Complement:Measurement The alternative pathway Specimen preparation and storage The As some els Fo hydrolysis of a small proportion of circulating C3.which serum.blood samples should arrive in the laboratory as permits binding of factor B in the presence of magnesium soon as possible after venepuncture.Blood should be ons.Factor D cleaves the bound The C3b g erated in this way can bind to surfaces via a thioester and,once attached,bind more factor B.The be aliquotted and stored at -70C as quickly as thioester bond preferentially reacts with hydroxyl groups practicable and adequate aliquots stored from each sample to form ester of alternative pathway- to prevent tha ng and reir reach assay.For use s Hand I Sialic acid is one a int known surface constituent which render surfaces resistant traacetic acid)(which prevents further complement activa ay-accele ples are su of on he d ed as fo to C3b on activating surfaces is cleaved by factor D to give ind C3bBbP car inhibitors such as phenylmethvlsul- s,thus amp bind C3 the below）. Haemolytic complement assays If the MACisa ne ofred blood The membrane attack complex(MAC) lls and po ur This phenomenon is used to assess the functional integrity of the ean be actnated by either cal whole of the class cal or alterative pathways with the respectively).Activated C5 binds to C6 and the remaining components,C7,C8 and C9.bind in turn.At least six and terminal complementco onents and de ends on the molecules o components being functionally active.The assay is 0 a cell on whi sas Two membrane-poundt roteins homo to proc Th logous restriction factor(HRF)and CD59,inhibit self- thway is initiated by IgM on the surface of sheep red inflicted cell lysis. blood cells(EA.antibody sensitized sheep erythrocytes) n the presence of calium and way activa Sheep red cells are inefficient at activating the alterative Measurement of Total Complement Activity thy ates cal but not n gn The Chsoand aphsoassays are used to determine whether way while permitting activation of the altemative or not a patient is genetically deficient in a complement pathway. These assays need to be ntrolled with positive contro a serum amp nown to co ,e.g.fres blood cel(classical pathway.CHor rabbit erytro control)which controls spontaneous lysis of the erythro cytes(alternative pathway,APHso).respectively.)Both the cytes.The amount of haemolysis(measured by the optical presence of a nonlunc a one-tube meth ernatively,using the 2 ENCYCLOPEDIA OF LIFE SCIENCES/2001 Nature Publishing Group/www.els.net The alternative pathway The alternative pathway of complement activation re￾quires C3, factor B, factor D and properdin. Activation of this pathway involves a constant, low level of spontaneous hydrolysis of a small proportion of circulating C3, which permits binding of factor B in the presence of magnesium ions. Factor D cleaves the bound factor B to form C3Bb and the latter can activate further C3 to form C3a and C3b. The C3b generated in this way can bind to surfaces via a thioester and, once attached, bind more factor B. The thioester bond preferentially reacts with hydroxyl groups to form ester bonds. Surfaces of alternative pathway￾activating particles bind C3b in a form that is resistant to the regulatory activities of factors H and I. Sialic acid is one known surface constituent which render surfaces resistant to alternative pathway activation. The membrane-asso￾ciated regulatory molecules decay-accelerating factor (DAF), CR1, and membrane cofactor protein (MCP) also mediate inactivation of C3b on host cells. Factor B bound to C3b on activating surfaces is cleaved by factor D to give C3bBb. Properdin stabilizes this complex and C3bBbP can cleave further C3 molecules, thus amplifying the process. Additional C3b can bind to the original C3b to alter the specificity of the enzyme to that of a C5 convertase (see below). The membrane attack complex (MAC) C5 can be activated by either the classical or alternative pathway by C5 convertases (C4bC3b2a and (C3b)nBbP respectively). Activated C5 binds to C6 and the remaining components, C7, C8 and C9, bind in turn. At least six molecules of C9 will bind to this complex and form a pore in the membrane of a cell on which it is present. This can lead to lysis of the cell, a property that is used in haemolytic assays. Two membrane-bound regulatory proteins, homo￾logous restriction factor (HRF) and CD59, inhibit self￾inflicted cell lysis. Measurement of Total Complement Activity The CH50 and APH50 assays are used to determine whether or not a patient is genetically deficient in a complement component. (CH50 and APH50 refer to the reciprocal dilution of a serum required to produce 50% haemolysis of a standard preparation of antibody sensitized sheep red blood cells (classical pathway, CH50) or rabbit erythro￾cytes (alternative pathway, APH50), respectively.) Both the deficiency of a component or the presence of a nonfunc￾tional component will be detected. Specimen preparation and storage As some components are extremely labile, incorrect storage of samples for complement assay can result in decreased levels. For assays of individual components in serum, blood samples should arrive in the laboratory as soon as possible after venepuncture. Blood should be allowed to clot at room temperature for 30 minutes, then the sample placed on ice for 1 hour for clot retraction to occur. Separation should occur at 2–48C. Samples should be aliquotted and stored at 2 708C as quickly as practicable and adequate aliquots stored from each sample to prevent thawing and refreezing for each assay. For use, samples should be thawed at 378C then immediately placed on ice. Blood collected into EDTA (ethylenediaminete￾traacetic acid) (which prevents further complement activa￾tion by chelating calcium and magnesium and, hence, such samples are suitable for activation product assays) should be kept on ice for as short a time as possible before being spun to produce platelet poor plasma which is stored as for serum. Samples from tissue culture may benefit from the addition of proteinase inhibitors such as phenylmethylsul￾fonyl fluoride (PMSF). Haemolytic complement assays If theMAC is assembled on the cell membrane of red blood cells and polymeric C9 inserted, lysis will occur. This phenomenon is used to assess the functional integrity of the whole of the classical or alternative pathways with the terminal pathway. The simplest of these assays is the CH50. This assay depends on a sample containing all the classical and terminal complement components and depends on the components being functionally active. The assay is quantitative, with the result expressed as the reciprocal of the serum dilution required to produce lysis of 50% of defined numbers red cells under standard conditions. The pathway is initiated by IgM on the surface of sheep red blood cells (EA, antibody sensitized sheep erythrocytes) and the assay is performed in the presence of calcium and magnesium ions (required for classical pathway activa￾tion). Sheep red cells are inefficient at activating the alternative pathway, so rabbit erythrocytes are used in APH50 assays. EGTA (ethyleneglycol bis(b-aminoethyl ether)-N,N-tetra￾acetic acid) chelates calcium, but not magnesium ions, and will prevent any concomitant activation of the classical pathway while permitting activation of the alternative pathway. These assays need to be controlled with positive control samples known to contain all the components, e.g. fresh normal human serum (NHS), and by buffer alone (negative control) which controls spontaneous lysis of the erythro￾cytes. The amount of haemolysis (measured by the optical density of the cell supernatant) can be compared with a known NHS in a one-tube method. Alternatively, using the Complement: Measurement 2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net