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Articles |
1 Market Development Group, PLUSCARE Division, Arkray Inc., 57 Nishi-aketa-cho, Higashi-kujo, Kyoto, Japan 601-8045.
aAuthor for correspondence. Fax 81-75-662-8961; e-mail tanakayos{at}arkray.co.jp.
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Abstract |
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Methods: For separation of blood cells, we devised a grooved, asymmetric, polysulfonate membrane impregnated with sucrose. We evaluated hemoglobin (Hb) concentrations in the serum, assay values for 15 frequently measured serum components (including glucose), and the stability of analytes in the device.
Results: In sera from the new device, the Hb concentration was 
0.43 mg/L. Recovered serum contained 65.0% ± 4.2% (mean ± SD; n = 41) of each of the serum components obtained by centrifugation. Serum components were stable in the device for 10 days at room temperature (25 °C).
Conclusions: The newly developed device allows recovery of 60% of serum components from microvolumes of blood by finger puncture with neither degradation of analytes at room temperature nor hemolysis.
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Introduction |
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Tests for liver function, kidney function, glucose (GLU), 1 and fat metabolism can be performed on serum separated from whole blood obtained by such methods as centrifugation (3). Because of requirements for prompt centrifugation, analysis, or chilling (4), this method cannot be used for blood samples that must be shipped to a distant medical facility for analysis.
Methods of sampling blood that use filter paper or other types of membranes have also been developed. Although several such methods have recently been described (e.g., for analysis of viral infections and tumor markers), the methods are inconvenient, and the serum may be contaminated with blood components (5)(6).
By applying pressure to a membrane, serum can be recovered efficiently from microvolume samples of blood (7)(8)(9). These methods, however, require a special device for applying external force on a membrane to separate blood cells forcibly and, therefore, cannot be implemented readily in a general household.
We have thus aimed at developing a blood-sampling device that enables blood cell separation easily at home, without the use of a centrifuge, and keeps the serum preserved without degradation of the activities of the most unstable enzymes (4) for 
7 days at ambient temperatures. Our method is based on spontaneous separation by an asymmetric membrane, and a modification was made to recover a maximum of serum.
An asymmetric membrane is a membrane that varies in membrane pore size and causes blood cells, bacteria, and other cell components to migrate through the membrane to achieve separation and filtration (10)(11). However, in the conventional methods that use such a membrane, there is excessive hemolysis with contamination of serum by hemoglobin (Hb), which affects the results of biochemical assays.
We have developed a blood cell separation and serum recovery method in which a groove is formed in an asymmetric membrane to partition the membrane. Blood cell separation and serum recovery are performed in this single, grooved membrane. To achieve our objective of stable preservation of the separated serum, we considered several methods of serum preservation, such as the use of sucrose or other disaccharides (12), the use of glycerol (13), and salting out methods using guanidine, urea, ammonium sulfate, or other compounds (14). Because disaccharide solutions are known to increase the thermal stability of enzymes and enhance stability on drying (15), sucrose was selected as the means of serum preservation.
Here we report the hemolysis-limiting effects, serum recovery, and serum stability of the new method and reagent set developed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-GTP J-HA Test Wako for -glutamyl transpeptidase (GGT), the GLUII-HA Test Wako for GLU, the GOT-HRII (GOT-7070) for aspartate aminotransferase (AST), the GPT-HRII (GPT-7070) for alanine aminotransferase (ALT), the TG E-HA Test Wako for triglycerides (TGs), the TPII-HA Test Wako for total protein (TP), and the LDHII-HA Test Wako for lactate dehydrogenase (LD). The following assay reagent sets were obtained from Daiichi Pure Chemicals: Clinimate ALB for albumin (ALB), Pure Auto CRE-N for creatinine (CRE), and Chorestest HDL for HDL-cholesterol. The T-CHO TC-KL reagent set for total cholesterol (TC) was obtained from International Reagents Corporation.
