- Email: [email protected]
SEPARATION OF VIABLE LYMPHOCYTES FROM HUMAN BLOOD
468 TABLE II-EFFECT OF INCREASING POTASSIUM CONCENTRATIONS OF SERUM ON LEVELS OF MICROCAPILLARY COLUMNS OF PRECIPITATE
in micro capillary columns of precipitate. These data suggest that capillary levels of precipitate are dependent upon serum-potassium concentrations. Other workers (Jancic 1955, Teeri and Sesin 1958) have used buffered ments
T.P.B. to remove
potassium from
serum
filtrates, though
buffered solution of T.P.B. seems not to render serum. proteins insoluble. This view is supported by our failure to demonstrate more than traces of biuret-reactive material in potassium precipitates. Direct treatment of serum with buffered T.P.B. circumvents the need for a
in duplicate with 30 sera in which the potassium-ion content ranged from 2-4 to 8-0 mEq. per litre. Differences between duplicate measurements of microcapillary columns of precipitate averaged 0-13 mm. (range 0-0-5 mm.). The degree of precision attained seemed unrelated to serum-potassium ion concentrations. Aliquots of 0-5 and 1-0 ml. of a solution containing 10 mEq. per litre of potassium chloride were taken to dryness in testtubes. 2-0 ml. of pooled serum of known potassium-ion content was then placed in each tube. Serum-potassium-chloride mixtures were treated with buffered T.P.B. according to the proposed technique. Addition of potassium chloride to pooled serum produced an increase in amounts of precipitate recovered in
microcapillary tubes (table II). Slightly more than a twofold precipitate columns was obtained by doubling the potassium content of pooled serum. Potassium was estimated by means of flame photometry in 101 sera collected from hospital patients. Aliquots of the sera were also treated with buffered T.P.B., and potassium precipitates were measured in microcapillary tubes to the nearest 0-1 mm. (fig. 2). Comparison of values obtained by the different methods yielded a correlation coefficient of 0-977. Using these data, an equation was derived from a line of best fit. increase in
Serum-potassium concentration= 0-73 x (mm. +0.64 (mEq. per litre).
of precipitate)
The equation permits conversion of precipitate column measureinto serum-potassium concentrations.
ments
Discussion
Direct treatment of serum with buffered T.P.B. seems potassium ion efficiently. When an equal volume or more of buffered T.P.B. was added to normal serum, analysis of supernatants by flame photometry failed to detect potassium. Also, similar serum-precipitant ratios yielded potassium-free supernatants from hyperkalæmic specimens. Hence, a serum-buffered T.P.B. ratio of 1:11 is satisfactory for isolation of potassium from serum. Larger amounts of precipitate were recovered when potassium chloride was added to normal serum. Increases in potassium concentration were reflected by step increto remove
relatively time-consuming procedures required to prepare filtrates. The close correlation between heights of microcapillary columns of precipitate in mm. with potassium concentra. tions in mEq. per litre permits derivation of a conversion equation. There is evidence that normal serum-potassium levels range from approximately 4 to 6 mEq. per litre (Teloh 1959). When the conversion equation is used, these values correspond to a capillary-precipitate column range of 4-6 to 7-3 mm. On this reckoning, potassium precipitates measuring 4-0 mm. or less point to hypokalsemia, whereas hyperkalæmic serum would yield precipitate columns in excess of 8-0 mm. The conversion equation was also applied to duplicate measurements of microcapillary columns of precipitate in 30 sera. Differences between duplicate measurements in terms of potassium concentration averaged only 0,095 mEq. per litre. Thus the technique is precise enough for most clinical purposes.
