Some standard content:
Pharmaceutical Industry Standard of the People's Republic of China
YY0329—2002
Leukocyte removal filters for single use
use2002-04-10 Issued
State Food and Drug Administration
2002-08-01 Implementation
YY0329—2002
Cited standards
Classification and naming
Inspection rules
Appendix A (Appendix to the standard)
Appendix B (Appendix to the standard)
Appendix C (Appendix to the standard)
Appendix D (Appendix to the standard)
Appendix E (Appendix to the standard)
Appendix F (Appendix to the standard)
Appendix G (Appendix to the standard)
Appendix H (Appendix to the standard)
Appendix I (Suggestive Appendix)
Appendix J (Suggestive Appendix)
Preparation of chemical performance test solution
Preparation method of bacterial endotoxin test extract. Method for determination of residual leukocyte count—ordinary optical microscope counting method·Method for determination of residual leukocyte count 1
—fluorescence microscope counting method (arbitration method)...Determination of free hemoglobin (o-tolidine method) Method for determination of red blood cell and platelet recovery rate Platelet hypotonic shock relative change rate test Hemolysis test
Application examples of leukocyte removal filter
Literature list
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Disposable leukocyte removal filter is a kind of equipment for preparing leukocyte-removed blood components for clinical blood transfusion, blood bank or blood center. Its main function is to filter out leukocytes in blood or blood components and agglomerates in banked blood. Appendices A to H of this standard are appendices of the standard, and Appendix 1 and Appendix G are appendices of reminder. This standard is proposed by the State Drug Administration. This standard is under the jurisdiction of the National Technical Committee for Standardization of Medical Infusion Equipment. The main drafting units of this standard are: Shanghai Blood Transfusion Technology Co., Ltd. and Jinan Medical Device Quality Supervision and Inspection Center of the State Drug Administration.
Participating drafting unit of this standard: Nanjing Sunway Industrial Company. Main drafters of this standard: Xu Yayong, You Shaohua, Zhang Qiang, Xie Rufeng. 1 Scope
Pharmaceutical Industry Standard of the People's Republic of China
Leukocyte removal filters for single use
Leukocyte removal filters for single useYY0329-2002
This standard specifies the requirements for leukocyte removal filters for single use. This product is connected with blood transfusion sets, blood collection-blood component separation systems to form leukocyte removal blood transfusion sets and blood banks, blood collection-leukocyte removal blood component separation type blood transfusion equipment. 2 Referenced standards
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard is published, the versions shown are valid. All standards will be revised, and the parties using this standard should explore the possibility of using the latest versions of the following standards. GB/T2828—1987 Batch inspection counting sample procedure and sampling table (applicable to inspection of continuous batches) GB8368—1998—Disposable infusion sets GB8369-1998—Disposable blood transfusion sets
GB/T14233.11998 Inspection methods for medical infusion, blood transfusion and injection equipment Part 1: Chemical analysis methods GB/T14233.2-1993 Inspection methods for medical infusion, blood transfusion and injection equipment Part 2: Biological test methods GB/T16886.1—2001 Biological evaluation of medical devices Part 1: Evaluation and testing YY/T0313-1998 Packaging, marking, transportation and storage of medical molecular products 3 Classification and nomenclature
Leukocyte removal filters are divided into whole blood or red blood cell suspension leukocyte removal filters and platelet suspension leukocyte removal filters according to the blood components they filter out.
Leukocyte-removing filters applicable to whole blood or red blood cell suspensions that meet the requirements of this standard are marked with: RF. Leukocyte-removing filters applicable to platelet suspensions that meet the requirements of this standard are marked with: PF. Note: Appendix 1 gives an application example of leukocyte-removing filters. 4 Materials
The materials and housing used to manufacture leukocyte-removing filters shall meet the requirements of Chapter 5. 5 Requirements
5.1 Physical properties
5.1.1 Appearance
When inspected with normal or corrected vision, the housing of the leukocyte-removing filter shall be smooth, free of obvious mechanical impurities and foreign matter, and the welding surface shall be uniform and free of bubbles.
5.1.2 Fit
One end of the leukocyte removal filter is sealed, and the other end is passed through a gas 50kPa higher than the atmospheric pressure, and placed in 20C~30C water for 2 minutes. Approved by the State Food and Drug Administration on April 10, 2002 and implemented on August 1, 2002
There should be no leakage.
Note: The fit test is only applicable to the welded surface inspection of the filter. 5.1.3 Connection firmness
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If the leukocyte removal filter is connected to other components, each connection should be able to withstand a static axial tensile force of 15N for 15s without breaking or falling off.
5.1.4 Particle content
When tested according to Appendix F of GB8368-1998 or other equivalent methods, the number of particles of 15μm~25um in 200mL of eluate of the leukocyte removal filter should not exceed 1.00/mL, and the number of particles larger than 25um should not exceed 0.50/mL. 5.1.5 Flow rate
When the leukocyte removal filter is connected to a blood transfusion set (see Figure 12) that meets the requirements of GB8369-1998, it should be able to transport not less than 700mL of 400g/L glucose aqueous solution within 30min at a solution temperature of 23℃±2C after complete immersion under a static pressure head of 1m. Note: Complete immersion means that after the filter is pressurized and started, the solution flowing through the filter can form a columnar liquid flow. 5.1.6 Protective cover
The protective cover at each end of the leukocyte removal filter should keep the connector and the inner surface of the filter sterile. 5.2 Chemical properties
The test solution prepared according to Appendix A shall meet the following requirements. 5.2.1 Reducing substances (easy oxidation substances)
When tested according to 5.2.2 of GB/T14233.1-1998, the difference in volume of potassium permanganate solution [c(KMnO,)=0.002mol/1,] consumed by the test solution and the blank solution shall not exceed 2.0mL. 5.2.2 Metal ions
When tested by atomic absorption spectrophotometry (AAS) according to 5.9.1 of GB/T14233.1-1998, the total content of chromium, copper, lead and tin in the test solution shall not exceed 1μg/ml. The content of cadmium shall not exceed 0.1μg/mL. When tested according to 5.6.1 of GB/T14233.1-1998, the color of the test solution should not exceed that of the standard reference solution with a mass concentration of p(Pb2+)=1ug/mL.
5.2.3 pH
When tested according to 5.4.1 of GB/T14233.1-1998, the pH difference between the test solution and the blank solution of the same batch should not exceed 1.5. 5.2.4 Evaporation residue
When tested according to 5.5 of GB/T14233.1-1998, the total weight of non-volatile matter in 50mL of the test solution shall not exceed 2mg. 5.2.5 Ultraviolet absorbance
When tested according to 5.7 of GB/T14233.1-1998, the absorbance of the test solution in the range of 250nm～320nm should not be greater than 0.3.
