ASTM F2567-06(2010)

Inappropriate activation of complement by blood-contacting medical devices may have serious acute or chronic effects on the host. Solid medical device materials may activate complement directly by the alternative pathway, or indirectly because of antigen-bound antibodies (as with immuno-adsorption columns) by the classical pathway. This practice is useful as a simple, inexpensive, function-based screening method for determining complement activation by solid materials in vitro by the classical pathway.

This practice is composed of two parts. In part A (Section 11), HS is exposed to a solid material. If complement activation occurs by the classical pathway, C4 will be depleted. Activation by the alternative pathway will not deplete C4. In part B (Section 12), C4 activity remaining in the serum after exposure to the test material is assayed by diluting the serum below the concentration needed to lyse antibody-coated sheep RBC on its own, then adding the diluted HS to C4(-)GPS (which is itself at a dilution where all complement components are in excess save the missing C4). Lacking C4, the C4(-)GPS does not lyse the antibody-coated sheep RBC unless C4 is present in the added HS. The proportion of lysis remaining in the material-exposed HS sample versus the 37°C control HS sample (which was not exposed to the test material) indicates the amount of C4 present in the HS, loss of which correlates with classical pathway activation.

This function-based in vitro test method for classical pathway complement activation is suitable for adoption in specifications and standards for screening solid materials for use in the construction of medical devices intended to be implanted in the human body or placed in contact with human blood outside the body. It is designed to be used in conjunction with Practice F1984 for function-based whole complement activation screening, and Practice F2065 for function-based alternative pathway activation screening.

Assessment of in vitro classical complement activation as described here provides one method for predicting potential complement activation by solid medical device materials intended for clinical application in humans when the material contacts the blood. Other test methods for complement activation are available, such as immunoassays for specific complement components (including C4) and their split products in human serum (see X1.3 and X1.4).

If nonspecific binding of certain complement components, including C4, to the materials occurs in part A of this practice, a false positive for classical pathway activation will be observed in step B. Classical pathway complement activation by the test material may be confirmed by demonstrating an absence of C4 bound to the material following removal of the serum, and/or production of complement split-products such as C4d in the serum (as determined by immunoassay). Although immunoassay could be done in place of this screening procedure, determination of C4d production alone may not be functionally significant. This practice does not detect trivial amounts of classical activation unable to affect functional lysis of sensitized RBC.

1.1 This practice provides a protocol for rapid, in vitro functional screening for classical pathway complement activating properties of solid materials used in the fabrication of medical devices that will contact blood.

1.2 This practice is intended to evaluate the acute in vitro classical pathway complement activating properties of solid materials intended for use in contact with blood. For this practice, “serum” is synonymous with “complement.”

1.3 This practice consists of two procedural parts. Procedure A describes exposure of solid materials to a standard lot of human serum [HS], using 0.1 mL serum per 13×100 mm disposable glass test tube. Procedure B describes assaying the exposed serum for significant functional classical pathway complement depletion (decrease in amount of C4) as compared to control serum samples not exposed to the material. The endpoint in Procedure B is lysis of sheep red blood cells (RBC) coated with antibody (hemolysin).

1.4 This practice does not address the use of plasma as a source of complement.

1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for other aspects of biocompatibility. Practice F1984 provides guidance for testing solid materials for whole complement activation in human serum, but does not discriminate between the classical or alternative pathway of activation. Practice F2065 provides guidance for testing solid materials for alternative pathway complement activation in serum.

1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.