British Society for Haematology, Slide session, Annual Scientific Meeting, Bournemouth, 2007.

Eight cases discussed by experts at the 2007 Annual Scientific Meeting of the British Society of Haematology are presented as at the meeting, with a discussion of the morphological features, digital information and differential diagnosis being followed by further information and a final diagnosis. Additionally, digital slides of two of the cases were available to be viewed by the internet with the opportunity for delegates to suggest diagnoses.

Targeted screening for genetic haemochromatosis: a combined phenotype/genotype approach.

OBJECTIVE: To evaluate the clinical utility of a targeted screening approach for the detection of genetic haemochromatosis. METHODS: Screening by measuring fasting serum transferrin saturation (TS) and gene testing was carried out in patients in whom a raised serum alanine amino transferase (ALT) activity and raised random serum TS had been found on routine blood testing. RESULTS: During the 29 month study period, 32 patients homozygous for the C282Y genotype were detected from a catchment population of 330,000 by screening blood samples referred initially for routine laboratory liver function tests. By comparison, during the same period of time and within the same population, only seven patients were found by clinical suspicion alone. The patients in the study, after treatment by venesection, have shown both clinical and biochemical improvement. CONCLUSIONS: The study shows that from a population of patients in whom a routine liver function profile had been requested, it is possible to detect subjects homozygous for the C282Y HFE genotype who have clinical or biochemical markers of iron overload.

Report on slide session, British Society for Haematology, 43rd Annual Scientific Meeting, Glasgow, 2003.

Seven patients who had a diagnostic problem were presented at the British Society for Haematology, Annual Scientific Meeting in 2003. The likely diagnosis was discussed on the basis of a synopsis of the history and blood film and trephine biopsy features and forms the basis of this report. Diagnostic problems dealt with included lymphoproliferative and myeloproliferative disorders and haemolytic anaemia.

Screening for genetic haemochromatosis in blood samples with raised alanine aminotransferase.

BACKGROUND: In the UK approximately 1 in 140 people are homozygous for the C282Y mutation of the HFE gene and are at risk from iron overload caused by genetic haemochromatosis (GH). Early detection can prevent organ damage secondary to iron deposition and increase life expectancy. AIM: To screen for GH in all blood samples sent to the laboratory for routine liver function tests in which raised serum alanine aminotransferase (ALT) activity was detected. METHODS: ALT was measured in sera sent to the laboratory for routine liver function tests. In those samples found to have raised activity, transferrin saturation and ferritin were measured followed by genetic testing when transferrin saturation was increased. RESULTS: Of the 35 069 serum samples assayed for routine liver function tests, 1490 (4.2%) had raised ALT levels (>50 u/l). Transferrin saturation and serum ferritin concentrations were measured in these patient samples, and in 56 transferrin saturation was >60%. Further blood samples were requested from these patients for genetic testing: 33 samples were obtained. There were nine patients homozygous for the C282Y mutation of the HFE gene and three compound heterozygotes (heterozygous for both C282Y and H63D mutations). CONCLUSIONS: The association of raised ALT activity and transferrin saturation of >60% could provide a simple, cost effective method for detecting individuals with clinical haemochromatosis. Although many patients with GH may have been missed, this study suggests that the clinical penetrance of the disorder may be much lower than is generally supposed and that genetic screening will identify many people who may never develop clinical haemochromatosis.

Treatment of Felty's syndrome with the haemopoietic growth factor granulocyte colony-stimulating factor (G-CSF).

Felty's syndrome (FS) (rheumatoid arthritis with neutropenia and splenomegaly) has a poor prognosis, largely because of the high risk of severe infection. Granulocyte colony-stimulating factor (G-CSF) is an emerging treatment for chronic neutropenia. We prospectively monitored its use in eight patients with recurrent infections or who required joint surgery. Significant side-effects were documented in five, including nausea, malaise, generalized joint pains, and in one patient, a vasculitic skin rash. In two patients treatment had to be stopped, and in these cases G-CSF had been started at full vial dosage (300 micrograms/ml filgrastim or 263 micrograms/ml lenograstim) alternate days or daily. G-CSF treatment was continued in three patients by restarting at reduced dose, and changing the proprietary formulation. G-CSF raised the neutrophil count, reduced severe infection, and allowed surgery to be performed. A combined clinical and laboratory index suggested that long-term treatment (up to 3.5 years) did not exacerbate the arthritis. Once on established treatment, it may be possible to use smaller weekly doses of G-CSF to maintain the same clinical benefit. One of the three patients whose FS was associated with a large granular T-cell lymphocytosis showed a reduction in this subset of lymphocytes during G-CSF treatment.

