An indirect Coombs test is performed to predict whether the fetus is at risk for

Antibodies are a part of your immune system. They fight germs, but sometimes they make a mistake and target your body's healthy cells instead. The Coombs test checks your blood for antibodies that attack red blood cells. You might also hear it called an antiglobulin test or red blood cell antibody screening.

Not everyone's red blood cells are alike. Your immune system will make antibodies if it finds ones that don't match yours. They're keyed to specific areas on the outside of the cell. Some of these antibodies are related to your blood type.

There are two types of Coombs tests. The direct test looks for antibodies that are stuck to red blood cells. The indirect test looks for antibodies floating in the liquid part of your blood, called serum.

Why You Get an Indirect Coombs Test

Doctors use the indirect Coombs test, also called IAT, to prevent problems.

They'll check your blood before you get a transfusion to make sure it doesn't have antibodies that would react badly to the donated blood. It's part of the "type and screen" process.

Pregnant women get a prenatal antibody screening with an indirect Coombs test. It checks the mother's blood to see if there are antibodies that could pass to and harm their unborn baby.

Why You Get a Direct Coombs Test

A direct Coombs test, or DAT, may help explain why you're not feeling great or have symptoms that suggest trouble related to your blood.

You might get sick after a blood transfusion if the donor's blood wasn't a good match. Your body may recognize those other blood cells as foreign and make antibodies to get rid of them, even though they're meant to help.

A blood disease called autoimmune hemolytic anemia happens when antibodies destroy your own red blood cells faster than your body can make them. You can get it because of:

• Diseases like lupus and leukemia
• Infections such as mononucleosis
• Medicines, including penicillin

Babies with yellowish skin and eyes may have hemolytic disease of the newborn [HDN]. Some antibodies from their mother could be attacking their red blood cells. This happens most often when the part of the baby's blood type inherited from the father doesn't mix well with the mother's.

How It's Done

A technician uses a needle to take a small sample of blood from a vein in your hand or arm. You may feel a small skin prick and have a little bleeding or bruising where the needle goes in. Then they'll send your blood to a lab.

Both the direct and indirect tests can look for simply the presence of antibodies in general or for a specific antibody.

Before a blood transfusion, each package of donated blood also needs to be tested.

Cross-matching is a special kind of IAT that may be done before a blood transfusion. The lab mixes your serum [where the antibodies are] with red blood cells from the donor.

What the Indirect Results Mean

A negative indirect Coombs test is good news. It usually means you don't have antibodies in your serum, so you:

• Can safely get blood from that donor
• Don't need to worry about trouble with your unborn baby

A positive result before a blood transfusion is a warning that the doctor will have to be careful when choosing donor blood. People who need a lot of blood transfusions may develop a lot of different antibodies and have a harder time finding blood that will work.

A positive indirect Coombs test during pregnancy means you may need to take steps to protect your baby. Not all antibodies the test finds are harmful, so depending on what the test was looking for, you may need more tests to narrow down which ones you have so your doctor will know what to do next.

What the Direct Results Mean

A positive direct Coombs test shows you have antibodies attached to your red blood cells, but it doesn't necessarily tell you which ones or why.

Regardless of the result of a direct Coombs test, you may need other tests to find the right diagnosis and treatment.

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.

9.1.4 Coombs test

Coombs test, also known as anti-globulin test, detects the antibodies that may agglutinate to the red blood cells and cause haemolysis [Ducrotoy et al., 2016]. Normally, no direct agglutination takes place with red cells coated with IgG or complement and red cells are said to be sensitized with complement or IgG. For an agglutination to occur, an additional antibody that reacts with the Fc portion of the IgG or with the C3b or C3d component of the complement must be added to the system forming a bridge between the antibodies or complement coated red cells resulting in agglutination [Beh, 1973; Beh & Lascelles, 1973; Cunningham et al., 1967]. A direct Coombs test is observed when antibodies are located on the red cells and haemolysis is mediated through the host immune system.

IgG-ELISA and Coombs test are reported to have good correlation; nonetheless, the ELISA and Coombs test remain positive more than other agglutination assays. Coombs test titre will remain high with chronic long-term Brucella infection, even before the diagnosis is made. In acute brucellosis, the Coombs titre remains 4–16 times higher than the STAT titres; whereas, the titres are 16–256 times higher in chronic patients without treatment [Ducrotoy et al., 2016].