Summary The reaction between buffered sodium tetraphenylborate (T.P.B.) and potassium makes possible rapid precipitation of the cation directly from serum. Amounts of potassium T.P.B. can be estimated in terms of microcapillary columns of precipitate, and can be converted into serum-potassium concentrations accurate enough for clinical purposes. The test is simple and requires little equipment, thereby facilitating detection and repeated assessment of serum-potassium levels in patients with severe electrolyte imbalance. REFERENCES
Amin,
A. M.
(1957) Chemist-Analyst, 46, 6. De La Rubia, P. J., Blasco, L. R. F. (1955) ibid. 44, 58. Gloss, G. H., Olson, B. (1954) ibid. 43, 80. Jancic, M. S. (1955) Bosne i Hercegovine, 3, 37. Teeri, A. E., Sesin, P. G. (1958) Amer. J. clin. Path. 29, 86. Teloh, H. A. (1960) Clinical Flame Photometry. Springfield.
SEPARATION OF VIABLE LYMPHOCYTES FROM HUMAN BLOOD A. S. COULSON B.A. Cantab. RESEARCH STUDENT
D. G. CHALMERS Cantab., M.B. Lond.
M.A.
UNIVERSITY HÆMATOLOGIST TO
ADDENBROOKE’S HOSPITAL, CAMBRIDGE
From the Department of Pathology, University of Cambrides THE following method may be useful to other workers wishing to separate viable lymphocytes from human blood. It is largely based on the work of Prof. R. Ceppelini
(1962).
Fig. 2—Correlation of microcapillary column serum-potassium concentrations.
measurements
and
Materials and Method Freshly drawn blood is defibrinated in the conventions! manner and is then mixed with a 3% w/v solution of gelatin n physiological saline (three parts of blood to one part of gelatin solution). The mixture is stood for half an hour a. 37°C in a well-siliconed vessel.
469
dilution of the supernatant
suspension gets
over
the
difficulty. A. S. C. is in
receipt of a grant from the Medical Research Council. REFERENCES
Brandt, L., Börjeson, J., Nordén, A., Olsson, I. (1962) Acta med. scand. 172, 459. Brent, L., Medawar, P. R. (1963) Brit. med. J. ii, 269. Cassen, B., Hitt, J., Hays, E. F. (1958) J. Lab. clin. Med. 52, 778. Ceppelini, R. (1962) Personal communication. Jago, M. (1956) Brit. J. Hœmat. 2, 439.
Preliminary
Communications
EFFECT OF DUODENAL CONTENTS ON THE GASTRIC MUCOSA UNDER EXPERIMENTAL CONDITIONS Leucocyte distribution before and after
treatment with
gelatin.
The gelatin solution should not be boiled during its preparation, and its temperature should not exceed 37°C when mixed with the blood. It can be sterilised by Seitz filtration. During the half-hour incubation there should be no air bubbles in the gelatin-blood mixture or at its surface, for bubbles reduce the purity of the final lymphocyte preparation. The gelatin solution must be freshly prepared for each separation: a solution more than a few hours old, or one that has been allowed to cool after preparation, will reduce the purity of the lymphocyte preparation.
annotation1 drew attention to the need for an investigation into the effects of duodenal contents on the gastric mucosa, and it was for this reason that the following investigation was undertaken. A
RECENT
MATERIAL AND METHODS
The sample used in the work described is batch no. 213 from the British Glue and Gelatine Research Association. It has a molecular weight of 190,000 and a log. viscosity no. of 586; it is derived from lime-processed hide. There is some variation between batches.
allow reflux of duodenal contents into the Operations stomach were carried out on 12 dogs. Gastric biopsy specimens were taken at the time of the operation, so that each dog acted The dogs were killed at comparable as its own control. intervals, and strips of mucosa were excised along the whole length of the stomach, and were rolled up after the method described by Stein2and examined histologically. This included measurements of the depth of the foveols and glands by means of an ocular micrometer, as well as by counts of the mitotic figures. Each measurement was an average of 10 random
Result At the end of thirty minutes the gelatin-blood mixture is found to have settled into two clearly defined layers. The lower darker layer (usually 45% of the total volume) contains nearly all the red cells, monocytes, and neutrophil polymorphonuclear leucocytes. The clear supernatant usually contains more than 2000 white cells per c.mm., of which 90-99% are lymphocytes. Eosinophil polymorphs form a significant proportion of the contaminating cells. The graph shows the leucocyte partition pattern obtained when 20 ml. of a blood-gelatin mixture were sedimented in a measuring cylinder. Successive 2 ml. aliquots were removed and examined. Viability of the lymphocytes, as measured by trypan-blue supravital staining, is 100%. Further evidence is provided by tissue culture with phytohsemagglutinin and diphtheria and tetanus toxoids, where cell transformation and mitotic figures are regularly observed. Finally, the lymphocytes have been seen moving in culture.
