2-10 CELL AND SERUM ACTIVITY

a. Background. Serum antibodies are not directly determined by gene activity, but are important phenotypic characteristics in the ABO system. Under most

circumstances, most individuals possess antibodies directed against the ABH antigens absent from their own cells. This predictable complementary relationship is the basis for using both serum and cell tests in blood grouping. The cells are observed for the presence or absence of agglutination by known anti-A and anti-B. Cell-testing is sometimes called direct-or forward-testing. The unknown serum is tested against known A and B cells, sometimes called reverse- or back-testing. Since the genetically determined cellular antigens are rarely altered by environmental modification, cell- testing alone is more reliable than serum-testing alone. Both should be routinely performed, partly because the tests serve to verify each other, and partly because investigation of discrepancies usually reveals a medically or serologically significant etiology. Table 2-5 shows the phenotypic products that develop when different genes from these independent but interacting systems are present.

Table 2-5. Red blood cell and salivary antigens determined by ABO, Hh, sese, and lele genes.

b. Development of Antibodies. The sugar-sugar linkages that confer A, B, and H activity on molecules in the red blood cell membrane also occur in other biologic materials. Many bacteria have structural products with the same sugar groups; the antigenic properties of these bacterial materials are similar to those of human red blood cells. Bacteria are everywhere in the environment, and it appears that dust, foodstuffs, and other widely distributed agents constitute a powerful, continuing antigenic stimulus. Persons with normal immune systems react to these stimuli by producing antibodies against those antigens foreign to their own system. Thus, anti-A occurs in serum from persons of group O or B, and anti-B in group O or A serum. AB persons, having both antigens, lack both antibodies. Except for the rare hh persons who lack H, everyone has some element of H in his cellular makeup. Anti-H appears regularly in the serum of hh individuals, but rarely in other serums. A few persons with strongly active A1, or A1

and B, transferases convert virtually all their H substance to A, or to A and B. These people may have a weak anti-H in their serum, but it never achieves the strength of the anti-H in hh persons, or of anti-A or anti-B.

(1) Time of appearance. Antibody production does not normally begin until after birth. Newborns have their mother's IgG antibodies, but these are passively

received through placental transfer, not actively produced. Anti-A and anti-B production begins in the first few months of life, with titers rising for the first 5 or 6 years, and then remaining functionally the same until late in adult life. In very old people, levels of anti-A and anti-B are significantly lower than in young adults.

(2) Antibody behavior. Agglutination is the most conspicuous "in vitro" serologic effect of anti-A and anti-B. Other effects can and do occur under appropriate circumstances. Hemolysis, an important "in vitro" effect, sometimes occurs under "in vitro" conditions, and should be sought in observing every serum test. ABH antibodies sometimes coat cells without causing agglutination. When coating and agglutinating antibodies of the same specificity are present in a serum, only agglutination is apparent, unless the agglutinating antibodies are neutralized or inactivated. Coating antibodies are usually IgG. They are clinically important in hemolytic disease of the newborn since, like all IgG antibodies, they readily cross the placenta. Small amounts of IgG anti- A or anti-B can be found in group B or group A serum, but they are seldom of clinical significance. Hemolytic disease of the newborn occurs primarily in the A or B offspring of group O mothers or, rarely, the B offspring of A2 mothers.

(3) Group O serum. In addition to separable anti-A and anti-B, serum from group O subjects may contain an antibody that reacts with either A cells or B cells. The two antibody specificities cannot be separated by differential absorption. Either A or B cells can be used for absorption, and the eluate from either will react with both A and B cells. This antibody has been called inseparable anti-A and B or anti-A, B. Anti-A, B from group O serum reacts more strongly with some variant examples of A and B antigens than does either individual anti-A or anti-B. Blood banks use an anti-A, B reagent, prepared from group O serum, to detect weakly reacting cells that are not agglutinated by anti-A or anti-B, and might otherwise be classified as group O.

(4) "Immune" antibodies. Agglutinating anti-A and anti-B develop so regularly after environmental exposure that they are considered naturally occurring;

for example, no recognizable immunizing event leads to their appearance. A person exposed to a specific immunizing event may produce antibodies of the same specificity but different biologic behavior. Immunizing events include pregnancy with an ABO-incompatible

fetus; transfusion of incompatible red blood cells or of plasma containing blood group substances; injection of purified blood group substances; or inoculation with viral or bacterial products containing blood-group-active materials. After immunization, the subject's antibody may increase in titer or avidity; develop powerful hemolyzing

properties; become more difficult to neutralize with soluble blood-group substances; or become more active at 37ºC. These changes are more common in group O subjects, but may occur in group A or B persons as well. Although the distinction is not always complete, naturally occurring antibodies tend to be IgM and "immune" activity is more often IgG. Serologic characteristics of IgG and IgM Anti-A and Anti-B are as follows:

(a) Properties in Common

Agglutinate red blood cells in saline suspension React well at room temperature

Present in serum of group O persons May cause hemolysis in "in-vitro" testing (b) Distinguishing Features

IgM

Antibody titer can be enhanced by use of enzymes,

cold temperature

Readily neutralized by soluble blood-group substances Inactivated by two-mercaptoethanol or dithiothreitol Predominant isoagglutinin in group A and B persons

IgG

Antibody titer can be enhanced by warm temperature and use of antiglobulin technique

Only partially neutralized by soluble blood group substances Unaffected by two-mercaptoethanol or dithiothreitol

Seldom conspicuous in unimmunized A or B persons 2-11. GENERAL PRINCIPLES FOR ALL SEROLOGIC TESTING

a. Document the strength, specificity, and behavioral characteristics of all reagents by a regular quality assurance program.

b. Label all tubes, slides, and so forth, completely enough that there can be no confusion about the identity of the unknown sample or the reagents in use.

c. Calibrate each centrifuge for the optimal time and speed needed for each type of procedure. Record these data on the centrifuge and follow the instructions for each test.

d. Record results immediately after observation.

e. Report interpretation, where appropriate, separately from recorded results. f. Record the identity of the person performing each test.