US 3645692 A
Process for the preparation, preservation and transportation of blood and serum samples for use in the determination of chemical and biochemical components therein, comprising applying onto an absorbent carrier having ion exchange properties samples of serum or blood, drying the carrier and sample and eluting the same just immediately prior to carrying out the indicated determination, the determination being carried out on the eluate.
Description (OCR text may contain errors)
United States Patent Stork et al.
[451 Feb. 29, 1972 PROCESS. FOR THE PREPARATION, PRESERVATION AND TRANSPORTATION OF BLOOD AND SERUM SAMPLES FOR USE IN CLINICAL ANALYSES lnventors: Harald Stork, Lampertheim/Hessen; Felix Ilelmut Schmidt, Mannheim-Neuostheim,
both of Germany Assignee: Boehringer Mannheim Gmbll, Mannheim,
Germany Filed: June 11, 1969 Appl. No.1 832,432
Foreign Application Priority Data July 15, 1968 Germany ..P I7 73 841.9 Mar. 19, 1969 Germany ..P 19 13 817.1
US. Cl ..23/230 B, 210/24, 23/258.5 Int. Cl ..G0ln 31/04, GOln 33/16 Field of Search ..23/230, 230 B, 258.5; 424/2;
 References Cited UNITED STATES PATENTS 3,494,744 2/1970 Zborowski ..23/ 230 3,380,888 4/l968 Numerof et al..... ....23/230 OTHER PUBLICATIONS Pileggi, V. J., J. Clin. Endocr. & Meta, Vol. 21, pp. I272- 1279(1961) Primary Examiner-Morris O. Wolk Assistant Examiner-Elliott A. Katz Attorney-Burgess, Dinklage & Sprung  ABSTRACT 10 Claims, No Drawings PROCESS FOR THE PREPARATION, PRESERVATION AND TRANSPORTATION OF BLOOD AND SERUM SAMPLES FOR USE IN CLINICAL ANALYSES The present invention relates to a process for the preparation, preservation and transportation of blood and serum samples for use thereof in the determination of chemical and biochemical components contained therein. More particularly the invention relates to a process for the preparation, preservation and transportation of blood and serum samples for use in the determination of chemical and biochemical components contained therein such as sugars, i.e., glucose and galactose, lipids, such as triglycerides and cholesterol, etc.
The qualitative detection and quantitative determination of components of blood or serum or of substances contained in blood or serum have, in recent years, assumed ever increasing importance in view of the reliance thereon by physicians in connection with medical diagnosis.
The analysis of, in particular, blood samples has heretofore involved considerable difficulties as the results of the analyses have not always been accurate not only due to changes taking place during the transportation of the sample to the laboratory but also in the storage thereof which is often unavoidable. Further since triglycerides and cholesterol can only be determined in serum and not in blood, in addition to the laborious and unpleasant procedure associated with vena puncture, the treatment of the blood sample to effect separation of the serum was also necessary. Furthermore, it was also necessary to introduce the blood samples into unbreakable and sterile containers for transportation thereof to the laboratory.
It has already been proposed to place the blood samples required for use in carryingout glucose determinations onto filter pape rs, followed by the drying thereof. However, as the blood glucose values of such samples decrease significantly even over the periods of 2 hours, it has been found to be necessary to stabilize the samples with formaldehyde vapor which involves a very laborious and time-consuming procedure (see Am. J. Clin. Path. 45, 297/1966). Furthermore, due to the action of the formaldehyde, the elution of the otherwise very readily soluble glucose is made so difficult that it is necessary to elute for at least minutes before the determination can be carried out.
In accordance with the invention, it has been found that blood samples suitable for use in the determination of chemical and biochemical components therein can be obtained without the disadvantages heretofore encountered by applying onto an absorbent carrier having ion exchange properties samples of blood or serum, drying the carrier and sample and eluting the same just immediately prior to carrying out the indicated determination.
Most surprisingly, in accordance with the invention, it has been found that a subsequent stabilization of the blood samples is rendered unnecessary.
Further surprisingly, the process according to the present invention permits the determination in blood not only of sugars, such as glucose and galactose, but also of lipids, such as triglycerides and cholesterol. Since all of the operations which are necessary for obtaining serum from whole blood are eliminated, the amount of blood necessary for the determinations can be reduced from 0.5 ml. to 0.05 ml. This is particularly important for large scale investigations as the small amount of blood needed can be drawn as capillary blood, vena-puncture no longer being necessary. A further advantage of the process according to the invention is that several blood samples can be taken from the same small laboratory animal in serial tests without causing any harm to the animal. This, of course, results in that the degree of accuracy of the results obtained is considerably increased.
