Advantages of negative isolation
Negative isolation with magnetobead-coupled anti-CD14 mAbs shows the following advantages. There is minimal contamination with macrophages (<2%) and other inflammatory cells (all <1%). Thus, although CD14 may be expressed to a lower degree than CD68 on RA synovial lining macrophages [36,39], the degree of CD14-positivity appears more than sufficient to conduct the isolation procedure. The positive fraction, in turn, contains virtually no SFB, indicating minimal cell loss, and thereby also excluding major subset selection. Another advantage is that negative isolation with Dynabeads® M-450 CD14 avoids direct contact of SFB with mAbs and/or magnetobeads, thereby reducing possible functional alterations of the cells. Finally, the anti-Thy-1 mAb AS02 (used in the parallel attempt to positively isolate SFB) identifies 91% of normal skin-FB, but not more than 74% of RA-SFB (Tables 1 and 4). In addition, the prolyl-4-hydroxylase+ cells obtained by negative isolation with Dynabeads® M-450 CD14 contain both a Thy-1+ and a Thy-1– fraction. Thy-1-independent isolation thus circumvents the danger of selecting for Thy-1+ SFB subpopulations, especially relevant since Thy-1+ and Thy-1- FB diverge in several characteristics potentially relevant to RA; for example, cytokine and matrix production, as well as antigen presentation [49] (reviewed in [50,51,52]).
The negative isolation technique, however, also bears possible disadvantages: the limited number of cells obtained (approximately 2.8 × 107), due to the lack of in vitro expansion, and the necessity for reliable access to fresh synovial specimens. On the other hand, the applicability of this procedure not only to joint replacement samples, but also to arthroscopic synovectomy samples, augments the potential sources of fresh synovial tissue.