Practice

The Case: A 72-year-old man withchronic lymphocytic leukemia presentsto the emergency department with a 3-month history of shortness of breath onexertion, lethargy, pallor and scleralicterus. Laboratory test results reveal ahemoglobin level of 90 g/L (normal

range 130–170 g/L), and a type andscreen is ordered. The blood bankphysician notifies the attending doctorthat the patient has a panagglutinatingIgG antibody with positive autocontroland suggests ordering a direct anti-globulin test and other biochemicalmarkers of hemolysis. How should theemergency doctor proceed?

The antiglobulin test was first de-scribed by Coombs in 1945 and is oftenreferred to as the Coombs test. It is per-formed to detect either erythrocyte-

directed IgG in plasma or IgG or com-plement coating on the surface of cir-culating erythrocytes. Neither of thesemolecules can cause direct agglutina-tion of erythrocytes, so, to detect theirpresence, monoclonal antihumanglobulin (AHG) with specificity for IgGor various complement proteins isadded to a suspension of erythrocytes.The AHG reagent is sufficiently large tocause agglutination of erythrocytes thatare coated with IgG or complement.Fig. 1 illustrates the steps of the directantiglobulin test (DAT), which detectsin vivo binding of either IgG or comple-D

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The role of the Coombs test

in evaluating hemolysis in

adults

Teaching Case Report

In vivo antibody coating of erythrocytes

Anti-IgG AHG reagent added after erythrocytes are washed

AHG reagent causes IgG-coated erythrocytes to agglutinate

Direct Antiglobulin Test

Indirect Antiglobulin Test

Antibodies in serum

Reagenterythrocyte

Erythrocyte

Antigen

Fig. 1: The direct antiglobulin test (DAT) and indirect antiglobulin test (IAT). AHG = antihuman globulin. A. The DAT reflects in vivo anti-body sensitization of erythrocytes. Erythrocytes are washed to remove any unbound antibodies, and anti-IgG AHG reagent is then added.IgG antibodies cannot cause direct erythrocyte agglutination, but if the erythrocytes are coated with IgG antibodies, the AHG reagent willcause them to agglutinate. This test can also be performed using anti-complement AHG reagent. If IgG antibodies are present, they canbe eluted off the erythrocytes for specificity determination. B. The IAT is used to detect the presence of IgG antibodies in serum (in vitrosensitization). Reagent erythrocytes are incubated in the presence of serum that potentially contains antibodies. If antibodies are pres-ent, they bind to their target antigens on the reagent erythrocytes. After the incubation period the erythrocytes are washed to remove un-bound antibodies. Anti-IgG AHG reagent is added and will cause IgG-coated erythrocytes to agglutinate.

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ment to erythrocyte antigens, and theindirect antiglobulin test (IAT), whichdetects serum antibodies that bind toerythrocytes during an incubation step.

Clinical use of the DAT

The DAT is useful in differentiating im-mune-mediated hemolysis from he-molysis caused by other factors (Box 1).It is important to note that a small butsignificant number of patients have apositive DAT result without shortenederythrocyte survival; a positive DAT re-sult without evidence of hemolysis hasbeen reported in 1:1–14 000 healthyblood donors and 1%–15% of in-hospi-tal patients.1,2 A positive DAT result inthe absence of hemolysis can also oc-cur in certain circumstances (Box 1). Inthese disease conditions, dysregulationof the immune system or a generalizedinflammatory state leads to nonspecificadsorption of antibodies to the erythro-cyte membrane. These IgG moleculesare not immunologically bound to theerythrocyte membrane (i.e., they do not

recognize and bind to specific erythro-cyte antigens) and do not usually causehemolysis. An eluate prepared fromDAT-positive erythrocytes can deter-mine whether the antibodies were im-munologically or nonspecifically at-tached to these cells.

When a DAT result is positive, it isimportant to determine whether thereis laboratory (Box 2) or clinical evi-dence of hemolysis. If there is no evi-dence of hemolysis, no additional in-vestigations are necessary.

However, if there is evidence of he-molysis, the physician needs to explainthe cause of the positive test result. Oneof the most common causes is autoim-mune hemolytic anemia (AIHA). AIHAis usually classified on the basis of thethermal amplitude of the autoantibody;warm AIHA is usually caused by an IgGantibody, whereas cold AIHA is typi-cally caused by an IgM antibody thatfixes complement to the surface of theerythrocyte. In the former case, theDAT result is positive with anti-IgGAHG reagent, and in the latter, reactiv-

ity with the anti-complement AHG re-agent is present (anti-IgM AHG re-agents are not readily available). Theantibody screen, as part of a type andscreen, is a useful way to begin differ-entiating warm from cold AIHA.

Many pharmacologic agents havebeen associated with a positive DAT re-sult, with or without hemolytic anemia.Many drugs bind to the membranes ofcirculating cells; antibodies elicited bythese medications may be directed ei-ther against a combination of the drugand certain membrane components oragainst epitopes of the drug moleculethat are bound tightly to the erythrocytesurface. Hemolytic anemias associatedwith these drugs (including penicillinsand other antibiotics) frequently re-solve once the agent is discontinuedand cleared from the circulation. Othermedications have been associated witha hemolytic anemia that persists evenafter the drug is cleared. These drugs,the archetype of which is α-methyl-dopa, are thought to cause hemolyticanemia by inducing the production ofanti-erythrocyte autoantibodies that donot require the presence of the drug tobind to erythrocytes.

