THE PRESERVATION OF BLOOD FOR CHEMICAL AN- ALYSIS BY THE USE OF SODIUM FLUORIDE.

BY JOSEPH H. ROE, OLIVER J. IRISH, AND JAMES I. BOYD.

(From the hpartment of Chemistry, George Washington University

Medical School, Washington.)

(Received for publication, September 23, 1927.)

Several preservative substances have been recommended for blood that must be kept for a time before an analysis can be performed. Denis and Aldrich (1) proposed the use of 1 drop of formaldehyde to 10 cc. of blood as a preservative for blood sugar. Bock (2) found that five out of six sam- ples of formaldehyde, when used as a blood preservative, gave increased values for blood sugar, the increase being more than 40 per cent in several cases. Ambard (3) advocated the use of a mixture of 2 parts of potassium dihydrogen phosphate and 1 part of sodium fluoride as a blood preservative. Aibara (4) found that 2 mg. of sodium fluoride per cc. of blood had an in- hibitory influence upon glycolysis. Sander (5) in a comprehensive investi- gation of blood preservation found that metallic salts, sodium cyanide, benzoic acid, boric acid, zinc chloride, ether, lysol, phenol, benzene, toluene, acetone, formaldehyde, and thymol, gave unsatisfactory results in maintaining unchanged the values for non-protein nitrogen, urea, uric acid, and sugar, for a period of 48 hours; and that creatinine and creatine remained constant for several days, whether a preservative was used or not. Sander also obtained unreliable results with sodium fluoride in concentra- tions of 10 mg. per cc. of blood when used alone, but, when combined with thymol, in proportions of 10 mg. of sodium fluoride and 1 mg. of thymol per cc. of blood, he obtained constant values after 10 days standing for sugar, uric acid, creatinine, and creatine, and satisfactory values for urea and non-protein nitrogen, though the latter increased slightly. Denis and Beven (6) used sodium fluoride as a preservative in proportions of 6 mg. per cc. of blood and noted that the concentrations of blood sugar, non- protein nitrogen, urea, creatinine, and uric acid remained unchanged after the samples had been allowed to stand for 48 hours at 28.531%. Major (7) recommended the use of 1 drop of a saturated solution of potassium fluoride to 5 cc. of blood as a preservative. Schwenkter (8) reported satisfactory preservation when a mixture of sodium fluoride and thymol in the propor- tions recommended by Sander was used as a preservative, but this author obtained values for blood sugar ranging from 7.5 to 50 per cent too high when the Lewis-Benedict picric acid method of analysis was used.

686

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686 Blood Preservation

The general conclusion, from the experience of the authors noted above, is that sodium fluoride is an efficient preservative of blood for chemical analysis. Further experimental work seemed desirable, however, upon (1) the concentration of sodium fluoride necessary for satisfactory preservation; (2) the advisability of sterilization when fluorides are used as preservatives; and (3) possible interference with standard methods of analysis. ‘It is the purpose of this paper to report the results of an investigation of these phases of the subject.

All of our experiments were carried out upon human blood. In the preservation tests the bloods were allowed to stand at the

TABLE I.

Amount of Sodium Fluoride Necessary to Prevent Blood Sugar Decomposition in Sterile Tubes.

No change in blood sugar resulted in 27 days in the tube containing 10 mg. of NaF per cc. of blood.

Mg. sodium fluoride per cc. blood.

-

1 2 4 6 8

10

Mg. sugar per 100 cc. blood.

No. of days standing.

0 1

I I

_____

93 90

___-- -- 93 90 18 11 18 11 90 90 90 44 35 90 44 35 85 85 85 47 40 85 47 40 90 90 90 93 89 90 93 89

103 103 103 102 108 103 102 108 95 95 90 90 96 90 90 96

4 5

__- -I---

30 30 24 24 90 87 90 87

108 105 108 105 95 99 95 99

T

6 6 13 13 20 20

-~- -~-

89 89 77 33 77 33 108 108 56 50 56 50

93 93 99 98 99 98

temperature of the laboratory, which ranged from 22-29°C. The

27

98

following methods of analysis were used: for sugar, the Rothberg- Evans (9) modification of the Folin-Wu method; for non-protein nitrogen, the method of Wong (10) ; for urea, the methods of Myers (ll), and of Roe and Irish (12) ; for creatinine, the Folin-Wu (13) method; for uric acid, the Folin (14) method; and for cholesterol, the Bloor (15) procedure.

