Methods for Obtaining Purified Lymphocytes, Glass-adherent

Blood, Vol. 40, No. 1 (July), 1972 77

Methods for Obtaining Purified Lymphocytes,

Glass-adherent Mononuclear Cells, and a Population

Containing Both Cell Types From Human Peripheral Blood

By William R. Levis and Jay H. Robbins

Methods are presented for obtainingsimultaneously or separately two popu-lations of cells from human peripheralblood, lymphocytes and monocytes,both of which are required to obtainblastogenesis and DNA synthesis inhuman leukocyte cultures. A simple5-mm centrifugation of heparinizedblood yields a mononuclear leukocyteculture fluid containing 70-90#{176}/olymph-ocytes with few granulocytes but withsufficient numbers of monocytes sothat the latter cell is not a limiting fac-tor in the blastogenic reaction. A

method is also presented for remov-ing both granulocytes and monocytesfrom lymphocyte populations in a man-ner that permits monitoring and choiceof the degree of lymphocyte purifica-tion. A method is also presented forobtaining glass-adherent mononuclearcells that do not undergo blastogene-sis but will enable suitably stimulated“purified” lymphocytes to undergoblastogenesis. Studies of the functionand morphology of these different cellpopulations are presented.

T HE HUMAN SMALL LYMPHOCYTE is the cell that undergoes blasto-

genesis and DNA synthesis in peripheral blood leukocyte cultures in re-

sponse to various blastogenic factors.’3 It has, however, recently been shown

that a glass-adherent (g-a) population of mononuclear cells, derived primarily

if not entirely from the blood monocyte, is necessary for the human pen-

pheral blood lymphocyte to undergo blastogenesis in response to specific

antigens47 and to allogeneic cells.4’5’8’2 C-a cells are also required to obtain

an early maximum blastogenic response to pokeweed mitogen13 and to all

concentrations of phytohemagglutinin (PHA).’4 To study the function of the

g-a cell and lymphocyte populations in the response of human peripheral

blood lymphocytes to blastogenic factors, it is useful to have techniques that

will adequately separate these cell types and make them available in separate

populations. We have utilized a method in which lymphocytes are separated

from other leukocytes in a manner that permits constant monitoring of the

degree of purification being attained, thereby providing a means for determin-

ing when the desired purity has been achieved.

The realization that g-a cells are required for the lymphocytes’ response to

blastogenic factors in leukocyte cultures and that granulocytes, the most

abundant leukocyte in human peripheral blood, perform no known useful

function and may even be injurious in such cultures’5 prompted us to employ

Pram the Dermatology Branch, National Cancer Institute, National Institutes of Health,

Bethesda. Md.

Submitted December 17, 1971; revised February 16, 1972; accepted February 17, 1972.

William R. Levis, M.D.: Senior Investigator, Dermatology Branch, National Cancer

Institute, National Institutes of Health, Bethesda, Md. Jay H. Robbins, M.D.: Senior

Investigator, Dermatology Branch, National Cancer Institute, National Institutes of Health,

Bethesda, Md.

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78 LEVIS AND ROBBINS

a simple and rapid method for obtaining plasma with sufficient quantities of

both lymphocytes and monocytes and as few granulocytes as possible for use

in routine, commonly employed leukocyte cultures. This method, which is a

simplified adaptation of that of Jago,’6 requires only a 5-mm centrifugation

of heparinized whole blood and yields a mononuclear leukocyte population

in which the ratio of monocytes to lymphocytes is sufficiently above that cur-

rently known to be a limiting factor in the lymphocytes’ response to blasto-

genic factors.

In this paper, we present in detail our methods for obtaining “purified”

lymphocytes, g-a cells, and a “mononuclear leukocyte culture fluid” containing

adequate numbers of both cell types.

