Journal of Clinical Laboratory Analysis 3:296-300 (1989)

Effects of Storage Conditions on Lymphocyte Phenotypes From Healthy and Diseased Persons Claramae H. Miller and Norman B. Levy

Department of Medical Pathology, University of California Davis, School of Medicine, Davis, California

Anticoagulant and room temperature stor- age of peripheral blood was evaluated in healthy and diseased persons (AIDS and CLL) for its effect upon lymphocyte pheno- typing by flow cytometry. Whole blood was stored in either Li heparin, EDTA, or ACD for up to 96 hours. Aliquots at 5 time intervals were evaluated using CD3, CD2, CD4, CD8, and CD19 monoclonal antibodies.

Blood from healthy donors stored in EDTA produced an apparent increase in T cells and

decrease in B cells most evident by 96 hours storage. Blood from diseased persons stored in Li heparin up to 96 hours showed insuffi- cient changes in phenotype to warrant a dif- ferent clinical interpretation.

We conclude that all three anticoagulants are adequate for short term (< 24 hr) room temperature transport and storage of periph- eral blood for flow cytometry. Li heparin or ACD are more appropriate if prolonged trans- port times are anticipated.

Key words: lymphocyte storage, flow cytometry, anticoagulant, leukocyte phenotype

INTRODUCTION

Clinical laboratories phenotyping peripheral blood lympho- cytes by monoclonal antibodies and flow cytometry often receive peripheral blood specimens for evaluation that are sev- eral days old. The effects of anticoagulants and storage con- ditions on cells that cannot be analyzed immediately should be understood in interpretation of the results.

The effect of a variety of storage conditions on the pheno- type of lymphocytes from normal healthy donors have been reported by several groups ( 1 -6). These studies used several monoclonal antibodies and flow cytometry on peripheral blood lymphocytes from healthy persons. In order to compare storage conditions on cells from both healthy and diseased persons, these studies were undertaken.

MATERIALS AND METHODS

Human peripheral blood lymphocytes were obtained by venipuncture from 10 healthy adults of both sexes, 4 males with AIDS, and an adult female with chronic lymphocytic leukemia (CLL). Blood from the healthy persons was drawn into vacutainers that contained Li (lithium) heparin, EDTA (ethylenediamine tetraacetate), or ACD (acid citrate dextrose). Blood from the AIDS patients and the CLL patient was obtained in Li heparin. All blood was stored at room temper- ature and aliquots were evaluated at time of venipuncture (0 time), at 24,48, 72, and 96 hours thereafter.

Lymphocytes were stained using an indirect fluorescence staining procedure on whole blood. Cells were Stained using the following commercially prepared primary monoclonal anti- bodies; CD2 (T1 l), CD19 (B4) (Coulter Immunology, Hia- leah, FL) and CD3 (Leu 4), CD4 (Leu 3a), CD8 (Leu 2a)

0 1989 Alan R. Liss, Inc.

(Becton-Dickinson, Inc., Mountain View, CA). One hundred (100) lambda of whole blood and 200 lambda of the primary monoclonal antibody were incubated for 10 minutes at 4°C. The cells were then washed twice with PBS and incubated (10 min 4°C) with 200 lambda of the secondary antibody, goat F (ab')z antimouse IgG-FITC (Tago, Inc., Burlingame, CA). Red blood cells were then lysed using Immuno-lyse (Coulter Immunology) and washed twice in PBS before being suspended in 1% paraformaldehyde. Cells were stored in the dark at 4°C until evaluation on a Coulter EPICS-C flow c ytometer.

The EPICS-C flow cytometer is equipped with a 2-watt argon laser, which emits excitation light at 488 nm at 400 mW. The lymphocyte area was gated by evaluation of the scattergram, which was obtained by plotting forward angle light scatter and 90" angle light scatter. Fluorescence data for each monoclonal antibody were collected on 5,000 gated lymphocytes.

RESULTS

Effect of Anticoagulant and Time on Lymphocyte Phenotype From Healthy Volunteers

Normal human peripheral blood collected in either Li hep- arin, ACD, or EDTA was stored for variable time periods at room temperature before staining for antibodies CD3 (Leu 4), CD2 (Tll) , CD19 (B4), CD4 (Leu 3a), and CD8 (Leu 2a).