Our first experiment was to determine the amount of sodium fluoride necessary to prevent decomposition of blood sugar in sterile containers. The results of this experiment are shown in Table I. Samples of blood were placed in sterile tubes containing

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1, 2; 4, 6, 8, and 10 mg. of NaF per cc. of blood. Blood sugar values were determined upon these samples, at first daily, later at weekly intervals, for 27 days, except where marked decomposition occurred. The samples containing 1, 2, and 4 mg. per cc. of blood showed unchanged values for 24 hours, but marked glycolysis at the end of the 2nd day; those containing 6 and 8 mg. per cc. showed practically constant values for 6 days but decomposition in 13 days; and the 10 mg. per cc. sample remained unchanged for 27 days, the analysis being discontinued at this point. As there are

TABLE II.

Preservative Injkence of Sodium Fluoride in Bloods Kept 10, 20, and 30 Days in Sealed, Sterile Tubes.

Kg. per 100 cc. blood.

Sample No.. . 1 2

Containing 10 mg. potassilnn Containing 10 mg. NaF per cc. oldate per cc.

Substance determined.

----------------

Non-protein N.. 37 40 40 41 34 36 42 42 47 51 27 42 44 57 22 54 Uric acid. . . 4.14.02.42.43.53.23.53.33.43.13.63.32.92.52.92.6 Creatinine 1.31.41.11.21.61.61.71.61.81.81.51.81.71.71.61.6 Sugar...........116 24162 20208195150144268254116110217214 90 97 Cholesterol. . 200 195 222 218 222 211 210 198 175 182 263 250

possibilities of contamination with organisms when removing from a tube samples of blood for analysis, we are unable to say that all of the specimens of blood in this experiment remained completely sterile. The practical conclusion from this experiment is that 10 mg. of NaF per cc. of blood is a satisfactory and desirable con- centration to prevent blood sugar decomposition when the blood is collected in a sterile container and efforts are made to prevent the introduction of organisms.

We next made a study of the preservative influence of 10 mg. of NaF per cc. of blood upon other constituents than sugar for pe-

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688 Blood Preservation

riods of 10, 20, and 30 days under sterile conditions. An experi- ment was carried out in which a series of duplicate samples of blood was collected in sterile tubes simultaneously from opposite arms of human subjects. One of each of the duplicate samples was analyzed at once and the other was kept sealed until the day of analysis. In this manner bloods were kept sterile for 10, 20, and 30 days, no possibility of contamination existing since the tubes were not opened until the time of analysis. The results are shown in Table II. In the two tubes containing potassium oxalate, defi- nite glycolysis occurred, but the other constituents remained un- changed for 10 days. The values upon the two samples containing

TABLE III.

Blood Sugar Values Obtained with Varying Amounts of Sorlium Fluoride in Bloods Inoculated with Bacillus coli communior.

20 mg. of NaF per cc. of blood prevented decomposition for 18 days.

Mg. 8ugar per 100 cc. blood.

No. of days standing.

0 3 7

11 18

Mg. NaF per CC.

10 15 20 25 30

104 187 152 111 270 85 188 146 111 260

0 153 152 111 268 155 152 113 258 80 153 258

NaF, which stood for 10 days before analysis, show satisfactory preservation of non-protein nitrogen, uric acid, creatinine, sugar, and cholesterol, within the limits of experimental error. In the bloods containing NaF, which stood for 20 and 30 days before analysis, the values for sugar, uric acid, creatinine, and choles- terol, remained essentially the same; but the non-protein nitrogen showed a definite increase in one of the 20 day bloods, and in both of the bloods that stood 30 days before analysis. The results of this experiment indicate that satisfactory preser- vation of sugar, non-protein nitrogen, uric acid, creatinine and cholesterol, may be expected in bloods collected in sterile tubes conta.ining 10 mg. of NaF per cc. of blood for at least 10 days, and

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J. H. Roe, 0. J. Irish, and J. I. Boyd 689

that in bloods submitted to the same conditions preservation of sugar, uric acid, creatinine, and cholesterol, but not non-protein nitrogen, will occur for 30 days.

