Separation of Mononuclear Leukocytes and Polymorphonuclear Leukocytes from Equine Blood S. P. Targowski*
ABSTRACT The present study describes a two step technique for the separation of mononuclear leukocytes or mononuclear and polymorphonuclear leukocytes from whole equine blood. First, the leukocyte rich plasma was obtained by sedimentation of erythrocytes in the undiluted blood. Subsequently, separation of the different populations of white blood cells was performed by centrifugation with different gradients overlaid with the leukocyte rich plasma. The optimal separation of the mononuclear cells was obtained by the centrifugation of the leukocyte rich plasma overlaying the gradient containing 24 parts of 9.5% ficoll and ten parts of 34% isopaque. The mononuclear leukocytes (95% lymphocytes and 5% monocytes) formed a monolayer band at the plasma-ficoll-isopaque interface and other blood cells migrated to the bottom of the tube. For the separation of mononuclear and granular leukocytes from the blood, the gradient containing 24 parts of 10% ficoll and ten parts of 34% isopaque was used. The separated monuclear leukocytes responded to stimulation with phytohemagglutin and viability of both mononuclear and polymorphonuclear leukocytes was not affected by ficollisopaque separation.
RESUMEk L'auteur decrit une technique a deux etapes visant a separer les leucocytes mononucleaires ou les leucocytes mono et polynucleaires du sang entier de cheval. II obtint d'abord le plas-
*School of Veterinary Science and Medicine, Purdue University, Lafayette, Indiana 47909. Present addTes: Department of Laboratory Animal Science, State University of New York, 408 Farber Hall, Buffalo, New York 14214. Submitted July 25, 1975.
Volume 40 -July, 1976
ma riche en leucocytes, en laissant sedimenter les hematies d'echantillons de sang non dilue. II separa ensuite les differentes varietes de leucocytes en centrifugeant avec divers gradients recouverts du plasma. Il obtint la separation optimale des leucocytes mononucleaires en centrifugeant le plasma ajoute 'a un gradient compose de 24 parties d'une solution de ficoll a 9.5% et de dix parties d'une solution d'isopaque 'a 34%. Les leucocytes mononucleaires (95% de lymphocytes et 5% de monocytes) s'accumulerent en une seule couche entre le plasma et le gradient et les autres cellules sanguines se deposerent au fond du tube. Pour separer du sang les leucocytes mononucleaires et les granulocytes, il utilisa un gradient compose' de 24 parties d'une solution de ficoll a 10% et de dix parties d'une solution d'isopaque ai 34%. Les leucocytes mononucleaires ainsi obtenus repondirent a la stimulation par la phytohetmagglutinine; la separation 'a l'aide d'un gradient 'a base de ficoll et d'isopaque n'affecta pas la viabilit6 des leucocytes mono et polynucle'aires.
INTRODUCTION Ficoll-isopaque is the gradient most commonly used to separate lymphocytes both for enumerating B and T cells and for functional tests from human and animal blood (1, 3, 6, 9). Two gradient components are advantageous because by adjusting the concentration or replacing either one of the components more desirable results can be obtained. Ficoll is prevalently used in separation of lymphocytes from blood by centrifugation because of its high solubility and relatively low viscosity. It can be replaced with a higher viscosity component such as methylcellu285
lose or dextran. Aqueous solutions of these polymers have a low osmolarity and density. In contrast, isopaque has a high density and osmolarity similar to blood plasma and it plays a major role in adjusting density of the gradient. Viscosity of the isopaque is low (6). The extensive studies of Boyum emphasize that numerous factors such as the cell content, volume and dilution of the sample, the pH, density and osmolarity of the gradient and the time, gravity and temperature of the centrifugation influence the results of separation (3). In addition, studies with animal blood have revealed that the sedimentation rate of blood cells plays an important role in the separation of white blood cells or their populations from erythrocytes. In ruminants the sedimentation rate is very slow, thus blood should be diluted to obtain a good separation of leukocytes (4). In contrast, equine erythrocytes tend to form rouleaux which greatly accelerates sedimentation of erythrocytes whereas white blood cells sediment less rapidly (5). Presently new diagnostic methods to measure cell mediated immunity (CMI) in vitro are available (2). By using these methods it is possible to demonstrate a correlation between some diseases of horses and their CMI deficiencies (7, 8). Some of these methods require a relatively pure population of white blood cells. Therefore, the purpose of this study was to determine a gradient for optimal separation of either mononuclear leukocytes or mononuclear and polymorphonuclear leukocytes from equine blood.