5.2.6 Ethylene oxide residue
The test solution prepared according to Appendix A should be immediately injected into the headspace bottle or Nessler colorimetric tube. Then, when tested according to Chapter 9 or Chapter 10 of GB/T14233.1-1998, the ethylene oxide residue of each set of leukocyte removal filter should not exceed 1.0 mg. 5.3 Biological performance
5.3.1 Sterility
The leukocyte removal filter should undergo a confirmed sterilization process. Note
1 For suitable sterilization methods, see Appendix 1.
2 GB/T14233.2 specifies the sterility test method, which is not suitable for factory inspection. 5.3.2 Bacterial endotoxins
Prepare the extract according to the method in Appendix B. The extraction medium injected into each set of leukocyte removal filter shall not exceed 80mL. Tested according to the method specified in GB/T14233.2, the bacterial endotoxin content shall be less than 0.5EU/mL. 2
5.4 Filtration performance
5.4.1 Residual leukocyte count
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When tested according to the provisions of Appendix C or Appendix D (Arbitration Law), the residual leukocyte count of 1 unit of whole blood or red blood cell suspension prepared by the RF type leukocyte removal filter shall be less than 2.5×10°; the number of residual white blood cells in 1 unit of single-donated platelet suspension or 10 units of mixed platelet suspension prepared by PF type leukocyte-removal filter shall be less than 1.0×10%. 5.4.2 Free hemoglobin
When tested according to Appendix E or other equivalent methods, the free hemoglobin in 1 unit of whole blood or red blood cell suspension prepared by RF type leukocyte-removal filter shall be less than 530mg/L.
5.4.3 Red blood cell/platelet recovery rate
When tested according to Appendix F or other equivalent methods, the red blood cell recovery rate after filtration of 1 unit of whole blood or red blood cell suspension prepared by RF type leukocyte-removal filter shall not be less than 85%; the platelet recovery rate after filtration of 1 unit of single-donated platelet suspension or 10 units of mixed platelet suspension prepared by PF type leukocyte-removal filter shall not be less than 80%. 5.4.4 Relative change rate of platelet hypotonic shock When tested according to Appendix G or other equivalent methods, the relative change rate of platelet hypotonic shock after filtration of 1 unit of single-collected platelet suspension or 10 units of mixed platelet suspension prepared by PF type leukocyte removal filter shall be less than 10%. 5.5 Biocompatibility Parts in contact with blood or blood components shall not release any substances that have adverse effects on the human body. The following items shall be evaluated in accordance with CB/T16886.1: a) pyrogen; b) hemolysis (hemolysis rate shall be less than 5%) c) acute systemic toxicity: d) cytotoxicity (should be no greater than grade 2); e) intradermal irritation; f) skin sensitization. GB/T14233.2 gives the relevant biological test methods. Appendix H gives the hemolytic test method. 6 Inspection rules
6.1 Type inspection
6.1.1 Type inspection should be carried out in the following cases: a) When a new product is put into production, the source of the filter material or the formula is changed; b) When there is a major change in the process or structure that may affect the performance of the leukocyte removal filter; c) At least once a year in continuous production; d) When production is resumed after suspension and rectification;
e) When required by the contract or the management department. 6.1.2 During type inspection, the biocompatibility evaluation shall be carried out in accordance with the provisions of GB/T16886.1-2001. Five sets of leukocyte removal filters shall be randomly inspected for physical properties, and three sets shall be randomly inspected for filtration performance. 6.1.3 If all type inspection items are qualified, the type inspection is passed. If the type inspection fails, mass production shall not be carried out. 6.2 Factory inspection (recommended)
6.2.1 Factory inspection shall be sampled in accordance with the provisions of GB/T2828. 6.2.2 The same post-processing amount of filter material constitutes a production batch. 6.2.3 The items of physical requirements for factory inspection, non-conforming classification, inspection level (IL) and qualified quality level (AQL) shall be specified in Table 1.3
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Table 1 Inspection level and qualified quality level table of leukocyte removal filter factory inspection Item
Fitness
Connection firmness
Particle content
Protective cover
Non-conforming classification
6.2.4 Each production batch shall also be tested for reducing substances (5.2.1), pH (5.2.3), ultraviolet absorbance (5.2.5) and bacterial endotoxins (5.3.2).
6.2.5 Products of the same sterilization process constitute a sterilization batch, and each sterilization batch shall be tested for Monitor the sterilization effect using a validated method (5.3.1). Products sterilized with ethylene oxide can only be shipped after the residual ethylene oxide content after sterilization is controlled below the specified value (5.2.6). 7 Marking
7.1 The single package should be marked with at least the following information: a) Text description of the contents:
b) Use the graphic symbol given in YY/T0313 to indicate that the leukocyte removal filter is sterile; c) Pyrogen-free:
d) Use the single-use graphic symbol given in YY/T0313 or equivalent instructions; e) Batch number, starting with the word "batch";
f) Expiration date and month (must be clearly identifiable); g) Product markings specified in Chapter 3;
h) Name and address of the manufacturer and/or distributor. 7.2 The packaging should be marked with at least the following information: a) Text description of the contents;
b) Number of leukocyte removal filters:
c) Use the graphic symbol given in YY/T0313 to indicate that the leukocyte removal filter is sterile; d) Batch number, starting with the word "batch";
e) Expiration date and month;
f) Product markings specified in Chapter 3;
g) Name and address of the manufacturer and/or distributor. 7.3 The information on the outer packaging should comply with YY/T0313. 8 Packaging
8.1 Leukocyte removal filters should be packaged individually to keep them sterile during the storage period. 8.2 Each set of leukocyte removal filters is a single package, and the single package should leave traces of opening after opening. 8.3 There should be no visible foreign matter in the single package. 4
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Appendix A
(Standard Appendix)
Preparation of Chemical Property Test Solution
Take three sets of sterilized samples and connect them to a 500mL glass flask to form a closed circulation system. Add 350ml of water to the flask and keep it at 37°C±1°C. Use a peristaltic pump to act on a medical silicone rubber pump tube as short as possible to circulate the water at a flow rate of 1L/h for 2h. Collect all the liquid and cool it to obtain the test solution. Take the same volume of water and place it in a glass bottle. Prepare a blank control solution in the same way without adding samples. Appendix B
(Standard Appendix)
Preparation of Extract Solution for Bacterial Endotoxin Test Under aseptic conditions, connect the pyrogen-free apparatus 1 (or sterile, pyrogen-free empty bag), the leukocyte removal filter 3 and the extract medium receiving container 2 (or sterile, pyrogen-free empty bag) in the manner shown in Figure B1, open the stop clamp 4, and inject no more than 80mL of pyrogen-free water into the inner cavity of each leukocyte removal filter. After repeated irrigation for 5 times, seal both ends and keep warm in a 37C±1C constant temperature box for 2 hours. The extract solution should not be stored for more than 2 hours. 1-Medium perfusion bag: 2-Medium receiving bag: 3-Leukocyte removal filter: 4-Stop flow diagram B1 Schematic diagram of bacterial endotoxin test extract preparation 5
C1 Principle
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Appendix C
(Standard appendix)
Method for determining the number of residual white blood cells - ordinary optical microscope counting method This method evaluates the leukocyte removal or adsorption effect of the filter by using an ordinary optical microscope to determine the number of residual white blood cells in the blood components after filtering through the leukocyte filter.
