JPWO2020032270A1 - Dry blood sample storage substrate - Google Patents

Abstract

Provided is a dry blood sample storage base material which can appropriately store a blood sample without being affected by the hematoclick value, and has a simple structure and does not require skill or labor. It has a base material plate 100 made of cellulose filter paper, an adhesive tape 200, and a blood holding disk 300 made of cellulose filter paper that holds the dropped blood sample in a dry state. The blood retention disc 300 is separated from the base plate 100 and attached to the base plate 100 by the adhesive tape 200. The base material plate 100 is provided with a plurality of openings 500, and the adhesive tape 200 has a gap (air gap) between the adhesive tape and the opening end at the opening end on one side of the opening 500 of the base material plate 100. ) 600 is attached so as to have.

Description

The present invention relates to a dry blood sample storage substrate for storing a blood sample in a dry state.

The dry blood sample storage base material is a paper card-shaped base material, and blood collected from the sample is dropped, left at room temperature for 2 to 3 hours to dry, and then the blood spot is hollowed out with a punch, and the hollowed out disk is It is taken into a tube or the like and used for blood tests (Non-Patent Document 1).

Blood tests using a dry blood sample storage substrate have the advantages that the amount of blood collected is relatively low, and that the drug is extracted from whole blood, eliminating the need for centrifugation for plasma separation. be. Furthermore, storage at room temperature eliminates the need for dry ice and a freezer, and has the advantage of being easily transported from a remote facility.

The dry blood sample storage substrate includes a partial punch analysis method in which the central portion of the dropped blood spot is punched out (Non-Patent Document 2). However, in this method, the blood component of the punched portion may fluctuate depending on the hematocrit value of the blood.

In addition, there is a punch analysis method in which a fixed amount of blood sample is spotted and the entire dropped blood spot is punched out (Non-Patent Document 2). Further, there is a pre-cut analysis method in which a fixed amount of blood sample is spotted on a pre-cut disc (Non-Patent Document 3). However, in these methods, it is necessary to drop a fixed amount of blood sample by a micropipette or the like, which requires skill and labor.

In addition, there is a quantitative absorption microsample method in which blood is absorbed into a small piece of filter paper and then dried and collected (Non-Patent Document 4). This method is not affected by the hematoclick value, and a certain amount of blood sample can be obtained simply by immersing a small piece of filter paper in the blood. However, this method is complicated in terms of equipment.

A.J. Wilhelm, J.C.G. den Burger, E.L. Swart, Therapeutic drug monitoring by dried blood spot: progress to date and future directions, Clin. Pharmacokinet. 53 (2014) 961-973. P.M. De Kesel, S. Capiau, W.E. Lambert, C.P. Stove, Current strategies for coping with the hematocrit problem in dried blood spot analysis, Bioanalysis. 6 (2014) 1871-1874. N. Youhnovski, A. Bergeron, M. Furtado, F. Garofolo, Pre-cut dried blood spot (PCDBS): an alternative to dried blood spot (DBS) technique to overcome hematocrit impact, Rapid Commun Mass Spectrom. 25 (2011) 2951-2958. Spooner N, Denniff P, Michielsen L, De Vries R, Ji QC, Arnold ME, Woods K, Woolf EJ, Xu Y, Boutet V, Zane P, Kushon S, Rudge JB. A device for dried blood microsampling in quantitative bioanalysis: overcoming the issues associated blood hematocrit. Bioanalysis. 7 (2015) 653-659.

The present invention has been made in view of the above problems, and is a dry blood sample storage base material that can appropriately store a blood sample without being affected by the hematocrit value, and has a simple structure and does not require skill or labor. The purpose is to provide.

The dry blood sample storage base material according to the present invention is a dry blood sample storage base material for storing a blood sample in a dry state, and holds a base material plate made of cellulose filter paper and a dropped blood sample in a dry state. It has a blood holding disc made of cellulose filter paper and a connecting portion for connecting the base material plate and the blood holding disc, and the blood holding disc is separated from the base material plate and the base material is separated from the base material plate by the connecting portion. Attached to the plate, the base metal plate is provided with a plurality of openings, and the blood retention disk is provided in the openings on the adhesive surface of the adhesive tape at intervals from the inner peripheral surface of the openings. It is characterized by being placed in. The dry blood sample storage base material is sometimes referred to as an air gap type base material. The air gap type base material is described as a round air gap type base material, a square air gap type base material, or the like, depending on the shape of the blood retention disc.

According to the present invention, a blood sample can be appropriately stored without being affected by the hematoclick value, and no skill or labor is required to store the blood sample.