Received July 14, 1988; accepted March 23, 1989.

Address reprint requests to Claramae H. Miller, Ph.D., University of Cali- fornia Davis, School of Medicine, Department of Medical Pathology MS-I, Davis. CA 95616.

Lymphocyte Phenotype: Effect of Storage 297

v) HEPARIN EDTA ACD

T

0 T

".. 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96

TIME (hours) N = 10 healthy peopie

Fig. 1. Percent of CD 3 positive cells during storage (ic t S.E.).

The data from freshly drawn whole blood lymphocytes were used as the reference point. No demonstrable change was observed with any of the anticoagulants up to 24 hours (Fig. 1; Table 1) . An apparent increase in T cells was found by 96 hours regardless of anticoagulant (P < 0.05, Student's t, Fig. 1 ) . The greatest apparent increase in T cells was found in those cells stored in EDTA (Fig. 1). For example, in hepa- rin, when comparing 0 time to 96 hours storage, the mean of positive cells staining with CD3 (Leu 4) rose from 69 to 79%, and CD2 (T-1 I ) rose from 80 to 91 %; cells stored in EDTA and stained with CD3 (Leu 4) changed from 69 to 91%, CD2 (T-1 1) from 79 to 87%. Cells stored in ACD changed during 0 time to 96 hours storage from 66 to 77% for staining by CD3 (Leu 4), to 80 to 83% for cells staining for CD2 (T-1 1). Storage in heparin or EDTA produced a drop in CD19 (€34) positive cells over 96 hours. This decrease was not observed in cells stored in ACD (Fig. 2).

The magnitude of the shift in mean helper (CD4)/suppressor (CD8) ratios was greater in cells stored in EDTA than in cells stored in either ACD or heparin (Fig. 3 ) . This change in ratio was demonstrable after 72 hours storage in EDTA and of much

TABLE I . Effect of Anticoagulant and 24-Hours Storage on Lymphocyte Phenotype of Healthy Persons ( % positive cells 2 SD)

Heparin EDTA ACD Antibodv 0" 24 0 24 0 24

CD3(Leu4) 6 9 t 8 . 2 6 8 t 1 1 . 4 6 9 t 1 4 . 1 7 1 2 1 1 6 6 2 1 2 . 5 5 8 t 1 0 . 4 CD4 (Leu3a) 47 t 6.3 46 2 7.3 47 k 1.6 48 t 7.7 45 2 7.8 46 t 6.5 CD8 (Leu2a) 22 t 7.6 21 t 6.8 22 t 6.0 22 t 6.5 23 ? 6.3 22 t 6.4 CD2 (TI 1) 80 t 6.5 81 t 6.7 79 2 6.0 82 & 6.8 80 ? 6.2 80 2 6.7 CD19 (B4) 12 t 2.9 13 t 2.4 12 t 2.5 I 1 2 2.6 12 t 2.6 12 k 3.6

"Time after draw (hours). N = 10

greater magnitude at 96 hours of storage, from a mean ratio of 2.2 at time 0 to 3.6 at 96 hours storage (Fig. 3).

The effect of storage and anticoagulant on WBC from normal persons is presented in Table 2. Although numerical variation did occur, the values were still within the reference interval and would be of no clinical significance.

Effect of Storage Time on Lymphocyte Phenotypes in Diseased Persons

Peripheral blood from 4 clinically ill, HIV positive AIDS patients and an 80-year-old female with CLL were drawn into Li heparin and stored at room temperature. Aliquots were removed at the same time intervals as previously stated. Helperkuppres- sor ratios and the absolute number of CD4 and CD8 cells from the 4 AIDS patients varied slightly over the total storage time but did not vary sufficiently over time to warrant a change in the clinical interpretations (Fig. 4; Table 3). In general, as was observed with total T cell numbers from healthy persons, there was an apparent increase in T cell percentages accompanied with an apparent decrease in B cell percentage (Table 4).

