Strategies to overcome the diagnostic challenges of autoimmune hemolytic anemias

Figures & data

Figure 1. The principle of the direct antiglobulin test (DAT).

Note: Auto-antibodies (ab) are bound to erythrocytes of the patient with AIHA. In the case of IgG an anti-IgG reagent is needed to promote the formation of a visible agglutination in the tube, by binding to the Fc portion of the auto-Ab. Similarly, anti-IgA or -complement fractions (c) antisera are essential to reveal their presence on erythrocytes. In the case of IgM auto-Ab, the anti-IgM antisera is not strictly necessary to cause agglutination, since IgM are pentamers, able to cause direct agglutination of erythrocytes. IgM strongly activate C and may detach from erythrocytes leaving on their surface C fractions only. In this case agglutination occurs with anti-C antisera, and the DAT is positive for C.

Figure 2. DAT in microcolumn (a) and solid phase (b).

Note: (a) Patient’s erythrocytes are tested in microcolumns containing a gel matrix with anti-human globulin reagents (anti-IgG, -IgA, IgM, or -C). Red cell agglutinates are trapped in the glass bead matrix during centrifugation (positive test), while unagglutinated cells form a pellet at the bottom of the column (negative test).

(b) Patient’s erythrocytes are tested in microplates coated with anti-human globulin reagents (anti-IgG, -IgA, IgM, or -c). Erythrocytes with auto-Ab or C bind to wells and give diffuse colored patter (positive test), while erythrocytes without form a pellet at the bottom of the plate (negative test).

Table 1. Further investigation for the evaluation of primary versus secondary autoimmune hemolytic anemia (AIHA) cases. IVIG intravenous immunoglobulin; CAD cold agglutinin disease; wAIHA warm type AIHA.

Test	sample	Comments
Immunoglobulin class quantification	Serum/plasma	Abnormal levels occur in primary immunodeficiencies, in monoclonal gammopathies, after IVIG or monoclonal antibodies treatment	If cold agglutinin is present, the specimen must be kept at 37–38°C from sampling until serum/plasma has been separated from clot/cells
Serum protein electrophoresis	Serum/plasma	Detect monoclonal immunoglobulin or hypogammaglobinemia
Immune fixation	Serum/plasma	Identify low-concentration monoclonal immune-globulin, in particular in CAD
ANA, ENA, anti-DNA autoantibodies	Serum	Identify non-organ specific autoantibodies typical of associated connective tissue diseases	Advised in patients with signs/symptoms of connective tissue diseases (serositis, arthralgia, myalgia, photosensitivity, etc.)
Organ specific autoantibodies	Serum	May reveal associated autoimmune diseases	Advised in patients with suspected autoimmune diseases (thyroid disorders, rheumatoid arthritis, Sjogren Syndrome, inflammatory bowel syndrome, etc.)
C3 and C4 levels	Serum/plasma	Identify complement consumption; may occur in primary AIHAs but also in those secondary to autoimmune diseases	Low levels of C4 and, often, C3 are regularly seen in CAD, but also in a fraction of wAIHA
Endogenous EPO	Serum/plasma	Identifies inappropriately low EPO level that may prompt use of recombinant EPO	Advised in CAD and wAIHA in case of inadequate reticulocytopenia or presence of chronic kidney disease
Bone marrow biopsy	Bone marrow	Detect and classify bone marrow disorders	Indicated in CAD at diagnosis. Indicated in warm AIHA in case of therapy failure or if a bone marrow disorder is suspected
Contrast enhanced whole body CT scan	-	Detect organomegalies (spleen, liver, lymph-nodes, thymus), infectious and neoplastic lesions	Indicated in CAD at diagnosis. Indicated in warm AIHA in case of therapy failure or if a lymphoproliferative or neoplastic disease is suspected
Anti-phospholipid antibodies (anti-cardiolipin, beta2glycoprotein 1, and LAC)	Serum	Their positivity in a patient with previous thrombosis or miscarriages may reveal an APS diagnosis	Advised in wAIHA and CAD at diagnosis since may be associated with increased thrombotic risk
Complete thrombophilia screening	Serum/plasma/full blood	The positivity in patients with previous thrombosis may lead to life-long anti-coagulation	To be considered in wAIHA and CAD experiencing thrombosis, particularly if unprovoked and in unusual sites
HBV, HCV, HIV, EBV, CMV serologies	Serum	May be indicative of recent infection and suggest secondary AIHA, or indicate previous infection	Advised in wAIHA and CAD at diagnosis and before starting immunosuppressive treatment; Positivity may prompt prophylaxis for HBV and eradication therapy for HCV.
HBV, HCV, HIV, EBV, CMV nucleic acid tests	Serum	Indicative of viral replication during primary infections or reactivations	Advised in case of positive serology to assess replicative status and before immunosuppressive therapy
TBC quantiferon	Serum	May be indicative of previous TBC contact/chronic infection	Advised in wAIHA and CAD before starting immunosuppressive treatment; along with chest Xray. Positivity may prompt prophylaxis.
Mycoplasma serology	Serum/plasma	IgM positivity may indicate recent infection suggestive for CAS diagnosis	Suggested in CAD at diagnosis in case of symptoms of sepsis/pneumonia

Table 2. Conditions associated with autoimmune hemolytic anemia (AIHA).

Associated conditions	Comments
Lymphoproliferative disorders	5–20% of chronic lymphocytic leukemias or non-Hodgkin lymphomas are complicated by AIHA.
Autoimmune diseases	Systemic lupus erythematosus may be associated with AIHA in up to 14% of cases; reports of AIHA have been published in patients with systemic sclerosis, autoimmune thyroiditis, sjogren Syndrome, inflammatory bowel diseases, autoimmune hepatitis/primary biliary cirrhosis.
Infections and vaccines	Several infections have been associated with AIHA development, including SARS-CoV-2, Parvovirus B19, Hepatitis C, Hepatitis A/B, HIV, Mycoplasma spp, Tubercolosis/Babesiosis, Brucellosis, Syphilis/Epstein-Barr virus/Respiratory Syncytial Virus. Vaccinations may be seldomly accompanied by AIHA development, including SARS-CoV-2.
Inborn errors of immunity	Autoimmune lymphoproliferative syndrome, common variable immunodeficiency, IgA deficiency, and Kabuki syndrome may be complicated by autoimmune phenomena in up to 70% of cases, most frequently AIHA.
Tumors	6 to 9% of ovarian and prostate cancer may be associated with AIHA; case reports of AIHA complicating thymoma have been reported.
Transplant	10–15% of hematopoietic stem cell transplant may be complicated by AIHA; the passenger lymphocyte syndrome is observed in solid organ transplant (kidney, liver, heart-lung).
Drugs	Several drugs have been associated with the development of AIHA, including penicillins, cephalosporins, tetracycline, tolbutamide, metformin, quinine, quinidine, amphotericin b, rifampicin, antazolinc, thiopental, tolmetin, probenecid, nomifensine, diclofenac, doxepin, tolmetin, α-methyldopa, L-dopa, mefenamic acid, teniposide, pentostatin, cladribine, fludarabine, lenalidomide, procainamide, platin, chlorpromazine, acetaminophen, ibuprofen, thiazides, nalidixic acid, and many others. Therapy with checkpoint inhibitors may be complicated by AIHA in 0.15–0.25%

Figure 3. Diagnostic algorithm of AIHA.

Note: AIHA: autoimmune hemolytic anemia; CAD: cold agglutinin disease; C: complement; LISS: low ionic salt solutions; MS-DAT: mitogen stimulated direct antiglobulin test; PNH paroxysmal nocturnal hemoglobinuria.