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

So Hee Lee; Hae In Bang; Yu Jeong Shin; Woo Yong Shin; Jieun Kim; Rojin Park; Jeong Won Shin; Tae Youn Choi

doi:10.3343/lmo.2019.9.2.57

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Lee, Bang, Shin, Shin, Kim, Park, Shin, and Choi: Evaluation of Time and Temperature Stability of Paroxysmal Nocturnal Hemoglobinuria Cells by Flow Cytometry

Original Article

Lab Med Online 2019;9(2):57-62.

Published online: 20 April 2019

DOI: https://doi.org/10.3343/lmo.2019.9.2.57

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

So Hee Lee, M.T., Hae In Bang, M.D., Yu Jeong Shin, M.T., Woo Yong Shin, M.D., Jieun Kim, M.D., Rojin Park, M.D., Jeong Won Shin, M.D., Tae Youn Choi, M.D.

Department of Laboratory Medicine, Soonchunhyang University Seoul Hospital, Seoul, Korea

Corresponding author: Hae In Bang, M.D. https://orcid.org/0000-0001-7854-3011 Department of Laboratory Medicine, Soonchunhyang University Seoul Hospiatal, 59 Daesakwan-ro, Yongsan-gu, Seoul 04401, Korea Tel: +82-2-709-9386, Fax: +82-2-710-3088, E-mail: genuine43@schmc.ac.kr

Received 26 March 2018 Revised 26 July 2018 Accepted 31 July 2018

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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˚C) and at 4˚C 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 (Cedar-lane, 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˚C 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˚C than at RT (P=0.014). Neutrophil PNH clones were stable until 48 hr and 72 hr at RT and 4˚C, 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.

Keywords: Flow cytometry, Paroxysmal nocturnal hemoglobinuria, Stability

References

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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.

Table 1.

Comparison of paroxysmal nocturnal hemoglobinuria (PNH) cells of two PNH patients according to time and temperature of specimen storage in flow cytometric analyses

		Case 1		Case 2
		Room temperature∗	4°C∗	Room temperature∗	4°C∗
Red blood cells	0 hr	49,914 (66.71)		11,129 (56.85)
	24 hr	52,777 (65.41)	13,646 (66.40)	18,252 (54.74)	51,736 (64.08)
	48 hr	5,380 (67.54)	4,283 (64.39)	10,061 (56.23)	9,923 (54.64)
	72 hr	119,438 (65.60)	92,789 (69.48)	80,937 (63.82)	120,066 (64.03)
	7 day	28,437 (62.03)	46,662 (65.71)	122,532 (65.78)	41,762 (58.54)
Neutrophils	0 hr	33,196 (99.93)		51,554 (94.59)
	24 hr	25,027 (99.92)	33,196 (99.93)	10,142 (92.21)	18,184 (93.71)
	48 hr	15,055 (99.80)	25,430 (99.84)	3,454 (92.70)	35,769 (94.81)
	72 hr	1,804 (96.68)	19,898 (99.71)	719 (84.99)	15,026 (95.16)
	7 day	0 (0)	2,601 (97.71)	44 (74.58)	319 (95.51)

^∗ Values are presented as N (%). N is the PNH cell number and % is the proportion of PNH cells to the number of total gated cells (RBCs or neutrophils) analyzed for each test.

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