このdraftはストックホルム会議(1996.4)ヨーク会議(1997.4)を経て, ISO 10993-4:1992/DRAFT AMMENDMENT 1として正式投票中である。 日本は賛成（コメントなし）とした。 Draft 6 11 May, 1995 Annex D - Evaluation of Hemolytics Properties of Medical Devices (INFORMATIVE) Annex D - Evaluation of Hemolytic Properties of Medical Devices Extensive literature exists describing blood/material interactions. Unfortunately, very few test methods exist which are reliable, reproducible, and predict clinical performance. This annex will review the known hemolysis test methods and discuss factors pertaining to their ability to characterize medical and dental materials and devices. [Consider moving to main section of document]. 1 Definitions 1.1 Colloidal oncotic pressure: The total influence of the proteins or other large molecular weight substances on the osmotic activity of plasma water.(1 1.2 Hemolysis: The rupture of the erythrocyte membrane with the liberation of hemoglobin, which diffuses into the fluid surrounding the erythrocytes. 1.3 Negative Reference Material: High Density Polyethylene (see ISO 10993-12). 1.4 Packed Red Cells: This component from a unit of human blood is obtained by removal of part of the plasma from whole blood, without further processing. (Properties of human red cells for transfusion: The erythrocyte volume fraction of the component is 0.65 to 0.75. The unit contains all of the original unit's red cells, the greater part of its leukocytes (about 2.5 to 3.0 x 10^9 cells) and a varying content of platelets depending on the method of centrifugation.) 1.5 Washed Red Cells: A red cell suspension obtained from one unit of whole blood after removal of plasma and washing in an isotonic solution. (Properties: This component is a red cell suspension from which most of the plasma, leukocytes and platelets have been removed. The amount of residual plasma will depend upon the washing protocol. Storage time should be as short as possible after washing and certainly not longer than 24 hours at 1C to 6C. 1.6 Whole Blood: Whole blood is unfractionated blood collected from a suitable donor. 2 Causes of hemolysis 2.1 Mechanical forces - pressure: The red blood cell membrane is a semipermeable membrane. A pressure differential will occur when two solutions of different concentrations are separated by such a membrane. Osmotic pressure occurs when the membrane is impermeable to passive solute movement. These pressure differentials can cause erythrocyte swelling and cell membrane rupture with release of free hemoglobin.(1 2.2 Mechanical forces - rheologic: Factors which influence velocity of blood flow, shear forces and other forces that can deform the red blood cell membrane can cause membrane rupture. 2.3 Biochemical factors: Changes to membrane structure on a molecular level can modify the strength and elastic properties of the erythrocyte membrane. A deficiency of nutritional factors or metabolic energy (ATP) can result in loss of the discoid shape and microvesiculation of hemoglobin. Bacterial toxins, and other chemicals can compromise the red cell membrane. These changes may cause membrane rupture at lower than expected osmotic pressures. A test to determine the pressure at which an erythrocyte membrane ruptures is termed osmotic fragility. 3 Clinical significance of hemolysis 3.1 Elevated levels of free plasma hemoglobin may induce toxic effects or initiate processes which may stress the kidneys or other organs.(1 3.2 Intravascular hemolysis may promote thrombosis by liberating phospholipids that activate the intrinsic coagulation pathway.(2 4 Medical devices and hemolysis50 Pass / Fail criteria: Hemolysis is a function of time, material properties such as, surface energy, surface morphology, and surface chemistry. Hemolysis is also a function of shear stress, cell-wall interaction, character of adsorbed protein layers, flow stability, air entrainment, and variations of blood source, age and chemistry.(3,4,5 These variables need to be adequately controlled for comparisons of hemolytic potential among materials and medical devices. The spectrum of methods for evaluating hemolysis vary from simplified to highly complicated models. Specific in vitro and in vivo models with flowing blood have been published. Studies of hemolytic potential are relative comparisons against materials or medical devices tested in the same model by a specific laboratory rather than absolute measures. In vitro methods may be able to quantify small amounts of hemolysis that may not be measurable in vivo and therefore possibly of no clinical consequence. It is not possible to define a universal level for acceptable and unacceptable amounts of hemolysis for all medical devices and applications. The effect