MATERIALS AND METHODS

Obtaining Mononuclear Leukocytes for Culture and as a Source of G-A Cells

Peripheral venous blood was drawn from normal donors whose last meal was

between 2 and 4 hr prior to venipuncture. Ten-milliliter quantities of this blood were

placed in i0-ml screw-capped conical centrifuge tubes (Cat. No. 2993-H25, A. H. Thomas)

each of which contained 100 U of preservative-free heparin (10 ,�l of Panheprin, Abbott

Lab.). The tubes were then gently inverted to mix the contents and were placed, within

10 mm, in Type 3 centrifuge carriages (International Equipment Co.) held on the

No. 269 rotor of the Model PRB centrifuge (International Equipment Co.). Centrifugation at

room temperature was begun by switching the speed control rapidly up nine notches from

its “off” position. As soon as the rotor attained a speed of 1500 rpm, the speed control

was reduced four notches to provide a setting at which the speed of rotation remained

at 1500 rpm. Under these conditions, a constant centrifugal force of approximately 300

g existed at the 7.5 ml level of each tube, which is the usual level of the resulting plasma-

erythrocyte interface. After 5 mm, the speed control was returned to its off position, and

the rotor permitted to slow down and stop of its own accord. After 1-2 mm, the tip of

a Pasteur pipette was inserted into the resulting supernatant plasma to a level approxi-

mately 1.0-1.5 mm above the buffy coat which is situated upon the packed erythrocytes.

By sucking up the top layer of the buffy coat into the pipette at this level with approxi-mately 1.5-2 ml of the plasma, a population of leukocytes was obtained that usually

contained 2-10 X 106 cells/ml of plasma and was comprised of at least 90% mononuclear

leukocytes. A “mononuclear leukocyte culture fluid” at a final concentration of 0.4-2 X 106

cells/ml was obtained by diluting this mononuclear-rich plasma with 4 volumes of

tissue culture medium 199 (Code 5477; Difco Laboratories) that contains 87.5 U/mi

of penicillin and 87.5 zg/ml of streptomycin (Code 51082; BBL, Division of BioQuest).

A mononuclear leukocyte culture fluid with a higher density of cells can be obtained

by recentrifuging the mononuclear-rich plasma for 6 mm at 900 rpm (150 g) and then

dispersing the resultant cell pellet in an appropriate volume of a mixture comprised of onepart of cell-free plasma and four parts of the medium 199.

The mononuclear leukocyte culture fluid was cultured at 38#{176}Cwith room air as the

gas phase in 0.5 ml aliquots in 12 X 35 mm screw-capped vials (No. 9802-C, A. H.

Thomas Co.) or in 0.4-2.0-ml aliquots in stoppered Leighton culture tubes (Cat. No.

1928, BelIco Class, Inc.) containing 9 X 9 mm glass cover slips. Stimulants used were

phytohemagglutmnin (PHA, 0.5-2.5 �zg/ml of culture fluid; batches E118 and E316A,

Burroughs Welcome and Co.) and tuberculin-purified protein derivative (PPD, lyophilized,without preservative, 1.25 �ug/ml of culture fluid, Parke-Davis). Mixed leukocyte cultures

were obtained by mixing equal parts of mononuclear leukocyte culture fluid from each

of tWo cell donors.

C-a cells were obtained attached to the glass cover slips after incubating the latter

in Leighton tubes with unstimulated mononuclear leukocyte culture fluid obtained directly




OBTAINING LYMPHOCYTES AND MONONUCLEAR CELLS 79

from the 5-mm centrifugation or after concentration of the cells by recentrifugation.

The cover slips were removed from the Leighton tubes after incubation periods of 2 hr-6

days. Nonadherent cells were removed from the g-a cells by dipping the cover slips into

three batches of 38#{176}Cmedium 199 and then subjecting each of the two surfaces of the

cover slips to a forceful flow of 2 ml of 38#{176}Cmedium 199 dispensed from a Pasteur

pipette. For morphologic studies of the g-a cells, the cover slips were air dried, and

their g-a cells stained with Wright’s stain. For studies on the function of the g-a cells,

the cover slips were recultured in cell-free culture fluid or added to Leighton tubes contain-

ing purified lymphocytes obtained as described below. Phagocytic activity was evaluated

by incubating the g-a cells with polystyrene latex particles (1.3 �tm in diameter; Dow

Chemical). Twenty-five microliters of a 1 :400 dilution of the original stock solution

of these particles was placed in each milliliter of culture fluid.