Having determined the amount of sodium fluoride necessary to give successful preservation of bloods kept in sterile containers, we next made a study of the influence of sodium fluoride under non-sterile conditions. An experiment was performed in which 10, 15, 20, 25, and 30 mg. of NaF per cc. of blood were used and each specimen was inoculated with a loopful of a pure culture of Bacillus coli communior. The results of this experiment are shown in Table III. Some decomposition occurred by the 3rd day in the

TABLE IV.

Influence of Sodium Fluoride upon Two Samples of Blood Containing 20 Mg. of NaF per Cc. When Inoculated with Bacillus coli communior.

No. of days standing.

0 142 129 3.73 3.67 36 37 4 143 129 3.42 3.40 36 47 7 145 127 3.27 3.26 47 51

10 141 125 3.57 3.63 44 54 17 141 126 3.57 3.49 62 63 24 137 123 3.63 3.55 66 76

Mg. per 100 cc. blood.

Sugar. Uric acid. Non-pmt.& N.

I ,

Tple Say.ple SaT.Ple Tple

Creatinine.

-___ 1.10 1.16 1.26 1.40 1.41 1.83

1.10 1.30 1.44 1.61 1.62 1.81

tube containing 10 mg. of NaF per cc. of blood and the sugar disappeared completely in t,his specimen by the 7th day. With 15 mg. of NaF per cc. the blood sugar value remained unchanged for 3 days, but decreased by the 7th day, and by the 18th day was less than half the initial value. The values remained practically the same in the samples containing 20, 25, and 30 mg. of NaF per cc. of blood. Similar results were obtained in an experiment in which samples of blood containing lo> 15, and 20 mg. of NaF per cc. were inoculated with Bacillus subtilis. In the presence of this organism the specimen containing 20 mg. of NaF per cc. showed unchanged blood sugar values for 28 days; and the samples

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690 Blood Preservation

containing 10 and 15 mg. per cc. showed undesirable decomposition in less than 7 days. These data demonstrate that 10 mg. of NaF per cc. of blood are inadequate to prevent decomposition of blood sugar in the presence of two common contaminating organisms, Bacik6s coli communior and Bacillus subtilis; and that at least 20 mg. of NaF per cc. of blood are necessary to maintain unchanged values for blood sugar in non-sterile blood.

In Table IV is shown the influence of sodium fluoride upon the non-protein nitrogen, uric acid, creatinine, and sugar, of bloods inoculated with Bacillus coli communior. As 20 mg. of NaF per cc. of blood had prevented decomposition of sugar in non-sterile bloods (Table III), this concentration was used to determine whether preservation of other constituents than sugar would result under non-sterile conditions. Two large pooled samples containing 20 mg. of NaF per cc. were inoculated with a pure culture of colon bacilli. Initial values for sugar, uric acid, non- protein nitrogen, and creatinine, were obtained, and the samples were then analyzed upon the 4th, 7th, lOth, 17th, and 24th days following collection. Table IV shows that sugar and uric acid were preserved satisfactorily for 24 days and that the non-protein nitrogen and the reducing substance determined as creatinine gradually increased. A similar increase in non-protein nitrogen was obtained in bloods inoculated with Bacillus subfilis. This increase in non-protein nitrogen was expected since a similar, though much less rapid, change had been noted in this constituent when the blood was kept under sterile conditions (Table II). Denis and Beven (6) also noted an increase in non-protein nitrogen in bloods treated with 6 mg. of NaF per cc. of blood after 96 hours standing. The increase in the value for creatinine is unusual and has not been noted by us in any other experiments than those in which the bloods were inoculated with colon bacilli. This ex- periment shows that 20 mg. of NaF per cc. of blood will prevent the decomposition of sugar and uric acid in bloods inoculated with Bacillus coli communior and that non-protein nitrogen and the substance determined as creatinine will gradually increase in bloods submitted to the same conditions.

The results we have obtained with sodium fluoride as a blood preservative are not in agreement with the work of Sander (5) in several respects. This author found that neither sodium fluor-

