Journal of Hematology, ISSN 1927-1212 print, 1927-1220 online, Open Access
Article copyright, the authors; Journal compilation copyright, J Hematol and Elmer Press Inc
Journal website http://www.thejh.org

Original Article

Volume 8, Number 3, September 2019, pages 111-120


Quantitative Assay of Mutated Nucleophosmin in Acute Myeloid Leukemia

Figures

Figure 1.
Figure 1. FACS analysis of HL60 and OCI-AML3 cells labeled with specific anti-NPM antibodies including anti-pan NPM and antibody for wild-type NPM, and their equivalent negative controls as labeled in the figure. NPM: nucleophosmin; FACS: fluorescence-activated cell sorting; AML: acute myeloid leukemia.
Figure 2.
Figure 2. FACS analysis of HL60 cells labeled with wtNPM, showing dual populations in the bivariate dot plots (upper panel), quiescent (red) and dividing (green), both cellular debris and dividing cells were gated as indicated in the figure, while the lower panel showing a humped peak single parameter histogram. wtNPM: wild-type nucleophosmin; FACS: fluorescence-activated cell sorting.
Figure 3.
Figure 3. FACS analysis of HL60 negative control (secondary antibody only), showing presence of single population in the bivariate dot plots (upper panel), debris was gated as indicated, and a single peak MF histogram in the lower panel. FACS: fluorescence-activated cell sorting.
Figure 4.
Figure 4. FACS analysis of wtNPM in OCI-AML3 cells, showing a single population in the bivariate dot plots (upper panel), debris was gated as indicated, and a single peak MF histogram in the lower panel. FACS: fluorescence-activated cell sorting; wtNPM: wild-type nucleophosmin; AML: acute myeloid leukemia.
Figure 5.
Figure 5. Immunostaining of wtNPM in (a) resting HL60 showing nucleolar localization (red arrowed), (b) dividing HL60 showing cytoplasmic localization in the arrowed cells, images were captured with the Hamamatasu camera and the nuclei were stained with DAPI (blue), (c) HL60 cells stained with wtNPM (white) image, grabbed with the Nikon Eclipse black and white camera, (d) HL60 negative control stained with Texas Red-conjugated secondary antibody only (no staining detected). wtNPM: wild-type nucleophosmin; DAPI: 4',6-diamidino-2-phenylindole.

Tables

Table 1. Numerical Values of Flow Cytometeric Analysis of Human Leukemia Cell Lines, HL60 and OCI-AML3 Labeled With wtNPM and Anti-Pan NPM, and Their Equivalent Negative Controls
 
Test/control% patientsSSC (mean)PE (mean)
wtNPM: wild-type nucleophosmin; SSC: side scatter; PE: phycoerythrin; AML: acute myeloid leukemia.
OCI-AML3 anti-pan NPM99.9131,48410,820
HL60-anti-pan NPM99.6129.0795,779
OCI-AML3 wtNPM99.0125,5669,855
HL60 wtNPM
  P198.7120,5079,522
  P218.4101,11323,654
HL60 control wt98.4120,4684,900
OCI-AML3 control wt99.8131,9257,748
OCI-AML3 control anti-pan99.7132,6287,509
HL60 control anti-pan98.6118,3124,007

 

Table 2. FACS Data of HL60 and OCI-AML3 Labeled With wtNPM, and HL60 Negative Control
 
Test% patientsFITC (mean)
wtNPM: wild-type nucleophosmin; FITC: fluorescein isothiocyanate; FACS: fluorescence-activated cell sorting; AML: acute myeloid leukemia.
HL60 wtNPM
  P188.917,282
  P224.828,436
HL60 control81.35,097
OCI-AML3 wtNPM87.515,539