Laboratory control of oral anticoagulant therapy: preservation of prothrombin time specimens using a polypropylene collection system.

Previous studies have shown a marked time and temperature dependent shortening of the prothrombin time (PT) when blood is exposed to borosilicate (glass) or siliconized borosilicate tubes. Current recommendations are that samples for PT estimation should be tested within 2 h of collection. In this study using polypropylene collection tubes, blood obtained from 30 patients on oral anticoagulant therapy showed no significant change in International Normalized Ratio (INR) value after 24 h storage--either at 4 degrees C or room temperature. After 48 h. changes in INR values from refrigerated samples were still clinically insignificant. After 48 h storage at room temperature, however, a minority of samples showed an increase in INR value which may be of clinical importance. The range of INRs studied was 1.0-9.1. In a second evaluation, replicate specimens from 22 orally anticoagulated patients with INRs ranging from 1.0 to 9.6 showed no significant change after 24 h at either temperature--even when samples had been subjected to 30 min of gentle agitation prior to storage and analysis. Overall, the results indicate that when polypropylene collection tubes are used, prothrombin time specimens can be successfully preserved for up to 24 h at room temperature or up to 48 h when refrigerated.

The prothrombin gene G20210A variant: prevalence in a U.K. anticoagulant clinic population.

We have investigated the prevalence of a recently reported genetic variation in the prothrombin gene (G20210A) in patients with an objectively confirmed history of venous thrombosis, 12/219 patients (5.5%) were found to be heterozygous carriers of the 20210A allele. The incidence of the 20210A allele in a group of 164 healthy controls was 1.2% (allele frequency 0.61%, 95% CI 0.08-2.19). When patients with a known alternative hereditary risk factor for venous thrombosis (factor V Leiden mutation or deficiency of antithrombin, protein C or protein S) were excluded, the G20210A variant was found to increase the risk for venous thrombosis by approximately 5-fold (odds ratio 5.4, 95% CI 1.16-25.0). This prothrombin gene sequence variation adds further to the list of recognized genetic risk factors for thrombophilia.

An unusual association of Felty syndrome and TCR gamma delta lymphocytosis.

Felty syndrome, comprised of neutropenia, rheumatoid arthritis and splenomegaly, occurs in approximately 1% of patients with rheumatoid arthritis. Up to one third of these patients have an increased number of large granular lymphocytes. The usual immunophenotype of these cells is CD3+, CD8+, CD57+, T cell receptor (TCR) alpha beta. A patient with Felty syndrome and large granular lymphocytosis, who had an unusual immunophenotype CD3+, CD4-, CD8-, TCR gamma delta, is described. Her neutropenia responded to treatment with granulocyte colony stimulating factor (G-CSF), which was given in order to raise her neutrophil count prior to bilateral knee replacement surgery. Thus, Felty syndrome with large granular lymphocytosis is a heterogeneous condition, one in which TCR gamma delta large granular lymphocytosis may be found, and also shows a response to treatment with G-CSF.

Large granular lymphocyte expansions in patients with Felty's syndrome: analysis using anti-T cell receptor V beta-specific monoclonal antibodies.