It is the schematic of the dry blood sample preservation base material which concerns on 1st Embodiment. It is the schematic of the dry blood sample preservation base material which concerns on 2nd Embodiment. It is the schematic of the dry blood sample preservation base material which concerns on 3rd Embodiment. It is the schematic of the dry blood sample preservation base material which concerns on 4th Embodiment. It is the schematic of the dry blood sample preservation base material which concerns on 5th Embodiment. It is a photographic diagram of the blood sample storage base material which concerns on Example 1, (A) the photographic diagram before the dripping of blood, (B) the photographic diagram after dripping blood, and (C) the photographic diagram of the detached disk. It is a figure which shows the influence of hematocrit on the blood retention of a round air gap type base material. It is a figure which shows the chromatogram of clozapine (CLZ), norcrozapine (DMC), clozapine N-oxide (CLZ-NO) and internal standard (IS). It is a figure which shows the calibration curve of clozapine (CLZ) and its metabolite, nor clozapine (DMC), clozapine N-oxide (CMO). It is a figure which shows the influence of hematocrit on the blood concentration of clozapine (CLZ) and its metabolites clozapine (DMC) and clozapine N-oxide (CNO) measured using a 4/6 mm round air gap type base material. It is a figure which shows the influence of hematocrit on the blood concentration of clozapine (CLZ) and its metabolites clozapine (DMC) and clozapine N-oxide (CNO) measured using a 6/8 mm round air gap type base material. It is a figure which shows the influence of hematocrit on the blood concentration of olanzapine. It is a figure which shows the influence of hematocrit on the blood concentration of risperidone. It is a figure which shows the influence of hematocrit on the blood concentration of aripiprazole. It is a photographic figure of the air gap-4 bridge type modified base material (Air gap-4 bridge), and shows 8/10 mm, 6/8 mm, and 4/6 mm from the left side. The effect of the hematocrit value on the blood retention volume of the round air gap 4-bridge type substrate is shown. The effect of hematocrit on the blood levels of clozapine and metabolites measured using a 4/6 mm round air gap 4-bridge substrate is shown. The effect of hematocrit on the blood levels of clozapine and metabolites measured using a 6/8 mm round air gap 4-bridge substrate is shown. The effect of hematocrit on the blood levels of clozapine and metabolites measured using an 8/10 mm round air gap 4-bridge substrate is shown. It is a photographic diagram of the blood sample storage base material which concerns on Example 4, (A) the photographic diagram before the dripping of blood, (B) the photographic diagram after dripping blood, and (C) the photographic diagram of the detached disk. It is a figure which shows the influence of hematocrit on the blood retention volume measured using the square air gap type base material. It is a photographic diagram of the blood sample storage base material according to Example 5, the left side is a photographic diagram before dripping blood, and the right side is a photographic diagram after dripping blood. It is a figure which shows the analysis result of the blood concentration of caffeine and its metabolite.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, but the embodiments are for facilitating understanding of the principles of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiment, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

(1) First Embodiment The dry blood sample storage base material 900 according to this embodiment is a round air gap type base material shown in FIG.

The dry blood sample storage base material 900 has a base material plate 100, an adhesive tape 200, and a blood retention disc 300.

The base plate 100 is made of cellulose filter paper and is provided with one or more openings 500. The dimensions of the base material plate 100 are not particularly limited, but are, for example, 65 mm to 85 mm in length, 95 mm to 115 mm in width, 0.87 mm to 1.07 mm in thickness, and preferably 75 mm in length, 105 mm in width, and 0.97 mm in thickness. be. The shape of the opening 500 is not particularly limited, and is, for example, a circle having a diameter of 6.0 mm to 14 mm, preferably a circle having a diameter of 10 mm. The shape of the opening is not limited to a circular shape, and may be, for example, an elliptical shape, an oval shape, a pear shape, or the like.

As the cellulose filter paper, it is preferable to use a filter paper having a large thickness and a high filtration rate. The cellulose filter paper is preferably made from cellulose fibers having 99% or more of α-cellulose. As the cellulose filter paper, for example, Advantech's paper chromatography filter paper can be adopted, for example, a mass of 285 g / m ³ and a filtration time of 20 seconds (according to JIS P 3801, using a Hertzberg filtration rate tester, 10 cm ^2). It is time to filter 100 mL of distilled water at a pressure of 0.98 Pa at 20 ° C.), 0.1% ash, and a wet burst strength of 10 kPa.

The water absorption of the cellulose filter paper is, for example, 17 cm / 10 minutes to 19 cm / 10 minutes at 20 ° C., preferably 18 cm / 10 minutes. The water absorption of the cellulose filter paper, for example, at 5 minutes 960~1000 (ml / m ^2), preferably 989ml / m ^2. In the case of a circular blood retention disk 300 having a diameter of 5.5 mm, the amount of water absorbed is 22.5 to 24.5 μl in 5 minutes, preferably 23.5 μl.

One or more adhesive tapes 200 are attached to one side of the opening end of the opening 500 of the base material plate 100. The shape of the adhesive tape is not particularly limited, but is, for example, a line segment shape. The adhesive tape is not particularly limited, but is, for example, cellophane tape, gum tape, or the like, and cellophane tape is preferable. As the adhesive tape, for example, 3M Japan's Scotch tape can be used, and as the product number, for example, No. 811 can be used.

A blood retention disc 300 is attached to the adhesive surface of the adhesive tape 200 so as to be arranged substantially in the center of the opening 500 at intervals from the inner peripheral surface of the opening 500 of the base material plate 100. Further, the adhesive tape 200 is attached to the opening end on one side of the opening 500 of the base material plate 100 so as to have a gap 600 between the adhesive tape 200 and the opening end. Since air can be passed through the gap portion 600 between the upper surface and the lower surface of the base material plate 100, the gap portion 600 can also be referred to as an air gap 600. In FIG. 1, one adhesive tape 200 is attached to the opening end on one side of the opening 500, and two adhesive tapes 200 are attached to the opening end on one side of the opening 500. The case is described. The blood retention disc 300 is made of, for example, a cellulose filter paper similar to the base plate 100, and holds the dropped blood sample in a dry state. The blood sample to be dropped is not particularly limited, and is, for example, venous blood or peripheral blood.

The shape of the blood retention disc 300 is not particularly limited, but is, for example, a circle having a diameter of 4.0 mm to 12 mm, preferably a circle having a diameter of 4.0 mm to 8.0 mm, and more preferably a circle having a diameter of 8.0 mm. .. The shape of the blood retention disc 300 is not limited to a circular shape, and may be, for example, an elliptical shape, an oval shape, a pear shape, or the like. It is preferable that the shape of the opening 500 and the shape of the blood retention disk 300 are the same. For example, the shape of the opening 500 can be a circle with a diameter of 10 mm, the shape of the blood holding disk 300 can be a circle with a diameter of 8.0 mm, and the gap 600 can be an arc shape with a width of 1.0 mm. For example, the shape of the opening 500 can be a circle with a diameter of 8.0 mm, the shape of the blood holding disk 300 can be a circle with a diameter of 6.0 mm, and the gap 600 can be an arc shape with a width of 1.0 mm. For example, the shape of the opening 500 can be a circle with a diameter of 6.0 mm, the shape of the blood retention disk 300 can be a circle with a diameter of 4.0 mm, and the gap 600 can be an arc shape with a width of 1.0 mm. The thickness of the blood retention disc 300 is, for example, 0.87 mm to 1.07 mm, preferably 0.97 mm. The distance between the inner peripheral surface of the opening 500 and the outer peripheral surface of the blood retention disk 300 (that is, the width of the arc-shaped gap 600) can be 0.8 mm to 1.2 mm, preferably 0.9 mm. It is ~ 1.1 mm, more preferably 1.0 mm.

