In which of the following clinical situations will the direct antiglobulin test be positive?

Gell and Coombs Type II: Antibody-Mediated Cytolytic Reactions

The type II immune reactions involve IgG, IgM, and to a lesser extent IgA, which are directed to cell-surface antigens on erythrocytes, neutrophils, platelets, and epithelial cells of glandular or mucosal surfaces or to antigens on tissues (e.g., basement membranes). The sensitizing antigens in these cases can be natural cell surface antigens, modified cell surface antigens, or haptens attached to cell surfaces. Three distinct immune reactions might be induced: The first occurs by opsonization, which is facilitated by complement activation; the second induces complement-mediated lysis; and the third is antibody-dependent cell-mediated cytotoxicity or ADCC. These mechanisms afford protection against infections and eradication of malignant cells but can also result in damage to various tissues associated with responses to self-antigens. An example of opsonization is phagocytic cell destruction of antibody-coated platelets, causing immune thrombocytopenia. The second category is demonstrated by penicillin binding to the surfaces of erythrocytes, creating a nonself-antigen composed of penicillin-modified erythrocyte cell surfaces. Antipenicilloyl antibodies, initially IgM and later IgG, fix to erythrocyte surfaces and concomitantly activate complement, leading to the lysis of the cell with penetration of the terminal hydrophobic complement membrane attack complex (MAC, C5 to C9). Clinically, this condition is known as penicillin-induced autoimmune hemolytic anemia. Other clinical examples of this reaction include quinidine-induced autoimmune thrombocytopenia and ceftriaxone-induced autoimmune hemolytic anemia.39 ADCC is the process by which NK cells and other cells recognize IgG bound to target cells, such as neoplastic cells, and triggers the release of cytotoxic granules.

Publisher Summary

This chapter describes the direct antiglobulin test (DAT) which is also known as Direct Coombs test (DCT). The test detects the presence of immunoglobulin G (IgG) or complement C3 and small amounts of IgG and complement are found on red blood cells (RBCs) of normal individuals. The DAT is mentioned to be useful in diagnosis of several situations that include: antibody identification, autoimmune hemolytic anemia, drug-induced hemolytic anemia and hemolytic diseases of the fetus and newborn. The detailed procedure of the test is discussed such as the specimen requirement, the methodology, evaluation of a positive DAT, eluate and panagglutinin. The clinical significance of a positive DAT is stated to be difficult to assess and among hospitalized patients, 0.7% have positive DATs, while 18% of AIDS patients have positive DATs. The significance of the DAT is assessed based on clinical and laboratory findings suggestive of decreased RBC survival, such as anemia, jaundice, hematuria and elevated lactate dehydrogenase. The false-negative DAT and false-positive DAT are further described.

Coombs' Test, Direct IgG—Serum

Indications

Normocytic Anemia

Because of the diverse nature of normocytic anemia, it is the most difficult type to evaluate. The reticulocyte count varies according to whether RBC production is increased, normal, or decreased. If erythropoiesis is increased, one must differentiate between hemorrhage and an increased rate of destruction. The blood smear may reveal a type of RBC shape that can be virtually diagnostic. Schistocytes are seen in microangiopathic hemolysis—as in the HELLP syndrome (hemolysis,elevatedliver enzymes,lowplatelets) and thrombotic thrombocytopenic purpura—and in association with prosthetic heart valves. Other types of poikilocytes that may be encountered on peripheral blood smear examination and that may suggest an etiology include sickle cells, target cells, stomatocytes, ovalocytes, spherocytes, elliptocytes, and acanthocytes.

The Coombs test differentiates immune from nonimmune causes of hemolysis. Immune hemolysis is related to alloantibodies, drug-induced antibodies, and autoantibodies. Nonimmune causes of hemolysis include various hereditary disorders such as hemoglobinopathies, disorders of the RBC membrane (hereditary spherocytosis and hereditary elliptocytosis), deficiency of an RBC enzyme, or the porphyrias, and acquired, nonimmune hemolytic anemias may be caused by PNH or lead poisoning.

Bone marrow examination can be helpful for evaluation of patients who have hypoproliferative anemias with normal iron studies, and folate and vitamin B12 levels. If increased ring sideroblasts are identified, both acquired and hereditary forms of sideroblastic anemia must be considered. If erythropoiesis is normoblastic, etiologic mechanisms fall into two major categories. The first category has myeloid-to-erythroid production ratios greater than 4 : 1 and includes red cell aplasia, primary marrow-based disorder (e.g., chronic myeloid leukemia), effects of chronic diseases, infection (e.g. parvovirus), and endocrine disorders such as hypothyroidism and hypopituitarism. In contrast, the myeloid-to-erythroid ratio is decreased (e.g., 2 : 1 or less) when erythroid hyperplasia is present, as with relatively acute hemolysis or myelodysplastic syndrome (MDS) if in conjunction with significant dysplasia. If there is megaloblastic erythropoiesis and erythroid hyperplasia, considerations include nutritional deficiencies such as folate and vitamin B12 deficiencies, MDS, drugs, particularly those that interfere with nucleotide synthesis, and toxins (benzene, arsenic).

