Persistent changes in circulating white blood cell populations after splenectomy.

The effect of splenectomy on the incidence of infections and thromboembolisms has been investigated thoroughly. Nevertheless, the long-term effects of splenectomy on immunological profile and circulating blood counts have not been described before. To study such long-term effects, we analysed several parameters in splenectomised trauma patients and compared the results of this group ("otherwise healthy patients") to patients with a specific underlying disease. We measured platelet count, leukocytes and differential, lymphocyte subsets, serum levels of immunoglobulins, and complement pathways in 113 patients. Indications to perform a splenectomy were trauma (n = 42), Hodgkin lymphoma (n = 24), hereditary spherocytosis (n = 21), and immune thrombocytopenia (n = 26). In trauma patients lymphocytes and lymphocytes subsets were particularly elevated compared to normal population values. Splenectomised patients with Hodgkin lymphoma had significant lower numbers of T lymphocytes than trauma patients. Significant increases in platelets, leukocytes, and monocytes were observed in patients with hereditary spherocytosis. Occurrence of MBL genotype was different in ITP patients than in other splenectomised groups and the normal population. In splenectomised patients (> 4 years), platelet counts and lymphocyte subsets are increased which persist over time. As a result, these blood counts in splenectomised patients differ from reference values in the normal population.

Detection of red blood cell antibodies in mitogen-stimulated cultures from patients with hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS. STUDY DESIGN AND METHODS: RBC antibody-bound immunoglobulin (Ig)G was investigated in 91 consecutive HS patients by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method able to magnify latent RBC antibody autoimmunity and related with hemolytic variables, previous splenectomy, and type of membrane defect. RESULTS: A total of 61% of HS cases had RBC antibodies by MS-DAT (29 Band 3, 17 spectrin deficiency, and nine no defined defect). The amount of RBC-bound IgG was greater in HS compared with controls (236 ± 192 ng/mL vs. 52 ± 29 ng/mL, p < 0.0001), although lower than that observed in autoimmune hemolytic anemia (AIHA; 634 ± 371 ng/mL vs. 236 ± 192 ng/mL, p < 0.0001). Western blot experiments showed that purified IgG fraction from MS-DAT-positive patients bind to α- and ß-spectrin, Band 3, and Band 4.9. Positive cases displayed increased reticulocytosis and slightly reduced hemoglobin (Hb) values compared to negative ones. Patients displaying RBC-bound IgG of more than 250 ng/mL (the positive threshold of AIHA) showed increased number of spherocytes and mainly had spectrin deficiency. RBC-bound IgG and free Hb increased over time after storage at 4°C, a surrogate of ex vivo aging, more evidently in HS than controls, and particularly in Band 3 deficiency. CONCLUSION: RBC autoantibodies were detected by MS-DAT in more than a half of HS patients. Positive cases showed a more evident hemolytic pattern suggesting a pathogenic role of these autoantibodies in RBC opsonization and splenic removal.

Comparison of 4 direct Coombs' test methods with polyclonal antiglobulins in anemic and nonanemic dogs for in-clinic or laboratory use.

BACKGROUND: Difficulties with the direct antiglobulin test (DAT) and its apparent lack of sensitivity and specificity for immune-mediated hemolytic anemia (IMHA) in dogs have raised skepticism regarding its diagnostic value. OBJECTIVE: To compare different DATs and other hematologic parameters in dogs. ANIMALS: Anticoagulated blood samples from 59 nonanemic and 46 anemic dogs (± IMHA) from a research colony and veterinary clinics. METHODS: Prospective observational study: Immunochromatographic strip, gel microcolumn, and capillary techniques were compared with standard microtiter DAT using 2 polyvalent antiglobulins. Spherocytosis, autoagglutination, osmotic fragility, and clinical data were assessed. RESULTS: Blood samples from all 59 nonanemic dogs were DAT-. Among 46 anemic dogs, 33 were suspected of IMHA, but only 20 were DAT+. Old and new DAT methods yielded comparable and consistent results even after storage of chilled blood samples for 1 week. Spherocytosis and autoagglutination (that did not persist after washing) were noted in 15 and 16 DAT+ dogs, respectively. The other 26 anemic dogs, including 21 previously transfused dogs and 4 with autoagglutination, tested DAT- by the other methods. Osmotic fragility was increased in 70% (19/27) of anemic and all 15 DAT+ dogs tested. Limited follow-up testing revealed DAT+ results for 3-70 days. CONCLUSIONS AND CLINICAL IMPORTANCE: The novel strip and capillary DAT methods are promising adjunct in-clinic tools. Despite prior immunosuppressive treatment and presence of autoagglutination, the DAT was positive in anemic dogs with IMHA. Transfusion did not cause false DAT+ results. Our results support DAT as a cornerstone in the diagnosis of canine IMHA.