MATERIALS AND METHODS EXPERIMENTAL ANIMALS
Eight normal horses were randomly selected from the herd at the University Farm. Four of the horses were nonpregnant mares ranging in age from four to eight years. Three of the mares were Quarterhorses and one mare was a Standardbred. Three Quarterhorse geldings and one Pinto gelding ranging between the ages of three to eight years were used in this study. There were no clinical or hematological abnormalities observed in the horses
selected. All of the horses were fed the same diet. Blood samples were taken before morning feeding and watering.
GRADIENTS The following three gradients (A, B, C) were prepared by dissolving either A, 9.0% of ficoll' (21.60 g of ficoll), B, 9.5% ficoll (22.80 g) or C, 10% ficoll (24.00 g) in 240 ml of distilled water and adding 100 ml of 34% isopaque' (sodium metrizoate) to each of the ficoll solutions. The ficoll-isopaque gradients were sterilized by autoclave at 121°C for ten min. Subsequently, the gradients were adjusted to the pH of 8 with 1 N sodium hydroxide and the solutions were kept at 40 C. The physical properties of the gradients are given in Table I.
SEPARATION OF WHITE BLOOD CELLS (WBC) Fifty milliliters of blood from each of the eight horses was drawn into a heparinized and siliconized syringe (20 i.u/ml) which was then supported in an upright position at ambient room temperature for 20 min to allow the erythrocytes to sediment. The leukocyte rich plasma was expressed through a bent needle into a 35 ml siliconized tube. The leukocyte rich plasma was thoroughly mixed and equally distributed into three siliconized, conical, 10 ml tubes, containing 3 ml of either A, B or C of the gradient. In addition, a sample of the leukocyte rich plasma was taken in order to count the total white and red cells as well as doing a differential count. Subsequently all of the tubes with gradients overlaid with leukocyte rich plasma were spun for 25 min at 800 x g at 200 C. Some of the white blood cells formed a band at the plasma ficoll-isopaque interface in the gradient or sedimented on the bottom of the tube. This banding process was affected by the properties of the gradients and by the types of cells. Each band of cells was removed separately with a Pasteur pipette and placed in a siliconized, conical tube.' Only the cells from the surface of the bottom layer were collected. The collected cells iPharmnacia Chemical,
Uppsala, Sweden. SNyegaard, Oslo, Norway.
Can. J. comp. Mod.
were diluted with 5 ml of autologous plasma and spun at 200 x g for ten min at 4°C. The supernatant fluid was discarded and cells were resuspended in 1 ml of plasma. TOTAL LEUKOCYTE AND DIFFERENTIAL COUNTS
Leukocyte counts were performed using a Coulter counter3. Differential counts were performed on peripheral blood smears, leukocyte rich plasma smears and on the separated different population of blood leukocyte smears stained with Wright's stain. One slide per sample was made and 300 cells were counted on each slide by the same observer throughout the study.
hours before harvesting. All of the cultures were incubated for 72 hours in 5% C02 at 37°C, washed, precipitated with 10% trichloroacetic acid and the incorporated isotope was counted (10). Incorporation of the isotope was expressed as an index of stimulation (disintegrations per minute of stimulated lymphocytes/ disintegrations per minute of nonstimulated lymphocytes). VIABILITY TEST
The separated cells were centrifuged and resuspended in 0.5 ml of Hank's balanced salt solution. Two drops of cell suspension were mixed with one drop of 1 % trypan blue in saline (9% NaCl). The number of unstained cells (alive) within a count of 200 cells was expressed as a percentage.
PREPARATION OF CULTURE
The separated cells were suspended in 5 ml of TC medium 199 and spun at 200 x g for ten min at 4°C. The supernatant fluid was discarded and cells were washed twice more and then resuspended in 10 ml of medium. The number of cells were counted using the standard procedure. For the culture, TC-medium 199 was supplemented with 0!.02 M L-glutamine, 100 U of penicillin4 per ml, 100 mg of streptomycin sulfate5 per ml and 10% fetal bovine serum6. Two sets of quadruplicate cultures were prepared from each of the blood samples. The concentration of cells was adjusted so that each culture contained 2 x 106 lymphocytes in each 2 ml of media within each test tube. Mitogen, phytohemagglutinin7 (PHA-M) was added to the one set of quadruplicate cultures in a previously determined optimal dose (5 ,ul/ml), while another set served as a control. One microcurie of thymidine'methyl-H3 with specific activity 3 C/m mole was added to each culture tube 18
3Model Fn, Counter Electronics Inc., Hialeah, Florida. 4E. R. Squibb and Sons, Inc., Princeton, New Jersey.