C2 Test instruments, reagents and materials
Optical microscope, 50μL sample volume counting plate, crystal violet staining solution [0.1g/L crystal violet dissolved in 1% acetic acid solution, i.e. Turksolution], hemolytic agent, adjustable micropipette (100μl.～1000μL, 20μL~100μL), micropipette tip (accurate to volume 40μL), plastic test tube, 9cm diameter culture III, filter paper. C3 Steps
C3.1 Blood sample preparation
For RF type, use 1 unit of whole blood or red blood cell suspension (200mL blood + anticoagulant) stored in the blood bank within 1d~7d; for PF type, use 1 unit of fresh platelet suspension (mixed platelets prepared by separation of 10 units of whole blood or single-donated platelets) stored in the blood bank within 1d~5d. Connect the leukocyte removal filter through a pipeline, and let the blood flow through the leukocyte filter under a net pressure difference of 1m. The blood that flows out is collected in a clean weighing container to obtain a blood sample, and the volume of the filtered blood sample is measured. Note: The nominal volume of a unit of blood, 200mL, does not include the content of blood anticoagulant. C3.2 Residual self-cell counting
Mix the blood sample filtered in C3.1. Take 100μL of whole blood or red blood cell suspension (its hematocrit [Hct] should be less than 60%) and add it to a plastic test tube containing 40μL of hemolytic agent, and repeatedly blow it several times until there are no more red blood cells attached to the micropipette tip. Then add 360L of crystal violet dye solution to the above mixture and mix it, and the final volume is 500μL.
Take 100μL of platelets and add them to a plastic test tube containing 400l of crystal violet dye solution, and repeatedly blow it several times until there are no more platelets attached to the micropipette tip, and the final volume is 500μL. Operate 3 tubes in parallel. Cover the counting plate with a glass slide and fill the counting chamber with the sample (do not overflow); then place the counting plate in the culture III with wet filter paper and cover it. After standing for 20 minutes, place the counting plate under a microscope to count the white blood cells. C4 Result Representation
Remaining white blood cell count:
- Average white blood cell count of 3 tubes of samples, pieces, where: n--
- Volume of blood components after filtration, mL; V-
V.—-Volume of blood sample for counting, 10μL; 10—conversion factor, 1mL=10μL.
1It is advisable to use talc-free gloves to avoid mistaking talc particles for white blood cells nv
2Blood components without white blood cells can be used as controls to confirm the results of crystal violet staining in the laboratory. 3Due to the variation of white blood cell morphology during the refrigeration process, the counting accuracy may be affected. 6
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4Demonstration of the accuracy of the counting method The sample can be serially diluted using a red blood cell suspension with a known white blood cell concentration (using a red blood cell suspension that contains almost no white blood cells to be filtered through two sets of filters), and the count value can be compared with the expected value. 5The accuracy of this method is not clear when the white blood cell concentration is less than 1 white blood cell/μL. Appendix D
(Standard Appendix)
Method for determination of residual leukocyte count
D1 Principle
Fluorescence microscopy counting method (arbitration method)
This method evaluates the leukocyte removal or adsorption effect of the filter by using a fluorescence microscope to determine the number of residual leukocytes in the blood components after filtering through the leukocyte filter.
D2 Test instruments, reagents and materials
D2.1 Fluorescence microscope: blue excitation light, wavelength 405nm~490nm. D2.250μL sample volume counting plate: the counting chamber volume is 50μL, and the total number of leukocytes in the upper and lower counting chambers is counted. D2.3 Fluorescence reagent (P1): 5mg of propidium iodide (PI), 100mg of sodium citrate and 30μL of TritonX-100. Dissolve in 100mL of steamed stuffing water and filter through a microporous filter membrane with a pore size of 0.22um to obtain the PI fluorescent reagent. Store at 4℃, dark. D3 Steps
D3.1 Preparation of blood samples
Same as C3.1.
D3.2 Count of remaining white blood cells
Take 100uL of the filtered and mixed blood sample and dilute it with PI fluorescent reagent at a ratio of 1:9 or an appropriate ratio, and let it stand for 20 minutes. Use a 50μl volume counting plate and count the white blood cells that emit red fluorescence under a fluorescence microscope at a wavelength of 405nm to 490nm. Operate 3 tubes in parallel. D4 Result Expression
Number of Remaining White Blood Cells
Where: n—average white blood cell count of 3 tubes, pieces; V—volume of filtered blood components, mL; Va——volume of blood sample for counting, 5μL, 10 conversion factor. 1mL=10°μL
E1 Principle
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Appendix E
(Standard Appendix)
Determination of Free Hemoglobin (O-Tolidine Method) Hemoglobin is a chromogenic protein with a relative molecular mass of 64.458. After being oxidized by O-Tolidine, it develops color. Its color development reaction is divided into two phases: the first phase oxidation product is blue, the reaction is carried out at about pH=4.6, and the absorption peak is at 630nm. The second phase oxidation product is yellow, the reaction is at pH=1.5, and the absorption peak is at 435nm.
This method is to pass blood through a filter, collect the blood components filtered by the filter, and centrifuge to obtain the upper plasma, and measure the hemoglobin in it to evaluate the damage of the filter to red blood cells. E2 Test instruments and reagents
E2.1 Instruments
Constant temperature water bath, spectrophotometer, centrifuge. E2.2 Reagents
o-Tolidine solution (weigh 0.25 g of o-Tolidine, dissolve in 90 mL of glacial acetic acid, and dilute to 100 mL with distilled water. The solution can be stored in the refrigerator for 8 to 12 weeks. The color will gradually darken during the storage period. If the color is too dark, it should be re-prepared), 1% by volume hydrogen peroxide solution (freshly prepared), 10% by volume acetic acid solution, and hemoglobin (Hb) standard bath solution (p(Hb) - 0.1 mg/LJ).
E3 Operating steps
E3.1 Sample preparation
Take 10 mL of whole blood or red blood cell suspension filtered by RF type leukocyte removal filter, centrifuge at 1190g1200g for 20 min, and aspirate the upper plasma or supernatant; take 20 μL of plasma or supernatant and place it in a plastic test tube. , add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix. Operate 3 tubes in parallel.
E3.2 Preparation of blank solution and standard solution
Take a plastic test tube and add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix, and prepare blank solution. Take another 3 plastic test tubes and add 20μL of human hemoglobin application standard solution Cp(Hb)=0.1mg/LJ, 1mL of o-tolidine solution, and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10% acetic acid 10mL of the solution was mixed to prepare a standard solution.
