Serelina Coluzzi

Immunological definition of acute promyelocytic leukemia (FAB M3) : a study of 39 cases

Acute promyelocytic leukemia (FAB‐M3) is a distinct entity among acute non‐lymphoid leukemias (ANLL) with peculiar morphological, biological, clinical and prognostic features. An atypical form of M3 (M3v) could be confused with other FAB ANLL and therefore the diagnosis of this variant requires ultrastructural analysis and/or cytogenetic study and/or selective gene rearrangement studies. The immunological phenotype of blast cells in 39 APL patients was studied at diagnosis. The diagnosis of M3 FAB type was ascertained in 32 and the diagnosis of M3v in 7 cases. Using a large series of monoclonal antibodies (mAb), the APL blast cells were B and T cell antigens‐negative, HLA‐DR constantly negative, CD 13‐ and/or CD33‐positive, CD9‐positive. Among ANLL this phenotype seems to be closely related to APL both in M3 type and M3v subtype. Because the diagnosis of APL (M3 or M3v) is important in order to establish the specific therapeutic approach, the discriminant capacity of the immunological typing to identify M3 and mainly M3v (hypogranular) could be determinant for a “quick” diagnosis.

Clinical effectiveness of platelets in additive solution treated with two commercial pathogen-reduction technologies

Two noninferiority, randomized, controlled trials were conducted in parallel comparing the safety and efficacy of platelets treated with Intercept or Mirasol pathogen‐reduction technologies versus standard platelets.

Hemolysis in patients with antibody deficiencies on immunoglobulin replacement treatment

Immunoglobulin (Ig)G replacement with intravenous or subcutaneous immunoglobulins is a lifelong substitutive therapy in patients with primary antibody deficiencies (PADs). Hemolysis after immunoglobulin therapy was described in patients receiving high immunoglobulin dosages. The issue of hemolysis after immunoglobulin administration at replacement doses has been considered of little clinical significance.

Recommendations for the prevention and treatment of haemolytic disease of the foetus and newborn

The publication of the second edition of the “Recommendations for the prevention and treatment of haemolytic disease of the foetus and newborn” is the result of collaboration between the Italian Society of Transfusion Medicine and Immunohaematology (SIMTI, Societa Italiana di Medicina Trasfusionale e Immunoematologia) and the Italian Society of Gynaecology and Obstetrics (SIGO, Societa Italiana di Ginecologia e Ostetricia). The recommendations published in 20061 have been revised in the light of current scientific evidence: the immunohaematological and instrumental investigations that should be performed in the antenatal and perinatal periods, the immunoprophylaxis (IP) to prevent the haemolytic disease of the foetus and newborn (HDFN due to RhD incompatibility and the treatment to use if HDFN develops are described. The recommendations are focused on the prevention and management of HDFN, in particular that one due to RhD incompatibility, the most serious form of this condition. Although IP has dramatically reduced the number of cases of HDFN, this disease continues to occur and engage specialists in Transfusion Medicine, Obstetrics and Neonatology. The recommendations are aimed at Transfusion Structures (TS) and all public facilities pertaining to Mother and Child Departments, Family Planning Clinics and private structures managing pregnancies, including those in which the woman gives birth at home. The prevention of HDFN must be guaranteed, through organisational models adapted to local circumstances, to all pregnant women for whom it is deemed necessary and the women must also be ensured adequate information. Besides HDFN due to RhD incompatibility, the recommendations also cover less frequent forms of the disease, caused by immunisation to other blood group antigens, and by ABO incompatibility, which is a more frequent laboratory finding, although of less importance from a clinical point of view. These recommendations will be periodically reviewed in the light of evolving scientific knowledge, technology and clinical practice. They were developed on the basis of an analysis of current scientific literature (identified through bibliographic searches of Medline/PubMed and Ovid databases) and were submitted to the consensus of experts from SIMTI and SIGO. Protocols jointly agreed upon by the Transfusion Medicine and Immunohaematology Services (SIMT, Servizio di Immunoematologia e Medicina Trasfusionale) and Obstetricians-Gynaecologists working in the same territory, including at a regional level, should be drawn up to promote compliance among pregnant women.