Obtaining Purified Lymphocytes

In the procedure for obtaining purified lymphocytes, seven 15-mi aliquots of peripheral

venous blood were placed in round-bottomed, 16 X 125 mm screw-capped glass tubes

(Cat. No. 9210-E44, A. H. Thomas Co.), each of which contained 150 U of the Panheprin.

The purified lymphocytes were subsequently obtained from the leukocyte-rich plasma

resulting after 1_lh/2 hr of gravity sedimentation of this heparinized blood at 38#{176}C.

This leukocyte-rich plasma was diluted with an equal volume of medium 199 and placed

in a separatory funnel held above a glass column 1 inch in diameter containing a tapered

opening at the lower end. The glass column was tightly packed to a height of 14 cm

with 12 gm of dry, sterile, nylon fiber17 (Leukopak, Fenwal Laboratories) that had

previously been washed in several batches of boiling, distilled water to remove soluble

contaminants. Up to 250 ml of the diluted leukocyte-rich plasma was then slowly dripped

from the separatory funnel frito the nylon fiber column followed by an equal volume of

medium 199. The flow rate from the separatory funnel was adjusted so that all the filtering

was completed within 1h/2�2 hr. The column effluent, which contaned small lympho-

cytes, erythrocytes, and platelets, was collected in an upright, 16 oz, glass prescription

bottle. The bottle was then tightly capped and laid horizontally at 38#{176}Cfor 2 hr on one

of its two flat sides to permit any contaminating g-a cells to adhere to the bottle while

the nonadherent lymphocytes remained in the supernate. Wright-stained smears of the

pellets from spun aliquots of the lymphocyte-rich supernate served to monitor the degree

of remaining contamination of the lymphocyte population with monocytes and/or

granulocytes. If any leukocytes other than lymphocytes were observed on these smears,

the still contaminated lymphocyte-rich supernate was gently decanted into clean, 16-oz

bottles, and the incubation process was repeated until the supernate was free of mor-

phologically indentifiable nonlymphoid leukocytes. Only occasionally was it necessary to

util’ize more than three such bottles.

When more than 99% of the resulting supernate’s leukocyte population had the

morphology of lymphocytes, the supernate was centrifuged at 150 g for 6 mm. The

cell pellets were dispersed to a final concentration of 0.5-3 X 106 lymphocytes/mi of a

fluid comprised of one part autologous cell-free plasma and four parts medium 199

containing the antibiotics. Four-tenths milliliter aliquots of these lymphocyte suspensions,

cultured in stoppered Leighton tubes with air as the gas phase, are hereafter referred

to as “purified” lymphocyte cultures. When glass cover slips containing g-a cells are

placed in the Leighton tubes with these purified lymphocytes, the cultures are referred

to as “reconstituted” cultures.

Evaluation of Radioactive Uridine and Thymidine Incorporation

DNA synthesis was measured by scintillation spectroscopy utilizing the Millipore

filter assay method’8 after the cells were incubated for 3 hr at 38#{176}Cwith thymidine-

methyl-3H (3HTdR, specific activity 17-22 Ci/mM, Amersham-Searle) �at a concentration

of 10 zCi/ml of culture fluid.

