Leishmaniasis is a protozoan disease caused by parasites of the genus Leishmania, transmitted to humans by sandflies. The diagnosis of leishmaniasis is often challenging as it mimics many other infectious or malignant diseases. The disease can present in three ways: cutaneous, mucocutaneous, or visceral leishmaniasis, which rarely occur together or consecutively. The patient was a 52 years old immunosuppressed Belgian woman with a long history of severe rheumatoid arthritis. She underwent bone marrow biopsy to explore thrombocytopenia. Diagnosis of visceral leishmaniasis was made by identification of Leishman Donovan (LD) bodies in macrophages. Treatment with liposomal amphotericin B was successful. She later developed cutaneous leishmaniasis treated with amphotericin B lipid complex. She next presented with relapsing cutaneous lesions followed by rapidly progressing lymphadenopathies. Biopsy confirmed the diagnosis of leishmaniasis. Treatments by miltefosine, amphotericin B, N-methyl-glucamine antimoniate were subsequently initiated. She later presented a recurrent bone marrow involvement treated with intramuscular paromomycin and miltefosine. She died two years later from leukemia. At the time of death, she presented with a mucosal destruction of the nose. A Leishmania-specific PCR (Polymerase Chain Reaction) identified L. infantum as etiological agent. Clinicians should be aware of the potential concomitant or sequential involvement of multiple anatomic localizations of Leishmania in immunosuppressed patients.
Le paludisme est une maladie potentiellement sévère sévissant particulièrement en Afrique.En Europe, les cas de paludisme proviennent majoritairement de voyageurs revenant de zones endémiques.La symptomatologie non spécifique peut ne pas alerter le clinicien si cette notion de voyage n'est pas abordée.Or, le diagnostic et l'instauration rapide d'un traitement empêchent l'évolution vers les formes graves, notamment en cas d'infection à Plasmodium falciparum, capable d'engager le pronostic vital en 24h.La microscopie sur frottis mince et en goutte épaisse est la méthode de référence pour le diagnostic mais certains automates d'hématologie ont démontré leur capacité à participer au diagnostic précoce.Nous décrivons deux cas illustrant la contribution de la chaine automatisée Sysmex XN-9100 dans le diagnostic de la malaria.Le premier cas clinique est celui d'un jeune homme infecté par de nombreux gamétocytes de Plasmodium falciparum.Ceux-ci forment une population additionnelle visualisable sur les scattergrammes des leucocytes WNR (numération des leucocytes) et WDF (formule leucocytaire).Le second cas concerne un homme atteint de neuropaludisme, avec une parasitémie élevée à Plasmodium falciparum.Les hématies parasitées forment une discrète double population sur le scattergramme des réticulocytes, située à la limite de discrimination des globules rouges matures et des réticulocytes.Les anomalies des scattergrammes, visualisables en quelques minutes, offre une anticipation du diagnostic de malaria en comparaison à la microscopie sur frottis mince et goutte épaisse, technique nécessitant un temps et une expertise non négligeable.
VEXAS syndrome is a new entity, described as the first one of a new class of hemato-inflammatory diseases. Through this article and based on the first case highlighted at the CHU of Liege, we offer you a review of the literature as well as an overview of different laboratory techniques used for the diagnosis of this syndrome.
There is a growing interest for flow and image cytometry analytes permitting the simultaneous discrimination of 6-10 fluorochromes. We have derived a strategy to optimize the factors affecting the sensitivity and specificity of multiple immunofluorescence analysis. Following a spectrophotometric analysis of 14 fluorochromes conjugated to streptavidin (SA), a set of 7 spectrally separable SA- dyes and appropriate filter combinations were selected for evaluation in image cytometry. The SA-dyes were bound to latex particles labeled with biotinilated mIgG1 and the emissions of all fluorochromes detected by each filter combination were measured. The resulting crosstalk matrix served as the basic tool for final selection of dyes, design of optimal trichroic beamsplitter and filter combination, modulation of illumination and mathematical correction of residual spectral overlap. Using this strategy we demonstrated that latex-bound SA conjugates of Cascade Blue, Lucifer Yellow, FITC, R-PE, Red613, PerCP and APC could be discriminated. More recently we extended the applicability of the technique by analyzing blood cells bound to glass slides. The same field could be initially measured for autofluorescence and non-specific IgG binding and then remeasured for specific binding of lineage markers. The ability to use paired measurements of background and total fluorescence is a significant advantage of image over flow cytometry.