A reinforcing plate made of kraft paper can be attached to the upper surface and the lower surface of the base material plate 100 and sandwiched. The reinforcing plate has a kraft opening so as to expose the opening 500 of the base plate 100. The shape of the kraft opening is not limited to a circular shape, and may be, for example, an elliptical shape.

Next, a usage embodiment of the dry blood sample storage base material 900 according to the present embodiment will be described.

The blood sample is dropped on the blood retention disk 300 with a dropper, for example, 1 to 3 drops. The total drop volume of the blood sample dropped with the dropper is not particularly limited, but is, for example, 25 to 75 μl. The dropped blood sample is absorbed and held by the blood holding disk 300. At this time, the excess blood sample passes through the gap between the outer peripheral surface of the blood holding disk 300 and the inner peripheral surface of the opening 500 of the base material plate 100, and passes through the gap between the base material plate 100 around the blood holding disc 300. Is absorbed by. Further, since there is a gap between the outer peripheral surface of the blood retention disk 300 and the inner peripheral surface of the opening 500 of the base material plate 100, even if the blood sample has a low hematoclick value, the blood retention disk 300 is excessively used. It is difficult to be absorbed by the surrounding base material plate 100.

As described above, in the dry blood sample storage base material 900 according to the present embodiment, only 1 to 3 drops of the blood sample are dropped onto the blood retention disk 300 with a dropper without being affected by the hematoclick value of the blood sample. , A certain amount of blood is held in the blood retention disk 300.

The base material plate 100 on which the excess blood sample is absorbed and the blood retention disk 300 on which the blood sample is dropped are, for example, dried at room temperature, stored and transported in a dry state. When analyzing a blood sample, the blood retention disc 300 is used by peeling it from the adhesive tape 200.

(2) Second Embodiment The dry blood sample storage base material 910 according to the present embodiment is a round air gap 4-bridge type modified base material shown in FIG. In the present embodiment, the reinforcing bridge portion 400 made of cellulose filter paper is attached to the adhesive surface of the adhesive tape 200 so as to connect the blood retention disk 300 and the base material plate 100. Other than that, it is the same as that of the first embodiment. In the dry blood sample storage base material 910 according to the second embodiment, since the reinforcing bridge portion 400 is attached to the adhesive surface of the adhesive tape 200, the adhesive tape 200 is protected from physical impact.

(3) Third Embodiment In this embodiment, unlike the first embodiment and the second embodiment, the adhesive tape 200 is not used. That is, the dry blood storage base material according to the present embodiment is a dry blood sample storage base material for storing a blood sample in a dry state, and is a base material plate provided with a plurality of openings made of cellulose filter paper. It has a blood holding disk made of cellulose filter paper that holds the dropped blood sample in a dry state, and a plurality of extremely small volume connecting portions made of cellulose filter paper that connect the blood holding disk and the base material plate. The plurality of connecting portions connect the blood holding disc and the base material plate so that the blood holding disc is arranged in the opening at intervals from the inner peripheral surface of the opening. According to the present embodiment, by pressing the blood holding disc holding the blood with, for example, a finger, the connection portion is broken and the connection between the blood holding disc and the base material plate is released, and the blood holding disc is easily released. It can be removed from the base plate.

More specifically, the dry blood sample storage base material 920 according to this embodiment is shown in FIG.

The connection portion 420 is a filter paper piece made of cellulose filter paper. A paraffin solution in which paraffin is dissolved in an organic solvent adheres to the connection portion 420, and then the organic solvent evaporates and dries to adhere the paraffin. Paraffin is a general term for alkanes having 20 or more carbon atoms. Paraffin is available, for example, under the trade names Lunaflex® (manufactured by Fuller) and Deawax® (manufactured by DEA Mineraloel AG). The organic solvent is not particularly limited, and examples thereof include heptane and hexane. In the present embodiment, the connection portion is a filter paper piece made of cellulose filter paper to which a paraffin solution is attached and the organic solvent is evaporated and dried. It can be used as long as it has paraffin attached.

The connection portion 420 connects the base material plate 100 made of cellulose filter paper and the blood retention disk 300 made of cellulose filter paper. The blood retention disk 300 is separated from the base material plate 100 and attached to the base material plate 100 by the connecting portion 420. The base material plate 100 is provided with a plurality of openings 500.

The blood retention disc 300 is arranged in the opening 500 at intervals from the inner peripheral surface of the opening 500. Therefore, there is a gap between the blood retention disk 300 and the inner peripheral surface of the opening 500 of the base plate 100. One or more connecting portions 420 are provided. In FIG. 3, the connecting portions 420 are evenly provided at intervals of 120 degrees. The connection portion 420 is provided integrally with the blood retention disk 300 and the base material plate 100. The connecting portion 420 is provided as a separate member from the blood holding disc 300 and the base material plate 100, and the base material plate 100 and the connecting portion 420 are adhered with an adhesive to bond the connecting portion 420 and the blood holding disc 300. By adhering with an adhesive, the connecting portion 420 can also connect the base material plate 100 made of cellulose filter paper and the blood retention disk 300 made of cellulose filter paper.

(4) Fourth Embodiment The dry blood sample storage base material 911 according to the present embodiment is a square air gap type base material shown in FIG. In the present embodiment, the shape of the opening in the plan view and the shape of the blood retention disk in the plan view are quadrangles such as a square and a rectangle, respectively.