Monocytopenia and infections in chronic lymphocytic leukemia (CLL)

To the Editor: The key role of monocytic/macrophage cells in the immune response is well known (1-2). These cells act as antigen-presenting cells, are involved in cytotoxic functions and are cytokine producers: therefore their importance in the immune defense is more crucial than that of granulocytes. Nevertheless, in hematological disease all therapeutic protocols define infectious risk in relationship to circulating granulocytes and do not consider monocyte levels

A metastatic breast carcinoma presenting as autoimmune hemolytic anemia.

The authors describe the case of a 75-year-old female who was hospitalized for anemia of unknown origin. Physical examination revealed a swelling in the right mammary region, where a mastectomy scar was present from surgery for a breast carcinoma. On admission, laboratory tests disclosed anemia (Hb, 8.5 g/dl), with a reticulocyte count of 65,000/mm3 and slightly increased bilirubin. Immunohematologic study revealed the presence of a red cell autoantibody with anti-D specificity in the serum and in the eluate from the patients erythrocytes. A biopsy of the swelling was performed and histologic examination showed the presence of metastatic cells of breast carcinoma. The patient was given chemotherapy and radiotherapy. At this writing the anemia was absent, the Immunohematologic study was negative, the swelling was greatly reduced, and no other metastatic lesions of breast carcinoma were present.

Autoimmune Hemolytic Anemia and Immune Thrombocytopenia as Unusual Presentations of Childhood Hodgkin Lymphoma: A Case Report and Review of the Literature

We discuss an unusual clinical presentation of childhood Hodgkin lymphoma; a 16-year-old girl was referred for Coombs-positive severe anemia, thrombocytopenia, and asymptomatic anterior mediastinal mass. Bone marrow examination showed no evidence of neoplastic disease. Biopsy of the mass was possible only after administration of both intravenous immunoglobulins and steroids resulting in a prompt rise of the platelet count and partial hemoglobin level stabilization. The identification of this clinical picture as a possible complication of an underlying Hodgkin lymphoma presents difficulties in diagnosis and management of the primary condition.

Lymphocyte T subsets and natural killer cells in Italian and Philippino blood donors.

Background and Objectives The characterization of lymphocyte subsets in blood donors has been utilized to determine the normal ranges that can be related to race. A study was performed in blood donors from two racial groups – Caucasian (Italians) and Asian (Philippinos) – to define respective T‐lymphocyte subsets and levels of cytokines.

Severe warm autoimmune haemolytic anaemia due to anti-Jk(a) autoantibody associated with Parvovirus B19 infection in a child.

