Successful use of topical retinoic acid in severe dry eye due to chronic graft-versus-host disease.

Topical retinoic acid has proved to be of variable benefit in a number of dry eye disorders of disparate aetiology, in which squamous metaplasia with keratinization of ocular epithelium is present. Its exact role in patients with dry eye however remains in dispute. We describe a case of severe dry eye due to chronic graft-versus-host disease, which was refractory to conventional therapy but which responded remarkably to topical retinoic acid with reversal of conjunctival keratinization and marked resolution of symptoms.

Thrombocytopenia following autologous bone marrow transplantation: evidence for autoimmune aetiology and B cell clonal involvement.

Thrombocytopenia outlasting anaemia and neutropenia is a well recognised sequel of autologous bone marrow transplantation (BMT) but the pathogenesis remains unclear. Autoimmune destruction of platelets has been suggested as a possible mechanism. We studied 5 patients who had undergone autologous BMT and were found to have persistent thrombocytopenia (< 150 x 10(9)/l) 6 months from transplantation with a normal haemoglobin level and granulocyte count. Apart from a mild reduction in the megakaryocyte numbers in one case, no other quantitative or qualitative defects of the megakaryocyte lineage were present to explain the peripheral thrombocytopenia. Two cases had positive anti-platelet autoantibodies. Immunoglobulin heavy chain gene rearrangement studies of peripheral blood and bone marrow mononuclear cells using the polymerase chain reaction showed evidence of clonal rearrangement in one of the two cases with positive anti-platelet autoantibodies. Our results support the previous reports that anti-platelet antibody-mediated destruction of platelets may play a role in the pathogenesis of post-autologous BMT thrombocytopenia. Furthermore, the demonstration of a clonal B cell expansion in one of the cases with anti-platelet antibodies suggests an aetiological link between clonal B cells, autoantibody production and thrombocytopenia.

An unusual cutaneous manifestation of myelodysplastic syndrome: "pseudo-Koebner phenomenon".

An unusual and hitherto unreported complication of myelodysplastic syndrome is reported: the "pseudo-Koebner phenomenon." The skin lesions were characterised by exuberant "fleshy" masses at the sites of intravenous cannulation and skin trauma, and by histological evidence of chronic inflammation with focal necrosis and abscess formation. No evidence of dermal infiltration by malignant haemopoietic cells was seen. The exact aetiopathology of the phenomenon is unclear but an inappropriate and exaggerated inflammatory response owing to aberrant mediator mechanisms that are known to occur in some cases of myelodysplastic syndrome may be implicated.

Fatal bone marrow embolism in a patient with sickle cell beta + thalassaemia.

Sickle cell beta + thalassaemia is regarded as the mildest of the sickle cell haemoglobinopathy syndromes with a benign natural course. In contrast to sickle cell disease, severe life threatening complications are not usually associated with this genotype. A case of a 30 year old West Indian man who, previously asymptomatic for 10 years, sustained a fatal pulmonary bone marrow embolism, is reported. This case report illustrates that serious, even fatal, complications may occur in patients with this 'benign' condition and bone marrow embolism should be included in the differential diagnosis of acute crisis in these patients.

Pure red cell aplasia associated with malignant thymoma, myasthenia gravis, polyclonal large granular lymphocytosis and clonal thymic T cell expansion.

A case with the triad of pure red cell aplasia (PRCA), myasthenia gravis, and malignant thymoma is reported. There was a clonal proliferation of T cells within the thymoma, as demonstrated by a T cell antigen receptor (TCR) delta chain gene rearrangement. However, despite a large granular lymphocytosis, clonality could not be shown in the peripheral blood either before or after thymectomy. There was no evidence of human T cell lymphotrophic virus type 7 (HTLV1) infection. It is postulated that the clonal thymic T cell population secreted cytokine(s), which stimulated the polyclonal proliferation of large granular lymphocytes, which in turn suppressed erythropoiesis. Thymectomy removed the stimulus to the large granular lymphocytes and hence there was a resurgence of erythropoiesis.

Unusual aetiology of persistent back pain in a patient with multiple myeloma: infectious discitis.

A 47 year old man with multiple myeloma presented with persistent back pain caused by infectious discitis. Aspiration of the affected vertebral disc space was carried out, guided by computed tomography, and microbiological examination of the aspirate revealed Staphylococcus aureus and Mycobacterium tuberculosis. Antituberculous and antistaphylococcal antibiotic treatment resulted in a dramatic clinical response with complete resolution of the vertebral abscess. Detailed radiological and microbiological investigations are necessary to diagnose unusual causes of chronic bone pain such as discitis or infectious bone disease in patients with multiple myeloma.

Lymphoma associated bone marrow necrosis with raised anticardiolipin antibody.

A case of high grade B cell lymphoma presented with bone marrow necrosis, followed by development of extensive marrow fibrosis, the evolution of which was documented by serial magnetic resonance imaging and bone marrow trephine histology. A markedly raised anticardiolipin antibody titre at diagnosis suggests that lymphoma associated antiphospholipid syndrome may have contributed to the aetiology of the bone marrow necrosis.

Clonality of CD3 negative large granular lymphocyte proliferations determined by PCR based X-inactivation studies.

