Detection of nickel and palladium contact hypersensitivity by a flow cytometric lymphocyte proliferation test.

We established a flow cytometric lymphocyte proliferation test (LPT) for the detection of nickel (Ni) and palladium (Pd) sensitization. Eighty-one consecutive patients with an indication for patch test (PT) were tested by LPT with Ni (NiSO4 ) and Pd (Na2 PdCl4 and PdCl2 ) salts. The imprecision of the LPT was low (coefficient of variation 7.2%). Using PT as a diagnostic reference, the sensitivity and specificity of LPT were 74.4% and 80% for NiSO4 , 74.4% and 78.3% for Na2 PdCl4 , and 57.2% and 85.4% for PdCl2 , respectively. For both Ni and Pd, the likelihood ratio for a positive PT markedly increased with increasing LPT value. With medical history as a reference, the sensitivity and specificity were 40.6% and 82.1% for LPT and 59.4% and 89.7% for PT, respectively. Combination of LPT and PT resulted in a higher specificity of 95%, albeit lower sensitivity of 34.4%. In conclusion, flow cytometric LPT represents a reliable and useful method for the detection of Ni and Pd sensitization. LPT values correlate with PT results and, when used in combination with PT, increase test specificity.

Skin prick test and basophil reactivity to cetuximab in patients with IgE to alpha-gal and allergy to red meat.

Severe hypersensitivity reactions to red meat with delay of several hours in patients with IgE to alpha-gal (galactose-alpha-1,3-galactose) have been reported. The diagnosis of meat allergy is difficult, because of the limited sensitivity of skin prick tests and specific IgE tests to meat extracts. These circumstances have been explained by the delayed expression of alpha-gal due to digestive processes. Because of the low sensitivity of skin prick tests to meat, we studied the possibility to perform skin prick tests with cetuximab, which carries the alpha-gal epitope. Skin prick and intradermal tests with cetuximab were clearly positive in 2 of 2 patients. As a further diagnostic step, we performed basophil activation tests with cetuximab. Skin prick tests and basophil activation test using cetuximab may be a more sensitive alternative in patients with an assumed allergy to meat.

Efficient mobilization of PBSC with vinorelbine/G-CSF in patients with malignant lymphoma.

High-dose chemotherapy (HDT) and hematopoietic SCT are effective in patients with relapsing or refractory malignant lymphoma. Collection of sufficient numbers of stem cells is a prerequisite for such a therapy. In a pilot trial, we evaluated the feasibility of stem cell mobilization with vinorelbine/G-CSF in patients with lymphoma, a regimen allowing precise timing and harvesting of sufficient stem cells in myeloma patients. Forty-five patients with lymphoma received vinorelbine 35 mg/m(2) i.v. on day 1 and G-CSF 10 microg/kg/day s.c., divided in two daily doses from day 4 until collection. Stem cell collection was successfully performed in 43 patients (96%) with a median of 3.6 x 10(6) CD34(+) cells/kg (range: 1.4-16) in the collected product. In 28 patients (62%), the first stem cell apheresis was performed on day 8, and for 28 patients a sufficient stem cell yield was reached with one apheresis only. All 43 patients underwent high-dose chemotherapy with BEAM and auto-SCT with hematological recovery on time and without unexpected toxicity. In conclusion, vinorelbine/G-CSF allows accurate timing and safe harvesting of sufficient stem cells in patients with malignant lymphoma.

[Paroxysmal nocturnal hemoglobinuria--consequences of a missing anchor]. / Paroxysmale nächtliche Hämoglobinurie--Folgen eines fehlenden Ankers.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder characterized by the clonal expansion and differentiation of a multi-potent hematopoietic stem cell carrying a somatic mutation in the X-linked PIG-A gene. As a consequence of this mutation, glycosylphosphatidylinositol (GPI)-anchored proteins are lacking on the surface of blood cells derived from the mutated stem cell. This may result clinically in hemolytic anemia and a tendency for venous thrombosis and serious infection. Bone marrow failure is a frequently observed phenomenon associated with PNH. Reliable diagnosis of PNH is currently best achieved by flow cytometric analysis of GPI-anchored proteins on peripheral blood cells. Both the clinically relevant size of the PNH clone and type of GPI deficiency (complete or partial) can be reproducibly determined by this method. Most patients will benefit from supportive measures, albeit that allogeneic hematopoietic stem cell transplantation is currently considered the only potentially curative therapy. The development of a new therapeutic monoclonal antibody that reduces intravascular hemolysis and progress in diagnostic flow cytometry using a new GPI-specific marker may provide further benefit for PNH patients in the future.