Fig. 1-Three cells in anaphase (oil immersion).
to
,
Discussion Previous methods of separating lymphocytes from human blood have relied on differential centrifugation, on filtration with glass wool or glass beads, or on the use of iron particles. Jago reviewed these methods up to 1956, and since then Cassen et al. (1958) have described a method in which the blood is shaken for thirty minutes with iron spherules and gum-arabic, and Brandt et al. (1962) have proposed using glass wool and gas under pressure. The drawback of these methods is that they involve much physical manipulation of the cells. Differential centrifugation imposes stress on the lymphocytes; filtration methods often include complex sterilisation procedures; and iron may have a cytotoxic action. In comparison, gelatin sedimentation is rapid and simple; sterilisation is easy and there is little risk of cell trauma. Brent and Medawar 1963) pointed out that gelatin solution is not fluid at room temperature; but for tissue cultures maintained at 37°C this objection does not arise, while at room temperature
counts, and each figure of mitotic activity was the total number of mitoses in 50 random high-power fields (x800). Fig. 1 illustrates three dividing cells, all in anaphase. RESULTS
The Normal Stomach
Harvey3 has described in detail the microscopic structure of the dog’s stomach, and only some relevant facts will therefore be discussed. Fig. 2 shows the histological appearance of the normal pyloric antrum. The foveolae extend about half the distance through the glandular layer towards the lamina muscularis mucosa, and table I indicates the average normal depth of foveolæ and glands. The mitotic count was between 0 and 4, with the maximum activity in the region of the neck cells. Occasionally the foveolee may be longer than the glands, but usually they are shorter by 005-029 mm. There 1. 2. 3.
Lancet, 1962, ii, 1039. Stein, H. B. S. Afr. J. med. Sci. 1937, 2, 117. Harvey, B. C. H. Amer. J. Anat. 1906, 6, 207.
in micro capillary columns of precipitate. These data suggest that capillary levels of precipitate are dependent upon serum-potassium concentrations. Other workers (Jancic 1955, Teeri and Sesin 1958) have used buffered ments
T.P.B. to remove
potassium from
serum
filtrates, though
buffered solution of T.P.B. seems not to render serum. proteins insoluble. This view is supported by our failure to demonstrate more than traces of biuret-reactive material in potassium precipitates. Direct treatment of serum with buffered T.P.B. circumvents the need for a
in duplicate with 30 sera in which the potassium-ion content ranged from 2-4 to 8-0 mEq. per litre. Differences between duplicate measurements of microcapillary columns of precipitate averaged 0-13 mm. (range 0-0-5 mm.). The degree of precision attained seemed unrelated to serum-potassium ion concentrations. Aliquots of 0-5 and 1-0 ml. of a solution containing 10 mEq. per litre of potassium chloride were taken to dryness in testtubes. 2-0 ml. of pooled serum of known potassium-ion content was then placed in each tube. Serum-potassium-chloride mixtures were treated with buffered T.P.B. according to the proposed technique. Addition of potassium chloride to pooled serum produced an increase in amounts of precipitate recovered in
microcapillary tubes (table II). Slightly more than a twofold precipitate columns was obtained by doubling the potassium content of pooled serum. Potassium was estimated by means of flame photometry in 101 sera collected from hospital patients. Aliquots of the sera were also treated with buffered T.P.B., and potassium precipitates were measured in microcapillary tubes to the nearest 0-1 mm. (fig. 2). Comparison of values obtained by the different methods yielded a correlation coefficient of 0-977. Using these data, an equation was derived from a line of best fit. increase in
Serum-potassium concentration= 0-73 x (mm. +0.64 (mEq. per litre).
of precipitate)
The equation permits conversion of precipitate column measureinto serum-potassium concentrations.