Compliance with immunizations in splenectomized individuals with hereditary spherocytosis.

Overwhelming infection by encapsulated bacteria is a significant risk of splenectomy. Strict adherence to immunizations against encapsulated organisms is recommended. We performed a cross-sectional study to determine immunization status after splenectomy in the hereditary spherocytosis (HS) population. As HS is most often autosomal dominant, we used a strategy of ascertaining older affected individuals by expanding pedigrees from patients in our pediatric hematology center. Only 26% of asplenic patients reported receiving all recommended post-splenectomy vaccines. This study demonstrates that pediatric hematology clinics have a unique opportunity to provide or recommend catch-up and booster vaccinations to splenectomized adult relatives of their patients.

Mode of splenectomy and immunogenicity of meningococcal vaccination in patients with hereditary spherocytosis.

BACKGROUND: Splenectomy predisposes patients to invasive disease from pneumococci, meningococci, and Haemophilus influenzae; immunization is mandatory. However, data on the impact of the splenectomy on vaccine immunogenicity are scarce. METHODS: A total of 41 children with hereditary spherocytosis (aged 5.8-14.4 years) had complete (16) or near-total (25) splenectomy. All received one dose of monovalent meningococcal C conjugate vaccine (MCV-C) and, 2 months later, a tetravalent meningococcal polysaccharide vaccine (MPV-ACWY). Serum bactericidal activity and antibodies against serogroups A and C were determined before and after they received MCV-C, and 4 weeks after they received MPV-ACWY. RESULTS: Before vaccination, only four of the 16 children who had a complete splenectomy were protected against serogroup A, compared with 15 of the 25 who had near-total splenectomy (P < 0.050), with the latter responding to immunization with significantly higher serogroup A serum bactericidal activity: geometric mean (95 per cent confidence interval) 1625.5 (49.9 to 3201.1) versus 980.6 (2.00 to 6204.1) (P < 0.050). All patients achieved putative protective serum bactericidal activity titres (at least 8) against serogroup C. CONCLUSION: Near-total splenectomy provides a favourable immunological basis for natural and vaccine-induced protection against meningococcal serogroup A and C infections. Sequential meningococcal vaccination is immunogenic in patients splenectomized for hereditary spherocytosis.

Abnormality of CD4(+)CD25(+) regulatory T cells in idiopathic thrombocytopenic purpura.

OBJECTIVES: The aim of this study was to explore the profile and function of CD4(+)CD25(+) regulatory T cells (Treg cells) in idiopathic thrombocytopenic purpura (ITP) patients. METHODS: Treg cell numbers were analyzed by flow cytometric analysis in peripheral blood mononuclear cells collected from healthy donors or patients with ITP. Quantification of cell proliferation was assayed by an enzyme-linked immunosorbent assay kit, based on the measurement of BrdU incorporation during DNA synthesis. RESULTS: The percentage of Treg cells was significantly decreased in ITP patients in active and non-remission state(5.79 +/- 1.22%) when compared with the patients in remission(11.63 +/- 4.56%) and to healthy subjects(12.68 +/- 3.59%). The suppressive activity of Treg cells in ITP patients was also found to be impaired. CONCLUSION: These results suggest that decreased number and function of Treg cells might be one of mechanisms that cause immune regulation dysfunction in ITP.

Immunogenicity of sequential pneumococcal vaccination in subjects splenectomised for hereditary spherocytosis.

Splenectomy predisposes for invasive pneumococcal disease. We investigated the immune response of splenectomised hereditary spherocytosis (HS) patients upon sequential pneumococcal vaccination. Thirty-nine HS-patients (2- to 18-year-old) had undergone near-total or total splenectomy. All received one dose of 7-valent pneumococcal conjugate vaccine (PCV-7) and 23-valent-pneumococcal-polysaccharide vaccine (PPV-23) 2 months apart. Pneumococcal antibodies against serotypes 5/6B/7/14/18C/19F/23F and immunoglobulin serum concentrations were determined before PCV-7 and 4 weeks after PPV-23. Significant rises in antibody geometric mean concentrations were observed after PCV-7 except for serotypes 5 and 7, which increased after PPV-23. We found no impact of the mode of splenectomy.