5Pfizer Laboratories Division, New York, New York.
6Filow Laboratories Rockville, Maryland. 7Lot D528-56 control 585786, Difco Laboratories, Detroit,
Michigan. SSchwarz/Mann, Orangeburg, New York.
Volume 40 -July, 1976
RESU LTS SEPARATION OF THE LEUKOCYTES
The separation of the different white blood cells was performed by spinning down the gradient overlaid with leukocyte rich plasma, which contained approximately 80%ff of the original count of white blood cells and 2%Sc of the original count of erythrocytes in blood samples. Differential counts of white cells in the leukocyte rich plasma and blood were similar. White blood cells were suspended in the gradient or sedimented to the bottom of the tube which was dependent upon the properties of the gradient and density of the different population of the leukocytes. In contrast, erythrocytes always sedimented to the bottom of the tube during centrifugation (Fig. 1). All cells and platelets sedimented to the bottom of tube with the gradient A, Fig. 1 (right tube). However, the rate of sedimentation was different. Thus erythrocytes and eosinophils formed the first layer from the bottom. The next layer consisted of a mixture of granulocytes, lymphocytes and a few erythrocytes. The last layer was formed by lymphocytes and platelets which migrated with the slowest speed. Lymphocytes and platelets formed the band at the plasma-ficoll-isopaque inter-
Fig. 1. Separation of equine white blood cells or mononuclear cells by centrifugation with gradients of different concentration of ficoll. Right tube contains gradient A, middle tube gradient B and left tube contains gradient C.
face in the gradient B, Fig. 1 (middle tube). Approximately 90%/, of lymphocytes of leukocyte rich plasma were recovered from this band. The band contained 95%c lymphocytes and 5% monocytes (Table II). The first layer from the bottom consisted of erythrocytes and eosinophils and the next one consisted of neutrophils with a few erythrocytes. In the gradient C, Fig. 1 (left tube) all white cells and platelets were suspended in the gradient except eosinophils which
sedimented together with erythrocytes on the bottom of the tube. The leukocytes from five horses formed three bands suspended in the gradient. The leukocytes from other horses were suspended in the gradient without distinctly defined bands. The majority of lymphocytes were suspended in the upper part of the gradient, whereas the majority of granulocytes were suspended in the lower part of the gradient. In the tube with gradient C, at least 0.5 ml layer of the clear gradient was observed between the bottom layer and the band of white blood cells and the platelets. Around 90% of mononuclear cells and 90%7- polymorphonuclear neutrophils of leukocyte rich plasma were suspended in the gradient C (Table II). Table II shows the distribution of the different white cells in the top band (formed at the plasma-gradient interface or in the gradient) and bottom layer (surface of the bottom layer) in each of three gradients. Results represent average data from the seven horses. The data from the Pinto horse was markedly different and was not included in the average. Recovery of the lymphocytes from the top layer of gradients B and C was only 50 % of its original count in blood from the Pinto horse, whereas recovery of the lymphocytes from the blood of other horses was around 70%ff0 of their original counts. Also part of the granulocytes and lymphocytes migrated through the gradient C to the bottom of the tube and formed a bottom layer in addition to the erythrocyte eosinophil layer. PHYSIOLOGICAL FUNCTION OF THE SEPARATED CELLS
Viability tests indicated that more than 99 % of the mononuclear cells and about
TABLE I. Some Properties of Different Ficoll-isopaque Gradients and Equine Plasma
Gradient A .......................... B .......................... C ..........................1 0.0% Plasma .........................