Twelve-year follow-up after discontinuation of preseasonal grass pollen immunotherapy in childhood.

BACKGROUND: In a previous controlled study, we demonstrated that preseasonal grass pollen immunotherapy for 3 years was effective in children. Moreover, a significant clinical benefit could still be observed 6 years after discontinuation of specific immunotherapy (SIT). In the current study, we examined the same group of patients again to investigate whether there is a prolonged benefit 12 years after SIT is stopped. METHODS: Twenty-two patients with previous SIT (from 1989 through 1991) or standardized seasonal pharmacotherapy only were prospectively followed during the grass pollen season of 2003. Primary end points were symptom score, medication use, and combined symptom and medication score. In addition, skin prick test reactivity, development of new sensitizations, and prevalence of seasonal asthma were evaluated. RESULTS: Total hay fever symptom score (P < 0.03), use of medication (P < 0.05), and combined symptom and medication score (P < 0.03) remained lower in patients with previous SIT when compared with the control group. Decreased immediate skin response to grass pollen returned 12 years after cessation of SIT. The percentage of new sensitization, however, continued to be significantly smaller in patients with previous SIT (58%) compared with the controls (100%, P < 0.05). There was a tendency for lower prevalence of seasonal asthma in the post-SIT group (P = 0.08). CONCLUSION: This prospective controlled prolonged follow-up study demonstrates the ongoing clinical benefit 12 years after discontinuation of SIT. Furthermore, the reduction in onset of new sensitization, which was found 6 years after discontinuation of SIT, is sustained 6 years later.

[Genetic analysis and immunophenotyping in the diagnosis of hematological disease]. / Genetische Analysen und Immunphänotypisierung in der Diagnostik hämatologischer Erkrankungen.

The developments in the fields of genetics and immunology and the application of these informations have significant consequences for the diagnosis of hematological diseases. The present article gives an introduction into the principles of several modern diagnostic techniques, which are applied in the diagnosis of hematological diseases. In addition, it summarizes the application of these techniques in the diagnosis of several acquired and inherited diseases. The most important method of immunophenotyping is FACS (fluorescence activated cell sorting) analysis, which is based on the automated recognition of fluorescently labelled monoclonal antibodies bound to specific antigens on the surface or in the cytoplasm of different cell populations of the immune system. Techniques from molecular biology and from cytogenetics are also relevant for the diagnosis of hematological diseases: they allow the identification of changes of the genetic material on the level of DNA (molecular biology) and chromosomes (cytogenetics). Molecular biological and cytogenetic methods coalesce in the field of molecular cytogenetics, which renders possible the identification of chromosome mutations, which are invisible by the classical cytogenetical approach, and difficult to detect by routine molecular biological analysis. Most hematological malignancies are associated with genomic changes, which can be identified by cytogenetic and/or molecular biological methods. These genetic changes usually correspond with a specific pattern of surface antigens of the tumour cells, which can be identified by FACS. The different mutations in different genes causing a large number of inherited hematological diseases can often be found by molecular analysis, too. For hematological neoplasias, the exact definition of the causative mutations is increasingly important for therapeutic decisions and follow-up analysis of minimal residual disease. For inherited diseases, the identification of mutations is often the basis for a correct genetic counselling of the family.

[Monoclonal gammopathies--from MGUS to myeloma]. / Monoklonale Gammopathien--vom MGUS zum Myelom.

Monoclonal gammopathies are characterized by the overproduction of a monoclonal immunoglobulin (M-Protein), which may be detected in serum or urine by protein electrophoresis and immunofixation. The presence of an M-Protein results from the proliferation of a single abnormal clone of differentiated B lymphocytes or plasma cells, and is associated with a variety of clinical conditions, ranging from asymptomatic to malignant disease. Recent years have witnessed considerable advances in the treatment of plasma cell myeloma, the most common malignant disorder of the monoclonal gammopathies. As compared with conventional-dose treatments, high-dose chemotherapy with autologous stem-cell transplantation increases response rates and overall survival of patients with myeloma who are younger than 65 years of age. Progress in supportive therapies and the development of promising new drugs such as proteasome inhibitors and thalidomide analogues may provide further benefits for myeloma patients in the future.