E3.3 Determination of free hemoglobin
Use a spectrophotometer at a wavelength of 435nm and take the blank solution as a reference to determine the absorbance of the standard solution and the sample respectively. E4 Calculation of results
Wherein: As is the average absorbance value of the sample;
AR is the average absorbance value of the standard solution;
100 is the hemoglobin content of the application standard solution, mg/mLCH
Free hemoglobin content of plasma or blood supernatant, mg/mL. (E1)
F1 Principle
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Appendix F
(Appendix to the standard)
Method for determining the recovery rate of red blood cells and platelets This method is to measure the recovery rate of whole blood or red blood cells. The adsorption of red blood cells and platelets by the filter is evaluated by the changes in the number of red blood cells and platelets in the blood components before and after filtration of the liquid and platelet suspension. F2 Test instruments and apparatus
Coulter blood cell counter, right-angle centrifuge, hematocrit tube (Wintrobe tube), capillary pipette. Note: The slender part of the capillary pipette must be more than 11cm, and the end of the tube can reach the bottom of the hematocrit tube (it can also be replaced by a 1mL syringe and a long puncture needle). F3 Blood samples and measurement steps
F3.1 Determination of blood cell concentration
For RF type, use 1 unit of whole blood (200mL blood + anticoagulant) stored in the blood bank within 1d to 7d. For PF type, use 1 unit of fresh platelet suspension for 1d to 5d (mixed platelets prepared by separation of 10 units of whole blood or 1 unit of single-collected platelets). Take 2 mL of blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter; then connect the whole blood or platelet suspension to the leukocyte removal filter through a pipeline, and let the whole blood or platelet suspension flow through the leukocyte filter under a static pressure difference of 1 m. Take 2 mL of the filtered and mixed blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter. The red blood cell concentration R of the blood sample before filtration = the reading value of the red blood cell concentration counter of the blood sample before filtration × 10*μL-1 The red blood cell concentration R of the blood sample after filtration = the reading value of the red blood cell concentration counter of the blood sample after filtration × 10°L-1 The platelet concentration P of the blood sample before filtration - the reading value of the platelet concentration counter of the blood sample before filtration × 10°μL-! The platelet concentration P of the blood sample after filtration = the reading value of the platelet concentration counter of the blood sample after filtration × 10°μL-1F3.2 Hematocrit determination
Use a capillary pipette to absorb 1mL of whole blood or red blood cell suspension before and after filtration, insert it into the bottom of the W tube, and then slowly inject the blood to the 100mm scale, paying attention to prevent the generation of bubbles. Centrifuge at a relative centrifugal force of 2264g for 30min in a right-angle centrifuge, read the number of millimeters of red blood cell sinking, and then centrifuge for 10min until the red blood cells will not drop. Read the number of millimeters occupied by red blood cells (based on the surface of the purple-black red blood cell layer) which is the number of liters of red blood cell volume in 100mL of blood. F3.3 Calculation of blood volume
Blood volume - (weight of blood containing blood bag - weight of empty blood bag) × blood density Note: ACD anticoagulated whole blood or red blood cell density = 1.040/mL: CPD anticoagulated whole blood or red blood cell density - 1.053g/mLt platelet density = 1.030g/mL.
F4 Calculation of red blood cell and platelet recovery rate
F4.1 Calculation method of red blood cell recovery rate 1
Red blood cell recovery rate (%):
Where: R red blood cell concentration of blood sample before filtration; R~red blood cell concentration of blood sample after filtration; V blood sample volume before filtration, mL;
Vh—blood sample volume after filtration, mL. F4.2 Calculation method of red blood cell recovery rate 22. Calculation method of red blood cell recovery rate2. Calculation method of red blood cell recovery rate2. Calculation method of red blood cell recovery rate2. Calculation method of red blood cell recovery rateWhen the accuracy is not clear. Appendix D
(Standard Appendix)
Method for determination of residual leukocyte count
D1 Principle
Fluorescence microscope counting method (arbitration method)
This method evaluates the leukocyte removal or adsorption effect of the filter by measuring the number of residual leukocytes in the blood components after filtering through the leukocyte filter with a fluorescence microscope.
D2 Test instruments, reagents and materials
D2.1 Fluorescence microscope: blue excitation light, wavelength 405nm~490nm. D2.250μL sample volume counting plate: the counting chamber volume is 50μL, and the total number of leukocytes in the upper and lower counting chambers is counted. D2.3 Fluorescence reagent (P1): 5mg of propidium iodide (PI), 100mg of sodium citrate and 30μL of TritonX-100. Dissolve in 100mL of steamed stuffing water and filter through a microporous filter with a pore size of 0.22um to obtain the PI fluorescent reagent. Store at 4℃, dark place. D3 Steps
D3.1 Preparation of blood sample
Same as C3.1.
D3.2 Count of remaining white blood cells
Take 100uL of the filtered and mixed blood sample and dilute it with PI fluorescent reagent at 1:9 or appropriate ratio, and let it stand for 20min. Use a 50μl volume counting plate to count the white blood cells that emit red fluorescence under a fluorescence microscope at a wavelength of 405nm~490nm. Operate 3 tubes in parallel. D4 Result Expression
Number of Remaining White Blood Cells
Where: n—average white blood cell count of 3 tubes, pieces; V—volume of filtered blood components, mL; Va——volume of blood sample for counting, 5μL, 10 conversion factor. 1mL=10°μL
E1 Principle
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Appendix E
(Standard Appendix)
Determination of Free Hemoglobin (O-Tolidine Method) Hemoglobin is a chromogenic protein with a relative molecular mass of 64.458. After being oxidized by O-Tolidine, it develops color. Its color development reaction is divided into two phases: the first phase oxidation product is blue, the reaction is carried out at about pH=4.6, and the absorption peak is at 630nm. The second phase oxidation product is yellow, the reaction is at pH=1.5, and the absorption peak is at 435nm.
This method is to pass blood through a filter, collect the blood components filtered by the filter, and centrifuge to obtain the upper plasma, and measure the hemoglobin in it to evaluate the damage of the filter to red blood cells. E2 Test instruments and reagents
E2.1 Instruments
Constant temperature water bath, spectrophotometer, centrifuge. E2.2 Reagents
o-Tolidine solution (weigh 0.25 g of o-Tolidine, dissolve in 90 mL of glacial acetic acid, and dilute to 100 mL with distilled water. The solution can be stored in the refrigerator for 8 to 12 weeks. The color will gradually darken during the storage period. If the color is too dark, it should be re-prepared), 1% by volume hydrogen peroxide solution (freshly prepared), 10% by volume acetic acid solution, and hemoglobin (Hb) standard bath solution (p(Hb) - 0.1 mg/LJ).
E3 Operating steps
E3.1 Sample preparation
Take 10 mL of whole blood or red blood cell suspension filtered by RF type leukocyte removal filter, centrifuge at 1190g1200g for 20 min, and aspirate the upper plasma or supernatant; take 20 μL of plasma or supernatant and place it in a plastic test tube. , add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix. Operate 3 tubes in parallel.