Radioautographic studies of 3H-uridine (uridine-5-T; specific activity; 16.3 Ci/mM;





1.25 izCi/ml of culture fluid, Nuclear Chicago Corp.) and 3HTdR incorporation into g-a

cells and into nonadherent leukocytes were performed after 2-24 hr of incubation with

the radioactive compounds. The incubated cells were then washed in three batches of

medium 199 after which the g-a cells, still on their cover slips, and the nonadherent

leukocytes, pelletized and then smeared on cover slips, were air dried and then fixed

in methanol for 10 mm. After a rinse in distilled water, the cover slips were air dried,

mounted cell-side up on regular 1 X 4 inch glass slides and coated with a thin layer of

43#{176}C,o.s% gelatin solution19 to promote adherence of the NTB-3 radioautographic

emulsion (Eastman Kodak) that was then applied. After exposure at 4#{176}Cfor periods

up to 3 wk, the radioautographs were developed and placed in running tap water for 30mm, rinsed in a 1 :1 methanol-Ciordano phosphate buffer solution (pH 6.4; Fisher Scientific

Co.), then in the Ciordano buffer alone, and then in absolute methyl alcohol. The slides

were then stained with Wright’s stain.

RESULTS

Mononuclear Leukocyte Culture Fluid

Morphology: Figure 1 shows a representative field containing the mono-

nuclear leukocytes obtained by the 5-mm centnifugation technique. Differen-

tial analyses of leukocytes obtained in 20 consecutively performed experiments

Table 1. Differential Analyses of the Leukocytes in the MononuclearLeukocyte-rich Plasma Obtained by the 5-Mm Centrifugation Method

Experiment No. 0/0 Lymphocytes’ o/o Monocytes’ �/o Granutocytes’

0.5 (±0.5)1 91.5 (±1.5) 8.0 (±1.0)

2 91.9 (±1.3) 4.9 (±0.4) 3.1 (±0.9)

3 79.9 (±0.9) 14.0 (±0.2) 6.2 (±1.0)

4 86.3 (±6.0) 12.7 (±5.3) 1.2 (±0.6)

5 71.5 (±4.3) 23.2 (±2.2) 2.6 (±0.8)

6 80.7 (±2.6) 16.4 (±3.3) 2.9 (±0.7)

7 77.7 (±0.7) 12.4 (±0.2) 10.0 (±0.5)

8 83.9 (±0.3) 13.0 (±0.0) 3.1 (±0.3)

9 82.5 (±2.4) 12.2 (±1.8) 5.4 (±0.5)

10 85.0 (±1.3) 7.6 (±1.8) 7.6 (±0.2)

11 84.8 (±1.2) 10.3 (±1.1) 5.0 (±0.2)12 81.1 (±1.9) 12.3 (±1.9) 6.7 (±0.1)13 88.8 (±0.7) 9.6 (±0.0) 1.7 (±0.7)14 82.9 (±2.7) 12.1 (±2.5) 4.9 (±0.3)15 90.1 (±0.2) 7.4 (±0.1) 2.5 (±0.1)

16 90.4 (±1.0) 7.7 (±0.9) 2.0 (±0.1)17 81.6 (±2.4) 14.2 (±2.6) 4.2 (±0.2)18 87.1 (±2.9) 15.2 (±2.2) 2.6 (±0.0)19 78.2 (±0.1) 17.7 (±0.8) 4.1 (±0.9)20 85.0 (±1.2) 12.6 (±0.6) 2.5 (±0.5)

Mean ± SD of differential cell countst 84.0±5.4 12.2±8.5 3.9±4.9

* Four cover-slip smears were prepared from each donor’s mononuclear leukocyte-richplasma obtained by the 5-mm centrifugation method. Total of 500 leukocytes wereanalyzed (125 from each of four cover slips), and percentages of these cells that werelymphocytes, monocytes, and granulocytes were calculated. At a later time, differentialcell counts were repeated on another 500 cells from same four cover slips without theanalyzer’s having knowledge of previous results. Results for each of 20 experimentsare expressed as mean percentage (±SE) of results of two 500-cell analyses.

t Average of mean percentages of differential cell counts of 20 experiments: this SDrepresents distribution of the 20 means about their average.





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Fig. 1. Mononuclear leukocytes obtained by 5-mm centrifugation method. Of97 Leukocytes in field, three are polymorphonuclear leukocytes (P), 19 aremonocytes (M), and 75 are lymphocytes (L). Platelets (PIt) and erythrocytes(E) are also present. X 160.

are presented in Table 1. Lymphocytes comprised, on the average, 84% (range

71.5-91.9%), monocytes 12.2% (range 4.9-23.2%), and granulocytes 3.9%

(range 0.5-10.1%).

