Lab Med Online.  2019 Apr;9(2):57-62. 10.3343/lmo.2019.9.2.57.

Evaluation of Time and Temperature Stability of Paroxysmal Nocturnal Hemoglobinuria Cells by Flow Cytometry

• Lee SH
• Bang HI
• Shin YJ
• Shin WY
• Kim J
• Park R
• Shin JW
• Choi TY

• KMID: 2441824
• DOI: http://doi.org/10.3343/lmo.2019.9.2.57

Abstract

BACKGROUND
Flow cytometry analysis of paroxysmal nocturnal hemoglobinuria (PNH) is significantly affected by the methodology used. The lack of data on the effect of age and refrigeration on PNH clone stability motivated us to study these aspects using flow cytometry.
METHODS
Peripheral blood was collected from six patients, of which two presented with PNH. All samples were tested immediately and stored at room temperature (RT, 20-25℃) and at 4℃ for re-analysis at 24, 48, 72 hr and 7 days. Anti-CD59-fluorescein isothiocyanate (Beckman Coulter, USA) and anti-CD235a-phycoerythrin (PE; Beckman Coulter) were used to stain red blood cells (RBCs). Fluorescein-labeled proaerolysin (Cedarlane, Canada), anti-CD15-PE (Beckman Coulter), anti-CD24-PE-cyanin 5 (Beckman Coulter), and anti-CD45-PE-cyanin 7 (Beckman Coulter) were used to stain granulocytes. Flow cytometry was performed using a FC500 flow cytometer (Beckman Coulter). The effects of time and temperature were analyzed using generalized estimating equations.
RESULTS
No significant differences in the gated percentage of RBCs and PNH clone size of RBCs were observed between the RT and 4℃ groups up to 7 days of testing. The percentage of gated neutrophils decreased with specimen age (P<0.001) and a better correlation with baseline was obtained at 4℃ than at RT (P=0.014). Neutrophil PNH clones were stable until 48 hr and 72 hr at RT and 4℃, respectively, and could not be analyzed at 7 days.
CONCLUSIONS
RBC analysis was successfully performed up to 7 days. For neutrophils, testing within 48 hr is recommended, because the number of gated cells decreases significantly with age.

Keyword

Flow cytometry; Paroxysmal nocturnal hemoglobinuria; Stability

MeSH Terms

Clone Cells
Erythrocytes
Flow Cytometry*
Granulocytes
Hemoglobinuria, Paroxysmal*
Humans
Neutrophils
Refrigeration

Figure

• 

  Fig. 1. Comparison of (A) gated RBCs (%), (B) paroxysmal nocturnal hemoglobinuria (PNH) clones of RBCs (%), (C) gated neutrophils (%), and (D) PNH clones of neutrophils (%) between the room temperature group and the 4°C group according to time in flow cytometry analysis. Generalized estimating equation tests were used.

• 

  Fig. 2. Paroxysmal nocturnal hemoglobinuria (PNH) red blood cell analysis according to testing time in a PNH patient (Case 2 in Table 1). A blood sample was tested within 1 hr of collection and stored at room temperature (A) and at 4°C (B). Reanalysis was performed at 24 hr, 48 hr, 72 hr, and 7 days. The size of PNH clone (II+III) of RBCs was 56.85% and showed no effect due to time or temperature. At 7 days, the PNH clone was 65.78% (room temperature) and 58.54% (4°C).

• 

  Fig. 3. Paroxysmal nocturnal hemoglobinuria (PNH) neutrophil analysis according to testing time in a PNH patient (Case 2 in Table 1). A blood sample was tested within 1 hr of collection and stored at room temperature (A) and at 4°C (B). Reanalysis was performed at 24 hr, 48 hr, 72 hr, and 7 days. The size of the PNH clone (II+III) of neutrophils was 94.59%. The clone was stable until 48 hr at room temperature and until 72 hr at 4°C.

Reference

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