of a device on hemolysis may be masked in the short term by the trauma of the surgical procedure. A device may cause a substantial amount of hemolysis, but be the only treatment available in a life- threatening situation. Intuitively, a blood compatible material is non-hemolytic. In practice, many devices cause hemolysis, but their clinical benefit outweighs the risk associated with the hemolysis. Therefore when a device causes hemolysis, it is important to confirm that the device provides a clinical benefit and that the hemolysis is within acceptable limits clinically. Acceptance criteria should be justified based on some form of Risk and Benefit assessment. The following questions are suggestions for developing such an assessment: i) What is the duration of exposure of the device to the patient? ii) How much hemolysis does the material or device cause? Does the hemolysis continue for the entire time the device is exposed to the patient? Does hemolysis continue after removal of the device? iii) What are the relative Risks and Benefits of other available methods for treating the condition? iv) What are the hemolytic properties of these known treatments? How does the device in question compare to these other treatments? v) How effective is the test device compared with other forms of treatment? A more effective device may cause more hemolysis during use but the additional effectiveness might increase the benefit to the patient. 5 Hemolysis Testing - General Considerations 5.1 Hemoglobin Assay - Classically, three analytical methods have been used to determine hemoglobin (Hb)(6.The first classical method, cyanmethemoglobin detection, is that recommended by the International Committee for Standardization in Haematology.(7 The cyanmethemoglobin (hemiglobincyanide; HiCN) analysis has the advantage of convenience, ease of automation, and the availability of a primary reference standard (HiCN). The method is based on the oxidation of Hb and subsequent formation of hemiglobincyanide which has a broad absorption maxima at 540 nm. Lysing agents such as detergents are used which, in addition to releasing Hb from the erythrocyte, decrease the turbidity (a source of interference as false absorbance at 540 nm) from protein precipitation (Note: A red cell lysing agent should be used for total hemoglobin measurements. Lysing agents should not be used for cell-free, plasma hemoglobin assays. False negatives may occur if the cells are fixed with glutaraldehyde or formaldehyde or the hemoglobin is precipitated.) The broad absorption band of HiCN in this region enables the use of simple filter type photometers as well as narrow band spectrophotometers for detection. Commercially available blood analyzers have automated quantitations based on either of the two strategies. The use of the HiCN reference standard provides comparability among all laboratories employing this method. The major disadvantage is the potential health risk in using the cyanide solutions. Cyano-reagents are themselves toxic by various routes of exposure, and additionally, release HCN upon acidification. Disposal of reagents and products has also become a considerable concern and expense. The other two classical methods are more cumbersome and not widely used today. The oxyhemoglobin method dependsontheformationofHbO2 during ammonia-hydroxide treatment, and spectrophotometric detection of this product. No stable reference standards exist for this method. The third classical method is based on determining the hemoglobin iron concentration in solution. Iron is first separated from Hb, usually by acid or by ashing. It is then titrated with TiCl3 or complexed with a reagent to develop color that can be measured photometrically. This method is too complex for routine work, and is rarely used. Recent techniques, although not necessarily superior analytically, may be inherently safer and more environmentally sound. There commended method for determining the concentration of hemoglobin is based on its catalytic effect on the oxidation of a benzidine derivative by hydrogen peroxide. The rate of formation of a colored product (photometrically detected at 600nm) is directly proportional to the hemoglobin concentration. Plasma should be prepared using anticoagulants other than EDTA or oxalates which interfere with H202 oxidation and should be avoided. The advantages of this method are ease of automation (commercial equipment), elimination of cyano-reagents, and the availability of Hb standard sets which are calibrated against the HiCN primary reference standards. Additionally, detection limits (as low as 1.0 mg/dL) are comparable to the hemiglobincyanide method.