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J. H. Roe, 0. J. Irish, and J. I. Boyd 691

ide nor thymol, when used alone, gave satisfactory preservation, but when these two substances were combined and used in a con- centration of 10 mg. of NaF and 1 mg. of thymol per cc. of blood, successful preservation resulted even in non-sterile containers. In our experiments, 10 mg. of NaF per cc. of blood gave very satisfactory preservation for periods of 10 days, or more, in bloods collected in sterile containers; but in non-sterile bloods neither 10 mg. of NaF, nor 10 mg. of NaF and 1 mg. of thymol, per cc. of blood, resulted in the preservation of blood sugar. The failure of a mixture of NaF and thymol to give preservation of sugar in non-sterile bloods was demonstrated in an experiment in which three samples of blood containing 10 mg. of NaF and 1 mg. of thymol per cc. were inoculated with Bacillus coli communior. Blood sugar estimations were carried out upon these bloods at once and upon the 3rd, 7th, and 11th days thereafter. Definite decomposition was shown in the 3rd and 7th day analyses and upon the 11th day the sugar had practically disappeared. These data indicate that thymol contributes little additional preserva- tive power to sodium fluoride when combined with the latter. Since thymol interferes with one of the standard methods of analy- sis for sugar (Schwenkter), its use as an adjunct to sodium fluoride seems contraindicated.

Sander (5) also states that: “It is evidently not necessary to draw blood under sterile conditions, except with regard to the patient, in order to preserve blood samples with fluoride and thymol.” Our experiments indicate that sterilization is very important in obtaining successful preservation of blood. With 10 mg. of NaF per cc. of blood we obtained entirely satisfactory pre- servation of sugar, non-protein nitrogen, uric acid, creatinine, and cholesterol, for at least 10 days, in sterile bloods; but only par- tially successful preservation was secured in non-sterile bloods when even twice as much NaF was used. We would emphasize that sterile conditions, while possibly not necessary to secure a certain degree of preservation, are certainly very desirable in connection with the use of sodium fluoride as a blood preservative.

We have found no interference by sodium fluoride in several times the concentration necessary as a preservative in the methods of analysis used, except in the determination of urea by urease hydrolysis. Sodium fluoride definitely retards the hydrolysis of

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692 Blood Preservation

urea to ammonium carbonate by urease action. This is not an unexpected finding since fluorides inhibit glycolysis and probably manifest other antienzymic powers in their action as preservatives.

The inhibition of urease action by sodium fluoride is best demonstrated by adding the latter to a pure urea solution and determining the urea by a urease method. With such experiments we found that NaF interferes in the determination of urea by urease hydrolysis in a degree proportional to the concentration of

TABLE V.

Influence of Sodium Fluoride upon Urease Action.

A pure urea solution containing 30 mg. of urea nitrogen per 100 cc., buffered with 0.25 M KzHPOI and KH2POa, was used. Each tube was treated with the same amount of the same urease solution and incubated 1 hour at 40°C.

Mg. sodium fluoride per CC. hydrolysete.

0 0 0

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 7.5

10.0

Mg. urea nitrogen per 100 cc. solution.

29.6 29.6 30.3 29.8 27.8 22.4 15.5 10.5

8.2 6.1 5.6 3.8 3.0

Not determinable. “ “

Per cent loss.

0 0 0 0

7.3 25.4 48.4 65.0 72.7 79.6 81.3 87.3 90.0

fluoride. This is shown in Table V. In this experiment a pure urea solution containing 30 mg. of urea nitrogen per 100 cc., buffered with 0.25 M KH2P04 and K2HP04, was used. Aliquot parts of this solution were placed in each of a series of tubes and water and NaF were added to each tube in amounts to make concentrations of 0.5 to 10 mg. per cc. of solution. The urea in each of these solutions was then determined by urease hydrolysis, the conditions of the hydrolysis being made identical in each

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J. H. Roe, 0. J. Irish, and J. I. Boyd 693

determination. The recoveries of urea obtained were in an inverse proportion to the concentration of NaF, the losses ranging from 7.3 per cent, where 1 mg. of NaF per cc. of hydrolysate was present, to about 99 per cent, where the concentration of NaF was 10 mg. per cc. of hydrolysate.

In attempting to overcome this objection to the use of NaF as a preservative, we found two procedures to be beneficial: (1) dilution of the blood; (2) precipitation of the fluoride ions with magnesium.