The dry blood sample storage base material 911 has a base material plate 101, an adhesive tape 201, and a blood retention disk 301.

The base plate 101 is made of cellulose filter paper and is provided with one or more openings 501. The dimensions of the base material plate 101 are not particularly limited, but are, for example, 65 mm to 85 mm in length, 95 mm to 115 mm in width, and 0.87 mm to 1.07 mm in thickness, preferably 75 mm in length and 75 mm in width, as in the first embodiment. It is 105 mm and 0.97 mm thick. In the present embodiment, the shape of the opening 501 is a square having a side of 4.0 mm to 12 mm, preferably a square having a side of 4.0 mm to 8.0 mm, and more preferably a square having a side of 6.0 mm. The shape of the opening is not limited to a square, and may be, for example, a rectangle, a parallelogram, a rhombus, or the like.

As the cellulose filter paper, a paper chromatography filter paper manufactured by Advantech Co., Ltd. can be adopted as in the first embodiment.

One or more adhesive tapes 201 are attached to one side of the opening end of the opening 501 of the base material plate 101. The shape of the adhesive tape is not particularly limited, but is, for example, a line segment shape. The adhesive tape is not particularly limited, but is, for example, a cellophane tape as in the first embodiment.

A blood retention disk 301 is attached to the adhesive surface of the adhesive tape 201 so as to be arranged substantially in the center of the opening 501 at intervals from the inner peripheral surface of the opening 501 of the base material plate 101. Further, the adhesive tape 201 is attached to the opening end on one side of the opening 501 of the base material plate 101 so as to have a gap 601 between the adhesive tape 201 and the opening end. Since air can be inserted between the upper surface and the lower surface of the base material plate 101 through the gap portion 601, the gap portion 601 can also be referred to as an air gap 601. In FIG. 4, one adhesive tape 201 is attached to the opening end on one side of the opening 501, and two adhesive tapes 201 are attached to the opening end on one side of the opening 501. The case is described. The blood retention disc 301 is made of a cellulose filter paper similar to, for example, the base material plate 101, and holds the dropped blood sample in a dry state. The blood sample to be dropped is not particularly limited, and is, for example, venous blood or peripheral blood.

The shape of the blood retention disk 301 is not particularly limited, but is, for example, a square having a side of 2.0 mm to 10 mm, preferably a square having a side of 4.0 mm. The shape of the blood retention disk 301 is not limited to a square, and may be, for example, a rectangle, a parallelogram, a rhombus, or the like. It is preferable that the shape of the opening 501 and the shape of the blood retention disk 301 are the same. For example, the opening 501 can be a square with a side of 12 mm, the blood retention disk 301 can be a square with a side of 10 mm, and the gap 601 can be a square with a width of 1.0 mm. For example, the opening 501 can be a square with a side of 10 mm, the blood retention disk 301 can be a square with a side of 8.0 mm, and the gap 601 can be a square with a width of 1.0 mm. For example, the opening 501 can be a square with a side of 8.0 mm, the blood retention disk 301 can be a square with a side of 6.0 mm, and the gap 601 can be a square with a width of 1.0 mm. For example, the opening 501 can be a square with a side of 6.0 mm, the blood retention disk 301 can be a square with a side of 4.0 mm, and the gap 601 can be a square with a width of 1.0 mm. For example, the opening 501 can be a square with a side of 4.0 mm, the blood retention disk 301 can be a square with a side of 2.0 mm, and the gap 601 can be a square with a width of 1.0 mm. The thickness of the blood retention disc 301 is, for example, 0.87 mm to 1.07 mm, preferably 0.97 mm, as in the first embodiment. The distance between the inner peripheral surface of the opening 501 and the outer peripheral surface of the blood retention disk 301 (ie, the width of the gap 601) can be 0.8 mm to 1.2 mm, preferably 0.9 mm to 1.1 mm. It is more preferably 1.0 mm.

A reinforcing plate made of kraft paper can be attached to the upper surface and the lower surface of the base material plate 101 and sandwiched. The reinforcing plate has a kraft opening so as to expose the opening 501 of the base plate 101. The shape of the craft opening is not limited to a square, and may be a rectangle, for example.

In the fourth embodiment, the shape of the opening in the plan view and the shape of the blood retention disk in the plan view are quadrangles such as a rectangle, a parallelogram, and a rhombus, respectively. It is also possible to use a polygon such as a triangle, a pentagon, and a hexagon.

(5) Fifth Embodiment The dry blood sample storage base material 912 according to the present embodiment is a square air gap 4-bridge type modified base material shown in FIG. In the present embodiment, the reinforcing bridge portion 401 made of cellulose filter paper is attached to the adhesive surface of the adhesive tape 201 so as to connect the blood retention disk 301 and the base material plate 101. Other than that, it is the same as that of the fourth embodiment. In the dry blood sample storage base material 912 according to the fifth embodiment, since the reinforcing bridge portion 401 is attached to the adhesive surface of the adhesive tape 201, the adhesive tape 201 is protected from physical impact.

1. Example 1
1-1. Preparation of dry blood sample storage base material As the cellulose filter paper, Advantech's paper chromatography filter paper No. 590 (thickness: 0.97 mm, water absorption rate: 18 cm / 10 minutes, 20 ° C) was used. .. Using this cellulose filter paper, a base material plate having a width of 105 mm, a length of 75 mm, and a thickness of 0.97 mm was prepared. Six circular openings with a diameter of 6 mm were created in a grid pattern on the base plate, and a circular blood retention disk with a diameter of 4 mm was placed in the center of the openings (4 / 6 mm round air gap type base material). .. In addition, six circular openings with a diameter of 8 mm were created in a grid pattern on the base plate, and a circular blood retention disk with a diameter of 6 mm was placed in the center of the openings (6 / 8 mm round air gap type base). Material). The width of the gap, that is, the air gap, was 1 mm. Scotch tape No.811 manufactured by 3M Japan Ltd. was used as the adhesive tape. The width of the adhesive tape was 1 mm. The adhesive tape was attached to the opening end on one side of the opening of the base material plate so as to have a gap between the adhesive tape and the opening end. The blood retention disc was placed in the center of the opening on the adhesive surface of the adhesive tape at intervals from the inner peripheral surface of the opening.

