Journal of Immunological Methods, 130 (1990) 15-18

15

Elsevier JIM05576

A method for isolating neutrophils from moderate volumes of human blood * Steve J. M c F a u l Letterman Army Institute of Research, Dioision of Blood Research, Presidio of San Francisco, CA 94129, U.S.A.

(Received 7 November 1989, revisedreceived2 January 1990, accepted 5 February 1990)

The Flow Laboratories procedure for isolating polymorphonuclear leukocytes (PMNs) from human blood whereby whole blood is centrifuged through Mono-Poly resolving medium (MPRM) has been modified. Using this modification, as much as 30 ml of whole blood could be processed in a single centrifuge tube, and erythrocyte contamination of the final PMN suspension was reduced significantly. PMN suspensions were composed of 96% neutrophils, and neutrophil recovery was 42-55% depending upon the volume of blood processed. Cell viability was 98% as determined by trypan blue exclusion. Greater than 99% of the neutrophils were spherical suggesting that the neutrophils were not primed during isolation. Key words: Neutrophil; Blood, human; Isolation; Priming; Shape change

Introduction The procedures employed most commonly for isolating neutrophils include dextran sedimentation and centrifugation through density gradients (Hafeman and Lucas, 1979; Segal et al., 1980; Petreccia et al., 1987; Wierusz-Wysocka et al.,

Correspondence to: S.J. McFaul, Letterman Army Institute of Research, Division of Blood Research, Presidio of San Francisco, CA 94129, U.S.A. * Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarilyendorsed by the U.S. Army. Abbreviations: EDTA, ethylenediaminetetraaceticacid; EU, endotoxin unit; HBSS, Hanks' balanced salt solution with ( + ) and without ( - ) Ca2÷ and Mg2+; FL, Flow Laboratories; MNL, mononuclear leukocyte; MPRM, Mono-Poly resolving medium.

1987). To minimize erythrocyte contamination, however, only small volumes ( < 10 ml) of buffy coat or diluted blood can be centrifuged through density gradients. Even so, neutrophil suspensions are contaminated with significant numbers of erythrocytes and thus are subjected to hypotonic shock in order to lyse the erythrocytes. However, hypotonic shock results in the loss of some neutrophils as well and may alter the functional characteristics of neutrophils (Haslett et al., 1985). Flow Laboratories (FL) markets a Ficoll-Hypaque medium called Mono-Poly resolving medium (MPRM) for the simultaneous isolation of mononuclear leukocytes (MNL) and PMNs from human blood. Centrifugation of whole blood through M P R M results in the separation of M N L and PMN bands. The procedure as described by FL is limited, however, to 7 ml of whole blood per tube. Furthermore, large numbers of erythrocytes remain suspended in the PMN band. This report describes a modification of the procedure described by FL whereby PMNs can be

16

isolated from 30 ml of whole blood in a single tube with as little as 1% erythrocyte contamination.

on a tube rocker at room temperature in order to prevent aggregation.

Cell counting and determination of shape change Materials and methods

MPRM was purchased from Flow Laboratories (McLean, VA). Hanks' balanced salt solution without phenol red, but with (HBSS +) and without ( H B S S - ) Ca 2+ and Mg 2+, were purchased from Gibco Laboratories (Grand Island, NY). Neutralbuffered formalin (10%) was purchased from Surgipath (Grayslake, IL).

PMNs and contaminating erythrocytes were counted simultaneously using either an electronic particle counter (Particle Data) or a hemocytometer. Shape change was determined on a hemocytometer on aliquots of cell suspensions that had been fixed by the addition of equal volumes of 10% neutral-buffered formalin and diluted with. HBSS-. Cells that were not spherical were counted as those that had undergone a shape change. At least 200 cells were examined.

