AIHA is essentially a laboratory diagnosis. The diagnostic pathway of AIHA should proceed in a stepwise fashion answering the following questions.

Diagnostic Steps

The first step is to establish the diagnosis of hemolytic anemia (see box on Four Important Questions for the Diagnosis and Management of Autoimmune Hemolytic Anemia). This diagnosis is established by the presence of the following pentad of findings: normocytic or macrocytic anemia (male Hb <13.0 to 14.0 g/dL; female <12.0 g/dL), reticulocytosis (corrected reticulocyte count >2% or absolute reticulocyte count >100,000/μL to 120,000/μL), low haptoglobin, elevated lactate dehydrogenase (LDH), and elevated unconjugated (indirect) bilirubin. Haptoglobin is an α2-globulin that binds Hb. This Hb–haptoglobin complex is degraded in the liver. Hemopexin is another plasma protein with a very high binding affinity to Hb. It scavenges heme released from RBCs and protects the organisms from the adverse effects of circulating Hb. The determination of hemopexin is not essential for the diagnosis of AIHA. Indirect bilirubin is usually not more than 5 mg/ dL except in associated liver disease (Epstein-Barr [EBV]-associated AIHA). Additional findings are increased urobilinogen in the urine and spherocytes in the blood smear (Fig.1). Leukoerythroblastosis occurs only in peracute AIHA, but microangiopathic hemolytic anemia should always be suspected in such cases. Bone marrow examination is usually not necessary except in patients in whom secondary AIHA, in particular lymphoma, is suspected. RBC survival is shortened, but its measurement with radioisotopes has no diagnostic value, not even for the prediction of the efficacy of splenectomy.

Fig1. BLOOD SMEAR SHOWING NUMEROUS SPHERO CYTES (ARROWS).

Step 1: Hemolytic anemia?

Among methodologic diagnostic problems, reticulocyte counting is the biggest because in many laboratories low-precision microscopic counts are still performed. Automatic-flow cytometric methods are more precise, reliable, and convenient. With flow cytometry, the number of highly fluorescent reticulocytes can also be measured. Falsely very high mean corpuscular volume (MCV) and mean corpus cular Hb concentration occur in some cases of cAIHA because RBC counts are falsely low because of agglutination of RBC at room temperature (Fig. 2). If a cAIHA is suspected, blood samples should be sent as quickly as possible to the laboratory in warmed containers.

Fig2. PERIPHERAL SMEAR IN COLD AGGLUTININ DISEASE. Low-power scan shows uneven distribution of red blood cells (RBCs) (A), which at slightly higher power (B) shows the RBCs to be clumped together or agglutinated. This must be distinguished from rouleaux formation. High-power scan shows nucleated RBCs (C), polychromatophilia, and microspherocytes (which are mostly seen in wAIHA) (D).

All of the findings of the pentad are not always present. Reticulocytosis is often (in ≈20%) not present at the onset of AIHA. This is mostly because of a delayed initial bone marrow response of erythropoiesis. After 1 week, most of these patients have reticulocytosis. In other patients (particularly in secondary cases), absence of reticulocytosis may be attributable to impairment of erythropoiesis caused by bone mar row infiltration, vitamin B9 or B12 deficiency or blunted erythropoiesis caused by an acute-phase reaction. If the reticulocyte count is very low, pure RBC aplasia (PRCA), either immune mediated or induced by a parvovirus (or HHV6) infection, should be suspected. Haptoglobin may be falsely normal or even slightly increased, particularly in patients with malignant or immune diseases, because haptoglobin is an acute phase protein. Haptoglobin may be falsely low in patients with a haplotype H₀H₀ and in patients with severe liver disease. Both increased bilirubin and elevated LDH have a limited specificity for AIHA.

Step 2: Autoimmune Hemolytic Anemia?

 The next step is to find out whether the hemolytic anemia is an AIHA. This is best done by the DAT (Fig. 3). In this test, washed RBCs of the patient (obtained from an ethylenediaminetetraacetic acid [EDTA] blood sample) are incubated in a tube with a polyspecific antibody to IgG and complement (C3d). If the RBCs agglutinate, the test result is positive. In many laboratories, the tube test has been replaced by the tube-gel test, which is easier to perform, more reliable, and probably more sensitive (Fig. 4). In the indirect antiglobulin test (IAT), patient plasma or serum is incubated with test RBCs and (after washing) RBC-bound IgG is detected with the DAT. IAT is usually not required for the diagnosis of AIHA except when a drug-dependent antibody is suspected. For the differentiation of drug-dependent antibodies and autoantibodies, an acid eluate of the patient’s RBCs should be made and tested in the IAT. If the IAT result is positive, the patient has autoantibodies. The severity of AIHA does not correlate with the strength of the DAT, but rather with the Ig subclass of the antibody (IgG1 or IgG3). The result of the DAT is not a reliable marker of treatment success because patients with a complete hematologic remission may remain DAT positive, and DAT positivity or negativity has only limited value to predict the duration of hematologic remission.