Table 2. DNA Synthesis in PHA-containing Mononuclear Leukocyte Culture Fluid

Donor No.

3HTdR Incorpora tion in cultures’

PHA

(cpm)

No Blastogenic Factor

(cpm)

1

2

3

4

5

678

9

10

87,901 (± 9,469)

208,487 (±29,843)

173,003 (± 613)

99,901 (± 731)

65,989 (± 4,863)

204,939 (±22,687)51,765 (± 3,849)

314,791 (±48,538)

66,543 (± 3,629)143,544 (± 1,250)

1057 (±178)

547 (±264)

808 (± 98)

399 (± 45)

2447 (±170)

206 (± 54)508 (± 98)178 (± 38)

304702

* Each culture contained 3-6 x 10� leukocytes and was assayed with 3-hr 3HTdRpulse on third or fourth day of culture. Values are expressed as mean (±SE) of cpmobtained from duplicate or triplicate cultures. In each of experiments 9 and 10, onlyone unstimulated control culture was assayed.





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Fig. 2. G-a cells attached to glass cover slips. (A). 36-hr culture; (B). 6-dayculture. Clean cover slips were incubated in Leighton tubes containing mono-nuclear leukocyte culture fluid for the stated periods, at which times coverslips were removed, dipped and sprayed, air dried, and stained. X 400.

Blasto genesis: When appropriately stimulated, lymphocytes in the mono-

nuclear leukocyte culture fluid developed into blastoid cells capable of DNA

synthesis and mitotic division. Table 2 shows the incorporation of 3HTdR

into DNA in PHA-containing mononuclear leukocyte culture fluids obtained

from ten consecutive donors. These mononuclear leukocyte culture fluids

responded well also to blastogenic factors other than PHA. For example, when

a 0.5 ml. mixture of culture fluids from donors 9 and 10 was incubated in the

absence of PHA, a mixed leukocyte reaction occurred that gave over 4000

cpm on the third day; in 0.5 ml aliquots of the culture fluid from donor 8

cultured with tetanus toxoid, 4300 cpm were obtained at the 96th hr of culture.

G-A Cells

Morphology: By the 36th hr of culture (Fig. 2A), all the g-a cells derived

from the centnifugation technique were mononuclear, and no intact poly-

morphonuclear leukocytes were seen. Some of the g-a cells already had the

peripheral, wedge-shaped nucleus and foamy cytoplasm characteristic of

macrophages. By the sixth day of culture (Fig. 2B) most of the g-a cells had

more abundant foamy cytoplasm.

RNA Synthesis: When 3H-uridine was added to the mononuclear leuko-

cyte culture fluid for the first 2 hr of culture, radioautograms revealed labeling





. L � Fig. 3. Radioautograms

a . 0 of incorporation of 3H-. uridine into g-a cells.

S (A). Predominantly flu-P . clear labeling found in

. g-a cells fixed after 2-hr

. . exposure to 3H-uridine at. . start of culture; (B). Nu-

, 0 #{149} clear and cytoplasmic. labeling in 5-day-old g-a

- cells fixed at end of 17-hr exposure to 3H-uri-

- dine. In latter experi-#{149} ment, polystyrene latex

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� other cell, even those g-a

cells that did not phago-cytose the polystyrene latex particles incorporated 3H-uridine. X 1000.

primarily over the nucleus in most of the cells (Fig. 3A), as has been reported

to occur in human macrophages, obtained by a different technique, when

fixed immediately after such a short pulse.2#{176}When 3H-uridine was added for

the last 17 hr of a 120-hr culture period, grains were found over both the

nucleus and cytoplasm in phagocytizing and nonphagocytizing g-a cells

(Fig. 3B).

