Articles on Medical Diseases and Conditions

Tag: Direct Coombs’ test

  • Antibody Detection Methods

    There are two methods of detecting and characterizing antibodies: (1) the direct Coombs’ test and (2) a group of procedures that try to determine if an antibody is present, and if present, attempt to identify the antibody by showing what the antibody will do in various controlled conditions.

    Direct Coombs’ test

    To prepare reagents for the Coombs’ test, human globulin, either gamma (IgG), nongamma (IgM), or a mixture of the two, is injected into rabbits. The rabbit produces antibodies against the injected human globulin. Rabbit serum containing these antihuman globulin antibodies is known as Coombs’ serum. Since human antibodies are globulin, usually gamma globulin, addition of Coombs’ serum (rabbit antibody against human gamma globulin) to anything containinghuman antibodies will result in the combination of the Coombs’ rabbit antibody with human antibody. There also has to be some indicator system that reveals that the reaction of the two antibodies has taken place. This can be seen visually if the Coombs’ rabbit antibody has been tagged with a fluorescent dye; or if the reaction takes place on the surface of RBCs, lysis or agglutination of the RBC can be produced.

    The direct Coombs’ test demonstrates that in vivo coating of RBCs by antibody has occurred. It does not identify the antibody responsible. It is a one-stage procedure. The Coombs’ serum reagent is simply added to a preparation of RBCs after the RBCs are washed to remove nonspecific serum proteins. If the RBCs are coated with antibody, the Coombs’ reagent will attack this antibody on the RBC and will cause the RBCs to agglutinate to one another, forming clumps. The antibody on the RBC is most often univalent but sometimes is polyvalent. Although antibodies on RBCs that are detected by the direct Coombs’ test are most often antibodies to RBC blood group antigens, certain medications (e.g., methyldopa and levodopa) in some patients may cause autoantibodies to beproduced against certain RBC antigens. Also, in some cases antibodies not directed against RBC antigens can attach to RBCs, such as antibodies developed in some patients against certain medications such as penicillin or autoantibodies formed in the rheumatoid-collagen diseases or in some patients with extensive cancer. In addition, some reports indicate an increased incidence of apparently nonspecific positive direct Coombs’ reactions in patients with elevated serum gamma globulin levels.

    The reagent for the direct Coombs’ test can be either polyspecific or monospecific. The polyspecific type detects not only gamma globulin but also the C3d subgroup of complement. Complement may be adsorbed onto RBCs in association with immune complexes generated in some patients with certain conditions, such as the rheumatoid-collagen diseases and certain medications, such as quinidine and phenacetin. Monospecific Coombs’ reagents are specific either for IgG immunoglobulin (and therefore, for antibody) or for complement C3d. If the polyspecific reagent produces a positive result, use of the monospecific reagents (plus elution techniques discussed later) can narrow down the possible etiologies.

    The direct Coombs’ test may be done by either a test tube or a slide method. The direct Coombs’ test must be done on clotted blood and the indirect Coombs’ test on serum, since laboratory anticoagulants may interfere. A false positive direct Coombs’ test result may be given by increased peripheral blood reticulocytes using the test tube method, although the slide technique will remain negative. Therefore, one should know which method the laboratoryuses for the direct Coombs’ test.

    In summary, positive direct Coombs’ test results can be due to blood group incompatibility, may be drug induced, may be seen after cardiac valve operations, and may appear in rheumatoid-collagen diseases, malignancy, idiopathic autoimmune hemolytic anemia, and other conditions. The overall incidence of a positive direct Coombs’ test result in hospitalized patients is reported to be about 7%-8% (range, 1%-15%).

    The main indications for the direct Coombs’ test include the following (most are discussed later in detail):

    1. The diagnosis of hemolytic disease of the newborn.
    2. The diagnosis of hemolytic anemia in adults. These diseases include manyof the acquired autoimmune hemolytic anemias of both idiopathic and secondary varieties. Results of the direct Coombs’ test at normal temperatures are usually negative with cold agglutinins.
    3. Investigation of hemolytic transfusion reactions.

    In these clinical situations the indirect Coombs’ test should not be done if the direct test result is negative, since one is interested only in those antibodies that are coating the RBCs (and thus precipitating clinical disease).

    Antibody detection and identification

    Indirect Coombs’ test. The indirect Coombs’ test is a two-stage procedure. The first stage takes place in vitro and may be done in either of two ways:

    1. RBCs of known antigenic makeup are exposed to serum containing unknown antibodies. If the antibody combines with the RBCs, as detected by the second stage, this proves that circulating antibody to one or more antigens on the RBC is present. Since the RBC antigens are known, this may help to identify that antibody more specifically.
    2. Serum containing known specific antibody is exposed to RBCs of unknown antigenic makeup. If the antibody combines with the RBCs, as detected by the second stage, this identifies the antigen on the RBCs.