Isolation of neutrophils Blood was collected from healthy donors by venepuncture into either EDTA (modified procedure) or heparin (FL procedure) vacutainer tubes and kept on a tube rocker until processed. All blood was processed within 60 min of collecting. Neutrophils were isolated using the procedure described by FL and the modified procedure described below. Modified procedure: MPRM was placed into either 15 ml or 50 ml conical polypropylene tubes, and whole blood that had been collected into EDTA was layered over the medium. The bloodto-MPRM volume ratio was 2:1. The tubes were centrifuged for 50 min at 750 × g in a swinging bucket rotor at room temperature. The PMN band (band 2, Fig. 1) was collected from each tube using an 18 gauge needle attached to a 10 ml syringe. The PMN suspensions were transferred to clean conical tubes, mixed gently by swirling, underlayered with fresh MPRM, and centrifuged for 15 min at 1200 × g in a swinging bucket rotor. 15 ml tubes and 1 ml of MPRM were used when centrifuging < 8 m l o f b a n d 2, and 50 ml tubes and 3 ml of MPRM were used when centrifuging > 8 ml of band 2. The entire upper fraction was collected and the cells were washed by diluting the upper fraction two-fold in HBSS- and centrifuging for 10 min at 300 x g . The PMN pellet was resuspended in 25 ml of HBSS- by gentle swirling, and the suspension centrifuged for 10 min at 150 × g. The PMN pellet was resuspended in 2 ml of HBSS ~, and the PMN suspension was maintained

Results

Fig. 1 shows that blood centrifuged through MPRM according to either the FL procedure or the modified procedure was separated into three bands. Band 1 contained platelets and MNL, and bands 2 and 3 contained PMNs and erythrocytes, respectively. The distribution of cells in band 2 averaged 96% + 0.69 neutrophils, 1.7% + 0.34 eosinophils, 0.62% _+ 0.2 monocytes, and 2.1% _+

Fig. 1. Photograph of tubes of blood processed as described in the materials and methods section. Blood (7.0 ml) was processed according to the ( a ) Flow Laboratories' procedure and the (b) modified procedure. Bands 1 (top), 2 (middle), and 3 (bottom) contained MNL, PMNs, and erthrocytes, respectively. The dark area between bands 2 and 3 in tube a was comprised of suspended erythrocytes.

17 TABLE I C O M P A R I S O N OF E R Y T H R O C Y T E C O N T A M I N A T I O N IN PMN SUSPENSIONS Erythrocyte contamination is expressed as percent of P M N concentration (number of erythrocytes/100 PMNs). Neutrophils were isolated according to the procedure described by Flow Laboratories (FLP) and the modified procedure (MP) described in the materials and methods section. Blood was drawn from different donors for the five experiments. Experiment

3.5 ml blood

7.0 ml blood

FLP

MP

FLP

MP

2 3 4 5

145 180 223 764 216

14 5.6 0.0 2.7 1.5

178 388 538 1468 242

4.0 15 9.4 14 1.8

Mean ± SEM

306±115

1

4.8±2.5

563±235

8.8+2.6

0.40 lymphocytes. Significant erythrocyte contamination was observed between bands 2 and 3 after processing blood according to the FL procedure (Fig. 1, tube a), whereas erythrocytes were not visible in this region after processing blood according to the modified procedure (Fig. 1, tube b). Furthermore, the final PMN suspension isolated using the FL procedure contained significantly more erythrocytes than did the suspension isolated using the modified procedure (Table I). Table I shows also that erythrocyte contamination

differed between donors irrespective of the isolation method employed. Table II shows that erythrocyte contamination of PMN suspensions was not affected significantly by the volume of blood processed, but was a function of the blood donor. However, the distance between M N L and PMN bands, increased as the volume of blood layered over the MPRM increased (Fig. 2). Furthermore, neutrophil recovery from whole blood was 42% + 4 (n = 16) when 12 ml of blood were centrifuged and 55% + 8 (n = 9) (significant at P < 0.05) when 16 ml to 30 ml of whole blood were centrifuged. The viability of neutrophils was > 98% by trypan blue exclusion, and neutrophils appeared to be spherical with < 1% (n = 12 isolations) of them exhibiting shape change.

