integrity of the DNA samples was verified by agarose gel electrophoresis after ethidium bromide staining under ultraviolet light. The suitability of the DNA obtained from clotted or anticoagulated blood was evaluated by PCR amplification of the HincII polymorphic region (exon 12) of the human low density lipoprotein receptor (LDLR) gene (9). Analysis of the washes between samples by DNA examination and PCR amplification did not show evidence of cross-contamination. Results were expressed as mean ± SD. Student’s t-test and regression analysis were used to evaluate differences of yield and purity between DNA extracts. Differences were considered significant at P <0.05. Clotted and whole blood provided similar yield and purity (A260/A280) of DNA (Table 1). These data are similar to those obtained when proteinase K treatment and organic extraction were used (4) and higher than reported for other salting-out procedures (1)(6). Concentration and A260/A280 ratio of DNA extracted from cryopreserved and fresh clotted blood and EDTA-anticoagulated blood. Mean ± SD. Number of samples. P <0.05. Concentration and A260/A280 ratio of DNA extracted from cryopreserved and fresh clotted blood and EDTA-anticoagulated blood. Mean ± SD. Number of samples. P <0.05. The reduction of the reagent volumes and replacement of Nonidet P40 with Triton X-100 (as described previously for DNA isolation from whole blood (10)) enhanced the feasibility and precision of the DNA isolation from fresh blood clot. The within-run CV of the DNA extraction from clotted blood was similar to that for the anticoagulated blood. The purity of DNA extracted from clot was comparable with whole blood (Table 1). All procedures can be performed at room temperature, providing similar results when compared with 4 °C (data not shown). We also could handle multiple samples in less time than with other protocols (4)(5)(6)(7)(8). These features make the procedure even more appropriate for routine laboratory work. The lower DNA recovery from cryopreserved clots (Table 1) probably reflected the higher number of wash steps necessary to remove hemoglobin from cryopreserved specimens (11). The DNA purity and concentration were similar to those reported by other authors (5). DNA concentrations recovered from blood clot and whole blood were correlated (P <0.05, Fig. 1 , top). A significant correlation (r= 0.86, P <0.05) was also found between leukocyte counts in blood and DNA concentrations (data not shown). Gel electrophoresis showed high molecular weight DNA in all samples (Fig. 1 , bottom). Moreover, the HincII region of the LDLR gene could be easily amplified from both DNA preparations. These results show that DNA can be efficiently extracted from cryopreserved samples, even after prolonged storage of the specimens. Comparison of DNA from clotted blood and EDTA-anticoagulated or whole blood. (Top) Correlation between concentrations of DNA extracted from fresh clotted blood (y) and EDTA-anticoagulated blood (x). (Bottom) Ethidium bromide-stained agarose gel electrophoresis of DNA extracted from clotted blood (lanes 1–3) and whole blood (lanes 4–6); lane M, DNA marker (1 Kb). In conclusion, the protocol described here enables molecular biologists to obtain DNA from blood clots by use of stable and nonhazardous reagents. The method is simple, fast, and reliable for obtaining high quantities of DNA suited for clinical molecular testing. Faculty of Pharmaceutical Sciences, University of São Paulo, CEP 05508-900, São Paulo, SP, Brazil fax 011-813-2197, e-mail mdchirta@usp.br We thank Carlos A. C. Sannazzaro, Francesca C. Theobaldo, and Regina C. Vendramini for assistance in collecting the samples, and Nga Y. Nguyen for primers used in the amplification of the LDLR gene. L.A.S. was the recipient of fellowship from the CNPq-Brazil.