Conc. of Ficoll
Densitya 1.078 1.079 1.080 1.026 + 0.003d
Viscosityb 3.19 3.36 3.56 1.76 + 0.12
Osmolalityc 305 304 306 276 + 5
gravity of the gradient was determined by weighing the bottle (22.4 ml) on the balance (Mettler, aSpecific Scientific Apparatus and Equipment, Syracuse, N.Y.)
bViscosity is expressed relative to viscosity of distilled water (Cannon-Fenske viscosimeter, Fisher Scientific cOsmolality was determined by the freezing point technique (Model 3L, Advance Instruments Inc., Newton
Highland, Ma.) dResults shown as the mean of 8 plasma values ± standard error
Can. J. comp. Med.
Table II. Effect of Increasing the Viscosity and Density of the Ficoll-isopaque Gradients on the Separation of Cells from Leukocyte Rich Plasma
91 + 2b
9 ± 1-
40 ±+ 8c
94 ± 3
5 i40.3 0
Top layer Bottom layer
Top layer Bottom layer
0.5 ± 0
89 ± 6c 0.5
1 52 ± 3
2.5 ± 0.3
N.D. 89 ± 4
10.5 ± 1 4.5
aNot determined bResults shown as the mean of counts ± standard error ePercentage of WBC recovered from leukocyte rich plasma. Total white cells in leukocyte rich plasma was only 80% of the original white cells in blood
98% of the granulocytes were alive. Ficollisopaque separated lymphocytes responded markedly to PHA stimulation and the average index of stimulation was 14.09 + 2.01.
DISCUSSION The results of the present study demonstrated that 9.5% of ficoll and 34% of isopaque is an optimal concentration in ficollisopaque gradient for separation of equine lymphocytes. Greater viscosity and slightly increased density of this gradient as compared with gradient A seems to be crucial for the optimal separation of the lymphocytes from blood. Boyum (3) has emphasized that a gradient density of 1.077 gm/ml or more ensures a separation of high yield mononuclear cells from human blood. The band that formed at the plasmaficoll-isopaque interface in B gradient contained around 95% lymphocytes and 50'% monocytes. The optimal separation of the human blood lymphocytes was observed with 9 % of ficoll and 33.9 % of isopaque concentration in the gradient (1). In contrast, the equine blood lymphocytes migrated through the 9 % of ficoll and 33.9 7 of isopaque gradient to the bottom of the tube. The yield of equine blood lymphocytes recovered from the band of the gradient B was around 70 % of their original count in blood but 95% of their count in leukocyte rich plasma. Similar results reported that the efficiency of the separation of
Volume 40-July, 1976
human blood lymphocytes by ficoll-isopaque gradient was greater than 70% which appeared to give genuine B to T
cell ratios (1). All leukocytes except eosinophils were suspended in the gradient C (10% ficoll) after centrifugation. The leukocytes from five of eight horses formed three bands. The first band from the top consisted of 96% lymphocytes and 4% monocytes, the second band consisted o 35% lymphocytes and 64% polymorphonuclear neutrophils and the third band consisted of 9 % lymphocytes and 90% polymorphonuclear neutrophils. This data suggests that the sedimentation rate of eosinophils was the most rapid, neutrophils and basophils were less rapid and lymphocytes and monocytes were the slowest to sediment of the equine blood leukocytes. Similar relationships in the sedimentation rate of different populations of human leukocytes were also demonstrated (3). The observations of the present study indicate a variation in separation of mononuclear and polymorphonuclear leukocytes from the blood of different horses. The leukocytes from three horses (Quarterhorse mare, and a Quarterhorse and Pinto gelding) formed only one very thick band instead of three bands as did the blood leukocytes from the other horses in gradient C. In addition, the yield of separation of mononuclear and polymorphonuclear cells was markedly lower from blood of the Pinto horse than others. Since selected horses for this study did not show any clinical and hematological abnormalities, these variations seem to be related to the 289
normal variations in the sedimentation rate of different blood leukocytes from different individuals rather than pathological changes. However, further studies on larger populations of horses are required to clarify these problems. The data of the present study has demonstrated that the viability of the cells was not affected by separation as judged by exclusion of the trypan blue dye by the cells. Also, the ability to replicate DNA of lymphocytes stimulated with PHA was not affected. Thus 9.5% of 10% of ficoll-isopaque gradient can be successfully used for separation of lymphocytes without disturbing the ability for replication of their DNA and their viability.
ACKNOWLEDGMENTS This paper is dedicated to Dr. Felix Milgrom of the State University of New York at Buffalo on the occasion of the thirtieth anniversary of his research activities.
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