DNA Synthesis: Cover slips containing g-a cells from 2- to 6-day-old

cultures of mononuclear leukocyte culture fluid were dipped and then sprayed

Table 3.Ability of G-A Cells to Stimulate Allogeneic Purified Lymphocytes

Experiment No.

3HTdR Incorporation’ in Culturest

(cpm)

Purified

Lymphocytes

Alone G-A cells Alone

Purified Lympho-

cytes Plus G-A Cells

12

34567

207 (± 9)123 (±13)

202194235 (±33)145 (±26)198 (± 4)

118 (± 39)

248 (±152)

140148

119 (± 1)217 (± 69)

184

2,820 (± 367)

18,828 (±1,062)

1,044

1,684

2,849 (±1,339)1,785 (± 585)

1,706 (± 146)

Values for �HTdR incorporatIon are expressed as mean (±SE) of cpm obtainedfrom duplicate, 0.4 ml cultures, except where only one culture was assayed. Thymidineincorporation was determined after incubating culture fluid with 4 /LCi of 3HTdR for 3-hrperiod sometime between 84th and 132nd hr of culture.

t Each culture containing g-a cells alone consisted of 0.4 ml of cell-free culture fluidoverlying a glass cover slip containing the g-a cells that had been washed by dipping andspraying procedure. Cultures of purified lymphocytes alone and plus g-a cells contained0.4 ml of culture fluid containing 0.4-1.0 x 106 pur!fied lymphocytes.





Table 4. Ability of Autologous G-A Cells to Restore DNA SynthesisIn Purified Lymphocyte Cultures in Response to PPD

Contents of Cultures’3HTdR Incorporation

(cpm)f

Lymphocytes alone 114 (± 18)Lymphocytes + PPDt 110 (± 14)Lymphocytes + g-a cells 254G-a cells alone 286G-a cells + PPD 417 (± 159)Lymphocytes + g-a cells + PPD 42,915 (±5227)

I Each culture with lymphocytes contained 106 purified lymphocytes.t Values for �HTdR incorporation are expressed as mean (±SE) of cpm obtained

from duplicate, 0.4 ml cultures, except where only one culture was assayed. ThymidineIncorporation was determined after incubating culture fluid with 4 �&Ci of 3HTdR for3-hr period beginning at 84th hr of culture.

* PPD, 1.25 ,ug of tuberculin-purified protein derivative/mI of culture fluid.

with medium 199 and reincubated in cell-free culture fluid containing PHA or

antigens. A 3-hr incubation with 3HTdR on the second, third, or fourth day

thereafter did not result in radioautographically detectable labeled g-a cells.

To determine whether any DNA-synthesizing g-a cells could become detached

from the cover slips and thereby escape radioautographic detection, the radio-

active culture fluid was passed through a Millipore filter to trap any such

detached cells. A small amount of thymidine incorporation (about 1100 cpm)

was thus found in response to PHA in only one of ten experiments in which

the cover slips had been dipped and sprayed. The necessity for spraying was

shown by the finding that considerable DNA synthesis was often detected

when the cover slips were dipped but not sprayed.

Stimulation of Allogeneic Lymphocytes: Table 3 shows that the purified

lymphocytes underwent DNA synthesis when cultured with g-a cells from

allogeneic donors. In other experiments with g-a cell and purified lymphocyte

mixtures in which the donors were of opposite sex, sex chromosome analysis

indicated that all the dividing cells were derived from the lymphocyte popu-

lation.”

Table 5. Ability of G-A Cells to Increase DNA Synthesis in Purified Lymphocyte Cultures

In Response to PHA

Contents of Cultures’3HTdR Incorporation

(cpm)t

Lymphocytes + PHAt 5,166 (± 152)Lymphocytes + PHM 106,602 (±7818)GA cells + PHA 52 (± 8)

#{149}Each culture with lymphocytes contained 1.1 x 106 lymphocytes. Glass coverslips with g-a cells, removed after 36-hr incubation in mononuclear leukocyte culturefluid, were dipped and sprayed; they were then added to purified lymphocytes or tocell-free culture fluid containing PHA.

t Values for 3HTdR incorporation are expressed as mean (±SE) of cpm obtainedfrom duplicate 0.4 ml cultures. 3HTdR incorporation was determined by incubatingculture fluid with 4 zCi of 3HTdR for 3-hr period beginning at 59th hr of culture.

