the acridine orange viability test applied to bone marrow - blood

The Acridine Orange Viability Test Applied to Bone

Marrow Cells I. Correlation with Trypan Blue

and Eosin Dye Exclusion and Tissue

Culture Transformation

By WILLIAM E. HATHAWAY, Loins A. NEWBY AND JOHN H. GITHENS

M ANY METHODS have been used to assay the viability of hematopoietic

cells during the course of procurement, preservation, and fransplanta-

tion of animal and human tissue. Tritiated thymidine is frequently used as a

cell marker in the study of hematopoietic proliferation.’ In vitro DNA

synthesis has been reported as an index of viability.2 Growth and transforma-

tion of living cells in tissue culture also indicate viability.3 Determination of

cell motility is used to detect living cells.4 The resistance of viable cells to

staining with trypan blue5 and eosin#{176} is probably the most frequently used

technic. The problems and limitations encountered in determining cell viabil-

ity by these and other in vitro methods have recently been reviewed.7 In the

field of bone marrow transplantation the most precise measurement of viabil-

ity is the ability of the tissue to transplant, proliferate, and protect a lethally

irradiated host. Because this in vivo method is not feasible prior to transplan-

tation with preserved human tissue, there is a need for a rapid and simple in

vitro estimation of cell viability. Following the suggestion of Pegg8 that the

acridine orange (A-O) staining reaction9’1#{176} may be correlated with the viability

of l)One marrow cells after preservation by freezing, the following studies were

done in order to establish an “A-O viability test of marrow cells.”

METHODS

Unless otherwise iIl(licated, the following conditions were carefully observed through-

out the experinlent.

Preparation of Marrow

The marrow was obtained froni the feniurs of freshly killed Balb/c mice �iiul suspended

in Puck’s tissue culture iriedia’ ‘ ( N-16 40 i)�r cent, saline F 30 per cent, fetal calf serum

30 per cent ) as single cells at an approximate concentration of 40-50 x 10� per ml.

fllis suspension was observed iillllle(liately after preparation and is called “fresh mar-

row. All other cell suspensions were at a similar concentration.

Frozen marrow samples were prepared by suspending marrow cells in Puck’s tissue

culture Illedia after passage through a 25 gauge needle to reduce particles to single cells

at a concentration of approxin�ately 50 x 1O� per ml. Glycerol or dililethylsulfoxide

( I)MSO ) was added prior to freezing in sealed 2 ml. glass ampules. The samples were

frozen in a C02-95 per cent ethanol I)ath l)Y slowly reducing the temperature 1 C. per

Illiflhlte to -40 C. and then rapidly to -79 C. The vials were tilen immersed in liquid

This work was �upported by American Cam�er Society Grants T138B and T255.From the Department of Pediatrics, University of Kentucky Medical Center, Lexingkrn,

Ky.

Submitted Sept. 4, 1963; accepted for publication Oct. 29, 1963.

517

BlooD, VOl.. 23, No. 4 (APRIL), 1964

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518 HATHAWAY, NEWBY AND G1THENS

nitrogen ( -196 C. ) for storage. Thawing was performed rapidly in a 37 C. water bath

and the specimen was examined immediately as described under “fresh marrow.”

Methods for Producing Injury to Ce&i

Aliquots of “fresh marrow” were treated in the following ways in order to note the

effects of various injurious agents on the staining characteristics with acridine orange.

a. pH-the pH of the cell suspensions were changed to 8.9 by addition of 0.1N sodium

hydroxide and to 6.0 by addition of 0.1N hydrochloric acid.’2 After incubation at 37 C. for

40 minutes, the cells were resuspended in fresh Puck’s media and observed.

b. Heat-Cell suspensions were heated to 60 C. for 30 minutes and observed.

c. Rapid freezing-Cell suspensions were frozen quickly by placing them in a freezer

at -20 C. without the use of a protective agent such as DMSO or glycerol. The cells

were then rapidly thawed and observed.

d. Aging of cells without tissue culture media or serum-Bone marrow cells were al-

lowed to stand in a balanced salt solution ( saline F ) for 6 hours at room temperature, andperiodic observations were made. The depletion of serum or tissue culture media from

the suspending fluid for long periods decreases the protective ability of bone marrow cells

being prepared for transplantation.’3

e. Poison.-0.25 ml. of a 700 mg. per cent solution of sodium cyanide was added to

1.5 ml. of marrow cell suspension for 1 hour at 37 C.14

Viability Test

a. Acridine orange: One drop of a murme marrow suspension was placed on a clean

microscope slide and mixed with a second drop of acridine orange suspension. A number

0 coverslip was then applied with firm pressure to produce a layer of single cell thickness.