(6,7 5.2 Blood and Blood Component Preservation - This section presents the best demonstrated practices for the preservation of human blood components by the American Association of Blood Banks and the Council of Europe.(8,15 Anticoagulant solutions have been developed for use in blood collection that prevent coagulation and permit storage of red cells for a certain interval of time. These solutions all contain sodium citrate, citric acid, and glucose; additionally, some contain adenine, guanosine and phosphate.(9,10.11,12,13,14 Blood clotting is prevented by citrate binding of calcium. Red blood cells metabolize glucose during storage. Two molecules of adenosine triphosphate (ATP) are generated by phosphorylation of adenosine diphosphate ADP for each glucose molecule metabolized via the Embden-Myerhoff anaerobic glycolysis cycle. The ATP molecules support the energy requirements of the erythrocyte in maintenance of membrane flexibility and certain membrane transport functions. Conversion of ATP to ADP releases the energy necessary to support these functions. In order to prolong storage time, alkalinity must be reduced by addition of citric acid to the anticoagulant solution. This provides a suitably high hydrogen ion concentration at the beginning of red blood cell storage at 4C. Increasing acidity during storage reduces the rate of glycolysis. The adenosine nucleotides (ATP, ADP, AMP) are depleted during storage and the addition of adenosine to the anticoagulant solution permits synthesis of replacement AMP, ADP, and ATP. A considerable portion of glucose and adenine is removed with plasma when red cell concentrates are prepared. Sufficient viability of the red cells can only be maintained after removal of plasma if the cells are not over-concentrated. Normal CPD-adenine red cell concentrates should not have an erythrocyte volume fraction greater than 0. 70. Even if more than 90% of the plasma is removed, red cell viability can be maintained by addition of an additive or suspension medium. Sodium chloride, adenine and glucose are necessary for viability while mannitol or sucrose may be used to further stabilize the cell membrane and prevent hemolysis.(15 The suitability of containers for the storage of blood products is evaluated by various methods that measure the quality of the blood product.(8,15 The container with blood product containing an appropriate anticoagulant is stored upright at lC to 6C under static conditions. At predetermined intervals, the amount of cell-free, plasma hemoglobin measured to assess the viability and quality of the stored product. The quality of the stored product may be enhanced by gentle mixing once a week. Evaluation of storage in the container indirectly evaluates the permeability of the container to waste carbon dioxide from red cell metabolism. 5.3 Red cell membrane stabilizing agents - In vivo, the red cell has a discoid shape. During in vitro storage, discocytes may become crenated and form echinocytes. Scanning electron micrographs of echinocytes show numerous microvesicles on the cell surface. Detachment of the microvesicles from the cell results in an increase in total plasma hemoglobin. The hemoglobin in the suspending medium during red blood cell storage is found as both free hemoglobin and microvesicles shed by the red blood cell. Spontaneous or osmotically induced red cell lysis proceeds through the transformation of discocytes to echinocytes. The cellular mechanism(s) supporting discocyte morphology of red cells is not known. Chemicals that are known to preserve discocyte morphology during in vitro storage are termed 'membrane stabilizers.' Membrane stabilizers reduce the amount of hemolysis which occurs during in vitro storage of whole blood and red cells. Agents known to stabilize red cell membranes include di(2-ethylhexyl)phthalate(DEHP) and lipid soluble anesthetics.(16,17,18,19 DEHP has been shown to reduce microvesiculation, hemolysis and the increase in osmotic fragility which red blood cells undergo during refrigerated storage.(20 The rate of microvesiculation can also be reduced by addition of sucrose, mannitol and sorbitol (3OmM final concentration each) to a SAG medium (Sodium, Adenine, Glucose- 150 mM NaCl, 50 mM glucose, 1.23mM adenine) for red blood cell storage.(21 5.4 Protection of employees handling blood-Written procedures are necessary for protecting employees receiving, handling, and working with potentially contaminated human blood (for example, as noted to be in compliance with 29 CFR, US Code of Federal Regulations - 1910.1030). Potentially contaminated materials include blood and other body fluids and products, equipment which has been or may have been in contact with blood or other body fluids, and materials used in the culturing of organisms causing blood borne infections.