The prepared and dried samples obtained by the process according to the present invention can simply be transported and stored in an envelope and, yet, can be used for the carrying out of quantitative determinations with a great degree of accuracy, even following the lapse of considerable time after the taking of the blood samples.
Not only in the case of samples drawn for use in the determination of glucose but also in the case of those intended for use in the determination of triglycerides, there has been determined a Pearson correlation coefficient of r=0.99 using in each case, 25 evaluations, in comparison with untreated samples.
As absorbent carriers having ion exchange properties, it is preferred to use ion exchange papers. As ion exchange paper, there can be used, depending upon the nature of the blood or serum components which are to be determined, not only cation exchangers but also anion exchangers. Glucose and galactose are preferably stabilized, i.e., preserved in the weakly acidic range, too low pH values having to be avoided in connection with carrying out glucose determination since otherwise glucose is split off from the cellulose forming part of the paper which would then inaccurately result in glucose values which are too high.
Since triglycerides are, in the course of enzymatic investigations, subjected to alkaline saponification, anion exchange papers have proved to be particularly well suited for the preparation and stabilization, i.e., preservation of samples intended for this purpose. Furthermore, samples intended for cholesterol determinations are also best stabilized on weakly basic ion exchange papers.
Of course, in place of filter paper, there can also be used a large variety of absorbent carriers, such as for instance compressed cellulose, hydrophilic synthetic resins, textile fabrics, wicks or layers of organic or inorganic powders or granulates having ion exchange properties fixed to suitable substrates by means of adhesives, such as by the addition of glue, it being immaterial whether the ion exchange properties depend upon the molecular structure of the absorbent carrier or are imparted to the carrier by a subsequent impregnation with suitable materials.
When blood is to be analyzed by the process according to the present invention, it is to be borne in mind that, due to the cellular components thereof, the results obtained are about 30 percent lower than those obtained when serum is used. However, it is not difficult to determine the necessary correction factor for each case by first carrying out comparative experiments.
The determination of galactose takes place, after elution of the absorbent carrier, utilizing enzyme galactose dehydrogenase, and using Warburg's optical test at 366 nm.
The determination of cholesterol is carried out, after elution of the absorbent carrier with a suitable organic solvent, preferably with a mixture of chloroform and methanol (2:1), using for the determination the analytical method described by Watson (D. Clin. Chim. Acta, 5, 637/ I960).
The following Examples are given for the purpose of illustrating the invention but are in nowise to be construed as limiting the scope thereof.
EXAMPLE 1 Serial determination of the blood glucose content in rabbits (with additional glucose loading).
Whatman cation exchanger paper WA 2 (AMBERLITE IR-C 50) was cut up into squares having 3 cm. edges.
Rabbits which had been subjected to glucose loading (lg. glucose/kg. body weight), were then subjected to removal of four 0.1 ml. blood samples over the course of 2 hours at time intervals of 30 and 60 minutes, respectively. Two of these samples were immediately applied onto the prepared ion exchange papers, while in the case of the two other samples, there were immediately carried out conventional determinations of the glucose content using the hexokinase/G-6-PDI-I and 60D] POD methods. The prepared squares of paper were, after drying, each eluted with 2 ml. 0.33N perchloric acid for 15 minutes, then decanted and the eluates centrifuged and thereafter also analyzed for glucose content using the hexdegree of correlation, as can be seen from the following Table TABLE 111 1:
TABLE I 5 Galactose Determination by Warburgs Method Galactoae in deprotein- Gnlactose in Hexokinase Method GOD/POD Method n blood sample mgpaper eluate mg. 1; Minutes glucose in glucose minutes in after deproteinin after deproteinin 56 0 56 l glucose ized blood paper glucose ized blood paper 10 adminisample eluate admini sample eluate stration (mg. (mg. 5) stration (mg. (mg. 12) 551 56.9
0 131 I32 0 I31 I35 30 328 308 30 336 333 15 Control investigations of blood samples deposited on ion I I 124 120 115 110 Control analyses were carried out on blood samples deposited onto ion exchange paper and showed, after 2 months storage, that the glucose content had not measurably changed.