Dear Sir, Autoimmune haemolytic anaemia (AIHA) is an uncommon clinical condition in which endogenous antibodies are directed against the patient’s own red blood cells coated by immunoglobulin and/or complement. Anaemia appears when the destruction of red blood cells overwhelms the bone marrow’s capacity to produce new cells. People of all ages can be affected, although some reports suggest that, in childhood, secondary cases are more common than idiopathic forms, and viral or bacterial agents are frequently the only recognisable stimuli. Indeed, AIHA follows viral infection or vaccination much more often in children than in adults. However, in the majority of cases of AIHA the pathogen remains undefined. Parvovirus B19 infections have frequently been implicated as a trigger of several forms of autoimmune diseases both in children and adults. Here we report the case of a 5-year old girl who was referred to our hospital because of anaemia and mild jaundice. The patient was previously healthy until 15 days prior to admission, when she developed a fever, weakness, lack of appetite, diarrhoea and vomiting for about 4 days. On admission, the main clinical signs and symptoms noted during a general physical examination were pallor, jaundice and tachycardia (heart rate: 150 bpm). Haematological tests showed a haemoglobin (Hb) level of 4.1 g/dL, mean corpuscular volume 83 fL, reticulocyte count 147×109/L and normal leucocyte and platelet counts. Marked polychromasia with spherocytosis and nucleated red blood cells were noted on the peripheral blood smear, without atypical cells. The serum lactate dehydrogenase (LDH) was raised at 1,047 IU/L, total bilirubin was 2.61 mg/dL, direct bilirubin 0.61 mg/dL, haptoglobin 10 mg/dL, C-reactive protein 10.8 mg/L, aspartate amino transferase 68 IU/L, alanine amino transferase 24 IU/L and ferritin level 354 ng/mL. Tests for anti-nuclear, anti-double-stranded DNA, and anti-smooth muscle antibodies and anti-phospholipids were negative. Abdominal ultrasonography revealed hepatosplenomegaly. An immunohaematological study was performed. A direct antiglobulin test (DAT) was performed with a broad-spectrum antiserum and with monospecific anti-IgG, -IgA, -IgM, -C3d and -C3b antisera, in liquid phase and by column agglutination (reagents from Ortho Clinical Diagnostics, Raritan, New Jersey, USA and Diamed, Cressier sur Morat, Switzerland). Eluate testing was performed by Rubin’s method and with low pH glycine buffer using a commercial kit (ELU-KIT™ II, Immucor, Norcross, Georgia, USA). An indirect antiglobulin test (IAT) with untreated and treated (ficin/papain) homologous red blood cells (Resolve C - Ortho Clinical Diagnostics and ID-Diamed Panel- DiaMed) was also performed. On admission, the DAT was strongly positive for an IgG autoantibody which was also present in the patient’s serum. Both the eluate and the serum, investigated using a broad panel of reagent red blood cells, showed an anti-Jka antibody. Kidd typing of the erythrocytes, performed using a monoclonal IgM reagent (Ortho Clinical Diagnostics), showed a Jk(a) positive, Jk(b) negative phenotype so the anti-Jka antibodies found in the blood of the patient were presumed to be autoantibodies. AIHA was diagnosed and therapy was started with intravenous methylprednisolone (20 mg/kg/die) and folic acid (20 mg/die). From the fifth day the steroid treatment was continued in the form of oral prednisone (2 mg/kg/die). Due to severe, symptomatic anaemia the child was transfused with a compatible unit (150 mL) of Jk(a) negative, Jk(b) positive red blood cells. Bacterial culture of stools for Campylobacter, Rotavirus, Yersinia, and Escherichia coli were negative, as was the search for lactate-positive, coagulase-negative Enterobacteria, Staphylococcus and Enterococci spp. Other causative agents were investigated: cytomegalovirus, Epstein-Barr virus, hepatitis B and C viruses, human immunodeficiency viruses 1/2, herpes simplex virus 1–2 were negative, while Parvovirus B19 IgM and IgG were found. The patient’s clinical conditions gradually improved and she was discharged from hospital. About 2 weeks after admission the immunohaematological tests became negative and the patient had a Hb level of 11.5 g/dL; LDH levels returned to the normal range. One month after the onset, the child developed a transient thrombocytopenia (platelet count of 96×109/L and 106×109/L on two different occasions). The prednisone treatment was maintained at a dose of 2 mg/kg/die for 15 days, and then gradually reduced to 2.5 mg/die and discontinued 40 days after the initial presentation because the patient had normal haematological findings. She remains healthy at follow-up (6 months). In paediatric patients AIHA is often associated with infections or organ-specific autoimmune diseases1–4. Human Parvovirus B19 may cause a widespread benign and self-limiting disease in children and adults, but this viral infection has also been associated with the production of autoantibodies against many autoantigens (nuclear antigens, neutrophil cytoplasmic antigens, phospholipids) and with rheumatoid factor.2. It has previously been shown that chronic B19 infection can induce anti-viral antibodies with autoantigen-binding properties. The autoimmune sequelae have a multifactorial and complex origin: the involvement of molecular mimicry between self-antigens and viral proteins, the induction of enhanced cytokine production via the viral transactivator protein NS1 and the phospholipase A2-like activity of the capsid protein VP1 seem to contribute to the induction of autoimmune diseases3. Parvovirus B19 interacts extensively with human red blood cells; in vitro studies have shown changes in capsid conformation following B19 binding to red blood cells, leading to exposure of a region (VP1 “unique region”) that seems to play a central role in the induction of autoimmune processes. Antibodies derived from the exposed VP1 “unique region” would not neutralise free infectious particles in the blood, but would instead target receptor-attached virus4. An interesting finding in our case was the rarely occurring specific complement-binding warm auto-antibodies against the Jk(a) antigen. Generally autoantibodies with single specificity are produced against Rh system antigens. Warm anti-Jka autoantibodies have been rarely described, in association or not with haemolysis; most of the cases reported in the literature were in patients with autoimmune disorders, such as ulcerative colitis or systemic lupus erythematosus. In our patient the simultaneous disappearance of the anti-Jka autoantibodies and the haemolysis strongly suggests that the anti-Jka was responsible for the haemolysis. It is noteworthy that the first manifestations of infection in our patient were in the gastrointestinal system and no infectious agent was identified other than the B19 virus. The gastrointestinal symptoms were followed 2 weeks later by acute haemolysis. Antigen sharing between the gastrointestinal tract and red blood cells has been described by Hinoue et al.5, who identified and characterised a Kidd antigen/UT-B urea transporter expressed in the human colon, encoded by the Slc14A1 gene, with a sequence identical to that reported for the Kidd/UT-B present on the red blood cells. In the light of these data, we can hypothesise a cross-reactivity of autoantibodies between autoantigens of the colon and the red blood cells. The severe, transient haemolysis observed in our patient occurred after the acute phase of infection. Our patient also showed a transient mild thrombocytopenia, while her leucocyte count remained normal. A meta-analysis that included 516 cases of childhood autoimmune thrombocytopenia and 246 healthy controls showed that human B19 infection is closely associated with childhood autoimmune thrombocytopenia. Parvovirus B19, which can be a primary cause of reticulocytopenic post-infection anaemia in childhood, is strongly suspected to have been responsible for the haemolytic anaemia in our patient. This case is unusual and interesting because of the association of B19 infection with warm AIHA and because of the rare specificity of the autoantibodies. Besides its scientific interest, the authors recommend identification of antibody specificities in AIHA since this will play an important role in the appropriate transfusion therapy if the severity of the clinical course is such as to impose this therapeutic strategy.