Since the initial description of anaplastic large cell lymphoma (ALCL) as a proliferation of large CD30+ lymphoid cells, the morphological spectrum of ALCL positive for anaplastic lymphoma kinase (ALCL, ALK+) has expanded, and beyond the common pattern most frequently encountered, several variants have been identified, including the lymphohistiocytic and the small cell patterns (Swerdlow et al, 2008). Recent data suggest that tumours with variant morphology may carry a worse prognosis than the common pattern, with a tendency to more frequent and early relapses (Lamant et al, 2007). Only ten ALK+ ALCL cell lines are currently available, and most were derived from tumours demonstrating the common type morphology (Drexler & MacLeod, 2004). We have established a novel cell line (CHIC) from the cerebrospinal fluid of a 32-year-old man with relapsing/refractory ALK+ ALCL whose initial tumour exhibited lymphohistiocytic features (Table I, Fig 1A–C). Morphology of the patient's lymphoma (A–D) and of CHIC cells (E–H). At diagnosis (October 2006), some areas in the lymph node biopsy comprised sheets of large atypical lymphoid cells, including hallmark cells (arrows), admixed with reactive lymphocytes (A, haematoxylin-eosin (HE), ×1000). Other areas were rich in histiocytes (arrows), with dispersed slightly smaller atypical cells (B, HE, ×1000). At relapses, the tumour infiltrate exhibited marked lymphohistiocytic features in the spleen (May 2007) (C, HE, ×1000), whereas the neoplastic cells in the cerebrospinal fluid (September 2007) were mainly large and pleomorphic (D, May-Grünwald-Giemsa (MGG), ×1000). Cultured CHIC cells showed an abundant, deeply basophilic cytoplasm and a large irregular nucleus. Occasional cells exhibited hallmark features (arrows) (E, MGG, ×1000). Subcutaneous tumours in NOD/SCID mice were composed of broad sheets of neoplastic cells with the typical morphological features of common ALCL and numerous mitotic figures. No significant histiocytic component was identified (F, HE, ×400). Immunohistochemistry confirmed CD30 expression (G, clone Ber-H2, ×400) and nuclear and cytoplasmic p-ALK positivity (H, clone ALK1, ×400). The cell line was derived with no feeder layer or addition of growth factors and, in contrast to others, did not require prior passage into immunodeficient animals. CHIC cells proliferated in permanent uninterrupted suspension culture for more than 12 months as single non-attached cells or in loose clumps. They were frozen and stored in liquid nitrogen for 1 year, and recovered after thawing without any loss of cellular features or cell viability. CHIC cells are large and pleomorphic, morphologically and immunophenotypically similar to the patient's malignancy (Table I, Fig 1D–E), showing positivity for CD30 and EMA, with nuclear and cytoplasmic expression of p-ALK. The cells are negative for T-cell associated markers, express CD43 and have a cytotoxic phenotype (perforin+, granzyme B+, TIA-1+). Expanded immunophenotyping demonstrated the expression of classical activation markers, such as CD25, CD26, CD45RO and HLA-DR. In situ hybridization confirmed the absence of Epstein-Barr virus (EBV). Cytogenetics revealed a complex karyotype including the t(2;5)(p23;q35) translocation, inducing NPM1-ALK fusion. The same TRG gene rearrangement was evidenced in CHIC cells, in xenografts (see below) and in the patient's tumour, demonstrating their T-cell origin and their derivation from one common clone. Subcutaneous xenografts of CHIC cells were tumourigenic to non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. The tumours growing at the inoculation sites were histologically and immunophenotypically similar to the patient's lymphoma and the cell line (Table I, Fig 1F–H). Autopsy showed no evidence of metastases. This xenograft mouse model was optimized by the use of Matrigel, enhancing engraftment of CHIC cells and allowing the development of tumours in a reasonable time frame (62% of mice