1-2. Quality and performance of base material
1-2-1. Weight of base material The weight of discs with diameters of 4 mm and 6 mm was weighed with a BD210 microbalance or Type 4431 ultra-micro balance (Zaritrius), and the measurement results are shown in Table 1. The weight of the disc is 4 mm: 3.44 ± 0.23 mg, 6 mm: 7.87 ± 0.47 mg (mean ± standard deviation, n = 15), and the relative standard deviation (% RSD), which indicates product variation, is close to 5%. The quality is uniform.

1-2-2. Blood density The waste blood transferred from the laboratory was anonymized and used. Blood was collected using a 2K-EDTA blood collection tube, and blood was collected 2-3 days after blood collection. Mix 10-20 bloods, 12,000
Centrifuge at rpm, 4 ° C. for 5 minutes, separated into serum and blood cells, and 30%, 40%, 50%, and 60% hematocrit blood was prepared by gravimetric method. For blood density, 5 ml of each blood was placed in a stoppered vial, aspirated with a 100 μl Gilson pipette, and the amount of decrease was weighed. The pipette volume was corrected with pure water (water density 0.99704 g / cm ³ , 25 ° C). The volume correction value of the pipette was 0.100772 mL.

Table 2 shows the blood densities of hematocrit, 30%, 40%, 50%, and 60%.

1-2-3. Volume of blood held on the substrate
Blood was spotted on each of the 4/6 mm round air gap type base material and the 6/8 mm round air gap type base material. A 1 ml disposable spoid was used to spot 1-2 drops on 4/6 mm and 2-3 drops on 6/8 mm. By this operation, blood spreads to the periphery of the base material. Five minutes after spotting, the disc was attached and detached, the weight thereof was measured, and the volume of blood held (blood content) was calculated from the following formula (Tables 3 and 4). The formula was blood retention volume (μl) = (gross weight (mg)? Empty weight (mg)) / blood density (g / mL). Note that FIG. 6 shows (A) a photographic diagram before dripping blood, (B) a photographic diagram after dripping blood, and (C) a photographic diagram of the detached disc.

From these results, the relative standard deviation of the blood content of the air gap type substrate is around 5%, and the coefficient of variation due to hematocrit is also within 5% excluding one value. Therefore, it was proved that a certain volume of blood can be collected without being affected by the hematocrit value only by dropping blood with a spoid using these base materials. The results are shown in FIG.

2. Example 2
Example 2 shows an example of application of the air gap type base material to a second-generation antipsychotic drug.

2-1. Therapeutic Drug Monitoring (TDM)
A consistent correlation was found between blood levels of antipsychotics and clinical efficacy, suggesting the effectiveness of blood level monitoring. In addition, the AGNP guidelines strongly recommend TDM due to the narrow therapeutic range of the drug and its serious side effects. Therefore, blood levels of second-generation antipsychotics, clozapine, olanzapine, risperidone, and aripiprazole, were measured to confirm the efficacy of the air-gap substrate. Regarding clozapine used for treatment-resistant schizophrenia, it is presumed that the use of this substrate has the advantage of relieving stress and anxiety in patients, such as avoiding venous blood sampling. investigated. Furthermore, the targeted antipsychotics account for the majority of drug treatment, and it is presumed that they can be used not only for inpatients but also for self-collection of outpatients and home-based patients.

2-2. Preparation of dry blood spots and extraction of drugs Hematocrit, 30%, 40%, 50%, 60% of blood was spiked with each drug to a final concentration of 250-2,000 ng / mL. 2-3 drops of blood were added dropwise to the substrate with a spoid and dried for 4 hours or overnight at room temperature.

Place the dry blood disc in a 2 ml Eppendorf tube with tweezers. Add 500 μl of 0.1 M aqueous sodium carbonate (pH 11.7) solution to this tube and stir with vortex. Add 20 μl of 1000 ng / ml internal standard to this tube, stir with vortex and sonicate for 5 minutes (clozapine, olanzapine) or 15 minutes (risperidone, aripiprazole). Add 800 μl of ethyl acetate to this Eppendorf tube, stir with vortex for 60 seconds, centrifuge (12,000 rpm, 5 minutes, 4 ° C), take 400 μl of supernatant and place in a sample tube. Completely evaporate the solvent in a nitrogen stream (40 ° C, 20 minutes). Add 60 μl of mobile phase solution to this tube, stir with vortex, and inject 20 μl into high performance liquid chromatography (HPLC) with an autosampler for measurement.

2-3. Analysis of blood drugs by HPLC-Coulochem II
HPLC separation columns include C18 reverse phase column, Eicompak ODS-C18, 5 μm, 150 x 4.5 mm, guard cell (ODS C-18) (Acom Co., Ltd.), or Gemini C18, 00F-4435-E0, 150 x 4.5 mm. , Guard cartridge C18, AJO-4287, 4.0mm x 3.0mmID, holder assembly kit KJO-4282, column inner diameter, 2.0mm-8.0mmID (Shimadzu GLC Co., Ltd.) was used. As the mobile phase, a mixed solution of methanol and 50 mM phosphate buffer solution (pH 2.1) was used. A Coulochem II (ESA) was used for drug quantification, and a Model 5011 analysis cell and a Model 5020 guard cell were attached. The separation conditions for each drug and the load voltage of Coulochem are shown in Table 5. Chromatography of clozapine and metabolites is shown in FIG.