preparation of grooved asymmetric membranes
A schematic illustration of the principles of our newly developed method for blood cell separation (hereafter called "new membrane method"), along with that of the conventional membrane method, is presented in Fig. 1
. In the new membrane method, 16 x 35 x 0.3-mm membrane sheets were prepared from BTS-SP. Each sheet was pressed from the large-pore side of the membrane to form a 0.5-mm-wide groove extending across the membrane (Fig. 1
, New Membrane Method), dividing it into the blood cell-separating part (hereafter called part A) and the serum recovery part (hereafter called part B). The groove was formed at a position of each membrane at which the ratio (A/B) of the area of part A to the area of part B would be 0.5 and the thickness of the membrane at the groove part would be 100 µm. The large membrane pores that allow the passage of blood cells collapse at the groove edge so that only the serum will move across the groove (Fig. 2
).
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Blood is dropped onto part A of the grooved membrane. The whole blood then moves in the transverse direction as well as into the asymmetric membrane. When the blood reaches the groove, the blood cells are blocked because of their large size, and only the serum components move across the groove into part B.
After adequate blood cell separation and drying, part B of the grooved membrane is removed from the membrane and immersed in PBS to elute the serum components from the membrane.
blood cell separation and serum recovery using a grooved asymmetric membrane
Fresh blood samples were collected by finger puncture from 41 healthy individuals (age range, 24–64 years) after we obtained their informed consent. The mean hematocrit (Ht) of the blood samples was 42.6% ± 3.6%. At 25 °C and 45% relative humidity, 100 µL of blood was dropped onto the center of part A of each grooved membrane. The blood was then left for 3 h to allow blood cell separation and drying of each membrane.
Part B of each grooved membrane was then removed by cutting and immersed in 300 µL of PBS in a 1.5-mL microcentrifuge tube. After the lid was closed, the microcentrifuge tube was left to stand for 20 min. The immersed membrane (part B) was then compressed against the bottom of the microcentrifuge tube with a glass rod [4 mm (diameter) x 75 mm]. The supernatant was then dispensed into a sample cup for the Hitachi 7070 General Purpose Automatic Analyzer for the biochemical assay, and the Hb concentration of the supernatant was measured using the assay reagents for the Automatic Analyzer.
For evaluation of serum recovery, sera were prepared in advance by the centrifugation method from blood samples obtained from the same individuals. Blood (200 µL) collected by finger puncture from the same individuals was centrifuged at 3000g to recover the serum. The biochemical assays were then performed on the sera thus obtained.
For each assay item, we calculated recovery as the percentage of the assay value (AVmemb.corr) determined from the serum recovered from the membrane and corrected for dilution with respect to the assay value (AVcent) determined from the serum obtained by centrifugation, as follows:
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Here, the dilution factor was calculated as follows:
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The AVmemb.corr/AVcent values were determined for each of the 15 biochemical assay items, and the mean AVmemb.corr/AVcent value was determined as the total mean of the AVmemb.corr/AVcent values of the respective assay items.
blood cell separation and serum recovery by the conventional membrane method using asymmetric membranes
Membranes with two overlapped layers, the blood cell separation layer and a serum recovery layer, were used (Fig. 1
, Conventional Membrane Method). Grooveless polysulfonate asymmetric membranes of the same type (BTS-SP) as used above were used for the conventional membrane method, and the total membrane area was made the same for both the new and conventional membrane methods.
Blood (100 µL) from each of the participants was dropped onto the center of the blood cell separation layer of each conventional asymmetric membrane, and after the blood was allowed to undergo adequate blood cell separation and drying in each membrane for 3 h, the lower half of each conventional membrane was removed and immersed in 300 µL of PBS in a 1.5-mL microcentrifuge tube. After the lid of the microcentrifuge tube was closed and the tube was allowed to stand for 20 min, the immersed membrane (lower half) was compressed against the bottom of the microcentrifuge tube with a glass rod [4 mm (diameter) x 75 mm]. The biochemical variables mentioned above and the Hb concentration of the supernatant were measured on the Hitachi 7070 General Purpose Automatic Analyzer.
stable preservation of serum components
A 160 x 210 x 0.3 mm sheet of the polysulfonate asymmetric membrane was prepared. Three liters of a 300 g/L aqueous solution of sucrose were prepared and placed in the bath of an ultrasonic cleaner (SV-150; Iuchi). The asymmetric membrane sheet was immersed in the sucrose solution and exposed to ultrasonic waves of 50 kHz for 15 min to allow the sucrose solution to permeate into the pores of the entire asymmetric membrane. The asymmetric membrane was then taken from the tank and left to dry at 25 °C and 45% relative humidity. After it dried, the membrane sheet was cut into smaller 16 x 35 x 0.3-mm sheets, and each of these membranes was grooved in the manner described above so that A/B would be 0.5. As negative controls, asymmetric membrane sheets of the same dimensions but not treated with sucrose were prepared and pressed as described above.