Fig. DIRECT ANTIGLOBULIN TEST FOR DETECTION OF (A) ERYTHROCYTE-BOUND C3d OR (B) IgG. Hemagglutination occurs when anti-C3d or anti-IgG can create a lattice structure by bridging sensitized RBCs. IgG, Immunoglobulin G; RBC, red blood cell. (Courtesy Cunningham MJ, Silberstein LE. Autoimmune hemolytic anemia. In: Hoffman R, Benz EJ Jr, Shattil SJ, et al., eds. Hematology: Basic Principles and Practice. 4th ed. Philadelphia, PA: Elsevier; 2005.)

Fig4. RESULT OF A DIRECT ANTIGLOBULIN TEST PERFOMED ON GEL COLUMNS, WITH A POSITIVE RESULT SHOWN WITH AN ANTI-IgG AND ANTI-C3d. ctl, Control; IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M.

Falsely Negative and Positive Direct Antiglobulin Test Results Without Hemolysis or Anemia

If the conventional DAT test result is negative, hemolytic anemia is defined as DAT-negative. This implies that positive DAT with anti IgA has been excluded as well as other causes or hereditary of acquired hemolytic anemia—in particular hereditary spherocytosis and paroxysmal nocturnal hemoglobinuria. However, AIHA cannot be definitely excluded because about 5% (2% to 11%) of AIHA patients are DAT negative. If AIHA is suspected for clinical grounds despite a negative DAT result, more sensitive quantitative tests are required to determine the amount of IgG on the RBCs. The threshold of positivity of the conventional DAT is 100 to 200 IgG molecules per RBC, but in some AIHA patients the RBC IgG is less than this amount. In about one-third of DAT-negative cases, one of the more sensitive test results (e.g., immunoradiometric tests) will be positive. However, the relationship between the amount of RBC IgG and hemolysis is not clear cut, and there is no “hemolysis threshold.”33 The reasons for these discrepancies between the in vitro and in vivo activity of RBC antibodies are largely unknown. Differences in macrophage activity may be one possible explanation. A search for antibodies in the RBC eluate in which antibodies are more concentrated is also useful. IgA antibodies are rare and sometimes not included in the analysis. Finally, there is the possibility of low-affinity antibodies. Such anti bodies are washed out when the washes are made with 37°C saline. A high rate of DAT-negative AIHA has been observed in AIHA induced by nucleoside analogues, but also in other secondary AIHA.

The DAT result is positive in 1 in 10,000 to 3 in 10,000 nor mal persons and 10% of hospital patients without anemia or signs of hemolysis.

Step 3: Warm or Cold Autoimmune Hemolytic Anemia?

In a further step, the DAT is carried out with monospecific antibodies to IgG and complement (C3d) to find out whether a warm or cold antibody is the cause of hemolysis. If the DAT result is positive with IgG alone or with IgG plus C3d, the AIHA is most probably a wAIHA. If the DAT result is positive with only C3d, the AIHA is most probably a cAIHA. The differentiation between wAIHAs and cAIHAs is extremely important for the choice of treatment.

Some special diagnostic problems exist when it comes to cAIHAs. If cAIHA is suspected, it must be taken care that the blood sent to the laboratory is kept at 37°C to get reliable results. Patients with only RBC C3d may (rarely) have wAIHAs when the amount of RBC IgG is very small. In patients with C3d positivity, the cold agglutinin titer should be determined. If it is equal or greater than 1:64 the diagnosis of a CAD is established. There is no consensual threshold titer that separates normal from abnormal. If the titer is less than 1:64, the thermal amplitude of the antibody must be determined. If the thermal amplitude is above 22°C, the diagnosis is CAD. Mixed type AIHA is rare. Frequently used criteria are a positive IgG DAT result and a positive eluate result plus a C3d-positive DAT result and the presence of CAs with a thermal amplitude of greater than 30°C or high titer Cas (>1:40). Many published cases of mixed-type antibodies do not fulfill these criteria.

