Single-color Multitarget Flow Cytometry Using Monoclonal Antibodies Labeled with Different Intensities of the Same Fluorochrome

Abstract

BackgroundWe developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay.MethodsWe selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples.ResultsCV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein α-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright, and MFC, in the 23 patient samples (B cells, T cells, Tcytotoxic cells, and Thelper cells; r2≥0.98, 0.99, 0.99, and 0.99, respectively; P<0.05).ConclusionsThe multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.

Baseline Immune Biomarkers as Predictors of MBSR(BC) Treatment Success in Off-Treatment Breast Cancer Patients

“…For instance, the number of lymphocytes averaged approximately 1,000/ml of blood (see Table 2), which is within the range of 490-6,000/ml (median of 2,000/ml) as reported by Park and Han (2012). The percentages of CD3þ lymphocytes (*80%), CD16þ/CD56þ cells (*10%), and CD19þ cells (*10%) were also in line with other reports (Abud-Mendoza et al, 2012; Carlson, Speca, Faris , & Patel, 2007;Park & Han, 2012). Regarding the ratio of Th1 to Th2 cells, others have reported ratios from 12.5 (Toldi et al, 2011) to 5.1 (Cseh et al, 2012), while we observed ratios ranging from 3.4 to 9.1 in the present study, depending upon the group.…”

“…Also, the baseline biomarker levels we obtained in the present study are similar to those other researchers have reported for similar populations. For instance, the number of lymphocytes averaged approximately 1,000/μl of blood (see Table 2), which is within the range of 490–6,000/μl (median of 2,000/μl) as reported by Park and Han (2012). The percentages of CD3+ lymphocytes (~80%), CD16+/CD56+ cells (~10%), and CD19+ cells (~10%) were also in line with other reports (Abud-Mendoza et al, 2012; Carlson, Speca, Faris, & Patel, 2007; Park & Han, 2012).…”

Flow cytometric discrimination of seven lineage markers by using two fluorochromes

“…Other reports have successfully combined few markers in two fluorochrome to positively identify and separate immune cell populations. [ 16 – 18 ]. Combining multiple antibodies in single channels is an accepted methodology that has also been implemented in clinical standardized protocols to identify malignant leukocytes [ 19 ].…”

pH controlled staining of CD4+ and CD19+ cells within functionalized microfluidic channel

“…They are indicated as TF1-antiCD4 and TF2-antiCD19 MAbs. [26][27][28] Thiophene fluorophores provide MAbs with attractive features for spectroscopic characterization. 28,29 First, the platforms were incubated with each single TF-labeled antibody at pH 5.0, to ensure that the proteins would be loaded via ion exchange mechanisms.…”

“…Anti-human CD4 and CD19 monoclonal antibodies (MAbs) were chosen for the detection of T and B lymphocytes, respectively. 27,28 These selected monoclonal antibodies were previously labeled with thiophene fluorophores (TFs). TFs are fluorescent probes characterized by high optical stability, bright fluorescence, and large Stokes shifts, particularly well-suited for multiplexing analyses.…”