E3.2 Preparation of blank solution and standard solution
Take a plastic test tube and add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix, and prepare blank solution. Take another 3 plastic test tubes and add 20μL of human hemoglobin application standard solution Cp(Hb)=0.1mg/LJ, 1mL of o-tolidine solution, and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10% acetic acid 10mL of the solution was mixed to prepare a standard solution.
E3.3 Determination of free hemoglobin
Use a spectrophotometer at a wavelength of 435nm and take the blank solution as a reference to determine the absorbance of the standard solution and the sample respectively. E4 Calculation of results
Wherein: As is the average absorbance value of the sample;
AR is the average absorbance value of the standard solution;
100 is the hemoglobin content of the application standard solution, mg/mLCH
Free hemoglobin content of plasma or blood supernatant, mg/mL. (E1)
F1 Principle
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Appendix F
(Appendix to the standard)
Method for determining the recovery rate of red blood cells and platelets This method is to measure the recovery rate of whole blood or red blood cells. The adsorption of red blood cells and platelets by the filter is evaluated by the changes in the number of red blood cells and platelets in the blood components before and after filtration of the liquid and platelet suspension. F2 Test instruments and apparatus
Coulter blood cell counter, right-angle centrifuge, hematocrit tube (Wintrobe tube), capillary pipette. Note: The slender part of the capillary pipette must be more than 11cm, and the end of the tube can reach the bottom of the hematocrit tube (it can also be replaced by a 1mL syringe and a long puncture needle). F3 Blood samples and measurement steps
F3.1 Determination of blood cell concentration
For RF type, use 1 unit of whole blood (200mL blood + anticoagulant) stored in the blood bank within 1d to 7d. For PF type, use 1 unit of fresh platelet suspension for 1d to 5d (mixed platelets prepared by separation of 10 units of whole blood or 1 unit of single-collected platelets). Take 2 mL of blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter; then connect the whole blood or platelet suspension to the leukocyte removal filter through a pipeline, and let the whole blood or platelet suspension flow through the leukocyte filter under a static pressure difference of 1 m. Take 2 mL of the filtered and mixed blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter. The red blood cell concentration R of the blood sample before filtration = the reading value of the red blood cell concentration counter of the blood sample before filtration × 10*μL-1 The red blood cell concentration R of the blood sample after filtration = the reading value of the red blood cell concentration counter of the blood sample after filtration × 10°L-1 The platelet concentration P of the blood sample before filtration - the reading value of the platelet concentration counter of the blood sample before filtration × 10°μL-! The platelet concentration P of the blood sample after filtration = the reading value of the platelet concentration counter of the blood sample after filtration × 10°μL-1F3.2 Hematocrit determination
Use a capillary pipette to absorb 1mL of whole blood or red blood cell suspension before and after filtration, insert it into the bottom of the W tube, and then slowly inject the blood to the 100mm scale, paying attention to prevent the generation of bubbles. Centrifuge at a relative centrifugal force of 2264g for 30min in a right-angle centrifuge, read the number of millimeters of red blood cell sinking, and then centrifuge for 10min until the red blood cells will not drop. Read the number of millimeters occupied by red blood cells (based on the surface of the purple-black red blood cell layer) which is the number of liters of red blood cell volume in 100mL of blood. F3.3 Calculation of blood volume
Blood volume - (weight of blood containing blood bag - weight of empty blood bag) × blood density Note: ACD anticoagulated whole blood or red blood cell density = 1.040/mL: CPD anticoagulated whole blood or red blood cell density - 1.053g/mLt platelet density = 1.030g/mL.
F4 Calculation of red blood cell and platelet recovery rate
F4.1 Calculation method of red blood cell recovery rate 1
Red blood cell recovery rate (%):
Where: R red blood cell concentration of blood sample before filtration; R~red blood cell concentration of blood sample after filtration; V blood sample volume before filtration, mL;
Vh—blood sample volume after filtration, mL. F4.2 Calculation method of red blood cell recovery rate 2When the accuracy is not clear. Appendix D
(Standard Appendix)
Method for determination of residual leukocyte count
D1 Principle
Fluorescence microscope counting method (arbitration method)
This method evaluates the leukocyte removal or adsorption effect of the filter by measuring the number of residual leukocytes in the blood components after filtering through the leukocyte filter with a fluorescence microscope.
D2 Test instruments, reagents and materials
D2.1 Fluorescence microscope: blue excitation light, wavelength 405nm~490nm. D2.250μL sample volume counting plate: the counting chamber volume is 50μL, and the total number of leukocytes in the upper and lower counting chambers is counted. D2.3 Fluorescence reagent (P1): 5mg of propidium iodide (PI), 100mg of sodium citrate and 30μL of TritonX-100. Dissolve in 100mL of steamed stuffing water and filter through a microporous filter with a pore size of 0.22um to obtain the PI fluorescent reagent. Store at 4℃, dark place. D3 Steps
D3.1 Preparation of blood sample
Same as C3.1.
D3.2 Count of remaining white blood cells
Take 100uL of the filtered and mixed blood sample and dilute it with PI fluorescent reagent at 1:9 or appropriate ratio, and let it stand for 20min. Use a 50μl volume counting plate to count the white blood cells that emit red fluorescence under a fluorescence microscope at a wavelength of 405nm~490nm. Operate 3 tubes in parallel. D4 Result Expression
Number of Remaining White Blood Cells
Where: n—average white blood cell count of 3 tubes, pieces; V—volume of filtered blood components, mL; Va——volume of blood sample for counting, 5μL, 10 conversion factor. 1mL=10°μL
E1 Principle
YY0329--2002
Appendix E
(Standard Appendix)
Determination of Free Hemoglobin (O-Tolidine Method) Hemoglobin is a chromogenic protein with a relative molecular mass of 64.458. After being oxidized by O-Tolidine, it develops color. Its color development reaction is divided into two phases: the first phase oxidation product is blue, the reaction is carried out at about pH=4.6, and the absorption peak is at 630nm. The second phase oxidation product is yellow, the reaction is at pH=1.5, and the absorption peak is at 435nm.
This method is to pass blood through a filter, collect the blood components filtered by the filter, and centrifuge to obtain the upper plasma, and measure the hemoglobin in it to evaluate the damage of the filter to red blood cells. E2 Test instruments and reagents
E2.1 Instruments
Constant temperature water bath, spectrophotometer, centrifuge. E2.2 Reagents
o-Tolidine solution (weigh 0.25 g of o-Tolidine, dissolve in 90 mL of glacial acetic acid, and dilute to 100 mL with distilled water. The solution can be stored in the refrigerator for 8 to 12 weeks. The color will gradually darken during the storage period. If the color is too dark, it should be re-prepared), 1% by volume hydrogen peroxide solution (freshly prepared), 10% by volume acetic acid solution, and hemoglobin (Hb) standard bath solution (p(Hb) - 0.1 mg/LJ).