Since the speed of urease hydrolysis in the presence of NaF is retarded in a degree proportional to the concentration of fluoride, it follows that dilution should favor completion of the hydrolysis, and thus bring about more complete recoveries of urea nitrogen in bloods preserved with this substance. This was demonstrated to be true in an experiment in which NaF was added to samples of the same blood in concentrations of 10,20,30,50,75, and 100 mg. per cc. of blood. These bloods were then diluted with 7 volumes of distilled water and their urea nitrogen was determined by urease hydrolysis. The samples containing lC, 20, and 30 mg. of NaF per cc. of blood gave the same value for urea as control samples containing oxalate only. Low recoveries were obtained with the samples containing concentrations of NaF higher than 30 mg. of NaF per cc. in this and other experiments. The inhibitory effect in this experiment was less marked than that noted in experiments with water solutions of urea. A concentration of 1 mg. of NaF per cc. of hydrolysate gave recoveries 7.3 per cent too low in the experiment with aqueous urea solution; but in the experiment with blood a concentration of 3.75 mg. of NaF per cc. of diluted blood was tolerated without interference. Less interference in the case of blood is due to the precipitation of fluoride ions by cal- cium and magnesium, and probably to chemical combination with other constituents of the blood.

Dilution with water is obviously limited in its possibilities, because above a certain limit increasing the volume of water makes the procedure impractical. However, for concentrations of NaF up to 30 mg. per cc. of blood dilution with 7 to 10 volumes of water is all the treatment that is necessary to secure complete hydroly- sis of urea. Above this concentration complete recoveries may be obtained by the addition of magnesium sulfate solution, the

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694 Blood Preservation

amount of magnesium added being inslight excess of that necessary to precipitate the fluoride ions. Of the fluoride precipitants ex- amined magnesium was found to be the most satisfactory. Calcium will precipitate fluoride ions, but cannot be used, because it precipi- tates the phosphate used as a buffer. Magnesium does not precipi- tate phosphate ions at the pH of the blood and, when added in mod- rate excess, does precipitate fluoride ions. Magnesium may thus beused to precipitate fluoride ions and remove the interfering action of the latter, but is not necessary unless concentrations greater than 30 mg. of NaF per cc. of blood are used. Below this con- centration of fluoride dilution with 7 to 10 volumes of water be- fore treatment with urease is adequate t,o secure complete re- coveries of blood urea by the urease methods of analysis.

SUMMARY.

1. It has been found that sodium fluoride in concentrations of 10 mg. per cc. of blood will prevent changes in the values for non- protein nitrogen, uric acid, creatinine, sugar, and cholesterol, in sterile blood for at least 10 days.

2. A concentrat,ion of at least 20 mg. of NaF per cc. of blood has been found necessary to prevent marked changes in non-sterile blood, but even increased amounts of fluoride do not give as satisfactory preservation as can be obtained with blood submitted to sterile conditions.

3. Sodium fluoride interferes with the determination of urea by methods involving urease hydrolysis. The interference is pro- portional to the concentration of fluoride in the hydrolysate. This difficulty can be overcome in bloods containing not over 30 mg. of NaF per cc. by diluting with 7 to 10 volumes of water previous to treatment with urease.

BIBLIOGRAPHY.

1. Denis, W., and Aldrich, M., J. Biol. Chem., 1920, xliv, 203. 2. Bock, J. C., J. Biol. Chem., 1924, lix, 73. 3. Ambard, L., Bull. Sot. chim. hiol., 1920, ii, 202. 4. Aibara, C., J. Biochem., 1922, i, 457. 5. Sander, F. V., J. Biol. Chem., 1923-24, lviii, 1. 6. Denis, W., and Beven, J. L., J. Lab. and Clin. Med., 1924, ix, 674. 7. Major, R. H., J. Am. Med. Assn., 1923, lxxxi, 1952. 8. Schwenkter, F. F., Arch. Path. and Lab. Med., 1927, iii, 410.

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9. Rothberg, V. E., and Evans, F. A., J. Biol. Chem., 1923-24, lviii, 435. 10. Wong, S. Y., J. Biol. Chem., 1923, Iv, 427. 11. Myers, V. C., Practical chemical analysis of blood, St. Louis, 2nd edi-

tion, 1924, 45. 12. Roe, J. H., and Irish, 0. J., J. Lab. and Clin. Med., 1926, xi, 1087. 13. Folin, O., and Wu, H., J. Biol. Chem., 1919, xxxviii, 81. 14. Folin, O., J. BioZ. Chews., 1922, liv, 153. 15. Bloor, W. R., J. BioZ. Chem., 1916, xxiv, 227.

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BoydJoseph H. Roe, Oliver J. Irish and James I.

SODIUM FLUORIDEOFCHEMICAL ANALYSIS BY THE USE

THE PRESERVATION OF BLOOD FOR

1927, 75:685-695.J. Biol. Chem. 

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