(22 5.5 Blood Collection (Phlebotomy) - While it is not possible to guarantee 100% sterility of the skin surface for phlebotomy, a strict, standardized procedure for preparation of the phlebotomy area should exist. It is especially important to allow the aseptic solution to dry on the skin surface prior to venepuncture and that no further contact is made with the skin surface before the phlebotomy needle has been inserted.(2 A closed container system is preferred for blood collection for the prevention of microbial contamination. Needle punctures in the rubber seal of the specimen vial should be completely closed after withdrawal of the needles, otherwise the partial vacuum created following cooling may draw in contaminated air.(2 Blood collected in an open system may be contaminated by exposure to room air and is not considered sterile. Microbial contamination is a known cause of hemolysis. 5.6 Species selection - Ideally, hemolysis testing should be done with human erythrocytes. However, several factors can make such a choice difficult or impossible. In some countries, human blood supplies are limited and must be reserved for human transfusion. All blood, regardless of species, must be handled carefully as there is always the risk of contamination. Health criteria for human and animal donors should also be considered. All blood has a limited "shelf life" and it may be more difficult to obtain human blood cells on a timely basis. If animal erythrocytes are used, attention should be paid to ensure one hundred percent hemolysis of positive controls due to differences in membrane stability among animal species. Negative controls should cause minimal hemolysis so that the activity of the test material is not masked. Rabbit and human erythrocytes are reported to have similar hemolytic properties whereas monkey erythrocytes are more sensitive and guinea pig erythrocytes less sensitive.(23 5.7 Evaluation of hemolysis: In vitro, ex vivo, and in vivo exposure to blood or blood components - Hemolysis may be evaluated by exposure of materials or devices under in vitro, in vivo, and ex vivo conditions. In vitro conditions are most often used to evaluate materials. Ex vivo and in vivo conditions are used to evaluate devices, which may contain more than one material. In vivo and ex vivo assessments in animal models or during clinical trials are possible. Justification may be made for either of the following study designs. In the first case, the test device is compared to reference control marketed devices with known acceptable levels of hemolysis. In the second case, the test subject is evaluated for clinically significant consequences of hemolysis. The purpose of in vivo or ex vivo tests is to characterize the hemolytic potential of a medical device. For ex vivo medical devices, the general practice is to recirculate blood through the device using conditions that simulate the intended clinical usage. The initial studies may use fresh or outdated human blood or blood from a nonhuman species. These investigations are followed by ex vivo simulations in an animal model for some medical devices or by limited, controlled studies in humans. The size of the medical device and the intended function influence the design of these studies. 5.8 Direct contact versus indirect methods - Some test methods call for direct contact of the device with red blood cells, while other methods describe the preparation of an extract which is then exposed to red blood cells. Test selection should be based upon the device itself and the conditions in which it will be used. Where elevated temperatures are used in the extraction process, the effect of these temperatures on the test article should be known to avoid obtaining extracts that would not be generated under physiological conditions. 6 Recommended Hemolysis Test Methods for Medical Devices (The following is primarily taken from ASTM F 756-93). 6. 1 In Vitro Methods. The purpose of in vitro tests is to evaluate the effect on isolated erythrocytes. Direct methods determine hemolysis due to physical interactions of red blood cells with either the specimen or the extractables from the test specimen. The dynamic method increases the physical interaction and, presumably, the amount of extraction that occurs. Indirect methods determine hemolysis due to extractables from the test specimen. Indirect methods permit separating the extraction phase from the test system, thereby offering opportunity to exaggerate extraction conditions and distinguish physical surface interactions from chemical effects. [Note: Equipment calibration can be achieved with a Reference Standard for hemoglobin analysis.