E3 Operating steps
E3.1 Sample preparation
Take 10 mL of whole blood or red blood cell suspension filtered by RF type leukocyte removal filter, centrifuge at 1190g1200g for 20 min, and aspirate the upper plasma or supernatant; take 20 μL of plasma or supernatant and place it in a plastic test tube. , add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix. Operate 3 tubes in parallel.
E3.2 Preparation of blank solution and standard solution
Take a plastic test tube and add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix, and prepare blank solution. Take another 3 plastic test tubes and add 20μL of human hemoglobin application standard solution Cp(Hb)=0.1mg/LJ, 1mL of o-tolidine solution, and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10% acetic acid 10mL of the solution was mixed to prepare a standard solution.
E3.3 Determination of free hemoglobin
Use a spectrophotometer at a wavelength of 435nm and take the blank solution as a reference to determine the absorbance of the standard solution and the sample respectively. E4 Calculation of results
Wherein: As is the average absorbance value of the sample;
AR is the average absorbance value of the standard solution;
100 is the hemoglobin content of the application standard solution, mg/mLCH
Free hemoglobin content of plasma or blood supernatant, mg/mL. (E1)
F1 Principle
YY0329-2002
Appendix F
(Appendix to the standard)
Method for determining the recovery rate of red blood cells and platelets This method is to measure the recovery rate of whole blood or red blood cells. The adsorption of red blood cells and platelets by the filter is evaluated by the changes in the number of red blood cells and platelets in the blood components before and after filtration of the liquid and platelet suspension. F2 Test instruments and apparatus
Coulter blood cell counter, right-angle centrifuge, hematocrit tube (Wintrobe tube), capillary pipette. Note: The slender part of the capillary pipette must be more than 11cm, and the end of the tube can reach the bottom of the hematocrit tube (it can also be replaced by a 1mL syringe and a long puncture needle). F3 Blood samples and measurement steps
F3.1 Determination of blood cell concentration
For RF type, use 1 unit of whole blood (200mL blood + anticoagulant) stored in the blood bank within 1d to 7d. For PF type, use 1 unit of fresh platelet suspension for 1d to 5d (mixed platelets prepared by separation of 10 units of whole blood or 1 unit of single-collected platelets). Take 2 mL of blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter; then connect the whole blood or platelet suspension to the leukocyte removal filter through a pipeline, and let the whole blood or platelet suspension flow through the leukocyte filter under a static pressure difference of 1 m. Take 2 mL of the filtered and mixed blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter. The red blood cell concentration R of the blood sample before filtration = the reading value of the red blood cell concentration counter of the blood sample before filtration × 10*μL-1 The red blood cell concentration R of the blood sample after filtration = the reading value of the red blood cell concentration counter of the blood sample after filtration × 10°L-1 The platelet concentration P of the blood sample before filtration - the reading value of the platelet concentration counter of the blood sample before filtration × 10°μL-! The platelet concentration P of the blood sample after filtration = the reading value of the platelet concentration counter of the blood sample after filtration × 10°μL-1F3.2 Hematocrit determination
Use a capillary pipette to absorb 1mL of whole blood or red blood cell suspension before and after filtration, insert it into the bottom of the W tube, and then slowly inject the blood to the 100mm scale, paying attention to prevent the generation of bubbles. Centrifuge at a relative centrifugal force of 2264g for 30min in a right-angle centrifuge, read the number of millimeters of red blood cell sinking, and then centrifuge for 10min until the red blood cells will not drop. Read the number of millimeters occupied by red blood cells (based on the surface of the purple-black red blood cell layer) which is the number of liters of red blood cell volume in 100mL of blood. F3.3 Calculation of blood volume
Blood volume - (weight of blood containing blood bag - weight of empty blood bag) × blood density Note: ACD anticoagulated whole blood or red blood cell density = 1.040/mL: CPD anticoagulated whole blood or red blood cell density - 1.053g/mLt platelet density = 1.030g/mL.
F4 Calculation of red blood cell and platelet recovery rate
F4.1 Calculation method of red blood cell recovery rate 1
Red blood cell recovery rate (%):
Where: R red blood cell concentration of blood sample before filtration; R~red blood cell concentration of blood sample after filtration; V blood sample volume before filtration, mL;
Vh—blood sample volume after filtration, mL. F4.2 Calculation method of red blood cell recovery rate 22 Remaining WBC Count
Take 100uL of the filtered and mixed blood sample and dilute it with PI fluorescent reagent at 1:9 or an appropriate ratio, and let it stand for 20 minutes. Use a 50μl sample counting plate to count the white blood cells that emit dull red fluorescence under a fluorescence microscope at a wavelength of 405nm~490nm. Operate 3 tubes in parallel. D4 Representation of results
Remaining WBC Count
Where: n—average white blood cell count of 3 tubes of samples, pieces; V—volume of filtered blood components, mL; Va——volume of blood sample for counting, 5μL, 10 conversion factor. 1mL=10°μL
E1 Principle
YY0329--2002
Appendix Ebzxz.net
(Standard Appendix)
Determination of free hemoglobin (o-tolidine method) Hemoglobin is a chromoprotein with a relative molecular mass of 64.458. After being oxidized by o-tolidine, the color development reaction is divided into two phases: the first phase oxidation product is blue, the reaction is carried out at about pH=4.6, and the absorption peak is at 630nm. The second phase oxidation product is yellow, the reaction is at pH=1.5, and the absorption peak is at 435nm.
This method is to pass the blood through the filter, collect the blood components filtered by the filter, and centrifuge to take the upper plasma, and measure the hemoglobin in it to evaluate the damage of the filter to the red blood cells. E2 Test instruments and reagents
E2.1 Instruments
Constant temperature water bath, spectrophotometer, centrifuge. E2.2 Reagents
o-Tolidine solution (weigh 0.25 g of o-Tolidine, dissolve in 90 mL of glacial acetic acid, and dilute to 100 mL with distilled water. The solution can be stored in the refrigerator for 8 to 12 weeks. The color will gradually darken during the storage period. If the color is too dark, it should be re-prepared), 1% by volume hydrogen peroxide solution (freshly prepared), 10% by volume acetic acid solution, and hemoglobin (Hb) standard bath solution (p(Hb) - 0.1 mg/LJ).
E3 Operating steps
E3.1 Sample preparation
Take 10 mL of whole blood or red blood cell suspension filtered by RF type leukocyte removal filter, centrifuge at 1190g1200g for 20 min, and aspirate the upper plasma or supernatant; take 20 μL of plasma or supernatant and place it in a plastic test tube. , add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix. Operate 3 tubes in parallel.