2-4. Calibration curve of clozapine and metabolites Clozapine (CLZ) and its metabolites, norcrozapine (DMC) and clozapine N-oxide (CMO) are spiked into blood, then dropped onto an air gap type substrate, dried, and dried. Their concentrations were measured after 3 days. The calibration curve is shown in FIG. Very good correlation (R> 0.99) between the drug and its signal was observed in the 4/6 mm and 6/8 mm air gap type substrates, and each substrate quantitatively retains blood. It was proved that there was no problem in measuring the blood concentration.

2-5. Effects of hematocrit on blood levels of clozapine and metabolites Clozapine (CLZ) and its metabolites, clozapine (DMC), clozapine N-oxide (CMO) in blood at hematocrit levels of 30, 40, 50 and 60% ) Was spiked at 750 ng / ml, respectively, and dropped onto 4/6 mm and 6/8 mm air gap type substrates. It was dried and analyzed 3 days later (FIGS. 10 and 11). From these results, no statistically significant difference was observed in the blood concentration of the drug depending on the hematocrit value, and the variation of hematocrit 30 to 60% was within ± 10%.
It was within the standard value of ± 15%. No change in blood concentration due to hematocrit was observed, but as shown in FIGS. 10 and 11, the blood concentrations of all the drugs tended to decrease as the hematocrit value increased. The cause of this was thought to be a decrease in the drug recovery rate in high hematocrit, so the extraction time and liquid-liquid extraction conditions were examined. As a result of extending the ultrasonic extraction time from 5 minutes to 15 minutes, no decrease in the blood concentration of the drug at a high hematocrit value was observed. From this, it is presumed that the effect of hematocrit can be ignored by extending the extraction time. In conclusion, the fluctuation range of the analytical value by the hematocrit value was within the allowable range, and it was confirmed that the air gap type substrate is effective for measuring the blood concentration of clozapine and its metabolites.

2-6. Effect of hematocrit on blood levels of olanzapine, risperidone and aripiprazole Add olanzapine, risperidone and aripiprazole to blood levels of 30, 40, 50 and 60% to a final concentration of 1000-2000 ng / ml. And dropped onto 4/6 mm and 6/8 mm air gap type substrates, and the blood concentration after drying was measured (FIGS. 12, 13, 14). From these results, it was confirmed that by using the air gap type blood sample dry storage substrate, the fluctuation range of the analytical value due to the hematocrit value is within the permissible range, and the variation of the measured value under the same conditions is suppressed to below the standard value. ..

3. Example 3
In Example 3, the reinforcing bridge portion made of cellulose filter paper can also be referred to as a dry blood sample storage base material (note that it is also referred to as an air gap bridge type modified base material) that connects the blood retention disc and the base material plate. , The air gap bridge type modified base material is, for example, a round air gap 4-bridge type modified base material, a square air gap 4-bridge type modified base material, and the like.)

3-1. Preparation of dry blood sample storage substrate As in Example 1, as a cellulose filter paper, a paper chromatography filter paper No. 590 (thickness: 0.97 mm, water absorption rate: 18 cm / 10 minutes) manufactured by Advantech Co., Ltd. , 20 ° C) was used. Then, in the same manner as in Example 1, this cellulose filter paper was used to prepare a base material plate having a width of 105 mm, a length of 75 mm, and a thickness of 0.97 mm. Six circular openings with a diameter of 6 mm were created in a grid pattern on the base material plate, and a circular blood retention disk with a diameter of 4 mm was placed in the center of the openings (4 / 6 mm air gap type base material). In addition, six circular openings with a diameter of 8 mm were created in a grid pattern on the base material plate, and a circular blood retention disk with a diameter of 6 mm was placed in the center of the openings (6 / 8 mm air gap type base material). .. In addition, six circular openings with a diameter of 10 mm were created in a grid pattern on the base material plate, and a circular blood retention disk with a diameter of 8 mm was placed in the center of the openings (8 / 10 mm air gap type base material). .. The width of the gap, that is, the air gap, was 1 mm. As in Example 1, Scotch Tape No. 811 manufactured by 3M Japan Ltd. was used as the adhesive tape. The width of the adhesive tape was 1 mm. The adhesive tape was attached to the opening end on one side of the opening of the base material plate so as to have a gap between the adhesive tape and the opening end. The blood retention disc was placed in the center of the opening on the adhesive surface of the adhesive tape at intervals from the inner peripheral surface of the opening. Then, unlike Example 1, the reinforcing bridge portion made of cellulose filter paper (paper chromatography filter paper No. 590 manufactured by Advantech Co., Ltd.) is attached to the adhesive surface of the adhesive tape so as to connect the blood retention disk and the base material plate. It was pasted (Fig. 15). FIG. 15 is a photographic view of a round air gap-4 bridge, showing 8/10 mm, 6/8 mm, and 4/6 mm from the left side.

3-1-1. Blood retention volume of round air gap 4-bridge modified substrate Spot blood with hematocrit (HCT) values of 30, 40, 50, 60% on the substrate shown in Fig. 15 and measure the weight after 5 minutes. , The blood content was determined (Tables 6, 7, and 8), and these results are shown in FIG. FIG. 16 shows the effect of the hematocrit value on the blood retention volume of the round air gap 4-bridge modified substrate.

3-1-2. Blood Concentrations of Clozapine and Its Metabolites Round Air Gap 4-Bridge Modified Substrate, 4/6 mm, 6/8 mm and 8/10 mm, 1000 ng / ml, 500 ng / ml, and 8/10 mm, respectively. Spot blood spiked with 250 ng / ml clozapine (CLZ), metabolites norclozapine (DMC), clozapine N-oxide (CNO), and hematocrit for blood levels of each compound. The effect of the values was investigated (FIGS. 17, 18, 19). FIG. 17 shows the effect of hematocrit on clozapine and metabolite blood levels measured using a 4/6 mm air gap 4-bridge modified substrate. FIG. 18 shows the effect of hematocrit on clozapine and metabolite blood levels measured using a 6/8 mm air gap 4-bridge modified substrate. FIG. 19 shows the effect of hematocrit on clozapine and metabolite blood levels measured using an 8/10 mm air gap 4-bridge modified substrate. From these results, for CLZ and CNO, the effect of hematocrit (HCT) was not observed on all the substrates used.