Blood samples were obtained by finger puncture from the 41 volunteers. At 25 °C and 45% relative humidity, 100 µL of blood was dropped at the center of part A of the sucrose-treated and untreated membranes. After being left for 3 h to separate the blood cells and dry, one set of treated and untreated membranes was stored at ambient temperature (25 °C). Other sets of membranes were stored in a constant-temperature bath (MIR-552; Sanyo Electric) at 10 and at 40 °C.
Initial AVmemb.corr/AVcent values were determined from a set of treated and untreated membranes immediately after blood separation and drying. In all cases, the serum was eluted from the membrane into 300 µL of PBS as described above. After the samples were stored at room temperature for 3 or 10 days, the serum components were measured by the methods described above, and the percentages of the initial values for the corresponding type of membrane (treated or untreated) were determined. In addition, ALT and CK concentrations were measured in the samples after storage at 10 and 40 °C for 1, 3, 5, and 10 days, and the percentages with respect to the initial values were determined.
trial serum recovery and preservation stability for samples from patients
Using the new membrane method described above, we obtained serum samples after consent from a total of 39 patients in a medical facility. These 39 patients consisted of 13 patients with diabetes, 9 with hyperlipemia, 10 with liver disorders, and 7 with kidney disorders. The serum Hb concentrations, assay values, and the stability at room temperature were evaluated in the same manner as for the healthy individuals.
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Results |
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Although the mean recovery of serum components by the new membrane method, 65.0% ± 4.2% (n = 41), was somewhat lower than that by the conventional method, 67.3% ± 5.7% (n = 41), the value for the conventional membrane method did not include AST and LD, which could not be measured because of hemolysis (Table 1
).
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These results demonstrate that the groove in the asymmetric membrane enables good blood cell separation, that serum can be recovered in part B of the grooved membrane with minimal hemolysis, and that recovery of serum components is comparable or superior to the conventional membrane method.
high stability of serum components during storage
The stabilities (percentages of initial recovery values) of serum components after 3 and 10 days of storage at room temperature for the sucrose-treated and untreated membranes are shown in Fig. 3
. Tables 2
and 3
show the stabilities of ALT and CK after 1, 3, 5, and 10 days of storage at 10 and 40 °C (percentages of initial assay values).
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TP, ALB, UN, and CRE were stable in both the presence and absence of sucrose. However, the presence of sucrose reduced degradation of HDL, ALT, ALP, LD, and CK by 25% or more compared with the conventional method with treated membranes. All components were maintained at 
85% of the initial values even after 10 days of storage.
At 40 °C, complete loss of activity of ALT was seen after 3 days with untreated membranes, whereas mean values of 71.2% and 56.3% of the initial activity were maintained after 3 and 10 days, respectively, with sucrose-treated membranes. Mean CK activity decreased 10.2% of the initial value after 3 days with untreated membranes, whereas 77.1% and 63.5% of the initial activity were maintained after 3 days and 10 days, respectively, with sucrose-treated membranes. With sucrose-treated membranes stored at 10 °C, ALT and CK activities were >90% of the initial activities even after 10 days.
results for patients with selected disorders
As shown in Table 4
, the recovery and stability at room temperature of each assay item after 10 days were equivalent to those of samples obtained from healthy individuals, indicating the method’s usefulness for the primary screening of disorders (Fig. 4
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7 µm moved out via the bottom surface of the asymmetric membrane during blood cell separation in spite of the small pore size of the bottom surface, 2 µm. The structure of the upper part of our new membrane is collapsed at the groove, and the pores that allow the passage of blood cells are thereby eliminated at this part. Furthermore, with the new membrane method, most of the blood moves transversely along the membrane rather than in the direction of the thickness of the membrane. Blood cells are stopped at the edge of the groove, leaving only serum in part B without contaminating blood cells, thus solving the problem of hemolysis that is seen in conventional methods.