Discussion By modifying the centrifugation conditions of the FL procedure and centrifuging a second time, neutrophils were isolated from as much as 30 ml of human blood per tube with very little erythrocyte contamination (Table II). The final level of

15-

I •~

T A B L E 11

12,

4

c 0~ u~

E R Y T H R O C Y T E C O N T A M I N A T I O N IN P M N SUSPENSIONS ISOLATED F R O M V A R I O U S V O L U M E S O F BLOOD Erythrocyte contamination is expressed as percent of PMN concentration (number of erythrocytes/100 PMNs). Blood was drawn form different donors for the five experiments. P M N s were isolated according to the modified procedure described in the materials and methods section. Experiment

1

2 3 4 5 M~n±SEM

P

-5

~

6'-

s~

Volume of blood 3.5 ml

7.0 ml

20 ml

30 ml

14 5.6 0.0 2.7 1.5

4.0 15 9.4 14 1.8

14 19 12 5.3 0.0

19 13 6.0 3.1 1.0

4.8±2.5

8.8±2.6

10±3.3

8.4±3.3

O---

10

-

-

15

20 Blood volume

25

~0

(ml)

Fig. 2. Relationship of distance between M N L and PMN bands and the volume of blood layered onto MPRM. Blood was processed according to the modified procedure as described in the materials and methods section. Data points represent the mean + SEM (n = 9 for all blood volumes except 24 ml where n = 3). r 2 = 0.991.

18 erythrocyte contamination differed among blood samples from different donors, but was relatively constant for the same donor. Since neutrophils could be isolated with as little as 1% erythrocyte contamination, there was no need for hypotonic shock treatment. Haslett et al. (1985) suggested that shape change was a sensitive indicator of neutrophil priming. By this criterion, neutrophils isolated using the modified procedure were not primed. Fewer than 1% of the neutrophils changed shape as long as neutrophil pellets were resuspended by gentle swirling. Recovery of neutrophils was greatest when at least 16 ml of blood was centrifuged in a 50 ml tube. This was probably due to the increased separation of the M N L and PMN bands which facilitated removal of the PMN band. The dependence of the separation between M N L and PMN bands on blood volume (Fig. 2) is consistent with the results of Ferrante and Thong (1982) who showed that the distance between M N L and PMN bands was a function of the height of the blood column above the MPRM. Thus, to recover the greatest number of neutrophils from whole blood, tube sizes should be selected such that the maximal blood column height is obtained. MPRM represents a significant improvement over other media for processing blood because it

permits simultaneous separation of M N L and PMNs from whole blood in a single centrifugation step. The procedure for using M P R M presented here should prove particularly useful in studies that require large numbers of neutrophils, such as enzyme characterization studies (Gabig and Lefker, 1986) and studies designed to test the effects of several treatments on different functional parameters of neutrophils.

References Ferrante, A. and Thong, Y.H. (1982) Separation of mononuclear and polymorphonuclear leucocytes from human blood by the one-step Hypaque-Ficollmethod is dependent on blood height. J. Immunol. Methods 48, 81. Gabig, T.G. and Lefker, B.A. (1986) NADPH oxidase from polymorphonuclear cells. In: G. DiSabato and J. Everse (Eds.), Methods in Enzymology,Vol. 132. Academic Press, New York, p. 355. Haslett, C., Guthrie, L.A., Kopaniak, M.M., Johnston, R.B. and Henson, P.M. (1985) Modulation of multiple PMN functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am. J. Pathol. 119, 101. Petreccia, D.C., Nauseef, W.M. and Clark, R.A. (1987) respiratory burst of normal human eosinophils. J. Leuk. Biol. 41, 283. Segal, A.W., Fortunato, A. and Herd, T. (1980) A rapid single centrifugation step method for the separation of erythrocytes, granulocytes, and mononuclear cells on continuous density gradients of percoll. J. Immunol. Methods 32, 209.