(8] 6.2 Preparation of erythrocyte suspension - Blood from one human donor is normally sufficient. Pooled blood from no more than three animal donors is normally sufficient to minimize variation between donors. A rationale for the selection of blood donor species should be provided in the report. Whole blood is collected in siliconized, borosilicate glass tubes containing anticoagulant. For the purpose of standardization, whole blood collected in a closed system containing ACD (acid citrate dextrose) or CPD (citrate phosphate dextrose) anticoagulant should be stored no more than 96 hours at lC to 6C. Blood collected in an open system should be used immediately. Heparinized whole blood collected in a closed system should be stored no more than 2 hours at lC to 6C. After dilution of whole blood or washed erythrocytes in crystalloid media, they shall be used within 2 hours because the crystalloid media does not have adequate nutrients for red cell preservation. The cell-free plasma hemoglobin content of whole blood or washed erythrocytes shall be less than 1.0 mg/mL (0.8%) to avoid artifacts. [Note: Sterile crystalloid solutions (saline or phosphate buffered saline) are not required for the assays because the duration of the test (less than 4 hours) is insufficient to allow for significant microbial growth unless the sample has been collected in an open system or grossly contaminated during phlebotomy.] 6.3 Method for Direct Contact with Diluted Blood - Dilute whole blood with isotonic saline (0.9%) to a total hemoglobin concentration of less than 25.0 +- 2.5 mg/mL using siliconized, borosilicate glass containers. This diluted suspension shall be used within 2 hours after its preparation when kept at lC to 6C or in an ice bath. Tests on each material and blood sample combination should be performed in triplicate. Prepare test specimens (10 mm by 50 mm strips or rods of approximately 10 mm length by 1 mm diameter) of material which has been subjected to the same manufacturing processes and sterilization as the final product. Prepare three tubes containing strips or rods of the test specimens and three tubes containing strips or rods of the Negative Reference Material. The amount of test specimens and Negative Reference Material shouldbe1500 mm2 for samples > 0.5 mm thickness and 3000 mm2for samples < 0.5mm thickness. (Note: The value of 15.7 cm2 in ASTM F 756-93 is based on 50 rods of 10 mm x 1 mm x 3.14). The change herein is consistent with ISO 10993-12.) Add 5 mL of diluted blood to each tube. Incubate with the test specimens and Negative Reference Material. Three test preparations are incubated for 4 hours at 37 +- 2C under static conditions and three are incubated for 1 hour at 37 +- 2C under dynamic conditions (mixing on a rocker plate at 30 +- 6 rocks per minute at a maximum of * 45oC from the vertical position. (Note: Dynamic mixing conditions should not cause frothing or protein denaturation which could contribute to red cell lysing.) At the end of the contact period, the test specimens are removed and the diluted blood samples are centrifuged at 100 to 200 G in a standard clinical centrifuge for 15 minutes. Remove and transfer the supernatant fraction to individual siliconized borosilicate glass tubes. Recentrifuge at 700 to 800 G in a standard clinical centrifuge for 5 minutes to precipitate any remaining erythrocytes. Remove and transfer the supernatant into a third borosilicate tube. Analyze the samples for free hemoglobin using the method in Section 1.5.1 6.4 Method for Indirect Contact with Diluted Blood - Saline extracts of the test specimen and Negative Reference Material are prepared. Prepare six replicate tubes containing 4 mL of saline extract and 5 mL of diluted blood with a hemoglobin concentration of 25 +- 2mg/mL. Three test preparations are incubated for 4 hours at 37 +- 2C under static conditions and three are incubated for 1 hour at 37 +- 2C under dynamic conditions. The Negative Reference Material should also undergo static and dynamic testing. At the end of the contact period, the test specimens are removed and the diluted blood samples are centrifuged at 100 to 200 G in a standard clinical centrifuge for 15 min. Remove and transfer the supernatant fraction to individual siliconized borosilicate glass tubes. Recentrifuge at 700 to 800 G in a standard clinical centrifuge for 5 min. to precipitate any remaining erythrocytes. Remove and transfer the supernatant into a third borosilicate tube. Analyze the samples for hemoglobin using the method in Section 1.5.1 (Note: It is important that the extracts be isotonic. Non-isotonic extracts may predispose cells to hemolysis.) 