The edges were sealed with petrolatum or Kronig’s cement. The acridine orange dye

( National Aniline Lot l4llp) was prepared fresh every 2 weeks as a 9 mg./100 ml.

suspension in Puck’s media buffered with tris(hydroxymethyl)-amino methane to pH

7.4. The slide-cover slip preparation was observed with fluorescent microscopy at 25-27 C.

in a partly darkened room and the necessary differential count or photograph made within

1 to 2 minutes of fluorescence per field.

The microscope was a Leitz Ortholux with the Osrarn HBO 200 mercury vapor lamp

as the light source. Two ( UGI ) exciter filters, one 4 mm., the other 2 mm., were used

with a Blau-absorption filter ( Leitz). Differential cell counts of the supravital preparations

were done with 430X and 970X magnification. Color transparencies were produced with

35 mm. Ektachrome, type B, color film with an exposure index of tungsten 100 and ex-

posure times of 30 to 60 seconds.

b. Eosin and trypan blue: Differential counts were done in the same manner ( one

drop of cell suspension plus one drop of dye ) under light microscopy with 1 per cent

and 0.1 per cent eosin Y and 0.05 per cent and 0.02 per cent trypan blue (in Puck’s

media at pH 7.4 ) . The percentage of unstained cells ( viable cells ) was determined. Thepreparations with eosin Y were examined within 5 minutes in order to avoid possible

photodynamic cytoxicity.’5C. Tissue culture transformation (TCT): A semiquantitative estimation of growth in

tissue culture was devised following the suggestion of Porterfield and Ashwood-Smith.’6

Aliquots of each specimen were plated on cover slips in Petri dishes at cell concentra-

tion of 12 x 10� per ml. When frozen specimens were used, the DMSO was reduoed

to 2 #{189}per cent. Puck’s culture media and a 5 per cent C02-95 per cent air incubator

( 37 C. ) were used. After 4 days, the cover slips were removed, air-dried, and stained with

Wright’s stain. The average number of “fibroblastic” or large “mononuclear” cells per

h.p.f. ( average of 25 random fields ) was recorded. The control was fresh murine marrow

in Puck’s media or 2’,� per cent DMSO in Puck’s media at the same cell concentration

(see figure 1).




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ACRIDINE ORANGE TEST AND BONE MARROW CELLS 519

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Fig. 1.-Above, photograph of Wright’s stained cover slip demonstrating the

tissue culture transformation into “fibroblasts” and “mononuclear cells” of freshInurine bone marrow cultured for 4 days. Below, photograph demonstrating poor

tissue culture transformation of bone marrow cells treated with sodium cyanide

prior to 4-day culture.




520 HATHAWAY, NEWBY AND GITHENS

RESULTS

With the factors of dye concentration, cell concentration, pH of the sus-

pending media, length of exposure to ultraviolet light, and thickness of the

preparation carfully controlled, a uniformly reproducible appearance of fresh

marine bone marrow cells was obtained. The fresh, “viable,” supravitally

stained nucleated cell displayed a bright, apple-green nucleus, and pale,

dull-green cytoplasm with varying amount of bright orange cytoplasmic

granulations present. With experience, a conventional differential count based

on the usual morphologic characteristics could be done even at 430X magnifi-

cation. In general, the supravital staining properties of mouse bone marrow17

cells are the same as for human marrow,’8 but decidedly different from fixed

preparations’9’2#{176} of either mouse or human marrow.

