Coombs' negative autoimmune hemolytic anemia diagnosed by flow cytometric analysis of autoantibodies to red blood cells.

Abstract Introduction. CD30, member of the TNF-receptor family, is expressed on activated T and B cells. Upon CD30 signaling and the downstream event of cytokine release of IL-4 and IL-13, CD30 is cleaved by a metalloprotease, resulting in soluble CD30 (sCD30). sCD30 has been shown to be a reliable proxy for Th2 activity and is elevated in Th2-polarized disorders such as atopic dermatitis, rheumatoid arthritis, and systemic lupus erythematosus (SLE) but is subnormal in patients with Th1-polarized disease such as X-linked agammaglobulinemia (Eur J Immunol2001; 31:1927–1934). Elevated sCD30 levels were also found in patients with Hodgkin’s lymphoma or anaplastic large cell lymphoma. We investigated sCD30 levels in patients with chronic idiopathic thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA). Method. We measured plasma sCD30 levels in 35 patients with ITP, 12 with AIHA, 8 with SLE, 9 with lymphoma and 18 healthy controls. sCD30 was measured by ELISA (Bender Medsystems) in platelet poor plasma from citrated blood. Results. Compared to controls (19.0 ± 6.2 units/mL), sCD30 levels were significantly higher in AIHA (mean 145.6, p=0.03) but not in ITP (22.7, p=0.1). sCD30 levels were also elevated in SLE (mean 73.6, p=0.001) and lymphoma (mean 107.4, p=0.04), confirming previously published studies. In AIHA, sCD30 levels were not significantly different in patients with active/chronic disease versus those in clinical remission, although levels trended higher during the acute episodes of hemolysis. In ITP, sCD30 levels were also similar between active disease versus remission and splenectomized versus unsplenectomized patients. Conclusion. Our results support the hypothesis that AIHA and SLE are Th2-polarized disorders, whereas ITP is not. Lack of significant differences in sCD30 between active disease and remission in AIHA and ITP may suggest that imbalances of Th1/Th2 immunity persist regardless of clinical course, consistent with Panitsas et al (BMC Blood Disord2004; 4:4). Discussion. Prior studies have examined serum cytokine concentrations such as IFN-γ and IL-4 in patients with ITP and reached inconclusive results. But recent studies using more sensitive techniques such as RT-PCR indicate that ITP is a Th1-polarized disorder (Blood2004; 103:2645–2647). Our findings of elevated sCD30 levels in AIHA but not ITP lead to a provocative question: what is it about the immunological destruction of platelets that makes it associated with a markedly different Th1/Th2 milieu in comparison to the destruction of red blood cells? We propose that the key differences are 1) platelets are more potent in modulating immunity via mechanisms including CD40L (CD154) release upon platelet activation (Immunity2003; 19:9–19) and 2) platelets express MHC I molecules whereas red blood cells do not and hence they are processed by macrophages in a different manner (Transfusion2002; 24:958–961).

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Reticulocyte-Gated Flow Cytometric Analysis of Red Blood Cells in Paroxysmal Nocturnal Hemoglobinuria

Laboratory Hematology ◽

10.1532/lh96.05026 ◽

2006 ◽

Vol 12 (2) ◽

pp. 82-85 ◽

Cited By ~ 4

Author(s):

Shoko Sato ◽

Yuichi Hasegawa ◽

Toshiro Nagasawa ◽

Haruhiko Ninomiya

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Flow Cytometric Analysis ◽

Flow Cytometric

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Soluble CD30 Levels Are Highest in Autoimmune Hemolytic Anemia (AIHA) and Lymphoma among the Hematologic Disorders, but Are Not Elevated in ITP.

Blood ◽

10.1182/blood.v104.11.3872.3872 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3872-3872

Author(s):

Eugene R. Ahn ◽

Eckhard R. Podack ◽

Gabriella Lander ◽

Carlos J. Bidot ◽

Wenche Jy ◽

...

