Immunomagnetic depletion of RBCS directly from whole blood without the need for hypotonic lysis or sedimentation

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• 作者：Carrie E. Peters^a ; Taylor N. Lee^a ; Steven M. Woodside^a ; Terry E. Thomas^a ; Allen C. Eaves^a ; ^b
• 刊名：Human Immunology
• 出版年：2015
• 出版时间：October 2015
• 年：2015
• 卷：76
• 期：supp_S
• 页码：64
• 全文大小：41 K

文摘

Sample analysis in HLA laboratories frequently requires red blood cell (RBC) depletion as a first step. However, hypotonic lysis of whole blood may be deleterious to certain cell types, and RBC removal by hetastarch sedimentation is inconsistent. Furthermore, RBC depletion with agents such as ammonium chloride is often ineffective, leaving samples that still contain many RBCs. We have developed an immunomagnetic method to deplete RBCs from whole blood samples that is rapid, effective, and leaves untouched total nucleated cells (TNC) ready for downstream assays.

Methods

RBCs were removed either magnetically using the EasySep™ RBC Depletion Reagent or using ammonium chloride lysis (control). To perform magnetic RBC depletion, whole blood was diluted 1:1 with buffer (phosphate buffered saline plus 2 mM EDTA), the EasySep™ RBC Depletion Reagent added and the sample placed in an EasySep™ magnet for 5 min. Magnetically labeled RBCs were retained in the magnet and TNC were simply poured or pipetted off. The Reagent addition, magnet incubation and pour-off steps were repeated once more, followed in some cases with one more final magnet incubation and pour-off. The number of TNC recovered were counted and the samples evaluated by flow cytometry for RBC content (CD45- / Glycophorin A+) and viability (7AAD-).

Results

4.2±1.3×10⁶$4.2\pm 1.3\times {10}^6$ TNC per mL of starting whole blood were recovered when using the EasySep™ RBC Depletion Reagent, compared to 5.5±2.1×10⁶$5.5\pm 2.1\times {10}^6$ TNC recovered from the same samples by ammonium chloride lysis (n = 8; p  <$<$0.05). RBC contamination of the TNC fraction was significantly lower when samples were treated with the EasySep™ RBC Depletion Reagent rather than ammonium chloride (1.2 ±$\pm$ 1.3% versus 19 ±$\pm$ 18% of total events were CD45- / Glycophorin A+ respectively; p  <$<$0.05). The viability of the TNC obtained by either method was 99 ±$\pm$ 1%.

Conclusions

RBCs can be immunomagnetically depleted from a sample of whole blood in 10 - 15 minutes, without exposing the cells to hypotonic lysis or the need for hetastarch sedimentation. The isolated nucleated cells are immediately ready for analysis or further downstream assays.

C.E. Peters:Employee; Company/Organization; STEMCELL Technologies Inc.T.N. Lee:Employee; Company/Organization; STEMCELL Technologies Inc.S.M. Woodside:Employee; Company/Organization; STEMCELL Technologies Inc.T.E. Thomas:Employee; Company/Organization; STEMCELL Technologies Inc.A.C. Eaves:Other (Identify); Company/Organization; Owner STEMCELL Technologies Inc.