    The second stage consists in adding Coombs’ serum to the RBCs after the RBCs have been washed to remove nonspecific unattached antibody or proteins. Ifspecific antibody has coated the RBCs, Coombs’ serum will attack this antibody and cause the cells to agglutinate. The second stage is thus essentially adirect Coombs’ test done on the products of the first stage.

    Therefore, the indirect Coombs’ test can be used either to detect free antibody in a patient’s serum or to identify certain RBC antigens, depending on how the test is done.

    The major indications for the indirect Coombs’ test are the following:

    1. Detection of certain weak antigens in RBCs, such as Du or certain RBC antigens whose antibodies are of the incomplete type, such as Duffy or Kidd (see antibody screen).
    2. Detection of incomplete antibodies in serum, either for pretransfusion screening or for purposes of titration.
    3. Demonstration of cold agglutinin autoantibodies.

    The indirect Coombs’ test is almost never needed routinely. In most situations, such as cold agglutinins or antibody identification, simply ordering atest for these substances will automatically cause an indirect Coombs’ test to be done. The indirect Coombs’ test should be thought of as a laboratory technique rather than as an actual laboratory test.

    False positives and false negatives may occur with either the direct or indirect Coombs’ technique due to mixup of patient specimens, clerical error when recording results, technical error (too much or not enough RBC washing; also failure to add reagents or adding the wrong reagent), contamination by 5% or 10% glucose in water (but not glucose in saline) from intravenous tubing, and, rarely, use of faulty commercial Coombs’ reagent.

    Antibody elution. When a direct Coombs’ test yields positive results, especially when thecause is thought to be a blood group–specific antibody, it is desirable to attempt elution (removal or detachment) of the antibody from the RBC to determine the antigen against which it is reacting. This is usually done by changing the physical conditions surrounding the antibody to neutralize the attachment forces. The most common current methods are heat, freeze-thaw, and chemical. Once the antibody is isolated from the RBCs, it can be tested with a panel of RBCs containing known antigens to establish its identity.

  • Laboratory Tests in Hemolytic Anemias

    Certain laboratory tests are extremely helpful in suggesting or demonstrating the presence of hemolytic anemia. Which tests give abnormal results, and to what degree, depends on the severity of the hemolytic process and possibly on its duration.

    Reticulocyte count. Reticulocyte counts are nearly always elevated in moderate or severe active hemolytic anemia, with the degree of reticulocytosis having some correlation with the degree of anemia. The highest counts appear after acute hemolytic episodes. Hemolytic anemia may be subclinical, detected only by RBC survival studies, or more overt but of minimal or mild intensity. In overt hemolytic anemia of mild intensity the reticulocyte count may or may not be elevated. Studies have found reticulocyte counts within reference range in 20%-25% of patients with hemolytic anemia, most often of the idiopathic autoimmune type. In one study of 35 patients with congenital spherocytosis, reticulocyte counts were normal in 8.5% of patients; and in one study of patients with thalassemia minor, reticulocyte counts were less than 3% in one half of the patients. Nevertheless, the reticulocyte count is a valuable screening test for active hemolytic anemia, and reticulocyte counts of more than 5% should suggest this diagnosis. Other conditions that give similar reticulocyte response are acute bleeding and deficiency anemias after initial treatment (sometimes the treatment may be dietary only). It usually takes 2 to 3 days after acute hemolysis or bleeding for the characteristic reticulocyte response to appear, and occasionally 4 or 5 days if the episode is relatively mild.

    Lactic dehydrogenase. Total serum lactic dehydrogenase (LDH) consists of a group of enzymes (isoenzymes) that appear in varying amounts in different tissues. The electrophoretically fast-migrating fraction LDH-1 is found in RBCs, myocardial muscle fibers, and renal cortex cells. RBC hemolysis releases LDH-1, which elevates LDH-1 values and usually increases total LDH values. The LDH measurement is a fairly sensitive screening test in hemolytic disease, probably as sensitive as the reticulocyte count, although some investigators believe that LDH results are too inconsistent and unreliable in mild disease. Other conditions that increase LDH-1 levels include artifactual hemolysis from improper venipuncture technique or specimen handling, megaloblastic anemia, and acute myocardial infarction. In addition, the total LDH value may be elevated due to an increase in one of the other LDH isoenzymes, especially the liver fraction. Therefore, nonspecificity has limited the usefulness of total LDH values in the diagnosis of hemolytic anemia. The LDH-1 assay is more helpful. A normal total LDH value, however, would assist in ruling out hemolytic anemia if the degree of anemia were substantial. The LDH-1/LDH-2 ratio is reported to be reversed in about 60% of patients with hemolytic anemia when the lab uses electrophoresis on cellulose acetate and may or may not occur using agarose gel, depending on the method. According to one study, reversed LDH-1/LDH-2 ratio is more likely to occur in hemolytic episodes if there is a substantial degree of reticulocytosis.