E3.2 Preparation of blank solution and standard solution
Take a plastic test tube and add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix, and prepare blank solution. Take another 3 plastic test tubes and add 20μL of human hemoglobin application standard solution Cp(Hb)=0.1mg/LJ, 1mL of o-tolidine solution, and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10% acetic acid 10mL of the solution was mixed to prepare a standard solution.
E3.3 Determination of free hemoglobin
Use a spectrophotometer at a wavelength of 435nm and take the blank solution as a reference to determine the absorbance of the standard solution and the sample respectively. E4 Calculation of results
Wherein: As is the average absorbance value of the sample;
AR is the average absorbance value of the standard solution;
100 is the hemoglobin content of the application standard solution, mg/mLCH
Free hemoglobin content of plasma or blood supernatant, mg/mL. (E1)
F1 Principle
YY0329-2002
Appendix F
(Appendix to the standard)
Method for determining the recovery rate of red blood cells and platelets This method is to measure the recovery rate of whole blood or red blood cells. The adsorption of red blood cells and platelets by the filter is evaluated by the changes in the number of red blood cells and platelets in the blood components before and after filtration of the liquid and platelet suspension. F2 Test instruments and apparatus
Coulter blood cell counter, right-angle centrifuge, hematocrit tube (Wintrobe tube), capillary pipette. Note: The slender part of the capillary pipette must be more than 11cm, and the end of the tube can reach the bottom of the hematocrit tube (it can also be replaced by a 1mL syringe and a long puncture needle). F3 Blood samples and measurement steps
F3.1 Determination of blood cell concentration
For RF type, use 1 unit of whole blood (200mL blood + anticoagulant) stored in the blood bank within 1d to 7d. For PF type, use 1 unit of fresh platelet suspension for 1d to 5d (mixed platelets prepared by separation of 10 units of whole blood or 1 unit of single-collected platelets). Take 2 mL of blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter; then connect the whole blood or platelet suspension to the leukocyte removal filter through a pipeline, and let the whole blood or platelet suspension flow through the leukocyte filter under a static pressure difference of 1 m. Take 2 mL of the filtered and mixed blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter. The red blood cell concentration R of the blood sample before filtration = the reading value of the red blood cell concentration counter of the blood sample before filtration × 10*μL-1 The red blood cell concentration R of the blood sample after filtration = the reading value of the red blood cell concentration counter of the blood sample after filtration × 10°L-1 The platelet concentration P of the blood sample before filtration - the reading value of the platelet concentration counter of the blood sample before filtration × 10°μL-! The platelet concentration P of the blood sample after filtration = the reading value of the platelet concentration counter of the blood sample after filtration × 10°μL-1F3.2 Hematocrit determination
Use a capillary pipette to absorb 1mL of whole blood or red blood cell suspension before and after filtration, insert it into the bottom of the W tube, and then slowly inject the blood to the 100mm scale, paying attention to prevent the generation of bubbles. Centrifuge at a relative centrifugal force of 2264g for 30min in a right-angle centrifuge, read the number of millimeters of red blood cell sinking, and then centrifuge for 10min until the red blood cells will not drop. Read the number of millimeters occupied by red blood cells (based on the surface of the purple-black red blood cell layer) which is the number of liters of red blood cell volume in 100mL of blood. F3.3 Calculation of blood volume
Blood volume - (weight of blood containing blood bag - weight of empty blood bag) × blood density Note: ACD anticoagulated whole blood or red blood cell density = 1.040/mL: CPD anticoagulated whole blood or red blood cell density - 1.053g/mLt platelet density = 1.030g/mL.
F4 Calculation of red blood cell and platelet recovery rate
F4.1 Calculation method of red blood cell recovery rate 1
Red blood cell recovery rate (%):
Where: R red blood cell concentration of blood sample before filtration; R~red blood cell concentration of blood sample after filtration; V blood sample volume before filtration, mL;
Vh—blood sample volume after filtration, mL. F4.2 Calculation method of red blood cell recovery rate 22 Remaining WBC Count
Take 100uL of the filtered and mixed blood sample and dilute it with PI fluorescent reagent at 1:9 or an appropriate ratio, and let it stand for 20 minutes. Use a 50μl sample counting plate to count the white blood cells that emit dull red fluorescence under a fluorescence microscope at a wavelength of 405nm~490nm. Operate 3 tubes in parallel. D4 Representation of results
Remaining WBC Count
Where: n—average white blood cell count of 3 tubes of samples, pieces; V—volume of filtered blood components, mL; Va——volume of blood sample for counting, 5μL, 10 conversion factor. 1mL=10°μL
E1 Principle
YY0329--2002
Appendix E
(Standard Appendix)
Determination of free hemoglobin (o-tolidine method) Hemoglobin is a chromoprotein with a relative molecular mass of 64.458. After being oxidized by o-tolidine, the color development reaction is divided into two phases: the first phase oxidation product is blue, the reaction is carried out at about pH=4.6, and the absorption peak is at 630nm. The second phase oxidation product is yellow, the reaction is at pH=1.5, and the absorption peak is at 435nm.
This method is to pass the blood through the filter, collect the blood components filtered by the filter, and centrifuge to take the upper plasma, and measure the hemoglobin in it to evaluate the damage of the filter to the red blood cells. E2 Test instruments and reagents
E2.1 Instruments
Constant temperature water bath, spectrophotometer, centrifuge. E2.2 Reagents
o-Tolidine solution (weigh 0.25 g of o-Tolidine, dissolve in 90 mL of glacial acetic acid, and dilute to 100 mL with distilled water. The solution can be stored in the refrigerator for 8 to 12 weeks. The color will gradually darken during the storage period. If the color is too dark, it should be re-prepared), 1% by volume hydrogen peroxide solution (freshly prepared), 10% by volume acetic acid solution, and hemoglobin (Hb) standard bath solution (p(Hb) - 0.1 mg/LJ).
E3 Operating steps
E3.1 Sample preparation
Take 10 mL of whole blood or red blood cell suspension filtered by RF type leukocyte removal filter, centrifuge at 1190g1200g for 20 min, and aspirate the upper plasma or supernatant; take 20 μL of plasma or supernatant and place it in a plastic test tube. , add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix. Operate 3 tubes in parallel.
E3.2 Preparation of blank solution and standard solution
Take a plastic test tube and add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10mL of 10% acetic acid solution to mix, and prepare blank solution. Take another 3 plastic test tubes and add 20μL of human hemoglobin application standard solution Cp(Hb)=0.1mg/LJ, 1mL of o-tolidine solution, and 1mL of 1% hydrogen peroxide solution respectively, let stand for 10min after mixing, then add 10% acetic acid 10mL of the solution was mixed to prepare a standard solution.
