tuberculosisis not influenced by anticoagulants and/or Ficoll. plasma, irrespective of type of anticoagulant or Ficoll present (r 0.85, p < 0.0001). However, the presence of ACD resulted in slightly lower values than those obtained with serum in both indirect (antibody reactivities to mycobacterial antigens) and Sandwich ELISAs (soluble CD14 measurements). Our data demonstrate that leftover plasma, regardless of made up of anticoagulants or Ficoll, can be used in TB antibody or other host protein biomarker studies but suggest the value of a correction factor when using ACD plasma interchangeably with serum in antibody binding studies. Keywords:EDTA, ACD, CPT, heparin, immunoglobulins, proteins == Introduction == Investigating human serum antibodies and other non-antibody host proteins is important for both discovery of tuberculosis (TB) biomarkers and assessment of correlates of protection against TB. Increasing evidence now supports a role of human serum antibodies in the protection against TB (reviewed in [13]). However, knowledge of protectiveM. tuberculosisantigens as well as non-sputum-based biomarkers for the prediction and diagnosis of early TB are urgently needed. The ability to utilize leftover plasma from other studies, regardless of made up of anticoagulants or Ficoll for blood separation, would greatly enhance opportunities for TB antibody and other host protein investigations. The anticoagulants EDTA, heparin, and acid citrate dextrose (ACD) are commonly used for the separation of plasma and blood cells. In addition, the use of BD Vacutainer Mononuclear Cell Preparation Tubes (CPT), made up of heparin sodium or sodium citrate and Ficoll-Paque, has greatly facilitated the separation and preparation of peripheral blood mononuclear cells (PBMCs) for research studies [46]. Ficoll, a neutral hydrophilic polysaccharide, dissolves in aqueous liquids. The use of CPT tubes is more convenient yet equally effective compared to the conventional Ficoll density gradient technique since the actions involved are easier to perform and are less time consuming. The less elaborate CPT isolation protocol also minimizes variations in processing and in specimen contamination, as well as eliminates the requirement for clinical sites TRx0237 (LMTX) mesylate with trained laboratory personnel or specialized laboratory gear [4,6]. For these reasons, CPT tubes are increasingly used for PBMC isolation in pediatric and adult populations. Serum, which, unlike plasma, lacks fibrinogen and clotting factors, is usually traditionally used for antibody reactivity and functional assays. While prior studies by us as well as others show that this anticoagulants heparin and EDTA in plasma have little effect on antibody reactivities toM. tuberculosisand other mycobacterial antigens, little is known about IP1 the effects of ACD and CPT plasma on antibody reactivities, especially in the context of TB [79]. Furthermore, we as well as others previously showed that antibodies toM. tuberculosisantigens can have Fc-gamma receptor (FcyR)-mediated functions, such as the uptake ofM. tuberculosisinto macrophages [13]. Whether anticoagulants could influence such antibody functions is usually unclear. Prior studies have shown that blood treated with citrate has decreased activity of some immune functions, such as activation of the complement system, neutrophil degranulation, and cytokine production [10,11]. Furthermore, serum antibodies from cholera patients have lower vibriocidal activity in the presence of ACD and EDTA compared to heparin or no anticoagulants [12]. Therefore, our objectives were to assess and to correlate the following with simultaneously obtained serum and plasma from heparinized, EDTA, ACD, and CPT tubes: i) IgG, IgM, and IgA antibody reactivities to two mycobacterial antigens, one polysaccharide and one protein; ii) antibody-mediated human macrophage phagocytosis ofM. tuberculosis; and iii) levels of the human TB biomarker host protein soluble CD14 (sCD14). == Subjects and Methods == == Subjects. == We enrolled 24 TRx0237 (LMTX) mesylate asymptomatic volunteers and patients; 11/24 (46%) had a history of a positive tuberculin skin-test, 18/24 (75%) had a history of receiving theM. bovisBacillus Calmette Guerin TRx0237 (LMTX) mesylate (BCG) vaccination, and 9/24 (38%) had a history of microbiologically confirmed TB, four of whom were HIV co-infected. Subjects median age TRx0237 (LMTX) mesylate was 42 years (range 25 to 69) and 14/ 24 (58%) were male. Blood samples were obtained using BD Vacutainer collection tubes: Serum Separation Tubes (SST), Heparin (lithium heparin), ACD, K2 EDTA, and Mononuclear Cell Preparation Tubes made up of sodium heparin and Ficoll (CPT; BD Life Sciences: Preanalytical Systems, Franklin Lakes, NJ). Of the 24 subjects, 16 had blood simultaneously obtained with all tubes, 7 with all except for ACD tubes, and one with all except for ACD and EDTA tubes. To allow for simultaneous processing of samples, the assessments ofM. tuberculosisphagocytosis and sCD14 were performed only with samples for the 16 subjects that had all plasma types available. Serum and plasma tubes were centrifuged for.