Immunophenotyping

Abstract

This unit presents basic techniques for immunophenotyping by flow cytometry, direct using a conjugated monoclonal antibody and indirect using an unconjugated primary antibody followed by a conjugated secondary antibody. Combinations of these methods are described for two‐, three‐, and four‐color staining. Analysis of data acquired from cells stained by these procedures is detailed. A procedure is given for the detection of the location of nonviable cells so that they can be gated out of the analysis. Keywords: flow cytometry; immunophenotyping; indirect staining; direct staining; multicolor staining; immunophenotypic analysis.

Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins

“…As cell surface proteins confer specific cellular functions and are easily accessible, they are often used as markers to classify cell types3 and as drug targets4. By using available antibodies against cell surface proteins, cells are thus often classified or immunophenotyped according to their cell-surface-protein expression profile5. This approach has been used to immunophenotype cells of the immune system, and for the development of the cluster of differentiation (CD) nomenclature for antibodies against cell surface molecules.…”

Flow Cytometry in Veterinary Oncology

“…Immunophenotyping of hemolymphatic cells pertains to use of a panel of antibodies directed to cell surface or cluster of differentiation (CD) antigens to characterize cells that appear microscopically similar. 73 Intracellular staining of antigens is often employed for detection of cytokines or cytoplasmic antigens but is more laborious than cell surface antigen detection and more prone to artifact. 66 To allow antibodies access to intracellular antigens, the cells have to be fixed and permeabilized.…”

A new adult AML case with an extremely complex karyotype, remission and relapse combined with high hyperdiploidy of a normal chromosome set in secondary AML

“…The abnormal cell population (60% in BM) was positive for CD45dim, CD34, HLADr, CD33, CD117, CD13 and CD11c. This cell population was negative for cyCD3, cyCD79a, CD14, CD64, CD32, CD7, CD19, CD10, and CD5.- The aCGH analysis revealed different genomic imbalances: deletion on 17q21.3; duplication of 3q26.1q29; and trisomy # 6).26 June −02 July 2016- (3 + 7) protocol:(Daunorubicin 60 mg/m 2 for 3 days and Cytarabine 200 mg/m 2 for 7 days)10 Jul 2016Peripheral blood (PB) showed cytopenia (WBC 0.4 × 10 9 /l), anemia (Hgb 9.5 g/dl); thrombocytopenia (Plt 12 × 10 9 /l).Serum creatinine value was 19.8 umol/l (normal 45–120) and serum total bilirubin value was 22.2 (normal value 2–21 umol/l), serum Ca + 2 value 2 (2.15–2.55 mmol/l), serum Na + value 132.3 (135–148 mmol/l).-BM smear showed almost 7% of blats.––26 Jul 2016-Complete remission (CR)PB showed: WBC (6.1 × 10 9 /l), Hgb (11.7 g/dl); Plt (303 × 10 9 /l).-BM smear showed almost 5% of blats46,XX [19]/HeH [2]11 Aug-17 Aug 2017Re-Induction: ICEcytrabin 200 mg/d Day1 ➔Day7etobside 100 mg/d Day1➔Day5idarubicin 20 mg/d Day1 ➔Day325 Sep 2016-Blurred vision in the right eye (retinal detachment sensory serous).-CR-PB showed: WBC (7.4 × 10 9 /l), Hgb (11.6 g/dl); Plt (183 × 10 9 /l).-BM smear showed almost 4% of blats46,XX [14]26 Sep-28 Sep 2017Consolidation1 - HAMCytrabin 3G/m 2 /d Day1➔Day3Methoxantron 20 mg/d D1,D215 Nov 2016Relapse.-Secondary treatment event: mass under vascular arch with splint edema of optical nerve of the right eye which causes to sever decrease of vision in the right eye.-BM smear showed almost 20–30% of blats.-Cerebrospinal fluid (CSF) was negative.PB showed: WBC (5.6 × 10 9 /l, with 98.5 of neutrophils), Hgb (11.6 g/dl); thrombocytopenia [Plt (70 × 10 9 /l)].-Serum creatinine value was 39 umol/l (normal 45–120) and serum Ca + 2 value was 1.94 (2.15–2.55 mmol/l).-CT scan of brain was normal.–17 Nov-19 Nov 2017Consolidation2 - HAMCytrabin 3G/d Day1➔Day3Methoxantron 20 mg/d D1,D230 Nov 2016PB showed: Cytopenia [WBC (0.1 × 10 9 /l)], anemia [Hgb (8.4 g/dl)]; thrombocytopenia [Plt (20 × 10 9 /l)].Serum creatinine value was 33 umol/l (normal 45–120), serum serum K + value 2.89 (3.5–5.2 mmol/l), and serum Na + value 134.6 (135–148 mmol/l).–The mass behind the retina of the right eye was still present03 Jan 2017-Disappeared the previous Mass behind retina.-Relapse.PB showed: WBC (7.5 × 10 9 /l, with 77.7 of neutrophils), Hgb (12 g/dl); Plt (178 × 10 9 /l).-BM smear showed almost 15% of blats- Post to chemotherapy treatment GTG-banding cytogenetics revealed a karyotype92,XXXX [4]/62,XX,+ 1,+ 4,+ 5,+ 5,+ 6, + 6, + 11, + 15, + 16, + 17, + 19, + 19, + 20, + 20,+ 21, + 22 [2]/46,XX [15]. FCM analysis of BM specimen post to chemotherapy treatment characterized this case as AML-M6 according to WHO classifications.…”

“…aCGH was performed using the Agilent Sure Print G3 Human Genome Microarray 180 K as previously described [15]. The aCGH analysis revealed different genomic imbalances (Fig.…”