E3.3 Determination of free hemoglobin
Use a spectrophotometer at a wavelength of 435nm and take the blank solution as a reference to determine the absorbance of the standard solution and the sample respectively. E4 Calculation of results
Wherein: As is the average absorbance value of the sample;
AR is the average absorbance value of the standard solution;
100 is the hemoglobin content of the application standard solution, mg/mLCH
Free hemoglobin content of plasma or blood supernatant, mg/mL. (E1)
F1 Principle
YY0329-2002
Appendix F
(Appendix to the standard)
Method for determining the recovery rate of red blood cells and platelets This method is to measure the recovery rate of whole blood or red blood cells. The adsorption of red blood cells and platelets by the filter is evaluated by the changes in the number of red blood cells and platelets in the blood components before and after filtration of the liquid and platelet suspension. F2 Test instruments and apparatus
Coulter blood cell counter, right-angle centrifuge, hematocrit tube (Wintrobe tube), capillary pipette. Note: The slender part of the capillary pipette must be more than 11cm, and the end of the tube can reach the bottom of the hematocrit tube (it can also be replaced by a 1mL syringe and a long puncture needle). F3 Blood samples and measurement steps
F3.1 Determination of blood cell concentration
For RF type, use 1 unit of whole blood (200mL blood + anticoagulant) stored in the blood bank within 1d to 7d. For PF type, use 1 unit of fresh platelet suspension for 1d to 5d (mixed platelets prepared by separation of 10 units of whole blood or 1 unit of single-collected platelets). Take 2 mL of blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter; then connect the whole blood or platelet suspension to the leukocyte removal filter through a pipeline, and let the whole blood or platelet suspension flow through the leukocyte filter under a static pressure difference of 1 m. Take 2 mL of the filtered and mixed blood sample and measure the red blood cell concentration or platelet concentration on the Coulter blood cell counter. The red blood cell concentration R of the blood sample before filtration = the reading value of the red blood cell concentration counter of the blood sample before filtration × 10*μL-1 The red blood cell concentration R of the blood sample after filtration = the reading value of the red blood cell concentration counter of the blood sample after filtration × 10°L-1 The platelet concentration P of the blood sample before filtration - the reading value of the platelet concentration counter of the blood sample before filtration × 10°μL-! The platelet concentration P of the blood sample after filtration = the reading value of the platelet concentration counter of the blood sample after filtration × 10°μL-1F3.2 Hematocrit determination
Use a capillary pipette to absorb 1mL of whole blood or red blood cell suspension before and after filtration, insert it into the bottom of the W tube, and then slowly inject the blood to the 100mm scale, paying attention to prevent the generation of bubbles. Centrifuge at a relative centrifugal force of 2264g for 30min in a right-angle centrifuge, read the number of millimeters of red blood cell sinking, and then centrifuge for 10min until the red blood cells will not drop. Read the number of millimeters occupied by red blood cells (based on the surface of the purple-black red blood cell layer) which is the number of liters of red blood cell volume in 100mL of blood. F3.3 Calculation of blood volume
Blood volume - (weight of blood containing blood bag - weight of empty blood bag) × blood density Note: ACD anticoagulated whole blood or red blood cell density = 1.040/mL: CPD anticoagulated whole blood or red blood cell density - 1.053g/mLt platelet density = 1.030g/mL.
F4 Calculation of red blood cell and platelet recovery rate
F4.1 Calculation method of red blood cell recovery rate 1
Red blood cell recovery rate (%):
Where: R red blood cell concentration of blood sample before filtration; R~red blood cell concentration of blood sample after filtration; V blood sample volume before filtration, mL;
Vh—blood sample volume after filtration, mL. F4.2 Calculation method of red blood cell recovery rate 21 Sample preparation
Take 10mL of whole blood or red blood cell suspension filtered by RF type leukocyte removal filter, centrifuge at 1190g1200g for 20min, and aspirate the upper plasma or supernatant; take 20μL of plasma or supernatant and place it in a plastic test tube, add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution, respectively, mix and let stand for 10min, then add 10mL of 10% acetic acid solution to mix. Operate 3 tubes in parallel.
E3.2 Preparation of blank solution and standard solution
Take a plastic test tube and add 1mL of o-tolidine solution and 1mL of 1% hydrogen peroxide solution, respectively, mix and let stand for 10min, then add 10mL of 10% acetic acid solution to mix, and prepare blank solution. Take another 3 plastic test tubes and add 20μL of human hemoglobin application standard solution Cp(Hb) = 0.1mg/L, 1mL of o-dimethylamine solution, and 1mL of 1% hydrogen peroxide solution respectively. After mixing, let it stand for 10min, then add 10mL of 10% acetic acid solution to mix and prepare the standard solution.
E3.3 Determination of free hemoglobin
Use a spectrophotometer at a wavelength of 435nm and use the blank solution as a reference to measure the absorbance of the standard solution and the sample respectively. E4 Result calculation
Where: As - average absorbance of the sample;
AR - average absorbance of the standard solution;
100 - hemoglobin content of the hemoglobin application standard solution, mg/mLCH
free hemoglobin content of plasma or blood supernatant, mg/mL. (E1)
F1 Principle
YY0329-2002
Appendix F
(Standard Appendix)
Method for determining the recovery rate of red blood cells and platelets This method is to evaluate the adsorption of red blood cells and platelets by the filter by measuring the changes in the number of red blood cells and platelets in the blood components of whole blood or red blood cell suspension and platelet suspension before and after filtration. F2 Test instruments and apparatus
Coulter blood cell counter, right-angle centrifuge, hematocrit tube (Wintrobe tube), capillary pipette. Note: The slender part of the capillary pipette must be more than 11cm, and the end of the tube can reach the bottom of the hematocrit tube (it can also be replaced by a 1mL syringe and a long puncture needle). F3 Blood sample and measurement steps
F3.1 Blood cell concentration measurement
For RF type, use 1 unit of whole blood (200mL blood + anticoagulant) stored in the blood bank within 1d to 7d. For PF type, use 1 unit of fresh platelet suspension (mixed platelets prepared by separation of 10 units of whole blood or 1 unit of single-donated platelets) for 1d to 5d. Take 2mL of blood sample and measure the red blood cell concentration or platelet concentration on Coulter blood cell counter; then connect the whole blood or platelet suspension to the leukocyte removal filter through a pipeline, and let the whole blood or platelet suspension flow through the leukocyte filter under a static pressure difference of 1m. Take 2mL of the filtered and mixed blood sample and measure the red blood cell concentration or platelet concentration on Coulter blood cell counter. The red blood cell concentration R of the blood sample before filtration = the reading value of the red blood cell concentration counter of the blood sample before filtration × 10*μL-1 The red blood cell concentration R of the blood sample after filtration = the reading value of the red blood cell concentration counter of the blood sample after filtration × 10°L-1 The platelet concentration P of the blood sample before filtration - the reading value of the platelet concentration counter of the blood sample before filtration × 10°μL-! The platelet concentration P of the blood sample after filtration = the reading value of the platelet concentration counter of the blood sample after filtration × 10°μL-1F3.2 Hematocrit determination|
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