The diagnosis of AIHA is based on the presence of hemolytic anemia and serologic evidence of anti-erythrocyte autoantibodies, detected by the DAT, and identified in the serum and/or eluate obtained from the patient’s red blood cells (RBCs) [44]. DAT tube, the traditional agglutination technique, is usually performed with broad-spectrum Coombs reagents. Clinically, it is important to perform the test with monospecific antisera, to distinguish “warm” (WAIHA), “cold” (cold hemoagglutinin disease, CHD), and “mixed” forms. The former are due to IgG which generally react at 37 °C, are usually directed against epitopes of the Rh system, and mainly determine extravascular hemolysis. Cold forms are due to IgM, which are able to fix complement more efficiently than other isotypes, have an optimal temperature of reaction at 4 °C, are directed against the I/i system, and prevalently cause intravascular hemolysis. The presence of cold IgM autoantibodies can easily be revealed by the spontaneous agglutination of RBCs at 20 °C. It should be reminded that there are rare cases of warm AIHA caused by IgM “warm” autoantibodies that have more severe hemolysis and more fatalities than patients with other types of AIHA. This may be related to the amount of RBC destruction by intravascular hemolysis, calculated in 200 mL of RBCs in one hour, versus extravascular hemolysis which is 10-fold lesser [44].

It is important to remind that DAT with polyspecific or anti-IgG and anti-C antisera may yield false-negative results due to the presence of IgA, low-affinity autoantibodies, or numbers of RBC-bound IgG molecules below the threshold of the test (400 molecules per RBC). For the former two conditions, the use of monospecific antisera against IgA and low ionic strength solutions (LISS) or cold washings can overcome the DAT negativity; small amounts of RBC-bound IgG can be detected employing more sensitive but less specific techniques, such as microcolumn and solid-phase antiglobulin tests that are suitable for automation, or other more sophisticated techniques, such as the complement fixation antibody consumption test, the enzyme-linked and radiolabeled tests, and the flow cytometry, that are not routine in the majority of laboratories. The latter deserves a particular mention because of its high sensitivity, being able to detect up to 30–40 molecules of anti-RBC autoantibodies. Finally, the mitogen-stimulated (MS) DAT has been proposed as a functional and quantitative method for the detection of anti-RBC antibodies in mitogen-stimulated whole-blood cultures, which may facilitate the diagnosis of some DAT-negative AIHA cases [45].

Despite the numerous tests available for the detection of antibodies against RBCs, and the development of additional more sensitive techniques, about 10 % of AIHA remains DAT negative, and the diagnosis is made after exclusion of other causes of hemolysis and on the basis of the clinical response to therapy [44].

It is worth reminding that DAT positivity may be due not only to autoantibodies, but also to alloantibodies, possibly present in transfused subjects and in multiparous females, and responsible severe hemolytic reactions in case of RBC transfusion. Moreover, false-positive DAT have been reported in patients with hypergammaglobulinaemia or following high-dose immunoglobulin therapy [46]. Finally, 0.007–0.1 % of the healthy population and 0.3–8 % of hospitalized patients have a positive DAT without AIHA.

The treatment of AIHA is still not evidence based as there are no randomized studies and only a few prospective phase 2 trials. Conventional therapy of WAIHA includes administration of corticosteroids as first-line therapy, which is expected to provide a response in 70–80 % of patients. Second-line therapy for APS-associated AIHA is mainly represented by immunosuppressive agents, such as azathioprine, cyclophosphamide, and cyclosporine, which generally provide a 40–60 % response rate. There is growing interest in the use of mycophenolate mofetil in patients with warm AIHA, particularly children; although limited to small series of patients, good responses are reported in almost all cases both idiopathic and secondary to SLE [47, 48]. More recently, rituximab (375 mg m²/weekly  × 4 weeks) has been shown to be effective in about 60–80 % of cases, either in monotherapy or in combination with corticosteroids and immunosuppressors, and regardless of prior therapy. The time to response varies considerably, with some patients responding very quickly and others taking weeks or even months to achieve their maximum response. Finally, rituximab re-treatment may be effective, and some patients responded to re-treatment more than once [49]. In attempt to minimize side effects and reduce costs, first-line treatment with low-dose rituximab (100 mg fixed dose × 4 weeks) plus a short course of steroids was reported equally effective as standard doses, with an overall response in 89 % (complete response 67 %) and 68 % relapse-free survival at 3 years [50, 51].

IVIG are frequently used in AIHA, alone or in combination with prednisone, probably because of their proven effectiveness in primary ITP and the relatively low incidence of adverse effects. However, only small case series have been reported [44, 52], and the only large retrospective study of 73 patients [53] showed a response in 40 % of cases, only 15 % achieving hemoglobin levels of 10 g/dL or greater.

It is noteworthy that steroids are poorly useful in cold forms, whereas rituximab is able to induce a complete response in 10–15 % and a partial in 50 % of patients; this compares favorably with the next best treatment regimens which give about 15–20 % of partial responses [54–56]. Recently, fludarabine-rituximab combination therapy was reported very effective, resulting in 75 % response rate, complete remissions in about 20 %, and more than 66 months estimated response duration [57].

The effectiveness of other options, such as plasmapheresis and danazol, is controversial and mostly anecdotal [54, 55]. High-dose cyclophosphamide was effective in 4/5 patients, all of whom were previously treated with corticosteroids [58]. Other “last option” treatments include alemtuzumab, bortezomib, and eculizumab which have been shown to be of some efficacy in few cases [54, 59–61]. Moreover, the administration of erythropoietin was successfully used in patients with therapy-refractory AIHA, particularly in the presence of reticulocytopenia [62]. Finally, both autologous and allogenic bone marrow transplantation were reported in immune cytopenias, including severe AIHA and Evans syndrome [63].

Splenectomy, although not performed in APS-associated cytopenias, is considered the most effective second-line treatment for primary warm AIHA, with early responses in around 70 % of cases and persistent remissions without need of medical intervention for years [54].

The most common treatment options for idiopathic AIHA are shown in Table 10.2.