4. Example 4
4-1. Preparation of dry blood sample storage base material As the cellulose filter paper, Advantech's paper chromatography filter paper No. 590 (thickness: 0.97 mm, water absorption rate: 18 cm / 10 minutes, 20 ° C) was used. .. Using this cellulose filter paper, a base material plate having a width of 105 mm, a length of 75 mm, and a thickness of 0.97 mm was prepared. A square opening with a side of 4 mm was created in a grid pattern on the base material plate, and a square blood retention disk with a side of 2 mm was placed in the center of the opening (2 / 4 mm square air gap type base material). A square opening with a side of 6 mm was created in a grid pattern on the base material plate, and a square blood retention disk with a side of 4 mm was placed in the center of the opening (4 / 6 mm square air gap type base material). A square opening with a side of 8 mm was created in a grid pattern on the base material plate, and a square blood retention disk with a side of 6 mm was placed in the center of the opening (6 / 8 mm square air gap type base material). A square opening with a side of 10 mm was created in a grid pattern on the base material plate, and a square blood retention disk with a side of 8 mm was placed in the center of the opening (8 / 10 mm square air gap type base material). A square opening with a side of 12 mm was created in a grid pattern on the base material plate, and a square blood retention disk with a side of 10 mm was placed in the center of the opening (10 / 12 mm square air gap type base material). The width of the gap, that is, the air gap, was 1 mm. Scotch tape No.811 manufactured by 3M Japan Ltd. was used as the adhesive tape. The width of the adhesive tape was 1 mm. The adhesive tape was attached to the opening end on one side of the opening of the base material plate so as to have a gap between the adhesive tape and the opening end. The blood retention disc was placed in the center of the opening on the adhesive surface of the adhesive tape at intervals from the inner peripheral surface of the opening.

4-2. Quality and performance of base material
4-2-1. Weight of base material The weight of a square blood retention disc with sides of 2 mm, 4 mm, 6 mm, 8 mm, and 10 mm was weighed with a BD210 microbalance or a Type 4431 ultra-micro balance (Zaritrius). The measurement results are shown in Table 9. The unit is mg. As shown in Table 9 below, the relative standard deviation (% RSD), which indicates the variation of the products, was about 5%, and the quality was uniform.

4-2-2. Blood density The waste blood transferred from the laboratory was anonymized and used. Blood was collected using a 2K-EDTA blood collection tube, and blood was collected 2-3 days after blood collection. Mix 10-20 bloods, 12,000
Centrifuge at rpm, 4 ° C. for 5 minutes, separated into serum and blood cells, and 30%, 40%, 50%, and 60% hematocrit blood was prepared by gravimetric method. For blood density, 7 ml of each blood was placed in a stoppered vial, aspirated with a 100 μl Gilson pipette, and the amount of decrease was weighed. The pipette volume was corrected with pure water (water density 0.99704 g / cm ³ , 25 ° C). The volume correction value of the pipette was 0.100772 mL. The blood densities of hematocrit, 30%, 40%, 50% and 60% are shown in Table 10.

4-2-3. Volume of blood held on the substrate
Blood was spotted on each of the 2/4 mm square air gap type base material, the 4/6 mm square air gap type base material, the 6/8 mm square air gap type base material, and the 8/10 mm square air gap type base material. Using a 1 ml disposable spoid, lightly touch the tip to 2/4 mm, 1 drop to 4/6 mm, 2 drops to 6/8 mm, 3 drops to 8/10 mm, 10/12 mm Spotted 3 drops. By this operation, blood spreads to the periphery of the base material. Five minutes after spotting, the disc was attached and detached, its weight was measured, and the volume of blood held (blood content) was calculated from the following formula (Tables 11, 12, 13, 14). The formula was blood retention volume (μl) = (gross weight (mg)? Empty weight (mg)) / blood density (g / mL). FIG. 20 is a photographic view of a square air gap type base material having a 4/6 mm square air gap type base material and a 6/8 mm square air gap type base material on one base material plate. FIG. 20 shows (A) a photographic diagram before dripping blood, (B) a photographic diagram after dripping blood, and (C) a photographic diagram of a detached disc.

Table 11 shows the effect of the hematocrit value on the blood retention volume of the 2/4 mm square air gap type substrate.

Table 12 shows the effect of the hematocrit value on the blood retention volume of the 4/6 mm square air gap type substrate.

Table 13 shows the effect of the hematocrit value on the blood retention volume of the 6/8 mm square air gap type substrate.

Table 14 shows the effect of the hematocrit value on the blood retention volume of the 8/10 mm square air gap type substrate.

FIG. 21 shows the hematoes for the blood retention volume in each of the 2/4 mm square air gap type base material, the 4/6 mm square air gap type base material, the 6/8 mm square air gap type base material, and the 8/10 mm square air gap type base material. It is a figure which shows the influence of a click. It is the most important condition of the substrate that the amount of blood retained in the substrate is not affected by hematocrit. As shown in FIG. 21, no significant effect of hematocrit on blood retention volume was observed.

5. Example 5
5-1. Preparation of dry blood sample storage substrate As in Example 1, as a cellulose filter paper, a paper chromatography filter paper No. 590 (thickness: 0.97 mm, water absorption rate: 18 cm / 10 minutes) manufactured by Advantech Co., Ltd. , 20 ° C) was used. Then, in the same manner as in Example 1, this cellulose filter paper was used to prepare a base material plate having a width of 105 mm, a length of 75 mm, and a thickness of 0.97 mm. Six circular openings with a diameter of 6 mm were created in a grid pattern on the base material plate, and a circular blood retention disk with a diameter of 4 mm was placed in the center of the openings (4 / 6 mm air gap type base material). The width of the gap, that is, the air gap, was 1 mm. The connecting portions for connecting the base material plate and the blood retention disc were evenly provided at three locations at 120-degree intervals. The shape of the connection part was a rectangular parallelepiped, the length in a plan view was 1 mm, and the width in a plan view was 0.3 mm.