Step 4: Primary or Secondary Autoimmune Hemolytic Anemia?

In the last diagnostic step, it must be determined whether the AIHA is primary or a complication of an underlying disease (secondary). About half of cases of AIHA (or probably even more) are secondary. The main causes are systemic immune diseases and malignancies and mostly B-cell lymphomas. Other underlying conditions are infections, primary immunodeficiencies, drugs, transplantation, and con genital defects. The decision regarding which diagnostic procedures should be used for this purpose depends mainly on the type of AIHA (wAIHAs or cAIHAs) and should include history, physical examination, laboratory tests, and imaging procedures if indicated. In children and younger patients with wAIHA, evidence for an infection should be sought. A list of all recent medication should be made. A history of weight loss, fever, or poor general condition, and arthritis points to a malignancy or systemic immune disease as the underlying condition. Palpable lymphadenopathy and important splenomegaly do not belong to the usual clinical picture of primary wAIHA. In this case, lymphoma (particularly splenic marginal zone lymphoma [SMZL]) should be suspected. Laboratory tests should include acute phase proteins, LDH, quantitative determination of Igs, antinuclear antibodies, and other tests guided by the clinical history. A bone mar row examination is not obligatory except when lymphoma is suspected. Abdominal ultrasonography or a CT scan is reasonable in all cases to look for a disproportionate splenomegaly, the remote (but important) possibility of an ovarian teratoma (in women), another solid tumor, lymphadenopathy, or solitary extranodal lymphomas. An activated partial thromboplastin time with a lupus-sensitive reagent may reveal an LA. Fluorescence-activated cell sorting or PCR for the detection of monotypic/monoclonal lymphocytes may be done, but the clinical usefulness of such tests to guide treatment decisions is uncertain.

In cAIHA, a history of a febrile illness should prompt a thoracic radiography, and if results are positive, a serologic test for mycoplasma pneumoniae is required. The quantitative determination of serum Igs and a search for a clonal Ig by immune fixation are important. If immune fixation findings are positive, a search for lymphoma is necessary, which may include bone marrow biopsy even when there is no lymphadenopathy. In addition to classical CAD, CAs occurring in association with other cancers, aggressive lymphomas, or infection should be termed cold agglutinin syndrome.

Other tests, including blood glucose, Hba1c , renal and hepatic function tests, HIV serology, hepatitis B antigen, and exclusion of latent tuberculosis are necessary to avoid complications of steroids (in wAIHAs) or rituximab (in cAIHAs and wAIHAs).

اخر الاخبار

اخبار العتبة العباسية المقدسة

دار علوم نهج البلاغة تقيم محفلها الأسبوعي في بغداد

قسم التطوير ومدينة الفردوس يبحثان آفاق التعاون في مجال التدريب

المجمع العلمي يواصل إقامة فعاليات برنامج يستبشرون القرآني

قسم الشؤون الفكرية يهدي كتاب شرح دعاء أبي حمزة الثمالي إلى جامعة وارث الأنبياء (عليه السلام)

المزيد

الآخبار الصحية

عادة نهارية شائعة قد تخفي مشكلات صحية كامنة

الخوف من العيادات الطبية.. أسباب نفسية خفية لدى البالغين

"علكة مبتكرة" تستهدف ميكروبات الفم المسببة للسرطان

دخان حرائق الغابات قد يرفع خطر الإصابة بأنواع من السرطان

المزيد

الآخبار التكنلوجية

منهج جديد باستخدام الساعات الذرية يعيد طرح سؤال: ما طبيعة الزمن؟

اكتشاف آلية خلوية تمنع تكوّن الألياف المرتبطة بألزهايمر

دراسة تكشف أدلة على احتمال وجود محيط قديم بالمريخ

تيم كوك ينهي 15 عاما في قيادة "أبل"

المزيد

مواضيع ذات صلة

Blood Coagulation Tests

Western Blot Immunoassays

Indirect Fluorescent Antibody Tests and Other Immunomicroscopic Methods

Enzyme-Linked Immunosorbent Assays

Laboratory Diagnosis of Thyrotoxicosis

Laboratory Findings of Beta-Thalassemia Syndromes

Complement Fixation Assays

Flocculation Tests

Particle Agglutination Tests

Direct Whole Pathogen Agglutination Assays

Separating IgM From IgG For Serologic Testing

Serodiagnosis of Infectious Diseases