PMC5294364_01
Kinetic of proteinuria (a) and CD20 cell count (b) in the first case. CD20 cell count is both displayed as absolute values (A) as well as percentage (B). Time points of Rituximab administrations are marked accordingly.
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fig1
The time course of proteinuria is shown in Figure 1(b). Surprisingly, we did not see a substantial suppression of CD20+ B-cells following the first two Rituximab applications, as seen in Figure 1(b).
CD20 expressing Daudi cells (a1) were incubated with urine for 20 min to enable suspected Rituximab to bind the CD20 antigen (a2). After three washes in PBS/BSA, cells were incubated with a PE-labeled anti-human IgG antibody and incubated for 20 min (a3). After three washes in PBS/BSA, cells were subjected to flow cytometry analysis. Controls were identically performed but by using Octagam solution instead of urine. Flow cytometric analysis of Daudi cells. Daudi cells were gated according to their FSC/SSC properties (b1). Histograms of Daudi after incubation with PBS (b2), Octagam (b3), urine of the first patient (b4), and peritoneal dialysate fluid of the second patient (b5) are shown. Cell numbers are shown on y-axis and relative fluorescence intensity of the secondary (anti-human IgG) antibody on x-axis.
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fig2
The principle of Rituximab detection by flow cytometry and representative FACS plots are shown in Figure 2. 10000 events of each sample were acquired after gating on Daudi cells according to their FCS/SSC properties.
Rituximab urine concentrations (a), urine Rituximab/creatinine ratios as well as IgG urine concentrations (c), and IgG/creatinine ratios in the first case.
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fig3
Results of Rituximab and IgG measurement in the first case are shown in Figure 3. Since the amount of urinary output and the urine concentration ability of the patient varied significantly between the different probes, we additionally determined urine creatinine and calculated Rituximab/creatinine ratios for all samples.  Figure 3(a) shows Rituximab urine concentrations and Figure 3(b) the corresponding Rituximab/creatinine urine ratios.
PMC5294364_02
Rituximab urine and peritoneal fluid Rituximab concentrations (a) and Rituximab/creatinine ratios (b), as well as IgG urine and peritoneal fluid concentrations (c), and IgG/creatinine ratios (d) of the second case. Since the second patient performed CAPD with three to four peritoneal fluid bag changes per day, the corresponding effluent bags are numbered in chronological order.
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fig4
Results are shown in Figure 4.