Keyword(s):

Red Blood Cells ◽

Hemolytic Anemia ◽

Sickle Cell ◽

Autoimmune Hemolytic Anemia ◽

Blood Cells ◽

Small Subset ◽

Hematologic Disorders ◽

Hematological Disorders ◽

Soluble Cd30 ◽

Negative Findings

Abstract Introduction. CD30, member of the TNF-receptor family, is expressed on activated T and B cells and is cleaved by a metalloprotease following CD30 signaling, but the function of CD30 remains elusive. CD30 was initially described on the surface of Reed-Sternberg cells, and elevated levels of soluble CD30 (sCD30) have been described in patients with lymphoma, HIV and autoimmune diseases. Higher levels have been associated with unfavorable prognosis in lymphoma and HIV, and are thought to represent a Th2-polarized response. Relatively little is known about sCD30 profiles in many hematologic disorders. Method . We measured plasma sCD30 levels in patients with 1) non-malignant [27 ITP, 12 autoimmune hemolytic anemia (AIHA), 5 sickle cell, 5 PNH, 8 SLE] and 2) malignant [13 lymphoma, 8 multiple myeloma (MM), 11 MDS, 19 MPD] hematological disorders, as well as 3) thrombotic disorders [11 DVT, 17 hypercoagulable states] and 18 healthy controls. sCD30 was measured by ELISA using platelet poor plasma from citrated blood as well as lysates of patient red blood cells following osmotic lysis with water (1:10 dilution) versus supernatant of RBC’s diluted in PBS control. Results . In non-malignant disorders, sCD30 was significantly higher in AIHA (142.7±195.1 u/ml, p=0.006) and SLE (73.6±33.9, p=0.0000003), but not in other diseases associated with hemolysis such as sickle cell (18.7±12.3, p=0.4) and PNH (40.8±41.1, p=0.06) nor in another autoimmune disease, ITP (23.1±16.5, p=0.2) when compared to controls (19.0±6.2). In malignant disorders, all diseases tested had significantly higher sCD30 versus control: lymphoma (94.3±108.2, p=0.003), MM (47.3±28.5, p=0.0002), MDS (47.1±35.1, p=0.001), and MPD (35.3±31.6, p=0.02). In thrombotic disorders, none were significantly different from control. The surprising difference between AIHA and ITP sCD30 levels was statistically significant as well (p=0.001). In AIHA, sCD30 was similar in both active disease and remission, although sCD30 trended higher during acute hemolysis. The source of sCD30 in AIHA was first evaluated by FACS analysis of peripheral lymphocytes, which was negative. A small subset of patients had glycophorin A+, CD30+ red blood cells, but it did not correlate with sCD30 levels. However, RBC lysates from patients with AIHA contained significant levels of CD30 by ELISA in 5 of 5 patients (10.7–170.4 u/ml) with AIHA versus 0 of 4 patients with ITP (0–9.0). Discussion . High sCD30 levels in AIHA is a novel finding, and it is of interest to find that sCD30 is not elevated in ITP, a disorder once thought to be similar in pathophysiology. The possible explanations are: 1) AIHA may reflect a highly polarized Th2 disease whereas ITP is a more Th1-mediated disorder 2) hemolyzed RBC’s in AIHA release CD30 acquired by absorption or pinocytosis or 3) CD30 is produced and stored by RBC precursors. The negative findings of sCD30 in other hemolytic diseases has yet to be explained. Further delineation of the role of CD30 in hematologic disorders will provide new light on its elusive function. The new finding of high CD30 in AIHA and intracellular CD30 in RBC’s offers a new avenue of research on CD30 in immune-mediated hemolysis.

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Predictors of autoimmune hemolytic anemia in beta-thalassemia patients with underlying red blood cells autoantibodies

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2019.102342 ◽

2019 ◽

Vol 79 ◽

pp. 102342 ◽

Cited By ~ 1

Author(s):

Monia Ben Khaled ◽

Monia Ouederni ◽

Nessrine Sahli ◽

Nawel Dhouib ◽

Ahmed Ben Abdelaziz ◽

...

Keyword(s):

Red Blood Cells ◽

Hemolytic Anemia ◽

Autoimmune Hemolytic Anemia ◽

Blood Cells ◽

Beta Thalassemia

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Expression levels of CD47, CD35, CD55, and CD59 on red blood cells and signal-regulatory protein-α,β on monocytes from patients with warm autoimmune hemolytic anemia

Transfusion ◽

10.1111/j.1537-2995.2008.01936.x ◽

2009 ◽

Vol 49 (1) ◽

pp. 154-160 ◽

Cited By ~ 26

Author(s):

Melca M.O. Barros ◽

Mihoko Yamamoto ◽

Maria S. Figueiredo ◽

Rodolfo Cançado ◽

Elisa Y.S. Kimura ◽

...