ments
Discussion
Direct treatment of serum with buffered T.P.B. seems potassium ion efficiently. When an equal volume or more of buffered T.P.B. was added to normal serum, analysis of supernatants by flame photometry failed to detect potassium. Also, similar serum-precipitant ratios yielded potassium-free supernatants from hyperkalæmic specimens. Hence, a serum-buffered T.P.B. ratio of 1:11 is satisfactory for isolation of potassium from serum. Larger amounts of precipitate were recovered when potassium chloride was added to normal serum. Increases in potassium concentration were reflected by step increto remove
relatively time-consuming procedures required to prepare filtrates. The close correlation between heights of microcapillary columns of precipitate in mm. with potassium concentra. tions in mEq. per litre permits derivation of a conversion equation. There is evidence that normal serum-potassium levels range from approximately 4 to 6 mEq. per litre (Teloh 1959). When the conversion equation is used, these values correspond to a capillary-precipitate column range of 4-6 to 7-3 mm. On this reckoning, potassium precipitates measuring 4-0 mm. or less point to hypokalsemia, whereas hyperkalæmic serum would yield precipitate columns in excess of 8-0 mm. The conversion equation was also applied to duplicate measurements of microcapillary columns of precipitate in 30 sera. Differences between duplicate measurements in terms of potassium concentration averaged only 0,095 mEq. per litre. Thus the technique is precise enough for most clinical purposes.
Summary The reaction between buffered sodium tetraphenylborate (T.P.B.) and potassium makes possible rapid precipitation of the cation directly from serum. Amounts of potassium T.P.B. can be estimated in terms of microcapillary columns of precipitate, and can be converted into serum-potassium concentrations accurate enough for clinical purposes. The test is simple and requires little equipment, thereby facilitating detection and repeated assessment of serum-potassium levels in patients with severe electrolyte imbalance. REFERENCES
Amin,
A. M.
(1957) Chemist-Analyst, 46, 6. De La Rubia, P. J., Blasco, L. R. F. (1955) ibid. 44, 58. Gloss, G. H., Olson, B. (1954) ibid. 43, 80. Jancic, M. S. (1955) Bosne i Hercegovine, 3, 37. Teeri, A. E., Sesin, P. G. (1958) Amer. J. clin. Path. 29, 86. Teloh, H. A. (1960) Clinical Flame Photometry. Springfield.
SEPARATION OF VIABLE LYMPHOCYTES FROM HUMAN BLOOD A. S. COULSON B.A. Cantab. RESEARCH STUDENT
D. G. CHALMERS Cantab., M.B. Lond.
M.A.
UNIVERSITY HÆMATOLOGIST TO
ADDENBROOKE’S HOSPITAL, CAMBRIDGE
From the Department of Pathology, University of Cambrides THE following method may be useful to other workers wishing to separate viable lymphocytes from human blood. It is largely based on the work of Prof. R. Ceppelini
(1962).
Fig. 2—Correlation of microcapillary column serum-potassium concentrations.
measurements
and
Materials and Method Freshly drawn blood is defibrinated in the conventions! manner and is then mixed with a 3% w/v solution of gelatin n physiological saline (three parts of blood to one part of gelatin solution). The mixture is stood for half an hour a. 37°C in a well-siliconed vessel.
469
dilution of the supernatant
suspension gets
over
the
difficulty. A. S. C. is in
receipt of a grant from the Medical Research Council. REFERENCES
Brandt, L., Börjeson, J., Nordén, A., Olsson, I. (1962) Acta med. scand. 172, 459. Brent, L., Medawar, P. R. (1963) Brit. med. J. ii, 269. Cassen, B., Hitt, J., Hays, E. F. (1958) J. Lab. clin. Med. 52, 778. Ceppelini, R. (1962) Personal communication. Jago, M. (1956) Brit. J. Hœmat. 2, 439.
Preliminary
Communications
EFFECT OF DUODENAL CONTENTS ON THE GASTRIC MUCOSA UNDER EXPERIMENTAL CONDITIONS Leucocyte distribution before and after
treatment with
gelatin.