Hypoxia-inducible factor-1α(Pro-582-Ser) polymorphism prevents iron deprivation in healthy blood donors.

BACKGROUND Frequent blood loss induces progressive depletion of iron stores, leading to iron deficiency and, ultimately, to overt iron-deficient anaemia. The erythropoietin-mediated bone marrow response to anaemia is under the control of hypoxia-inducible factors (HIF), the master regulators of oxygen and iron homeostasis. Since the HIF-1α(Pro-582-Ser) variant is associated with elevated trans-activation capacity of hypoxia responsive elements of target genes, we investigated whether the HIF-1α(Pro-582-Ser) polymorphism might influence the response to repeated blood withdrawals. MATERIALS AND METHODS Using polymerase chain reaction analysis and DNA sequencing, we retrospectively investigated the presence of HIF-1α(Pro-582-Ser) in a series of 163 blood donors. Haematological findings, serum ferritin levels and frequency of donations were compared according to the mutational status of the HIF-1α gene. RESULTS We found that male carriers of the HIF-1α(Pro-582-Ser) polymorphism had higher haemoglobin and ferritin levels than individuals homozygous for the wild-type allele. Moreover, the HIF-1α(Pro-582-Ser) polymorphism protected regular blood donors from developing iron deficiency and anaemia and predicted uninterrupted donation activity. DISCUSSION These findings show for the first time that the HIF-1α(Pro-582-Ser) polymorphism significantly affects red blood cell and iron homeostasis after blood loss, conferring to male carriers a resistance to anaemia. Regarding the female gender, large series of individuals should be investigated to establish whether there is an effect of the HIF-1α(Pro-582-Ser) polymorphism in this population. Although these data need to be confirmed in prospective studies, they could have important implications in blood donor selection and donation procedures.