sucrose stabilizes serum components during storage
In serum stored in a liquid state at room temperature, TP, ALB, TC, and AMY are stable for 1 week and UN and CRE are stable for 3 days (4). However, our results show that our new membrane method and the use of sucrose as a stabilizer enables stable preservation for >1 week for UN and CRE as a result of the drying of the serum components. In addition, the sucrose molecules, which lack reduced terminal groups, reduce the consumption of GLU by the saccharification of ALB and other protein molecules (Maylard reaction), thus making stable preservation possible.
The lipids TG, TC, and HDL and the enzymes AMY, AST, ALT, GGT, ALP, LD, and CK were considered to have retained their conformation because of the hydration water of sucrose.
The results also showed that sucrose preserved ALT and CK, which degrade readily, indicating that the sucrose treatment restricts conformational changes of enzyme molecules by thermal denaturation.
scattering and validity of measured values
In our experiments, separately obtained Ht values were used to determine the dilution factor for correction of the assay values determined with a membrane. At present, we are examining a correction method that makes use of the metal ions in the serum to determine the dilution factor and have obtained satisfactory results with this method, which does not require separate measurement of the Ht values (manuscript in preparation).
With both the new and conventional membrane methods, measurement errors attributable to dilution, which occurs when the sample is absorbed into the membrane and the extract solution is measured, are unavoidable. This becomes especially important in the case reported here where samples that exhibit values at the lower limit of the reference interval are measured. In such cases, there is a high possibility that measurements are made near the measurement limits of the instruments and reagents, inevitably lowering the precision of the measured values. The precision of measurement can be improved by either (a) including reagents in part B or (b) increasing the sensitivities of the instruments and reagents. With the former method, because the serum reacts with the reagents immediately after blood cell separation, extremely high precision can be anticipated. However, if, for example, a sample is stored for 1 week after the reaction, components for which the rate of reaction is observed will not be measurable because the reaction will progress during the storage period. Furthermore, the fixing of reagents in the membrane, the stabilization of color pigments, and other factors are extremely difficult to achieve in terms of technology and cost, making this method high in development cost.
We are performing further examinations of the latter method because it is highly feasible in terms of technology. With regard to the measurement reagents, we are examining changes of the sample-to-reagent ratios and the use of coloring agents with higher molar absorptivity. We are also examining improvements in the precision of the sampling and optical systems for determining assay values.
future developments and possible uses of the new blood-sampling device
The new membrane method makes use of the development of blood in the transverse direction of a developing medium. Immunochromatography testing devices are well known as existing devices that make use of the method of developing the sample in the transverse direction (16)(17)(18). Because the serum that is separated by our new blood-sampling device is also developed under the same mechanism, further possibilities can be realized through the provision of biochemical and immunologic reaction systems in the serum-developing part of the membrane.
Application of this method to blood-sampling devices, which require less blood than the commercially available immunochromatography test devices for the CK isoenzyme MB fraction, myoglobin, and other blood components, is also a possibility. Applications are not limited to blood, and the new membrane method should be applicable to urine and other body fluids as well.
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Acknowledgments |
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Footnotes |
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-glutamyl transpeptidase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TG, triglyceride; TP, total protein; LD, lactate dehydrogenase; ALB, albumin; CRE, creatinine; TC, total cholesterol; and Ht, hematocrit. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  G. A. Maguire and C. N. Hales Use of Sodium Measurement as a Surrogate Volume Measurement in Unmetered-Blood Collection Devices Clin. Chem., March 1, 2002; 48(3): 592 - 593. [Full Text] [PDF]
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, untreated membrane, 3 days later; 
, untreated membrane, 10 days later; •, sucrose-treated membrane, 3 days later; 
, sucrose treated membrane, 10 days later. Error bars, SD.