Coombs' Test, Direct IgG—Serum

4.1.1.3.3 Coombs test

The Coombs test, named after the British Immunologist Robin R. Coombs, together with his colleagues, first described it in 1945 [Coombs, Mourant, & Race, 1945]. There are two types of Coombs test, the direct and indirect antiglobulin test [DAT, IAT]. Both utilize rabbit anti-human globulin that is also referred to as Coombs reagent. Common uses of this test include suspicion of autoimmune hemolytic anaemia, prenatal compatibility testing, and cross-typing for blood transfusions. The direct Coombs test assesses antibodies adherent to the patient's own RBCs, while the indirect test measures free serum antibodies against RBCs of a fetus during pregnancy or recipient of a blood transfusion.

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].

Differential Diagnosis

•

History and Coombs test critical for all differential diagnoses

ITP

Patients have thrombocytopenia but are usually not anemic

The coexistence of ITP and hemolytic anemia is called Evans syndrome

Antiplatelet antibodies are present in plasma and on patient's platelets

Histologically, “dirty cords” are due to phagocytosis of platelets [best seen in touch preparations], but there is no evident erythrophagocytosis

Congestive splenomegaly

Patients have portal hypertension and usually have liver disease

Spleen shows an increased stroma content [fibrocongestive splenomegaly]

Coombs test negative

Hereditary spherocytosis

Family history

Lifelong hemolytic anemia

Abnormal red blood cell morphology

Coombs test negative

Direct Coombs Test

The direct Coombs test identifies the presence of antibodies or complement on RBCs. The antibodies and complement on an RBC may or may not be directed toward the RBC itself and may or may not damage the RBC. The screening test is a “polyvalent direct antiglobulin test” in which a polyvalent Coombs reagent is mixed with the patient's RBCs. This reagent contains species-specific antibodies against various classes of antibodies and complement. If the patient's RBCs have enough antibody or complement to be detected and the ratio of these antibodies to the antiglobulin is in the proper proportion, gross or microscopic hemagglutination occurs [i.e., a positive reaction]. Some laboratories use monovalent antiglobulin to classify IMHA as either immunoglobulin M [IgM] or immunoglobulin G [IgG] types and detect the presence of complement on RBCs. The pattern found in 48 dogs at the University of Minnesota was: 71% IgG positive, 10% IgG and IgM positive, and 19% IgG, IgM, and complement positive. The effect of steroid treatment in vivo is unpredictable. Dogs remain Coombs-positive for variable lengths of time during corticosteroid treatment of IMHA.

The direct Coombs test is neither highly specific nor sensitive for IMHA. The test is positive in only 60% to 70% of canine IMHA cases. Possible reasons for false-negative results include insufficient quantity of antibody on RBCs, temperature at which the test was performed, improper antigen : antibody ratio, and elution of low-avidity antibodies from RBCs during washing. To ensure that the proper antigen : antibody ratio is achieved, serial dilutions of the antiglobulin reagent should be routinely used. Over time, antibody and complement elute off the RBCs in blood samples. The time that an EDTA blood sample may be stored before a Coombs test is unknown. Therefore results obtained from samples sent to laboratories by mail are questionable. Alsever's solution is good for storing antibody-coated RBCs but is not readily available to most veterinarians. Positive Coombs reactions are expected in RBC parasite infections, incompatible blood transfusions, and drug reactions, which cause RBCs to appear foreign to the immune system.

The specificity of the Coombs test for IMHA is often considered good, but positive reactions frequently occur in the absence of evidence of IMHA. These false positives are not detected as often as false-negative results, because a Coombs test is usually not requested unless the patient has signs of IMHA. Of Coombs-positive anemias in 134 dogs, half were positive for the third component of complement [C3], but there was infrequent evidence of intravascular or extravascular hemolytic anemia.21 Therefore a positive Coombs test should be supported by other strong evidence of IMHA.

Diagnosis

•

Use Coombs test with whole blood [EDTA] to help confirm an immune reaction. Note: Close examination of the sample may reveal autoagglutination [presence of clumps], in which case a Coombs test is not needed for confirmation.

Liver function is frequently affected and not always correlated with severity of NI.

Some cases, mostly those with one or more transfusions, may have progressive liver failure, possibly associate with iron overload, even after recovering from the hemolytic aspect of NI.

What to Do