Splenectomy in haemophagocytic lymphohistiocytosis: report of histopathological changes with CD19+ B-cell depletion and therapeutic results.

The pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in children without a known familial pattern of inheritance is often difficult to establish. Splenic enlargement, one of the main clinical findings in this disorder, has led to the use of splenectomy for uncontrollable coagulopathy, persistent cytopenia or both. This procedure is also thought to be a useful tool in making a differential diagnosis in cases of the immunochemotherapy-resistant HLH. We report here five cases of splenectomized childhood HLH, in which subsets of mononuclear spleen cells were analysed either by flow cytometry or immunohistochemistry, and the results were compared with those from cases of hereditary spherocytosis (controls). There was a statistically significant depletion of CD19+ B cells in the HLH cases (3.8 +/- 3.2% vs. 52.6 +/- 4.5%, P < 0. 0001) associated with an increase of T cells in three cases and of natural killer cells in another. The histopathological findings included atrophic white pulps, B-cell depletion with fibrosis and haemosiderosis in all five cases. Despite temporary therapeutic benefits, three of the HLH patients had a rapidly deteriorating post-splenectomy course and all three eventually died. These results demonstrate striking depletion of B cells in the enlarged spleens of children with HLH, which may be an intrinsic feature of HLH pathogenesis. Further study is needed to establish the therapeutic value of splenectomy in this disease.

Heavy transfusions and presence of an anti-protein 4.2 antibody in 4. 2(-) hereditary spherocytosis (949delG).

BACKGROUND AND OBJECTIVE: A patient with hereditary spherocytosis (HS) was found not to have red cell membrane protein 4.2. This rare form of HS, or 4.2 (-) HS, stems from mutations within the ELB42 or the EPB3 genes. The patient had long suffered from a gastric ulcer and impaired liver function. He had had several dramatic episodes of gastrointestinal tract bleeding and had received numerous transfusions. An antibody against a high frequency, undefined antigen was found, creating a transfusional deadlock. We elucidated the responsible mutation and searched for an anti-protein 4.2 antibody. DESIGN AND METHODS: Red cell membranes were analyzed by SDS-PAGE and by Western blotting. Nucleotide sequencing was performed after reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: The not previously described mutation was a single base deletion: 949delG (CGCAECC, exon 7, codon 317) in the homozygous state. It was called protein 4.2 Nancy. The deletion placed a non-sense codon shortly downstream so that no viable polypeptide could be synthesized. The patient carried a strong antibody against protein 4.2 as shown by Western blotting. INTERPRETATION AND CONCLUSIONS: The manifestations resulting from the mutation described were compared with the picture of HS stemming from other ELB42 gene mutations. We discuss the mechanism through which the anti-protein 4.2 antibody developed. There was no way to establish or to rule out whether the antibody participated in the transfusional deadlock found in our patient.

Intravenous immunoglobulin G and anti-D as therapeutic interventions in immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) is a disorder caused by accelerated destruction of antibody-coated platelets in the reticuloendothelial system (RES), especially the spleen. Inhibition of RES function following intravenous administration of high-dose immunoglobulin G (IVIG) or intravenous anti-D leads to rapid, albeit usually temporary, reversal of thrombocytopenia in the majority of children and adults with ITP. In emergency situations high-dose IVIG is preferred over anti-D because of the more rapid rate of platelet response; for maintenance therapy in Rh positive ITP patients (e.g. children with chronic ITP pre-splenectomy) anti-D is preferred because of its comparable efficacy to IVIG plus ease of administration and lower cost. In children with typical acute ITP and platelet counts < 20 x 10(9)/L IVIG is preferred over anti-D; however other approaches in this patient cohort should be considered before high-dose IVIG, specifically careful observation alone with therapy given only to children with clinically significant haemorrhage or short course oral prednisone at a starting dose of approximately 4 mg/kg/day. Studies are required to define the short and longer term effects of both IVIG and anti-D on the immune system in order to plan more rational use of these immunomodulatory therapies in this model autoimmune disorder.

T lymphocyte subsets, natural killer cell cytotoxicity and autologous mixed lymphocyte response in children after splenectomy because of hereditary spherocytosis.

