1-13. THE ALTERNATIVE COMPLEMENT PATHWAY
a. The alternative pathway bypasses C1, C2, C4, and enters the classic
pathway at the C3 stage. It can be activated by several substances including
aggregated immunoglobulins and endotoxin. Thus, an antigen-antibody reaction is not
necessary to initiate the cascade.
b. This pathway is not fully understood at present, but several factors necessary
for its reaction have been isolated. Once C3 is activated in this pathway, the molecular
consequences are identical to the classic pathway. The alternative pathway has not
been incriminated in many immunohematologic problems yet, but it is of interest to note
that the red -blood cells of patients suffering with paroxysmal nocturnal hemoglobinuria
(PNH) have been shown to hemolyse through this pathway.
1-14. STABILITY OF COMPLEMENT WITH PARTICULAR REFERENCE TO THE
DETECTION OF BLOOD-GROUP ANTIBODIES
a. In order to demonstrate hemolysis or RBC bound complement by the
antiglobulin test, one must sensitize RBCs with complement-fixing antibodies in the
presence of complement, or in a two-stage technique, in which cells are first incubated
with antibody, washed, and then incubated with a source of complement. The most
practical source of complement is human serum, but in order to utilize this source, one
must have information about the stability of complement under varying conditions of
storage. A study by Garratty was specifically designed to determine the effect of
storage on the activity of complement with a reference to the detection of blood-group
antibodies. In this study, normal serums were stored at -90C, -55C,
-20C, 22C, and 37C from 24 hours to 3 months. Complement activity was assessed
by a standard hemolytic assay and also by antiglobulin test assay, which measured the
ability of the stored serums to serve as a source of complement in the detection of
blood-group antibodies by the antiglobulin test. Hemolytic assays closely paralleled
antiglobulin assay. At levels below 60 percent complement activity, there was a danger
of missing weak complement-binding antibodies. An average of these assays showed:
at 37C, activity fell to 30 percent In 24 hours; at room temperature, activity was 40
percent at 48 hours, 8O percent at 24 hours, zero at 72 hours; at 4C, as 90 percent at
72 hours and 60 percent at two weeks. At -20C, activity was more than 60 percent for
2 months, and at -55C and -90C, activity was retained at three months.
b. It should be stressed that these studies were carried out on normal serums,
and that serums from hospital patients may be deficient in certain complement
components before storage, or they may develop anticomplementary properties faster
than normal serums. The Standards of the American Association of Blood Banks states
that tests for compatibility should be performed on nonactivated, refrigerator-stored
serum, collected within 72 hours of performance test.
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