By comparing the acridine orange staining characteristics with the degree

of viability demonstrated by the various marrow preparations in tissue culture,

it was possible to recognize several staining patterns which indicated cell

death or injury. It became evident that the typical “injured” or “dead” cell

nucleus and cytoplasm stained diffusely bright red, and could be readily dis-

tinguished following injury by pH change, NaCN poisoning, and aging

without culture media. In addition, certain cells displayed an increase in red

granulation of the cytoplasm and an orange hue to the nucleus. These cells

were considered to be injured or damaged to a lesser degree than the “dead”

cells.

A second type of “dead” cell staining reaction was observed in the cells

killed by rapid freezing or heating. The nuclei of cells disrupted by quick

freezing stained a deep, dull-green. They could be distinguished from fresh

cells by the absence of intact cytoplasm and total loss of red granulations.

In a similar manner, the bare nuclei produced by mechanical trauma in

preparation of the suspensions could be distinguished by a dull-green stain and

absence of cytoplasmic granulations. Heated cells showed a diffuse, dull-green

staining reaction. Figure 2 illustrates the difference in the above designated

A-O staining properties of “live,” “injured,” and “dead” cells.

The results of the experiments designed to produce injured or “dead” cells

are summarized and compared with the eosin and trypan blue dye exclusion

test and tissue culture transformation in table 1.

a. pH-Change of pH to 8.9 revealed decreased numbers of viable cells

by the A-O method and TCT which was not indicated by eosin or trypan.

However, pH 6.0 effected the A-O test but not the TCT or dye exclusion tests.

b. Heat-All methods indicated death of most of the cells. The A-O

stained cells were diffusely dull-green.

3. Quick freezing toithout protective agent-The A-O cells were stained

a dull-green, easily differentiated from “live” cells. Most of the cells were

stained with eosin and trypan blue and showed little growth in tissue culture.

d. Aging “without” serum or tis.s’u� culture media-The TCT indicated de-

creased viability of cells suspended for 6 hours in saline F. This observation

correlated well with the A-O test but poorly with the dye exclusion tests.

e. Sodium cyanide-A-O cells were diffusely red and orange. The TCT also




1

3

5

2

4

6

ACRIDINE ORANGE TEST AND BONE MARROW CELLS I 521

�. � 4t.4. �#{149}

, � #{149}5’� �I

4 1.

Fig. 2.-i. Fresh marrow suspension, X430. 2. Fresh marrow suspension, X900( oil immersion) . Note cluster of “dead” cells in center. 3. Fresh marrow, X900,

predominately granulocvtic elements. 4. Fresh marrow, X900. Note megakaryocyte.

5. Non-viable cells, X900. 6. “Injured” cells, mixed with two “live” cells, X900.

iIldiC�1tC(I decreased viability which was not apparent with eosin and trypan

blue.

f. Varying amounts of l)rotecticc agent ( DMSO )-Reduction of DMSO

concentration below 10 per cent gave evidence of decreased viability by all

the tests employed.

The results of the application of the A-O viability test to fresh and freshly

thawed specimens of murine marrow preserved by freezing in DMSO or

glycerol, and the comparison with eosin and trypan dyes is shown in table 2.

With A-O, the cells staining diffusely red or red-orange and those with bare




522 HATHAWAY, NEWBY AND GITHEN5

Table 1.-Percentage of Viable Cells as Determined by Supravital_________ Staining and Tissue Culture_Transformation

Treatment of Marrow Staining Methods Tissue Culture

% Viable cells Average--�� numberof

1.0% 0.1% .05% .02% live cells perModality used A-O Rosin Rosin TB. TB. h.p.f.

Control ( fresh marrow) 97 90 93 98 94 16-18

Heat (60 C.-30 mm.) 0 0-i 0 0-1 0-i 0

Rapid freezing 5 3 0 6 8 0-1

pH 6.0 (40 mm.) 62 98 92 97 96 17

pH 8.9 (40 mm.) 10 93 97 99.5 98 8-9

NaCN (1 hr.) 11 54 82 80 92 2

Aging without serum

(6 hr.) 30 92 99 99 98 10

Freezing-2�/� % DMSO

(14 days) 48 5 83 30 70 1-2

Freezing-5% DMSO

( 14 days ) 74 65 90 73 81 6

Free7ing-10% DMSO

(14 days) 80 82 97 80 91 8

Freezing-12#{189} % DMSO

(14 days) 82 65 97 80 90 8-10

Freezing-15% DMSO

(14 days) � - 68 � � � 79 92 8

- #{176}T.B. = Trypan blue.

green nuclei were considered non-viable in the differential count. In general,

the A-O stain gave a consistent result from specimen to specimen and cor-

related well with the dye exclusion tests. One per cent ( 1 per cent ) eosin

occasionally gave markedly different values in comparison to the other stains.