* PHA, 2.5 �g/ml of culture fluid.




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Fig. 4. Lymphocytes during different stages of their purification. (A). Smearof centrifuged aliquot of column effluent prior to incubation on inner surfaces ofbottles. At this magnification (X 160), there are no detectable contaminatingpolymorphonuclear leukocytes among the hundreds of lymphocytes. Paler-stained erythrocytes are also evident at this magnification. (B). Higher powerview (X 400) of smear from aliquot of another batch of purified lymphocytesafter 2-hr incubation in glass bottle. All leukocytes are lymphocytes, most ofwhich appear morphologically intact. Erythrocytes (E) and platelets (PIt) arealso present.

Purified Lymphocytes

The leukocytes obtained from the nylon fiber columns were comprised

almost exclusively of small lymphocytes (Fig. 4A). Occasionally a contaminat-

ing polymorphonuclear leukocyte or monocyte was observed, but most were

removed by incubating the lymphocyte preparations in the glass bottles (Fig.

4B).

Two additional techniques were employed that provided more sensitive

indications of minor degrees of contamination than the use of cell smears.

The first consisted simply of incubating 4 X � or more purified lymphocytes

for up to several days at 38#{176}Cin Leighton culture tubes containing clean

cover slips, which serve as “concentrating” attachment sites on which con-

taminating monocytes eventually settle. At the end of the incubation period

the cover slips were removed, air dried, stained, and examined for the presence

of g-a cells. One or more macrophages could thereby be detected among4 X i05 lymphocytes, despite the fact that in smears of the original lympho-

cyte population no monocytes had been observed.

The most sensitive index of “purity” was a functional one and consisted of

determining the degree of impairment of the blastogenic responses to specific

antigens and PHA. Thus, lymphocytes of sufficient purity failed to give any





detectable blastogenic response to antigens but did respond when cultured in

the presence of a glass cover slip containing autologous g-a cells (Table 4).

Purified lymphocytes still gave some response to highly blastogenic concen-

trations of PHA (Table 5, line 1). However, addition of both PHA and g-a

cells to these lymphocytes resulted in a much greater response (Table 5, line 2).

DISCUSSION

One of the first steps frequently required in obtaining leukocytes for in vitro

studies is to effect a substantial separation of the leukocytes from erythro-

cytes. It was for this purpose that phytohemagglutinin was used,2124 prior to

the discovery25 that PHA preparations also contained a blastogenic factor(s).

Numerous other substances26 have been utilized to separate erythrocytes from

leukocytes, e.g., fibrinogen, dextran, polyvinylpyrrolidone, gelatin, and gum

acacia. The methods we use involve either a simple centrifugation or a gravity

sedimentation of the erythrocytes, thereby obviating the necessity of exposing

the leukocytes to any of these substances.

Investigators have utilized several methods that separate lymphocytes

from phagocytic cells (monocytes and polymorphonuclear leukocytes).

In some of these methods the phagocytic cells were removed after they

had ingested iron particles.273’ In most other methods, however, the separa-

tion is based on the tendency of the phagocytic cells, but not the lymphocytes,

to be retained by certain surfaces and matrices, e.g., siliconized glass

beads,3234 glass wool,33’35 nylon fiber,436 and cotton wool.37’38 None of these

methods is well suited to providing cells for the study of blastogenesis, since

all of them may remove most of the monocyte-containing fraction (which con-

tains the g-a cells essential for the blastogenic response) together with the

polymorphonuclear leukocytes that perform no known useful function in, and

may be detrimental to, blastogenic responses in vitro.15’39 Another disadvan-

tage of many of these methods, when utilized for obtaining purified lympho-

cytes, is that the methods may not reliably produce the required high degree

of separation and generally do not provide a method for monitoring and thus

determining when the desired purity has been attained. Each of these disad-

vantages can be easily circumvented by the cell preparation methods we have

described herein and discuss below.