If the patient has received a transfu-sion within the previous 3 months, apositive DAT result may indicate al-loimmunization to an antigen on thetransfused cells that is not present onthe recipient’s own erythrocytes. In thiscase only the donor’s antigen-positivecells, which stimulated the recipient’simmune response, would be coatedwith the newly formed antibody. Therecipient’s autologous cells, which lackthe antigen, would not be agglutinatedby anti-IgG reagents nor hemolyzed bythis antibody. An unexpected erythro-cyte antibody might appear as early as7–10 days after transfusion, or earlier ifthe patient was previously alloimmu-nized and is re-exposed to the sameantigen in a subsequent transfusion.

A less common cause of a positiveDAT result is recent administration ofintravenous immune globulin, whichcan contain antibodies directed againstABO, Rh or other erythrocyte antigens.The titre of these passively acquired an-tibodies diminishes over time, and theDAT result will eventually become neg-ative. In patients who have undergone

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Box 1: Differential diagnosis of hemolysis

Conditions associated with hemolysis and a positive DAT result

• Hemolytic disease of the newborn

• Drug-induced hemolytic anemias

• Acute hemolytic transfusion reaction

• Delayed hemolytic transfusion reaction

• Autoimmune hemolytic anemia (warm autoimmune hemolytic anemia, coldagglutinin syndrome, paroxysmal cold hemoglobinuria, mixed-type autoimmunehemolytic anemia)

Conditions associated with a positive DAT result, with or without hemolysis

• Exogenous immune globulin administration

• Recent hematopoietic stem-cell transplantation

• Recent solid organ transplantation

• Systemic lupus erythematosus

• Infectious mononucleosis

• Some hematologic diseases, including lymphoproliferative diseases

Conditions associated with hemolysis and a negative DAT result

• Microangiopathic hemolytic anemias (thrombotic thrombocytopenic purpura,disseminated intravascular coagulation)

• Hypersplenism

• Liver disease

• Hemoglobinopathies (sickle cell disease, thalassemia)

• Erythrocyte membranopathies (spherocytosis)

• Deficiencies of erythrocyte enzymes (G-6-PD deficiency, pyruvate kinasedeficiency)

• Infectious diseases (Clostridium difficile infection)

• Erythrocyte trauma (mechanical heart valves, improper use of blood warmers)

either recent hematopoietic stem celltransplantation or solid organ trans-plantation, donor lymphocytes accom-panying the allograft organ or stemcells may produce antibodies withspecificity for recipient erythrocyte an-tigens. In these latter cases, the extentof the chimerism between the anti-body-producing lymphocytes and therecipient will dictate how long the DATresult remains positive.

The indirect antiglobulin test andits clinical applications

The IAT is used by the blood bank todetect unexpected erythrocyte antibod-ies in the patient’s serum or plasma.The IAT is the final phase of the anti-

body screen and serologic crossmatchprocedures. In an antibody screen therecipient’s serum is incubated with 2 or3 different type O erythrocytes that ex-press clinically significant antigens.This erythrocyte and serum mixture isthen incubated with anti-IgG AHG andobserved for agglutination. If aggluti-nation occurs, the screen is consideredpositive, and further testing is per-formed to determine the specificity ofthe IgG antibody. The serologic cross-match procedure is also an IAT proce-dure and is performed on patients withunexpected erythrocyte antibodies toverify that the potential donor erythro-cyte unit lacks the antigen correspon-ding to the recipient’s antibody. In thisprocedure, the patient’s serum is incu-bated with erythrocytes from a poten-tial erythrocyte donor unit, and anti-IgG reagent is added. Agglutinationwould indicate an incompatibility be-tween the donor erythrocytes and therecipient’s IgG antibody. Thus the re-sults of the IAT reflect in vitroerythrocyte sensitization. The anti-complement AHG reagent is generallynot used in antibody screening orcrossmatch procedures.

Case resolution

The emergency physician arranges forthe patient to be admitted to hospital,

and the testing is arranged. The pa-tient’s DAT result is strongly positivewith anti-IgG AHG reagent only. Theeluate prepared from his erythrocytesagglutinates all reagent erythrocytes, afinding that, when combined with theother blood bank findings and the pos-itive biochemical markers of hemoly-sis, confirms the diagnosis of AIHA.

J. Manuel ZarandonaResidency Training ProgramDepartment of PathologyUniversity of PittsburghPittsburgh, Pa.Mark H. YazerMedical DirectorRBC Serology Reference LaboratoryCentralized Transfusion ServiceInstitute for Transfusion MedicineDepartment of PathologyUniversity of PittsburghPittsburgh, Pa.

REFERENCES1. Brecher ME, editor. American Association of Blood

Banks technical manual. 14th ed. Bethesda, MD:American Association of Blood Banks Press; 2002.

2. Freedman J. The significance of complementon the red cell surface. Transfus Med Rev 1987;1:58-70.

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Acknowledgements: We thank Dr. Darrell Triulzifor his thoughtful discussion and critical reviewof the manuscript.

Competing interests: None declared.

Box 2: Biochemicalevidence of hemolysis

• Hemoglobinuria

Elevated

• Reticulocyte count

• Lactate dehydrogenaselevel

• Unconjugated bilirubinlevel

Decreased

• Hemoglobin level

• Haptoglobin level

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