Lymphocyte subpopulations and autologous mixed lymphocyte reaction (AMLR) studied in 13 hereditary sphereocytosis and 21 postsplenectomy patients; the operation was performed because of hereditary spherocytosis. Significant increases of Th cells and the Th/Ts ratio have been noted in children with hereditary spherocytosis. Splenectomy because of spherocytosis has resulted in an increase in Ts cell number and diminution of Th/Ts ratio. Hereditary spherocytosis patients showed both diminished natural killer cell activity and AMLR proliferation. Splenectomy normalized the above parameters. The presented data demonstrated disturbances in T lymphocyte number and functions both before and after splenectomy in children with hereditary spherocytosis.

Association of hereditary spherocytosis and idiopathic hemochromatosis. A synergistic effect in determining iron overload.

Two siblings, both splenectomized at an early age for hereditary spherocytosis, had a severe hemochromatosis develop. The human leukocyte antigen (HLA) system typing showed that they were half HLA identical. All the other members of the family who did not have evidence of hereditary spherocytosis, including those who displayed identical HLA haplotypes with the two patients, did not have any increase in iron stores. These results suggest that the two siblings are heterozygous for idiopathic hemochromatosis and that the coexistence of this condition with hereditary spherocytosis can cause a severe iron overload.

[Regulating effect of bone marrow cells on human T-lymphocyte function]. / Reguliruiushchee vliianie kletok kostnogo mozga na funktsiiu T-limfotsitov cheloveka.

A study was made of the regulatory effect of human bone marrow cells in two experimental systems: lymphocyte proliferation in response to PHA, and spontaneous and PHA-induced production of macrophage migration inhibition factor (MIF) by peripheral blood lymphocytes. It was shown that bone marrow cells inhibit the proliferative activity of stimulated peripheral blood lymphocytes and induced MIF production. The effect of bone marrow cells on spontaneous MIF production was found to be inconclusive.

Antibody response to pneumococcal vaccine in splenectomized adults and adolescents.

The antibody response of 67 splenectomized adults and adolescents with benign underlying diseases to a 14-valent pneumococcal capsular polysaccharide vaccine was determined by an enzyme-linked immunosorbent assay. It was not significantly different from that of 12 healthy non-splenectomized adult volunteers for 13 of the 14 polysaccharide antigens studied. Residual splenic tissue as detected by Tc-scintigraphy was without any influence on the vaccination response. In comparison 5 untreated splenectomized adults with malignant diseases and 11 splenectomized adults receiving immunosuppressive therapy exhibited a significantly reduced combined geometric mean of their postvaccination antibody concentrations (all 14 antigens added) and of their combined geometric mean antibody fold increase as compared to the healthy non-splenectomized adults. The reduction in antibody response was most pronounced in the group of immunosuppressed patients. Immunogenicity of each of the 14 vaccine antigens varied considerably as judged by the geometric means of the postvaccination arbitrary antibody concentrations. Also individual variation in postvaccination antibody concentration against each antigen was large.

Utilization of a cell separation technique to evaluate patients with a positive direct antiglobulin test.

Density distribution curves of red blood cells (RBC) from patients with a positive direct antiglobulin test (DAT) were compared to a standardized curve constructed from cell column measurements of centrifuged microcapillary tubes filled with RBC and phthalate ester mixtures encompassing a specific gravity range of 1.078 to 1.114. Shortened survival resulted in a loss of older RBC and a shift of the curve to the right over the high specific gravity ester range. Reticulocytosis resulted in a downward shift of the curve over the low specific gravity range. In patients with a positive DAT due to an autoantibody or drug, the density distribution curve was either normal or demonstrated evidence of shortened RBC survival. In patients with a positive DAT due to an alloantibody, however, evidence of shortened survival was not seen. The distribution of antibody on young and old RBC harvested from the appropriate microcapillary tubes depended upon the etiology of that antibody. In patients with a positive DAT due to to an alloantibody or drug, both the young and old RBC gave an equally reactive DAT, while in patients with a positive DAT due to an alloantibody the young cells were weakly reactive or nonreactive and the older cells were more strongly reactive. When used together, the position of the density distribution curve and the pattern of distribution of antibody coating on young and old RBC provide important diagnostic information about the etiology and clinical status in a patient with a positive DAT and allow for the recognition of an alloantibody and autoantibody when both are present in the same patient.