6.5 Hemolysis Tests for Blood Containers - An in vitro hemolysis assay which uses an extract of the blood container materials is recommended in the European Pharmacopeia(24 and in ISO Standard (Plastics Collapsible Containers for Human Blood and Blood Components).(25 The extracts are prepared at 121C for one hour; these are the sterilization conditions of blood containers with liquid anticoagulants. These assays are focused on the container materials and do not assess gas permeability or other quality assurance parameters essential for storage of whole blood and blood products (see section 1.5.2.1 for further discussion of this subject). 7 Publications: 7.1 Current Published Standards containing Hemolysis Test Methods 1. September 1980: Transfusionsbehaltnisse und Zubehor. DIN 58361 Teil 4. 2. 1984: Sterile Plastic Containers for Human Blood and Blood Components. European Pharmacopoeia, Second Edition Part II, Second Fascicule, VI.2.2.2. 1. 3. 1993: Standard Practice for Assessment of Hemolytic Properties of Materials. ASTM Designation: F 756-93 4. 1993: Plastics collapsible containers for human blood and blood components. ISO 3826. 7.2 Published standards that include a requirement for hemolysis testing: 1. February 1981: Materiel Medico-chirurgical - Oxygenateurs. NF S 90-300. 2. December 1985: Medical Surgical Equipment - Single Use Sterile Hemodialyzers and Hemofilters. NF S 90-302. 3. 1992: Selection of Tests for Interactions with Blood. BSEN 30993/4 Bibliography 1. Taber's Cyclopedic Medical Dictionary, 17th Edition, F.A. Davis Company, Philadelphia, PA, 1993. 2. Hoch, J.R., Silver, D., Hemostasis and Thrombosis, in "Vascular Surgery: A Comprehensive Review," 3rd edition, W.S. Moore, ed., W.B. Saunders, Philadelphia, 63-79, 1991. 3. Offeman, R.D., Williams, M.C., Material effects in shear-induced hemolysis, Biomat. Med. Dev. Art. Org., 7:359-391, 1979. 4. Lampert, R.H., Williams, M.C., Effect of surface materials on shear-induced hemolysis, J. Biomed. Mater. Res., 6:499-532, 1972. 5. Obeng, E.K., Cadwallader, D.E., In vivo dynamic method for evaluating the hemolytic potential of intravenous solutions. J. Parenteral Sci. Technol., 43:167-173, 1989. 6. Henry, J. B., Hematology and Coagulation, In: Clinical Diagnosis & Management By Laboratory Methods, 18th Ed., W.B. Saunders Co., Philadelphia, PA, USA, 556-603, 1991. 7. International Committee for Standardization in Haematology (ICSH): Recommendations for reference method for hemoglobinometry in human blood. (ICSH Standard EP 6/2: 1977) and specifications for international hemiglobin cyanide reference preparation (ICSH Standard EP 6/3: 1977). J. Clin. Path., 31:139, 1978. 8. Standards for Blood Banks and Transfusion Services, 16th ed. Bethesda, MD: American Association of Blood Banks, 1994. 9. Anticoagulant Citrate Dextrose Solution. U.S. Pharmacopeia 23:119 (1995). 10. Anticoagulant Acid Citrate Dextrose Solutions (ACD). European Pharmacopoeia 2:209-210 (1989). 11. Anticoagulant Citrate Phosphate Dextrose Solution. U.S. Pharmacopeia 23:119-120 (1995). 12. Anticoagulant Citrate Phosphate Dextrose Adenine Solution, U.S. Pharmacopeia 23:121-122 (1995). 13. Anticoagulant Heparin Solution. U.S. Pharmacopeia 23:122 (1995). 14. Anticoagulant Sodium Citrate Solution. U.S. Pharmacopeia 23:122 (1995). 15. Guide to the Preparation, Use and Quality Assurance of Blood Components. Strasbourg: Council of Europe Publishing and Documentation Service, 1992, pp. 37-38. 16. Estep, T., Pederson, R.A., Miller, T.J., Stupar, K.R., Blood, 64:1270-1276 (1984). 17. Horowitz, B., Stryker, M.H., Waldman, A.A., et al., Vox Sanguinis, 48:150-155 (1985). 18. Rock, G., Tocchi, M., Ganz, P.R., Tackaberry, E.S., Transfusion 24:493-498 (1984). 19. Roth S. Seeman P. Nature new Biol. 231:284-285 1971 . 20. Greenwalt, T.J., McGuinness, C.G., Dumaswala, U.J., Studies in red-blood-cell preservation. 4. Plasma vesicle hemoglobin exceeds free hemoglobin. Vox Sanguinis, 61:14-17, 1991. 21. Stibenz, D., Preservation of resuspended red cell concentrates. Rate of vesiculation and of spontaneous hemolysis. Folia Haematol. Int. Mag. Klin. Morphol. Blutforsch (Germany, East), 114:469-470, 1987. 22. 29 CFR, Code of Federal Regulations - 1910. 1030, "Bloodborne Pathogens." 23. Wennberg, A., Hensten-Pettersen, A., Sensitivity of erythrocytes from various species to in vitro hemolyzation, J. Biomed. Mater. Res., 15:433-435, 1981. 24. European Pharmacopeia VI. 2.2.2. 1, Sterile Plastic Containers for Human Blood and Blood Components. 25. ISO Standard 3826 (Plastics Collapsible Containers for Human Blood and Blood Components).