Blood pathogen inactivation method

Abstract

The present disclosure provides a pathogen inactivation method, which is low-frequency sonication together with illumination of a photosensitizer-containing blood sample; and the low-frequency sonication is conducted at a frequency of 15-500 KHz. Through the combination of sonication and photochemical pathogen inactivation technology that enhance and complement each other, the blood pathogen inactivation method provided by the present disclosure enhances a pathogen inactivation effect, reduces a dosage of the photosensitizer, photosensitizer-related blood quality damage, energy demand for the illumination, and pathogen inactivation treatment time, increases the blood illumination thickness for effective pathogen inactivation, saves illumination bag materials, shortens the size of illumination equipment, saves costs, and helps the pathogen inactivation technology go to the market.

Claims

1. A blood pathogen inactivation method, comprising the following steps: drawing blood, adding a photosensitizer, and conducting illumination and low-frequency sonication simultaneously, wherein the low-frequency sonication is conducted at a frequency of 15-500 KHz.

2. The blood pathogen inactivation method according to claim 1, wherein the photosensitizer is riboflavin.

3. The blood pathogen inactivation method according to claim 2, wherein a concentration of the riboflavin in a blood sample is 10-100 .Math.mol/L, and preferably 50 .Math.mol/L.

4. The blood pathogen inactivation method according to claim 3, wherein the riboflavin is dissolved in normal saline and added with the blood sample.

5. The blood pathogen inactivation method according to claim 4, wherein a content of the riboflavin in the normal saline is 50-500 .Math.mol/L, and preferably 500 pmol/L.

6. The blood pathogen inactivation method according to claim 1, wherein the low-frequency sonication and the illumination last for 3 min to 2 h.

7. The blood pathogen inactivation method according to claim 6, wherein the low-frequency sonication and the illumination last for 10-30 min.

8. The blood pathogen inactivation method according to claim 1, wherein the low-frequency sonication is conducted at frequency of 30 KHz.

9. The blood pathogen inactivation method according to claim 6, wherein the low-frequency sonication is conducted at frequency of 30 KHz.

10. The blood pathogen inactivation method according to claim 7, wherein the low-frequency sonication is conducted at frequency of 30 KHz.

11. The blood pathogen inactivation method according to claim 1, wherein the illumination is conducted at a wavelength of 311 ± 50 nm.

12. The blood pathogen inactivation method according to claim 6, wherein the illumination is conducted at a wavelength of 311 ± 50 nm.

13. The blood pathogen inactivation method according to claim 7, wherein the illumination is conducted at a wavelength of 311 ± 50 nm.

14. The blood pathogen inactivation method according to claim 1, wherein the blood sample is one selected from the group consisting of plasma, platelets, suspended red blood cells, and whole blood.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 illustrates the growth of S. aureus after treatment with different combinations of sonication, ultraviolet (UV) light and riboflavin (in log, n = 6).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1. Blood Pathogen Inactivation Method Provided by the Present Disclosure

[0024] Step 1, whole blood was taken, and a 500 .Math.mol/L riboflavin normal saline solution was added to make the concentration of riboflavin in the whole blood be 50 .Math.mol/L; and

[0025] Step 2, the whole blood containing riboflavin in step 1 was placed in an illumination environment at a wavelength of 311 ± 50 nm, and simultaneously sonicated at 15 KHz for 30 min to obtain pathogen-inactivated whole blood.

Example 2. Blood Pathogen Inactivation Method Provided by the Present Disclosure

[0026] Step 1, plasma was taken, and a 200 .Math.mol/L riboflavin normal saline solution was added to make the concentration of riboflavin in the plasma be 50 .Math.mol/L; and

[0027] Step 2, the plasma containing riboflavin in step 1 was placed in an illumination environment at a wavelength of 311 ± 50 nm, and simultaneously sonicated at 30 KHz for 20 min to obtain pathogen-inactivated plasma.

Example 3. Blood Pathogen Inactivation Method Provided by the Present Disclosure

[0028] Step 1, platelets were taken, and a 100 .Math.mol/L riboflavin normal saline solution was added to make the concentration of riboflavin in the platelets be 25 .Math.mol/L; and

[0029] Step 2, the platelets containing riboflavin in step 1 were placed in an illumination environment at a wavelength of 311 ± 50 nm, and simultaneously sonicated at 50 KHz for 10 min to obtain pathogen-inactivated platelets.

Example 4. Blood Pathogen Inactivation Method Provided by the Present Disclosure

[0030] Step 1, suspended red blood cells were taken, a 50 .Math.mol/L riboflavin normal saline solution was added to make the concentration of riboflavin in the suspended red blood cells be 25 .Math.mol/L; and

[0031] Step 2, the suspended red blood cells containing riboflavin in step 1 were placed in an illumination environment at a wavelength of 311 ± 50 nm, and simultaneously sonicated at 30 KHz for 3 min to obtain pathogen-inactivated suspended red blood cells.