Our 5-mm centrifugation of freshly drawn heparinized peripheral blood

and collection of the leukocytes in the upper portion of the resulting buffy

coat are conducted almost exactly as described by Jago.’#{176}However, her sub-

sequent 30-mm, 37#{176}Csedimentation of the leukocytes is entirely omitted

from our procedure, which yields a leukocyte population (Table 1) containing

approximately 70-90% lymphocytes, 5-20% monocytes, and less than 10%

polymorphonuclear leukocytes, thus essentially eliminating the unnecessary

and possibly deleterious latter cell type. Because of the high percentage of

mononuclear leukocytes in this leukocyte preparation, we refer to the culture

fluid resulting from its addition to medium 199 as the “mononuclear leukocyte

culture fluid.” In view of its very adequate responses to PHA (Table 2) and

other blastogenic factors, it is apparent that this mononuclear leukocyte cul-

ture fluid contains sufficient numbers of viable lymphocytes, as well as suffi-





cient numbers of the required g-a cells, the latter presumably being derived

from the monocytes.

Jago reported’#{176} obtaining in 16 of 23 experiments leukocyte populations

comprised 100% of lymphocytes. Mangi and Mardiney,4#{176} using an adaptation

of Jago’s technique, stated that they almost invariably obtained lymphocyte

purity greater than 96% and at times attained a purity of 99%. It is known

that when populations of leukocytes are comprised of approximately 98-

100% lymphocytes, the blastogenic response to antigensfr7 allogeneic

cells,4’5’8’2 and PHA’4 will be limited by the extremely low numbers of g-a

cells. The technique, as employed precisely as described by Jago’6 and by

Mangi and Mardiney,4#{176} will, in view of their reported results, in many in-

stances provide insufficient numbers of g-a cells to permit the leukocyte popu-

lations to be used as measures of the lymphocytes’ ability to respond to blasto-

genic factors. In our adaptation of Jago’s technique, we have never obtained

a leukocyte population in which the lymphocytes comprised more than 92%

of the cells (Table 1), a degree of purity that is below that at which the per

cent of g-a cells is known to become a limiting factor.

Our method for obtaining purified lymphocytes from human peripheral

blood differs from most other methods in the following respect: we utilize

two procedures in the purification process, the latter of which permits monitor-

ing and choice of the purity attained. Thus, the partially purified column

effluent is incubated on the inner surfaces of glass bottles to permit the

remaining g-a cells to adhere to the glass. By removing periodically tiny

aliquots of the nonadherent supernatant lymphocyte population, concentrat-

ing them via centrifugation, and making smears of the resulting pellets for

microscopic examination, it is possible to monitor the degree of purification

being achieved and to continue the purification process until the desired purity

has been attained.

Most, if not all, of the cells in the g-a mononuclear leukocyte population

that restores detectable blastogenic responses in cultures of purified lympho-

cytes are derived from the monocytes. However, it is not known whether it is

these monocyte-derived cells that restore the response or whether the g-a cell

restoring the response is derived from a lymphoid cell that had, or developed,

glass-adherent characteristics. In either event, removal of virtually all the

monocytes, i.e., achieving a leukocyte population composed more than 99%

of lymphocytes, results in the depletion of the required g-a cell, indicating

that even if the required cell is not derived from the monocyte it is removed

when the monocytes are removed.

ACKNOWLEDGMENT

We thank Miss Rosemary Cuddy for helpful discussion and Mrs. Elaine Cowles for

typing the manuscript.

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1972 40: 77-89

William R. Levis and Jay H. Robbins Blood

PeripheralCells, and a Population Containing Both Cell Types From Human Methods for Obtaining Purified Lymphocytes, Glass-adherent Mononuclear

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