Ann Lab Med.  2019 Mar;39(2):150-157. 10.3343/alm.2019.39.2.150.

Comparison of High Sensitivity and Conventional Flow Cytometry for Diagnosing Overt Paroxysmal Nocturnal Hemoglobinuria and Detecting Minor Paroxysmal Nocturnal Hemoglobinuria Clones

Affiliations
  • 1Department of Laboratory Medicine, University of Ulsan College of Medicine, Ulsan University Hospital, Ulsan, Korea. uucm77@gmail.com
  • 2Department of Laboratory Medicine, Pusan National University Yangsan Hospital, Yangsan, Korea.
  • 3Department of Hematology and Cellular Therapy, University of Ulsan College of Medicine, Ulsan University Hospital, Ulsan, Korea.

Abstract

BACKGROUND
High sensitivity flow cytometry (HS-FCM) was recently developed for diagnosing paroxysmal nocturnal hemoglobinuria (PNH). We compared its performance with conventional flow cytometry (C-FCM) for diagnosing overt PNH and detecting minor (0.1-1%) PNH clones in aplastic anemia (AA)/low-grade myelodysplastic syndrome (MDS) patients.
METHODS
C-FCM and HS-FCM were performed simultaneously on 41 samples from healthy controls and 23 peripheral blood samples from 15 AA/low-grade MDS and eight PNH patients, using a Navios flow cytometer (Beckman Coulter, Miami, FL, USA). Results were compared.
RESULTS
No healthy control samples had PNH clone size >0.01%. For granulocytes, C-FCM detected a smaller PNH clone size than HS-FCM (mean difference: 0.7-1.7%). In AA/low-grade MDS patients, three samples showed >1% PNH clones with C-FCM but not with HS-FCM. Seven samples showed minor PNH clones by C-FCM, but HS-FCM showed negative results for all these samples. In PNH patients, C-FCM detected a smaller PNH clone size than HS-FCM (mean difference: 1.9-5.0%). For red blood cells, C-FCM detected a greater PNH clone size than HS-FCM (mean difference: 1.5%). In AA/low-grade MDS patients, C-FCM showed >1% PNH clones in six samples, but HS-FCM showed >1% PNH clones in none of the samples. C-FCM detected minor PNH clones in nine samples, but six of them were negative by HS-FCM. In PNH patients, C-FCM detected a greater PNH clone size than HS-FCM (mean difference: 2.5%).
CONCLUSIONS
HS-FCM can sensitively detect minor PNH clones and reduce false-positive C-FCM minor PNH clone cases in AA/low-grade MDS patients.

Keyword

Flow cytometry; High sensitivity; Paroxysmal nocturnal hemoglobinuria; Performance; Clone

MeSH Terms

Anemia, Aplastic
Clone Cells*
Erythrocytes
Flow Cytometry*
Granulocytes
Hemoglobinuria, Paroxysmal*
Humans
Myelodysplastic Syndromes

Figure

  • Fig. 1 Comparison of high sensitivity flow cytometry results obtained from three cell lineages (granulocytes, monocytes, and RBCs). Results are compared using Passing-Bablok regression analysis (A-C) and Bland-Altman analysis (D-F). Regression equations, correlation coefficients, and 95% CIs for the slope and intercept of the equations are shown in A-C. In the Bland-Altman analysis, mean difference (indicated by the blue horizontal line), lower and upper limits of agreement (indicated by the red dotted horizontal lines), and their 95% CIs (indicated by the cyan vertical bars) are presented in D-F.Abbreviations: RBC, red blood cell; PNH, paroxysmal nocturnal hemoglobinuria; CI, confidence interval.

  • Fig. 2 Comparison of C-FCM granulocyte PNH clone size results with three different GPI markers and HS-FCM. Results are compared using Passing-Bablok regression analysis (A-C) and Bland-Altman analysis (D-F). Regression equations, correlation coefficients, and 95% CIs for the slope and intercept of the equations are shown in (A-C). In the Bland-Altman analysis, mean difference (indicated by the blue horizontal line), lower and upper limits of agreement (indicated by the red dotted horizontal lines), and their 95% CIs (indicated by the cyan vertical bars) are presented in (D-F).Abbreviations: HS-FCM, high sensitivity flow cytometry; C-FCM, conventional flow cytometry; PNH, paroxysmal nocturnal hemoglobinuria; FLAER, fluorescein-labeled proaerolysin; CD, cluster of differentiations; CI, confidence interval.

  • Fig. 3 Comparison of C-FCM RBC PNH clone size results with scatterplot and histogram and HS-FCM. Results were compared by means of Passing-Bablok regression analysis (A-B) and Bland-Altman analysis (C-D). Regression equations, correlation coefficients, and 95% CIs for the slope and intercept of the equations are shown in (A) and (B). In the Bland-Altman analysis, mean difference (indicated by the blue horizontal line), lower and upper limits of agreement (indicated by the red dotted horizontal lines), and their 95% CIs (indicated by the cyan vertical bars) are presented in (C) and (D).Abbreviations: RBC, red blood cell; HS-FCM, high sensitivity flow cytometry; C-FCM, conventional flow cytometry; PNH, paroxysmal nocturnal hemoglobinuria; CI, confidence interval.


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