Example 5. Blood Pathogen Inactivation Method Provided by the Present Disclosure

[0032] Step 1, whole blood was taken, and a 500 .Math.mol/L riboflavin normal saline solution was added to make the concentration of riboflavin in the whole blood be 50 .Math.mol/L; and

[0033] Step 2, the whole blood containing riboflavin in step 1 was placed in an illumination environment at a wavelength of 311 ± 50 nm, and simultaneously sonicated at 30 KHz for 30 min to obtain pathogen-inactivated whole blood.

[0034] The beneficial effects of the present disclosure will be described below through the test example.

[0035] Test Example 1 Evaluation of the optimal combination of low-frequency sonication and photochemical method

1. Methods

[0036] 1.1 Two bags of ABO hemolytic fresh frozen plasma (approximately 400 mL in total) were thawed and mixed well.

[0037] 1.2 No more than 10% of the indicator pathogen (S. aureus or VSV) was added, and the final concentration of the pathogen was approximately 5-6log.

[0038] 1.3 The samples were equally divided into two aliquots, one was added with normal saline (10% of the final sample), and the other one was added with 500 .Math.mol/L riboflavin normal saline solution (10% of the final sample), where the concentration was 50 .Math.mol/L after adding plasma.

[0039] 1.4 The two aliquots of samples were dispensed into sterile culture plates with a diameter of 3.5 cm, 3 mL per well, and 6 wells per plate; each sample contained 9 plates finally, and there were a total of 18 plates.

[0040] 1.5 The samples were divided into three groups to treat for different illumination time (with energy); each group contained 6 plates of samples (3 plates of riboflavin-containing samples, and 3 plates of riboflavin-free samples), and the treatment conditions of each group of 6 plates were as follows: [0041] a. riboflavin-containing sample + illumination + low-frequency sonication; [0042] b. riboflavin-containing sample + illumination; [0043] c. riboflavin-containing sample + low-frequency sonication; [0044] d. riboflavin-free sample + illumination + low-frequency sonication; [0045] e. riboflavin-free sample + illumination; and [0046] f. riboflavin-free sample + low-frequency sonication; [0047] the treatments for different illumination time (with energy) were as follows: the light wavelength was 311 ± 50 nm, and the illumination time (energy) was 10, 20, and 30 min (light energy was 0.27, 0.54, and 0.81 J/mL, respectively) for the three groups, respectively; [0048] the frequency of the low-frequency sonication was 30 KHz; in the three groups, the sonication time was synchronized with the illumination time, which was 10, 20, and 30 min, respectively; and [0049] the controls were the riboflavin-containing sample without illumination and the riboflavin-free sample without illumination, and the number of replicates for each group of data was N = 6.

[0050] 1.6 After inactivation treatment, samples were taken, diluted 1:10, and cultured, and the pathogen growth concentration was calculated according to the Reed-Muench method.

[0051] 1.7 The pathogen inactivation effect of the optimal combination of low-frequency sonication and photochemical method was analyzed.

2. Results

[0052] The detailed results are shown in Table 1 and FIG. 1.

[0053] Table 1 illustrates the growth of S. aureus after treatment with different combinations of sonication, UV light and riboflavin (in log, n = 6)

TABLE-US-00001 Light energy (J/mL) Sonication + UV light + riboflavin Sonication + UV light UV light + riboflavin UV light Riboflavin-containing sample + low-frequency sonication Low-frequency sonication Control 0.27 4.48 ± 0.46 5.80 ± 0.17 5.41 ± 0.029 5.95 ± 0.18 5.84 ± 0.18 6.00 ± 0.19 5.87 ± 0.32 0.54 2.04 ± 0.71 5.69 ± 0.38 5.66 ± 0.09 5.80 ± 0.31 5.72 ± 0.30 5.87 ± 0.27 5.87 ± 0.32 0.81 1.28 ± 0.66 4.04 ± 0.53 5.23 ± 0.16 5.76 ± 0.06 5.67 ± 0.32 5.93 ± 0.17 5.87 ± 0.32

[0054] According to the results: the sonication + UV light + riboflavin group has a strong pathogen inactivation effect; when the light intensity reaches 0.81 J/mL, the growth of S. aureus is almost close to the detection limit level, 1.28 ± 0.66log (due to the limitation of the determination method, when the bacterial concentration of the sample is lower than 0.5log, the undetermined pathogen concentration of the sample is marked as 0.5log), while the sonication + UV light group has a poor inactivation effect when the light energy reaches 0.81 J/mL; when the light intensity reaches 0.81 J/mL, the UV light + riboflavin group, the sonication + riboflavin group, and the UV light group have no inactivation effect. It is shown that sonication can enhance the pathogen inactivation effect of the UV light, and substantially enhance the photochemical pathogen inactivation effect of UV light and riboflavin. Therefore, in the present disclosure, the combination of low-frequency sonication and riboflavin photochemical method has a synergistic effect.

[0055] In conclusion, through the combination of sonication and photochemical pathogen inactivation technology that enhance and complement each other, the blood pathogen inactivation method provided by the present disclosure enhances the pathogen inactivation effect, saves the cost of pathogen inactivation, and has practical popularization and application value.