Keyword(s):

Red Blood Cells ◽

Hemolytic Anemia ◽

Autoimmune Hemolytic Anemia ◽

Blood Cells ◽

Regulatory Protein ◽

Expression Levels

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Flow cytometric analysis of the oxidative status of normal and thalassemic red blood cells

Cytometry ◽

10.1002/cyto.a.20017 ◽

2004 ◽

Vol 60A (1) ◽

pp. 73-80 ◽

Cited By ~ 68

Author(s):

Johnny Amer ◽

Ada Goldfarb ◽

Eitan Fibach

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Flow Cytometric Analysis ◽

Oxidative Status ◽

Flow Cytometric

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An Unusual Case of Autoimmune Hemolytic Anemia-Thinking Outside the Box for Management

Blood ◽

10.1182/blood-2018-99-111196 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5075-5075

Author(s):

Paul C Williams ◽

Ileana Lopez-Plaza

Keyword(s):

Red Blood Cells ◽

Hemolytic Anemia ◽

Autoimmune Hemolytic Anemia ◽

Blood Cells ◽

High Dose ◽

Quadrant Pain ◽

Lower Quadrant ◽

Packed Red Blood Cells ◽

Splenic Embolization ◽

Positive Direct

Abstract A 27 year old male with no significant past medical history presented to an outside hospital for chief complaints of nausea/vomiting, epigastric pain, and acute jaundice. Initial laboratory workup revealed a hemoglobin of 5.5 g/dl, normal WBC and platelet count, elevated direct bilirubin (10 mg/dl), elevated lactate dehydrogenase (969 IU/L), low haptoglobin (<8 mg/dl), a peripheral smear showing rare polychromasia and spherocytes, and positive direct antiglobulin test (DAT: BS +, IgG +, C3d -) with presence of warm autoantibodies. All infectious disease testing was negative. Initial CT abdomen showed a normal sized spleen. The patient was diagnosed with idiopathic warm autoimmune hemolytic anemia and was treated with pulsed steroids, one dose of Rituximab, and transfused with up to 13 units of packed red blood cells. The patient did not respond to medical treatment, and after one week, the patient was transferred to our institution for escalation of care. At our institution, the patient presented with similar symptoms. Due to lack of response to packed red blood cell transfusions, As the patient continued a downtrend in the hemoglobin despite continued transfusion support, Transfusion Medicine advised to only transfuse phenotypically matched, least incompatible, packed red blood cells for significantly symptomatic anemia or hemodynamic instability. High dose steroid were continued and IVIG, Cytoxan and Vincristine were added to his warm autoimmune hemolytic anemia (WAIHA) therapy. Despite these efforts, the patient's hemoglobin continued to decrease despite an additional 9 RBC units transfused at our institution, reaching a hemoglobin nadir of of 2.1 g/dL. In addition, the patient developed thrombocytopenia, NSTEMI, and left lower quadrant pain accompanied by bloody bowel movements. A repeat CT scan of the abdomen was performed, which revealed an enlarged spleen with a span of 16.5 cm in its maximum dimension. Due to concerns for splenic infarction and/or rupture, and since the patient was not a candidate for surgery, partial splenic artery embolization was performed. Thirty percent residual viable spleen remained. Within hours post embolization, a dramatic increase in hemoglobin was observed without any additional transfusion support. Shortly after splenic embolization the patient developed severe left upper quadrant abdominal pain with a peri-splenic fluid measuring slightly greater than simple fluid attenuation on CT which was managed medically. After close observation the patient was discharged home 2 weeks after admission. At home the patient was doing well with the exception of persistent left upper quadrant pain. He had no clinical or laboratory signs of recurrent hemolysis. Three months post discharge, due to persistent left upper quadrant pain and presence of a splenic cyst, the patient underwent a laparoscopic splenectomy that was performed without complications. During this hospitalization, in preparation for surgery, compatibility testing showed no autoantibodies. However new clinically significant alloantibodies were identified. Hypersplenism and/or reticulocytopenia are infrequent complications of a warm autoimmune hemolytic anemia, constituting a life threatening complication that may not respond to standard therapy and may not qualify for surgical intervention. The above patient did not improve with any medical therapies and displayed a decreasing trend in hemoglobin that was accompanied by increasing lactate levels and NSTEMI. Splenic embolization achieved a dramatic response that was maintained through follow up. Due to recurrent pain from the embolization, the patient eventually received a splenectomy once he was stable. Four months after initial presentation, post infusion of 4 doses of Rituxan and steroid therapy, the autoantibodies were not detectable. However, the patient developed 3 alloantibodies post multiple transfusions. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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