    Serum haptoglobin. Haptoglobin is an alpha-2 globulin produced by the liver that binds any free hemoglobin released into the blood from intravascular or extravascular RBC destruction. Haptoglobin can be estimated in terms of haptoglobin-binding capacity or measured by using antihaptoglobin antibody techniques (Chapter 11). Under ordinary conditions a decreased serum haptoglobin level suggests that hemolysis has lowered available haptoglobin through binding of free hemoglobin. Total haptoglobin levels decrease within 8 hours after onset of hemolysis.

    The usefulness of serum haptoglobin levels in the diagnosis of hemolytic conditions is somewhat controversial, although the haptoglobin level is generally considered to have a sensitivity equal to or better than that of the reticulocyte count. The actual sensitivity for minimal or mild hemolytic disease is not well established. There are reports that haptoglobin values may be normal in 10%-20% of cases. Serum haptoglobin levels have been used to differentiate reticulocytosis due to hemolytic anemia from reticulocytosis due to acute bleeding or iron deficiency anemia under therapy. However, some reports indicate that occasionally haptoglobin levels may be mildly decreased in patients with iron deficiency anemia not known to have hemolytic anemia. Most patients with megaloblastic anemia have decreased haptoglobin levels. Haptoglobin levels also may be decreased in severe liver disease, from extravascular hematomas (due to absorption of hemoglobin into the vascular system), and with estrogen therapy or pregnancy. Congenital absence of haptoglobin occurs in approximately 3% of African Americans and about 1% (range, less than 1%-2%) of Europeans. About 80%-90% of newborns lack haptoglobin after the first day of life until 1-6 months of age. Haptoglobin is one of the “acute-phase reaction” serum proteins that are increased in conditions such as severe infection, tissue destruction, acute myocardial infarction, and burns, and in some patients with cancer; these conditions may increase the haptoglobin level sufficiently to mask the effect of hemolytic anemia or a hemolytic episode.

    Plasma methemalbumin. After the binding capacity of haptoglobin is exhausted, free hemoglobin combines with albumin to form a compound known as methemalbumin. This can be demonstrated with a spectroscope. The presence of methemalbumin means that intravascular hemolysis has occurred to a considerable extent. It also suggests that the episode was either continuing or relatively recent, because otherwise the haptoglobins would be replenished and would once again take over the hemoglobin removal duty from albumin.

    Free hemoglobin in plasma or urine. Circulating free hemoglobin occurs when all of the plasma protein-binding capacity for free hemoglobin is exhausted, including albumin. Normally there is a small amount of free hemoglobin in the plasma, probably because some artifactual hemolysis is unavoidable in drawing blood and processing the specimen. This is less when plasma is used instead of serum. If increased amounts of free hemoglobin are found in plasma, and if artifactual hemolysis due to poor blood-drawing technique (very frequent, unfortunately) can be ruled out, a relatively severe degree of intravascular hemolysis is probable. Marked hemolysis is often accompanied by free hemoglobin in the urine (hemoglobinuria). In chronic hemolysis, the urine may contain hemosiderin, located in urothelial cells or casts.

    Direct Coombs’ test. This test is helpful when a hemolytic process is suspected or demonstrated. It detects a wide variety of both isoantibodies and autoantibodies that have attached to the patient’s RBCs (see Chapter 9). The indirect Coombs’ test is often wrongly ordered in such situations. The indirect Coombs’ test belongs to a set of special techniques for antibody identification and by itself is usually not helpful in most clinical situations. If antibody is demonstrated by the direct Coombs’ test, an antibody identification test should be requested. The laboratory will decide what techniques to use, depending on the situation.

    Serum unconjugated (indirect-acting) bilirubin. The serum unconjugated bilirubin level is often elevated in hemolysis of at least moderate degree. Slight or mild degrees of hemolysis often show no elevation. The direct-acting (conjugated) fraction is usually elevated to less than 1.2 mg/100 ml (2.05 µmol/L) and less than 30% of total bilirubin unless the patient has coexisting liver disease. Except in blood bank problems, serum bilirubin is not as helpful in diagnosis of hemolytic anemias as most of the other tests and often shows equivocal results.

    Red blood cell survival studies. RBC survival can be estimated in vivo by tagging some of the patient’s RBCs with a radioactive isotope, such as chromium 51, drawing blood samples daily for isotope counting, and determining how long it takes for the tagged cells to disappear from the circulation. Survival studies are most useful to demonstrate low-grade hemolytic anemias, situations in which bone marrow production is able to keep pace with RBC destruction but is not able to keep the RBC count at normal levels. Low-grade hemolysis often presents as anemia whose etiology cannot be demonstrated by the usual methods. There are, however, certain drawbacks to this procedure. If anemia is actually due to chronic occult extravascular blood loss, radioisotope-labeled RBCs will disappear from the circulation by this route and simulate decreased intravascular survival. A minor difficulty is the fact that survival data are only approximate, because certain technical aspects of isotope RBC tagging limit the accuracy of measurement.