In the dry blood sample storage base material according to this example, the connection portion was provided integrally with the blood retention disk and the base material plate. The dry blood sample storage base material according to this embodiment corresponds to, for example, the arcuate convex portions evenly arranged at intervals of 120 degrees against the rectangular cellulosic filter paper in a plan view. Each part is punched out from a rectangular cellulosic filter paper in a plan view, whereby the blood retention disk is separated from the base material plate and attached to the base material plate by a connecting portion.

Then, paraffin Lunaflex (registered trademark) (manufactured by Fuller) was dissolved in heptane (n-Heptane) to prepare a solution having a paraffin concentration in which paraffin was saturated at room temperature. 0.5-1 μL of this solution was pipetted to each connection and then air dried (Fig. 22, left side). Paraffin became invisible when dried, but spotting a blood sample on a blood retention disc was found to block blood flow from the blood retention disc beyond the connection (Fig. 22, right side).

5-2. Volume of blood retained on the substrate After confirming the drying of the spotted blood sample, the blood retention disk was separated from the base plate with scissors, and the concentrations of caffeine and its metabolites were measured (Fig.). 23). Blood concentrations of all compounds were in perfect agreement in the blood of 30% and 60% hematocrit. It was proved that this method collects a certain amount of blood without being affected by hematocrit by a simple operation of dropping blood with a dropper.

It can be used for storage and transportation of dried blood samples.

100,101: Base plate 200,201: Adhesive tape 300,301: Blood retention disc 400,401: Reinforcing bridge part 420: Connection part 500,501: Opening part 600,601: Void part (air gap)
900,901,911,912: Dry blood sample storage substrate

Claims (9)

A dry blood sample storage base material that stores blood samples in a dry state.
Cellulose filter paper base plate and
It has a blood retention disk made of cellulose filter paper that holds the dropped blood sample in a dry state, and a connecting portion that connects the base material plate and the blood retention disk.
The blood retention disc is separated from the base material plate and attached to the base material plate by the connection portion.
The base material plate is provided with a plurality of openings.
A dry blood sample storage base material, wherein the blood retention disc is arranged in the opening at intervals from the inner peripheral surface of the opening. It is a dry blood sample storage base material for storing a blood sample in a dry state, and the connection portion is an adhesive tape.
Cellulose filter paper base plate, adhesive tape,
It has a blood retention disc made of cellulose filter paper that holds the dropped blood sample in a dry state.
The blood retention disc is separated from the base material plate and attached to the base material plate by the adhesive tape.
The base material plate is provided with a plurality of openings.
The adhesive tape is one or a plurality of single-sided tapes having an adhesive surface on one side, and the adhesive tape has the adhesive tape and the opening end at the opening end on one side of the opening of the base material plate. It is pasted so that there is a gap between the two
The dry blood sample storage according to claim 1, wherein the blood retention disc is arranged in the adhesive surface of the adhesive tape at a distance from the inner peripheral surface of the opening. Base material. The dry blood sample storage base material according to claim 1 or 2, wherein the filter paper forming the blood retention disc has a water absorption rate of 18 cm / 10 minutes and 20 ° C. The blood retention disc is substantially circular in plan view, the diameter of the blood retention disc is 4.0 mm to 12 mm, and the distance between the inner peripheral surface of the opening and the outer peripheral surface of the blood retention disc is 0.8 mm to. The dry blood sample storage base material according to any one of claims 1 to 3, which is 1.2 mm. The dry blood sample storage base material according to claim 4, wherein the blood retention disc has a diameter of 4.0 mm to 8.0 mm. The blood retention disc is substantially square in plan view, one side of the blood retention disc is 2.0 mm to 10 mm, and the distance between the inner peripheral surface of the opening and the outer peripheral surface of the blood retention disc is 0.8 mm to. The dry blood sample storage base material according to any one of claims 1 to 3, which is 1.2 mm. The dry blood sample storage base material according to claim 6, wherein one side of the blood retention disc is 4.0 mm to 8.0 mm. A dry blood sample storage base material that stores blood samples in a dry state.
The connection portion is a filter paper piece made of cell roll filter paper to which a paraffin solution in which paraffin is dissolved in an organic solvent is attached and the organic solvent is evaporated and dried.
Cellulose filter paper base plate and
It has a blood retention disk made of cellulose filter paper that holds the dropped blood sample in a dry state, and a connecting portion that connects the base material plate and the blood retention disk.
The blood retention disc is separated from the base material plate and attached to the base material plate by the connection portion.
The base material plate is provided with a plurality of openings.
The dry blood sample storage base material according to claim 1, wherein the blood retention disc is arranged in the opening at intervals from the inner peripheral surface of the opening. A dry blood sample storage base material that stores blood samples in a dry state.
Cellulose filter paper base plate and
Adhesive tape and
It has a blood retention disc made of cellulose filter paper that holds the dropped blood sample in a dry state.
The base material plate is provided with a plurality of openings.
The adhesive tape is one or a plurality of single-sided tapes having an adhesive surface on one side, and the adhesive tape has the adhesive tape and the opening end at the opening end on one side of the opening of the base material plate. It is pasted so that there is a gap between the two
The blood retention disc is arranged in the opening on the adhesive surface of the adhesive tape at a distance from the inner peripheral surface of the opening.
A dry blood sample storage base material, wherein a reinforcing bridge portion made of cellulose filter paper is attached to an adhesive surface of the adhesive tape so as to connect the blood holding disk and the base material plate.

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