Toxicology tests-Biological evaluation of medical devices - Hemocompatibility - Activation of the complement system (UNE-EN ISO 10993: 4: Part 4-Selection of tests for blood interactions) and ASTM F1984-99 (Standard Practice for Testing for Whole Complement Activation in Serum by Solid Materials)

 

Test accredited by ENAC (National Acreditation Entitiy).

Test with the Certificate of Good Laboratory Practices (GLPs).

 

            Activation of the complement system is one of the hemocompability tests indicated by the UNE-EN ISO 10993-4 standard: Biological evaluation of medical devices. Part 4: Selection of trials for blood interactions.

            This Standard guideline include for control:

  • Blood storage and administration equipment, products for blood collection, products for extension equipment.
  • Catheters placed for more than 24 hours (e.g., intravascular endoscopes, intravascular ultrasound, laser systems, retrograde coronary perfusion catheters.
  • Cellular preservatives.
  • Products for absorption of specific substances from the blood.
  • Equipment for donation and therapeutic apheresis and blood cell separation systems.
  • Hemodialysis/hemofiltration equipment.
  • Leukocyte retention filter.
  • Annuloplasty rings, mechanical heart valves.
  • Intraortic balloon pumps, total artificial hearts, products for intraventricular assistance.
  • Products for embolization.
  • Endovascular grafts.
  • Defibrillators and implantable cardioverter.
  • Pacemaker drivers.
  • Prosthetic (synthetic) grafts and patches, including arteriovenous shunts.
  • Vascular prostheses ("stens").
  • Tissue cardiac valves.
  • Grafts and vascular patches of tissue, including arteriovenous shunts
  • Vena cava filters.  

            The complement system is a biological system of interaction of several proteins that are activated sequentially after the activation of some initial components (factors). There are three possible activation pathways, each with the activation of different initial components (factors). The three activation pathways are known as: 1) classic activation pathway; 2) Alternative activation pathway, previously called the properdin pathway; and 3) Lectins or mannoses pathway.

In the classical activation pathway there is an interaction of a specific antibody with its antigen. This interaction leads to changes in the Fc region of the antibody molecule that binds the three different subcomponents (C1q, C1r and C1s) of the C1 component (factor) of the complement system. After this fixation an enzymatic activity (C1 esterase) appears, which acts on the C4 and C2 components, cleaving them. From the generated fragments (C4a, C4b, C2a and C2b), the C4b and C2a fragments join together and generate the so-called C3 factor convertase (C3 convertase), which cleaves to the C3 factor, generating the C3a and C3b fragments. The latter, C3b, binds to the previous sequence and generates the convertase of the C5 factor (C5 convertase), which cleaves the C5 factor generating C5a and C5b, of which the former is free, while C5b sequentially activates C6, C7, C8 and C9. The activation of the entire chain of factors, causes the activity of the complement system whose ultimate goal is to degrade the membranes on which the activation occurred. However, throughout the activation, fragments are generated that have biological activity by themselves, such as the increase in vascular permeability, the attraction of inflammatory cells, the opsonization of foreign molecules that facilitates phagocytosis, the activation of factors of the coagulation system, etc. This classic activation pathway is not considered to be of interest for hemocompatibility studies, because it requires the presence of specific antibodies.

The alternative activation pathway of the complement system (formerly called properdin pathway), however, does not require the presence of specific antibodies, and is activated when some molecules are able to perpetuate the spontaneously occurring activation of factor C3. This causes the sequence of factors of the activation cascade of the complement system to activate without the participation of C1, nor of C4, nor of C2. The C3b fragment, spontaneously generated by C3 cleavage, and a factor B, which would bind to the previous one (C3bB), activate to give rise to C3bBb. This dimer is very unstable and to become the C5 convertase of the alternate path, it requires it to stabilize. Stabilization was first observed with properdin (C3bBbP), and therefore this activation pathway was called the properdin pathway. Subsequently, other molecules that can perform the same effect have been found. This route is of interest in the case of hemocompatibility tests because it could be the case that some components of medical devices could perpetuate the activation of C3, generating responses of increased vascular permeability, inflammatory cell attraction and activation of the blood coagulation.

The lectin or mannose pathway begins activation, also without the presence of antibodies, or the participation of C1, but begins with the activation of C4 and C2, by compounds such as mannose, fucose, glucosamine, which bind to a protein called mannose binding protein (serine protease associated with mannose), which once activated by binding to certain molecules, can carry out the cleavage of C4 and C2, giving rise to the C3 convertase , which initiates the activation of C3, and subsequently the cleavage of C5, as we have previously commented, activating the rest of the activation cascade of the complement system, as happened with the classical pathway. This route can also participate in the activation of the complement system generated by medical device components.

     Tests to determine the activation of the complement system.

  • The overall activity of the complement system can be determined by calculating the 50% hemolytic dose (CH50). This test determines the complete amount of complement system present in a plasma sample. Logically, if the system has been activated after exposure to a product (medical device) the hemolytic dose 50% will decrease, compared to that found in the respective controls, not exposed to the product (medical device). A decrease of CH50 is an indicator of total complement consumption. The classical method used to determine the hemolytic dose (CH50) is useful with human, bovine, porcine and rabbit serum. 
  • Quantify each of the factors present, or of the fragments of cleavage that may have been generated: C3a, C5a, Bb, iC3b, C4d, C5b-9. Elevated levels of any of these complement components indicates activation of the complement system, with the corresponding release of the excision factors cited. The measurement of the complement cleavage products has the drawback of specificity according to the species and of needing high basal levels when the measurement is made after the in vitro test. 

      Tests carried out in IVAMI

  • Determination of the ability of a medical device to activate the complement system by calculating 50% hemolytic dose (CH50), both in plasma exposed to the device evaluated, and their respective controls. The method is carried out in accordance with ASTM Standard F1984-99 (Standard Practice for Testing for Whole Complement Activation in Serum by Solid Materials). This method is recommended to detect the activating properties of solid materials used in the manufacture of medical devices that may come into contact with blood.