EXAMPLE 2 Triglyceride determination in rabbit blood Whatman cation exchange paper AMBERLITE IRA-400 (chloride form) was cut up into squares having sides measuring 3 cm.
Following the application of 0.1 ml. blood samples and drying of the papers, the individual samples were each hydrolyzed for 1 hour at 70 C., with 0.1 ml. 0.9 percent sodium chloride solution and 0.5 ml. 0.5N ethanolic potassium hydroxide solution using small tubes which closed by means of ground stoppers. The determination of the triglycerides was thereafter carried out, using the enzymatic neutral fat determination method (see F. H. Schmidt, Z. Klin. Chem. u. Klin. Biochem., 6, 156/ 1968). The values obtained for the paper eluate were multiplied by the empirical factor 1.515 in order that they could be compared with the'values obtained with serum. The results of the determinations are set out in Table II which follows:
TABLE II Paper eluate Paper eluate X Serum (compar- Percent Galactose determination in rat blood 0.75 g./kg. galactose was first administered to a rat and, 15 minutes thereafter, five 0.05 ml. blood samples were taken which, immediately following their drawing were applied onto the ion exchange papers cut from the AMBERLITE IR-C 50 type paper which has been described in Example 1.
For control purposes, a further 0.2 ml. blood sample was taken and, after deproteinization thereof, a conventional galactose determination was carried out on this sample using Warburg's optical test.
The prepared pieces of paper were eluted by the method described in Example I, and thereafter the eluate was also analyzed using the Warburgs optical test.
The results which were obtained are set out in Table III which follows:
exchange paper showed, after storage for several weeks, that the galactose content had not changed significantly.
EXAMPLE 4 Cholesterol determination in rabbit blood Cation exchange papers of the AMBERLlTE IRA-400 type (chloride form) were cut up into squares havingsides measuring 3 cm.
Following the application of 0.1 ml. samples of rabbit blood thereto, the individual samples were dried and then eluted for at least 10 hours in small tubes which had been closed with ground stoppers, using 5 ml. of Folch-Sperry extraction mixture (chloroform-methanol 2:1) for the extraction. After the elution was completed, the paper was removed from the tube and the solvent evaporated. The residue which was obtained was mixed with 0.1 ml. water, shaken up and the resultant solution used for carrying out the cholesterol determination according to Watsons method in the same manner as the same is used for blood serum determinations.
In Table IV, which follows, the results of 8 individual determinations carried out on serum and paper eluate, respectively, are set out.
TABLE IV Cholesterol in rabbit serum mg. 26
Cholesterol in paper eluate mg. I:
Control analyses on blood samples which had been applied onto ion exchange papers and stored for several weeks, showed that the cholesterol content had not changed significantly. The mentioned ion-exchange papers named Whatman Amberlitev JRC 50 and JRA 400 are cellulose filter papers containing poly-methacrylic acid resins and polystyrene resins with quarternary ammonio groups. They are manufactured by Whatman and Balstone Ltd. Maidstone, England.
1. Process for the preparation, preservation and transportation of blood and serum samples for use in the analytical determination of chemical and biochemical components contained therein, which comprises applying onto an absorbent carrier having ion exchange properties a sample of serum or blood and drying the absorbent carrier and sample.
2. Process according to claim 1 wherein said absorbent carrier is a member selected from the group consisting of paper, compressed cellulose, textile fabrics and hydrophilic synthetic resins.
3. Process according to claim 1 wherein said absorbent carrier has weakly acidic cation exchange properties.
4. Process according to claim 1 wherein said absorbent carrier has anion exchange properties.
5. Process according to claim 1 wherein following said drying and just prior to carrying out the analytical determination, the dried absorbent carrier and sample is eluted with an eluting agent for said chemical or biochemical component and said determination is carried out with said eluent.
6. Process according to claim 5 wherein said chemical or biochemical component is glucose and said absorbent carrier has weakly acidic cation exchange properties.
7. Process according to claim 5 wherein said chemical or biochemical component is triglyceride and said absorbent carrier has anionic exchange properties.
' longed periods for the subsequent determination of chemical and biochemical components contained therein, which comprises applying on an absorbent carrier having ion exchange properties a sample of serum or blood, drying the absorbent carrier and sample and storing the dried absorbent carrier serum or blood sample until needed for said determination.