had developed tumours at 2 weeks after injection and 81% at 8 weeks). We explored, in vitro and in vivo, whether CHIC cells could reproduce mixed morphological patterns as observed in the donor patient, by considering (i) the size of the tumour cells (smaller in the lymphohistiocytic variant), and (ii) the reactive histiocytic infiltrate (typical of the lymphohistiocytic variant). Flow cytometric data provided no evidence for two distinct cell populations, in spite of a relatively broad forward- and side-scatter distribution, suggesting that the latter was reflective of one tumour cell population exhibiting marked pleomorphism. Accordingly, the tumours developed from xenografts were pleomorphic and comprised many hallmark cells but no small cell component. In order to examine whether CHIC cells secreted bioactive products involved in the constitution of a histiocyte-rich microenvironment, we generated their cytokine profile by using the Human Cytokine Array kit (R&D Systems, Minneapolis, MN, USA), and compared it to that of three cell lines derived from common pattern ALCLs (i.e. SUP-M2, Karpas 299, SU-DHL-1). We identified three cytokines (MIF, CCL3/MIP-1α and serpin E1/plasminogen activator inhibitor (PAI)-1) described to have a potential role in macrophage recruitment (Gregory et al, 2006; Menten et al, 2002; Peiretti et al, 1996). These cytokines, however, were not specific to the CHIC cells but were also secreted by the common ALCL cell lines. In line with these considerations, the xenografts in NOD/SCID mice showed no evidence of histiocytic infiltration. This observation, however, must be balanced by the known macrophage dysfunction in NOD/SCID mice. The cytokine profiles also demonstrated production of sICAM-1, CXCL8/IL-8 and IL-10 by the CHIC cells and by some of the other ALCL cell lines. Interleukin (IL)-8 and IL-10 have been previously reported in ALCL (Hsu et al, 1995; Bouland et al, 1998). IL-8 is a pro-inflammatory and pro-angiogenic cytokine that affects tumour-cell proliferation and metastasis. IL-10 has been reported to rescue tumour cells from apoptosis and to inhibit antitumour cytotoxic T-cell activities. The secretion of the soluble form of ICAM-1 (sICAM-1) by ALCL cells, documented here for the first time, could be involved in immunosurveillance escape and tumour cell dissemination: as the expression of ICAM-1 on leukaemic cells renders them susceptible to natural killer (NK) cell cytolytic activities (Barber et al, 2004), the shedding of ICAM-1 by ALCL cells could give them a selective advantage. Altogether, our findings indicate that CHIC cells are derived from an end-stage pleomorphic cell population evolving from an initial mixed histological pattern ALCL and having lost the ability to recruit histiocytes. Similar observations have been reported for another ALCL cell line (COST), derived from a small cell variant but progressing towards two distinct cell populations (a small one and a large one) in the patient and in the xenografted mice (Lamant et al, 2004). In summary, a new ALCL cell line, established from relapsing/refractory ALK+ ALCL with a t(2;5)(p23;q35) translocation, is now made available to the scientific community. In addition to multiple in vitro applications, the tumourigenic capacity of these cells represents a useful property for in vivo drug testing. The authors would like to thank Stéphanie Maquet, Christelle Deceuninck and Luc Duwez for their excellent technical assistance, and Sandra Ormenese and the GIGA 'Cell imaging and flow cytometry' facility. This work was supported by a grant from the Belgian National Fund for Scientific Research (F.N.R.S.) (convention 3.4582.05), by the Fondation Léon Fredericq, and by the Centre Anticancéreux près l'Université de Liège. L. de Leval is a Senior Research Associate, C. Thielen, B. Bisig and S. Gofflot are Scientific Research Workers-Télévie of the F.N.R.S. None of the authors has any conflict of interest in relation to this work.