AIMS: To examine persistent CD3-large granular lymphocytosis (LGL) cases for clonality, both by lineage specific (T cell receptor) and lineage independent (X-inactivation) molecular methods; and to find out whether X-inactivation studies are more appropriate than gene rearrangement studies for this subset of LGL disorders. METHODS: Patients were selected who had LGL of more than six months' duration and identified as CD3- by immunophenotyping. T cell receptor studies and, where possible, X-inactivation studies of the phosphoglycerate kinase (PGK) gene were carried out. Analysis of subpopulations was carried out on cases heterozygous for PGK by the use of a polymerase chain reaction (PCR) method for X-inactivation. RESULTS: Of 17 CD3- LGL cases studied, all were found to be germline for beta, gamma, and delta T cell receptor studies, and immunoglobulin heavy chain genes. However, six of these were analysed by X-inactivation of the PGK gene and two cases gave clonal band patterns but only within the CD3- subpopulation. CONCLUSIONS: Clonal analysis by the lineage independent method of X-inactivation allows clonal expansion undetected by T and B cell specific markers to be identified. It is therefore a more appropriate method for the analysis of CD3- LGL. This has implications for diagnosis in CD3- LGL disorders.

Trisomy 21 in transient myeloproliferative disorder.

Transient leukemia in phenotypically normal children is rare. A newborn child in whom fever and tachypnea developed at age 2 days had a white blood cell count of 20.1 x 10(9)/L and many abnormal blast cells. Chromosome analysis of spontaneously dividing cells from the blood showed these to have trisomy 21, and 80% of cells in the marrow were also trisomic. No trisomic cells were present in skin fibroblast cultures. At age 6 months, at which time the blood film appeared normal, trisomic cells were no longer present.

Persistent clonal expansions of CD3+TCR gamma delta+ and CD3+TCR alpha beta+CD4-CD8- lymphocytes associated with neutropenia.

This communication reports the clinical and cellular features of five elderly female patients with persistent moderate to severe neutropenia and concomitant relative expansions of CD3+TCR gamma delta+ (n = 4) or CD3+TCR alpha beta+CD4-CD8- (n = 1) lymphocyte populations. In clinical terms, severe neutropenia was the main contributing factor to patient symptoms although two additionally had long-standing histories of rheumatoid arthritis. The absolute lymphocyte counts did not exceed the normal upper limit in these patients, and morphologically the lymphocytes were not typically of large granular lymphocyte (LGL) type although LGL-associated BLT-esterase staining was consistently increased. Expression of NK-associated (NKa) membrane determinants (CD16, CD56 and CD57) were variable but there was an apparent correlation between weak membrane CD8 and CD16 expression. DNA genotypic studies confirmed that the four CD3+TCR gamma delta+ cases were clonal in nature and add further support to an emerging impression that expansions of these lymphocyte subpopulations may frequently be clinically associated with autoimmune phenomena in general and neutropenia in particular.

Validation of capillary electrophoresis method for determination of N-methylpyrrolidine in cefepime for injection.

The present study relates to a new capillary electrophoresis method for the determination of N-methylpyrrolidine, an impurity considered to be toxic and also potential degradation impurity in cefepime hydrochloride drug substance. The newly developed capillary electrophoresis method for determining the content of N-methylpyrrolidine in cefepime for injection has been validated as per International Conference on Harmonization (ICH) guidelines to prove the selectivity, sensitivity, suitability, robustness, and ruggedness of the method. This simple, efficient, and rapid methodology may be used by pharmaceutical industry for routine analysis as well as during stability studies. The newly developed capillary electrophoresis method to determine the content of N-methylpyrrolidine in cefepime for injection requires 10 min for data acquisition, and uses an indirect UV photometry method to detect the analyte signal at 240 nm against the reference signal at 210 nm. The electrophoretic system is optimized to get stable base line, higher signal to noise ratio and peaks with narrow peak width. The method employs bare fused silica capillary with extended light path, effective length of capillary is 56 cm and inner diameter of capillary is 50 µm, 5 mmole of imidazole buffer adjusted to pH 5.1 with 3 molar acetic acid solution is used as background electrolyte. The sample is introduced in hydrodynamic mode employing pressure of 50 mbar for 5 s, and the desired separation is achieved with constant applied voltage of 25 kV at ambient temperature (~25°C).

Validation of a sensitive ion chromatography method for determination of monoethylsulfate in Indinavir sulfate drug substance.

The present study relates to the optimization of an ion chromatography method to determine the content of monoethylsulfate at very low levels in Indinavir sulfate drug substance, and subsequent validation of the method to prove its suitability, reliability and sensitivity. Monoethylsulfate is a potential impurity of Indinavir sulfate, and may forms during the preparation as well as during storage. The ion chromatography method was developed in such a way that to enhance the detection level by introducing suppressor, and minimizing acquisition time by using suitable buffer of 3.2 mmole of sodium carbonate and 1 mmole of sodium hydrogen carbonate in water as eluent. The retention time of monoethylsulfate was about 9.5 min and the total acquisition time was 25 min. The optimized method was validated to prove its performance characteristics by demonstrating selectivity, sensitivity (limit of detection and quantification), linearity, precision and accuracy. The established limit of detection and quantification of monoethylsulfate in Indinavir sulfate by this method was found to be 24 ng/ml and 74 ng/ml respectively, and the overall percent accuracy (recovery) of samples evaluated at different concentration levels was found to be 97.1, indicating the sensitivity and accuracy of this optimized ion chromatography method.