Peripheral blood from a CLL patient stored for 96 hours at room temperature in Li heparin demonstrated little change in percent positive staining cells regardless of monoclonal anti- body used (Table 5). The variation in percent positive CD 19 (€34) staining cells did not change sufficiently to alter the clini- cal interpretation, although the percentage of CD19 (B4) pos- itive cells did drop from 92% at time 0 to 79% after 96 hour of storage.

Effect of Anticoagulant and Storage Time on the Light Scatter Measurement

Storage in ACD or heparin had minimal effect on the for- ward and right angle light scattering characteristics of all leu-

298 Miller and Levy

100 I

60- LIl

5 40- 0 a &?

2

20

1 HEPARIN

-

Y Lu 8ol /

4 -

3 -

2 -

0 c U

- a

9 r

:3 A w Leu Leu 4 3a

0 Leu 2a - + 6 4

-P

ACD

t-t- v

O i l I ’ I I l l I I I I

0 24 48 72 96 0 24 48 72 96 0 24 48 72 96

TIME (hours) N = 10 healthy people

Fig. 2. Changes in T cell subsets and B cell percent during storage (ir).

T

T A Heparin

5 0 EDTA ‘r mACD

1

’ t 0 I 1 I I I

0 24 48 72 96

N = 10 healthy people TIME (hours)

Fig. 3. Changes in helperisuppressor cell ratio during storage (ir 2 S.E.)

TABLE 2. Effect of Storage on WBC Count from 10 Healthy Persons

cocyte populations, allowing detection and separation of the lymphocyte, monocyte, and granulocyte regions. However, by 48 hours of storage in EDTA, neutrophils in 1 of the 10

0 24 48 72 96 normal specimens demonstrated a drop in the PMN area to a Heparin 9,500” 8,800 8,900 8,600 8,800 region parallel to the lymphocytes. By 72 hours, 3 of the 10 EDTA 10,500 9,400 9,700 7,800 9,200 samples stored in EDTA had the PMN region parallel to ACD 10,300 9,100 8,700 7,800 8,100 or below the lymphocyte region. By 96 hours 6 of the 10

Time (Hours)

”Mean cells/mm3.

Lymphocyte Phenotype: Effect of Storage 299

1.5

1.2

0

U 4 F 0.9

Patient 1 - Patient 2 0 Patient 3 A Patient 4

0 24 48 72 96 TIME (hours)

Fig. 4. Changes in helperisuppressor cell ratio in AIDS patients during heparin storage.

TABLE 3. Effect of Heparin Storage on CD4 and CD8 Cells in 4 AIDS Patients

Storage Patient 1 Patient 2 Patient 3 Patient 4 Time (Hr) CD4 CD8 CD4 CD8 CD4 CD8 CD4 CD8

0 644(32)a 623(33) 5 l(6) 527(62) 102(12) 400(47) 892(14) 3121 (49) 24 724(36) 704(35) 59(7) 578(68) 34(4) 50 l(59) 1146(18) 2548(40) 48 724(36) 704(35) 686) 637(75) 51(6) 561 (66) 892(14) 2801(44) 72 764(38) m ( 3 2 ) 94(11) 620(73) W 7 ) 476(56) 955(15) 2102(43) 96 925(46) 684(32) W 7 ) 5 lO(60) 51(6) 535(63) 1337(21) 2220(33)

"Absolute number positive cells (% positive). TABLE 5. Effect of Time on Phenotyping of CLL Cells Stored

TABLE 4. Total T and B Cell Percentages in 4 AIDS Patients in Heparin During 96-Hours Storage at Room Temperature in Heparin Time (hr) CD3 CD4 CD8 CD2 CD19 Monoclonal Antibody (% Positive Cells) (WBC X lo4) (Leu 4) (Leu 3a) (Leu 2a) (TI 1) (B4)

Antibody (% Positive)

Time 0 24 Hr 48 Hr 72 Hr 96Hr 0 6 3 3 7 92

24 8 5 1 7 89

6 3 1 7 86

5 2 0 8 94

Patient CD2 CD19 CD2 CD19 CD2 CD19 CD2 CD19 CD2 CD19 (13.5)

1 77 15 78 10 79 10 80 6 84 4 (13.4) 2 82 0 81 1 92 1 92 1 90 0 48 3 75 7 81 4 87 5 84 4 85 3 (13.4) 4 88 1 84 2 89 2 82 1 91 I 72

(14.1) samples stored in EDTA demonstrated a marked drop in the 96 5 3 0 6 79

PMN area. Gating of the lymphocyte area in these 6 samples was difficult.