DisCuSsioN

Our findings regarding the A-O staining reaction of healthy and damaged

marrow cells are in accord with the studies of Wolf and Aronson2’ for other

tissues. They have described the fluorescence and metachromasy of fibroblasts

from chick embryo hearts, rabbit lens epithelium and chorioidal melanocytes

cultured in the presence of acridine orange. They have demonstrated that

relatively healthy cells stained orthochromatically ( deoxyribonucleic acid

hues of green; ribonucleic acid hues of red ) ; more diffuse metachromatic

staining ( red and orange ) accompanied cell injury; and complete cytoplasmic

metachromasy accompanied irreversible injury ( red ) . In accord also, is the

finding of diffuse green staining ( loss of metachromasy ) at an early stage of

degeneration. This finding was demonstrated in our study by the staining

reaction of the cells damaged by heat. Wolf and Aronson have emphasized

that the “A-O viability test” must be interpreted along with a study of

morphology and history of the cells being tested.

Emphasis should be placed on factors which need to be carefully controlled

in order to have reproducible results when employing the A-O viability test.

( 1 ) The cell concentration:stain concentration ratio should be kept constant.

The number of “dead” cells at the usual concentration of 40-50 x i0� cells per




ACRIDINE ORANGE TEST AND BONE MARROW CELLS I 523

Table 2.-Comparison of A-O Staining and Dye Exclusion to Indicate Viability of

Murine Marrow Preserved_by Freezing for Varying Periods of Time

Per Cent Viable Cells

Length of Eosin Trypan blueStorage

Protective Agent (daye) A-O 1% 0.1% 0.05% 0.02%

Fresh ( average 5samples) 93 94 97 98 97

10% DMSO 7 76 42 85 80 73

10% DMSO 14 83 74 84 - 73

10% DMSO 22 81 76 98 88 9310% DMSO 31 90 84 98 85 96

10% DMSO 48 77 79 88 91 8210% DMSO 78 83 77 98 88 91

15% DMSO 180 89 44 90 - 88

15e/( Glycerol 90 51 48 42 55 77

ml. and 9 mg. per cent A-O stain is appreciably fewer than would be noted

when the cell concentration is 10-20 x i0� cells per ml. (2) The pH of the

suspending media is critical and easily controlled by use of the tris buffer.

( :; ) The cell preparations should be promptly examined under the ultraviolet

microscope with as little U-V radiation per field as possible in order to avoid

photodynamic effect on cell viability. Khodas22 has emphasized the importance

of these factors in studies of acridine orange staining of peripheral blood cells

after X-irradiation.

The results of the present study indicate that the A-O test correlated well

with the TCT for all types of marrow cell injury, although the A-O staining

results suggested greater injury with pH change than was apparent from the

tissue culture growth. The A-O test compared favorably with the 0.1 per cent

(‘OSill and 0.02 per cent trypan blue dye exclusion tests in determining the

viability of fresh hemic cells and those damaged by heat, and by rapid or

slow freezing. The 1 per cent eosin and 0.05 per cent trypan blue occasionally

gave results that correlated much less well with the other methods than the

0. 1 per cent eosin and 0.02 per cent trypan blue.

Furthermore, cell damage due to injury of metabolic processes associated

with change of pH, depletion of serum, and treatment with sodium cyanide

was indicated by the acridine orange test and confirmed by tissue culture, but

was not indicated by the eosin or trypan blue dye exclusion tests. Eaton et al.�

have shown that the tumor-producing capacity of Ehrlich ascites cells injured

with NaCN or viruses tends to diminish at a somewhat greater rate than

cellular respiration or the ability to exclude eosin or trypan blue dyes. Cells

which have been frozen and preserved at low temperatures for long periods

of time probably undergo biochemical denaturation.24

Although in the present study eosin and trypan blue results correlated well

with the A-O and TCT in revealing cell death of marrow frozen up to 180

days, it has been shown that the eosin exclusion test does not correlate with

the ability of long-term frozen cells to transplant.25 Tullis has shown that the

eosin exclusion test is not sensitive in determining viability when compared

to amoeboid or phagoc�tic activity of preserved leukocytes�26 Whether the




524 HATHAWAY, NEWBY AND GITHENS

A-O test is any more sensitive in indicating damaged or dead cells after long-

term freezing, or in indicating marrow which will not transplant, remains to

be shown.