62 What is the difference between the direct and indirect Coombs tests?

Coombs serum is rabbit antihuman immunoglobulin.

Direct test: Coombs serum is added directly to a patient's washed RBCs. The occurrence of agglutination means that the patient's RBCs have been sensitized in vivo by the antibody. Direct Coombs testing is vital for diagnosing autoimmune hemolytic anemias.

Indirect test: This involves incubating a patient's serum with RBCs of a known type and adding Coombs serum. If in vitro sensitization occurs, agglutination will result, which indicates that antibodies are present against the known blood type. Indirect testing is key for blood crossmatching.

Hemolytic Transfusion Reactions

A DAT is performed on the post transfusion sample in order to evaluate a possible acute or delayed hemolytic transfusion reaction (see Chapters 61 and 62Chapter 61Chapter 62). A positive DAT result should be compared with that of the pretransfusion sample. If the DAT is positive for IgG, then an eluate should be performed to determine the specificity of the antibody coating the RBCs. If the patient is non-group O, then the eluate should be tested with group O reagent cells as well as group A and group B cells. A mixed field appearance in the post transfusion DAT (i.e. agglutination of donor RBCs and no agglutination of the patient’s RBCs) may or may not be seen. The DAT may be negative in a hemolytic transfusion reaction if all the transfused RBCs have been hemolyzed. Therefore, a negative DAT does not rule out a hemolytic transfusion reaction.

Antiglobulin Test

The antiglobulin test (Coombs test) was introduced by Coombs and colleagues in 194538 as a method for detecting ‘incomplete’ Rh antibodies (i.e. IgG antibodies capable of sensitizing red cells but incapable of causing agglutination of the same cells suspended in saline), as opposed to ‘complete’ IgM antibodies, which do agglutinate saline-suspended red cells.

Direct and indirect antiglobulin tests can be carried out. In the direct antiglobulin test (DAT), the patient's cells, after careful washing, are tested for sensitization that has occurred in vivo; in the indirect antiglobulin test (IAT), normal red cells are incubated with a serum suspected of containing an antibody and subsequently tested, after washing, for in vitro-bound antibody.

The antiglobulin test is probably the most important test in the serologist's repertoire. The DAT is used to demonstrate in vivo attachment of antibodies to red cells, as in autoimmune haemolytic anaemia (see p. 275), alloimmune HDN (see p. 535) and alloimmune haemolysis following an incompatible transfusion (see p. 542). The IAT has wide application in blood transfusion serology, including antibody screening and identification and crossmatching.

Antiglobulin Test

The antiglobulin test (Coombs test) was introduced by Coombs, Mourant, and Race in 194541 as a method for detecting “incomplete” Rh antibodies (i.e., IgG antibodies capable of sensitising red cells but incapable of causing agglutination of the same cells suspended in saline) as opposed to “complete” IgM antibodies, which do agglutinate saline-suspended red cells.

Direct and indirect antiglobulin tests can be carried out. In the direct antiglobulin test (DAT), the patient's cells, after careful washing, are tested for sensitisation that has occurred in vivo; in the indirect antiglobulin test (IAT), normal red cells are incubated with a serum suspected of containing an antibody and subsequently tested, after washing, for in vitro–bound antibody.

The antiglobulin test is probably the most important test in the serologist's repertoire. The DAT is used to demonstrate in vivo attachment of antibodies to red cells, as in autoimmune haemolytic anaemia (p. 246), alloimmune HDN (p. 540), and alloimmune haemolysis following an incompatible transfusion (p. 549). The IAT has wide application in blood transfusion serology, including antibody screening and identification and crossmatching.

Antiglobulin test

The antiglobulin test (Coombs test) was introduced by Coombs and colleagues in 194531 as a method for detecting ‘incomplete’ Rh antibodies (i.e. IgG antibodies capable of sensitising red cells but incapable of causing agglutination of the same cells suspended in saline), as opposed to ‘complete’ IgM antibodies, which do agglutinate saline- suspended red cells.

Direct and indirect antiglobulin tests can be carried out. In the direct antiglobulin test (DAT), the patient’s cells, after careful washing, are tested for sensitisation that has occurred in vivo; in the indirect antiglobulin test (IAT), normal red cells are incubated with a serum suspected of containing an antibody and subsequently tested, after washing, for in vitro-bound antibody.

The antiglobulin test is probably the most important test in the serologist’s repertoire. The DAT is used to demonstrate in vivo attachment of antibodies to red cells, as in autoimmune haemolytic anaemia (see p. 255), alloimmune HDN (see p. 491) and alloimmune haemolysis following an incompatible transfusion (see p. 488). The IAT has wide application in blood transfusion serology, including antibody screening and identification and crossmatching.