DISCUSSION

(11 .4)

perature storage on lymphocyte phenotype of cells from healthy and diseased persons. Peripheral blood was stored in 3 anti- coagulants, Li heparin, EDTA, and ACD, up to 96 hours. Storage of whole blood was maintained only at room temper- ature because the effect of storage at 4°C has been shown by others to result in a decrease in total T cells and in helper T cell numbers (2,3,4).

It is often necessary to transport peripheral blood samples considerable distances to regional laboratories for lympho- cyte phenotyping, and several days can pass before the cells are analyzed. The experiments reported here were performed to evaluate the effect of anticoagulant on prolonged room tem-

300 Miller and Levy

We determined that for normal peripheral blood lympho- cytes, all anticoagulants provided adequate preservation of cell phenotype and structure for 24 hours (Table 1). However, all specimens demonstrated an apparent increase in T cells by 96 hours storage (P < 0.05, Student’s t, Fig. 1). This apparent increase in T cell percentages was associated with increased proportions of CD4 (Leu 3a) positive cells and a decrease in percentage of CD19 (B4) labelled cells (Fig. 2). In contrast, Shield et al. (2) and Nicholson et al. ( 5 ) found that when using ACD as an anticoagulant they were able to maintain consistent percentages of CD3 (T3) labeled cells throughout the 96-hour storage, whereas only the percentage of CD2 (T1 1) labeled cells dropped significantly at 96 hours.

Our data obtained from cells stored in EDTA is consistent with that of Shield et al. (2), Nicholson et al. (S), and Thornthwaite et a]. (6). We observed that the light scatter of PMN areas shifted considerably as the cells aged, making gating of the lymphocyte area quite difficult. Further we observed that not only were the apparent increases in T cell numbers greater in EDTA storage than ACD or heparin (Fig. l), but the magnitude of the changes in helper/suppressor ratios of cells from healthy persons was greater in EDTA (Fig. 3).

The helper/suppressor ratios and absolute numbers of CD4 and CD8 cells from AIDS patients whole blood stored in hep- arin for 96 hours did not change sufficiently to warrant a change in clinical interpretation (Fig. 4; Table 3). Further, blood from a CLL patient stored for 96 hours in heparin varied for B4 marking cells from 92% at 0 time to 79% positive at 96 hours

(Table 5 ) . These changes also would not warrant a different clinical interpretation.

Our results have indicated that when using an indirect flu- orescent technique, EDTA is not an appropriate anticoagu- lant for lymphocyte phenotyping if the whole blood samples are stored at room temperature longer than 24 hours. ACD or heparin are both much better anticoagulants if extended stor- age is anticipated. It was felt that use of an indirect rather than a direct fluorescent procedure would not bias the data since the manufacturers do not recommend one technique over the other. Heparin is readily available in most laboratories and is an appropriate anticoagulant for cell phenotyping of whole blood from both healthy and diseased persons.

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Hensleigh PA, Waters VB, Herzenberg LA: Human T lymphocyte dif- ferentiation antigens. Effects of blood sample storage of Leu antibody binding. Cytometry 3:453-455, 1983. Shield 111 CF, Marlett P, Smith A, Gunter L, Goldstein G: Stability of human differentiation antigens when stored at room temperature. Jlmmunol

Weibler BJ, Debell K. Valeri CR: “Acquired immunodeficiency” of blood stored overnight. NEJM 309:793, 1983. Dzik WH, Necken L: Lymphocyte subpopulations altered during blood storage. NEJM 309:435, 1983. Nicholson JKA, Jones BM, Cross GD, McDougal JS: Comparison of T and B cell analysis on fresh and aged blood. J Immunol Methods 73:29-40, 1984. Thornthwaite JT, Rosenthal PK, Vasquez DA, Seckinger D: The effects of anticoagulant and temperature on the measurement of helper and sup- pressor cells. DiagnosticImm 2:167-174, 1984.

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