An additional advantage of the acridine orange test is that the viability of

specific types of cells can be noted by performing cell type differential

counts simultaneously with the “live-dead” determination. Cell differentia-

tion is not possible with the eosin and trypan blue dyes.

SUMMARY

Suspensions of murine bone marrow cells were stained with acridine orange

( A-O ) and observed under fluorescent microscopy after treatment with

various injurious agents in order to establish the staining characteristics of

“live” and “dead” cells. The percentage of viable cells demonstrated by the

“A-O viability test” were correlated with eosin and trypan blue dye exclusion

and tissue culture transformation viability tests. In general, the A-O test

demonstrated the viability of cells preserved by freezing as effectively as the

other in vitro tests. In addition, the A-O test may be more sensitive in

determining the viability of cells where metabolic processes have been injured

by poisons or change in pH.

SUMMARIO IN INTERLINGUA

Suspensiones de murin cellulas de medulla ossee esseva tincturate con

orange acridinic ( 0-A ) e observate per microscopia fluorescente post tracta-

mento con vane agentes nocive, con le objectivo de establir le characteristicas

tincturatori de cellulas “vive” e “morte.” Le procentage de cellulas viabile

demonstrate per le “test de viabilitate a 0-A” esseva correlationate con tests

de viabilitate a exclusion de eosina e trypan blau e a transformation de cultura

de tissu. A generalmente parlar, le test a 0-A demonstrava le viabilitate de

cellulas preservate per congelation tanto efficacemente como le altere tests in

vitro. In plus, le test a 0-A es possibilemente plus sensibile in determinar le

viabilitate de cellulas in casos in que le processos metabolic es lesionate per

venenos o per alterationes del pH.

REFERENCES

1. Cronkite, E. P., Bond, V. P., Fliedner,

T. �sI., and Kiliman, S.-A.: The Use

of tritiated thymidine in the study of

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Ciba Foundation Symposium on

Haemopoiesis. Cell Production and

Its Regulation. G. E. W. Wolsten-

holme and M. O’Connor, eds. Boston,

Little, Brown & Co., 1960, pp. 70-98.

2. Nlannick, J. A., Lochte, H. L., Jr.,

Thomas, E. D., and Ferrebee, J. W.:

In Vitro and in vivo assessment of

the viability of dog marrow after

storage. Blood 15:517, 1960.

3. Puck, T. T., and Marcus, P. I. : Action

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4. Proteous, 1. B.: Persistence of motility

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28:389, 1936.

7. -: Slide-chamber method to measure

sensitivity of cells to toxic agents.

A.M.A. Arch. Path. 66:569, 1958.




A�RIDINE ORANGE TEST AND BONE MARROW CELLS I 525

8. Pegg, I). E.: Experience in the preserva-

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9. Strugger, S. : Fluorcszenzmikroskopie’

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\Villiam E. Hathaway, M.D., As�tistant Professor of Pediatrics,

University of Kentucky Medical Center, Lexington, Ky.

Louis A. Newby, B.S., Research Assistant, Department of Pedi-

atrics, University of Kentucky Medical Center, Lexington, Ky.

John H. Githens, M.D., formerly Professor arid Chairman,

Department of Pediatrics, University of Kentucky Medical

Center, Lexington, Ky.; presently Professor of Pediafrics,University of Colorado Medical Center, Denver, Cob.




1964 23: 517-525

WILLIAM E. HATHAWAY, LOUIS A. NEWBY and JOHN H. GITHENS TransformationCorrelation with Trypan Blue and Eosin Dye Exclusion and Tissue Culture The Acridine Orange Viability Test Applied to Bone Marrow Cells I.

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