The gelatin solution should not be boiled during its preparation, and its temperature should not exceed 37°C when mixed with the blood. It can be sterilised by Seitz filtration. During the half-hour incubation there should be no air bubbles in the gelatin-blood mixture or at its surface, for bubbles reduce the purity of the final lymphocyte preparation. The gelatin solution must be freshly prepared for each separation: a solution more than a few hours old, or one that has been allowed to cool after preparation, will reduce the purity of the lymphocyte preparation.
annotation1 drew attention to the need for an investigation into the effects of duodenal contents on the gastric mucosa, and it was for this reason that the following investigation was undertaken. A
RECENT
MATERIAL AND METHODS
The sample used in the work described is batch no. 213 from the British Glue and Gelatine Research Association. It has a molecular weight of 190,000 and a log. viscosity no. of 586; it is derived from lime-processed hide. There is some variation between batches.
allow reflux of duodenal contents into the Operations stomach were carried out on 12 dogs. Gastric biopsy specimens were taken at the time of the operation, so that each dog acted The dogs were killed at comparable as its own control. intervals, and strips of mucosa were excised along the whole length of the stomach, and were rolled up after the method described by Stein2and examined histologically. This included measurements of the depth of the foveols and glands by means of an ocular micrometer, as well as by counts of the mitotic figures. Each measurement was an average of 10 random
Result At the end of thirty minutes the gelatin-blood mixture is found to have settled into two clearly defined layers. The lower darker layer (usually 45% of the total volume) contains nearly all the red cells, monocytes, and neutrophil polymorphonuclear leucocytes. The clear supernatant usually contains more than 2000 white cells per c.mm., of which 90-99% are lymphocytes. Eosinophil polymorphs form a significant proportion of the contaminating cells. The graph shows the leucocyte partition pattern obtained when 20 ml. of a blood-gelatin mixture were sedimented in a measuring cylinder. Successive 2 ml. aliquots were removed and examined. Viability of the lymphocytes, as measured by trypan-blue supravital staining, is 100%. Further evidence is provided by tissue culture with phytohsemagglutinin and diphtheria and tetanus toxoids, where cell transformation and mitotic figures are regularly observed. Finally, the lymphocytes have been seen moving in culture.
Fig. 1-Three cells in anaphase (oil immersion).
to
,
Discussion Previous methods of separating lymphocytes from human blood have relied on differential centrifugation, on filtration with glass wool or glass beads, or on the use of iron particles. Jago reviewed these methods up to 1956, and since then Cassen et al. (1958) have described a method in which the blood is shaken for thirty minutes with iron spherules and gum-arabic, and Brandt et al. (1962) have proposed using glass wool and gas under pressure. The drawback of these methods is that they involve much physical manipulation of the cells. Differential centrifugation imposes stress on the lymphocytes; filtration methods often include complex sterilisation procedures; and iron may have a cytotoxic action. In comparison, gelatin sedimentation is rapid and simple; sterilisation is easy and there is little risk of cell trauma. Brent and Medawar 1963) pointed out that gelatin solution is not fluid at room temperature; but for tissue cultures maintained at 37°C this objection does not arise, while at room temperature
counts, and each figure of mitotic activity was the total number of mitoses in 50 random high-power fields (x800). Fig. 1 illustrates three dividing cells, all in anaphase. RESULTS
The Normal Stomach
Harvey3 has described in detail the microscopic structure of the dog’s stomach, and only some relevant facts will therefore be discussed. Fig. 2 shows the histological appearance of the normal pyloric antrum. The foveolae extend about half the distance through the glandular layer towards the lamina muscularis mucosa, and table I indicates the average normal depth of foveolæ and glands. The mitotic count was between 0 and 4, with the maximum activity in the region of the neck cells. Occasionally the foveolee may be longer than the glands, but usually they are shorter by 005-029 mm. There 1. 2. 3.
Lancet, 1962, ii, 1039. Stein, H. B. S. Afr. J. med. Sci. 1937, 2, 117. Harvey, B. C. H. Amer. J. Anat. 1906, 6, 207.