Felty's syndrome (FS), the association of rheumatoid arthritis (RA) and idiopathic neutropenia, remains an unexplained phenomenon. HLA-DR4 is found in over 90% of cases. Patients with FS may have a T cell lymphocytosis of CD3+CD8+CD57+ large granular lymphocytes (LGL syndrome). In this study of 47 patients with FS, 19% had clear evidence for LGL expansions, while in total 42% had variable evidence for the LGL syndrome using currently available techniques. Of these T cell expansions, 76% were clonal, as demonstrated by Southern blotting and analysis with T cell receptor (TCR) beta chain constant region probes. This technique may fail to detect clonal populations in some patients. Cytofluorographic analysis using antibodies specific for TCR V beta chains identified patients with clonal LGL expansions with results comparable to those obtained with Southern blotting. No evidence for shared V beta usage among expansions from different patients was seen. The role of LGL in RA and FS is currently unclear, but this technique offers a practical and accessible means of identifying patients with LGL expansions, as a starting point for further investigation.

The large granular lymphocyte syndrome with rheumatoid arthritis. Immunogenetic evidence for a broader definition of Felty's syndrome.

OBJECTIVE: To assess whether the HLA-DR4 association found in rheumatoid arthritis (RA) is also seen in the large granular lymphocyte (LGL) syndrome. METHODS: HLA-DR genotyping was performed using restriction fragment length polymorphism and polymerase chain reaction analysis. RESULTS: LGL syndrome patients with RA showed the same HLA-DR4 association seen in RA/Felty's syndrome (FS), while LGL syndrome patients without arthritis did not. CONCLUSION: It is proposed that FS and the LGL syndrome represent different variants of a broader syndrome comprising RA, neutropenia, LGL expansions, HLA-DR4 positivity, and splenomegaly.

Serum soluble CD23 in patients with hypogammaglobulinaemia.

Serum levels of the soluble form of the low-affinity receptor for IgE (FcERII, CD23) (sCD23) are elevated in autoimmune conditions associated with hypergammaglobulinaemia and B cell hyperactivity. Very high levels of sCD23 are found in patients with B-chronic lymphatic leukaemia (B-CLL) who are, however, frequently hypogammaglobulinaemic. We therefore compared the serum levels of sCD23 in healthy controls (n = 33) with three conditions associated with hypogammaglobulinaemia (HGG) and varying B cell numbers: X-linked agammaglobulinaemia (XLA, n = 12), common variable immunodeficiency (CVI, n = 20) and B-chronic lymphatic leukaemia (n = 33). Serum levels of sCD23 showed a significant correlation with the CD19+ B cell count in both normals and patients with CVI (r = 0.65, P < 0.0001). Amongst the different clinical groups, serum levels of sCD23 were increased in the order XLA < CVI < normals < CLL (medians 2.5, 7.7, 11.1 and 540, respectively; P < 0.001 for all comparisons except CVI versus normals P < 0.03 in a one-tailed test). In the CVI group, serum sCD23 was lowest amongst four patients with low B cell numbers. There was no overlap in sCD23 between patients with XLA and this subgroup of CVI patients. Serum sCD23 is, therefore, derived predominantly from B cells, and is significantly related to the peripheral blood B cell count.

Therapy-related acute promyelocytic leukaemia.

We report three patients with acute promyelocytic leukaemia (APL) occurring after treatment for other malignant disorders. One patient had had razoxane (a drug affecting DNA topoisomerase II) for cancer of the colon, and the other two had had treatment for cancer of the breast. Two out of the three patients went into complete remission. We review the published literature on therapy-related acute promyelocytic leukaemia (t-APL) and suggest that it is a genuine clinical entity which may be caused by drugs affecting DNA topoisomerase II, and has a prognosis similar to de novo APL.

Identification of nine novel mutations in type I antithrombin deficiency by heteroduplex screening.

We have utilized DNA heteroduplex detection as a method for screening sequences of the antithrombin (AT) gene for the presence of mutations. Affected individuals from 41 kindreds with type Ia antithrombin deficiency were investigated. Heteroduplexes were detected in 12 cases; direct sequencing of the appropriate exons revealed nine cases with novel mutations, and two with previously described mutations. In addition, a new polymorphism in the 5' untranslated region was characterized. The defects included minor insertions and deletions which lead to the removal of intact codons or premature termination, and single base substitutions leading to premature termination or amino acid substitution. In all cases, the affected individuals were heterozygous for the defect and variant AT protein was not detected. In keeping with previous reports the defects associated with type Ia AT deficiency are extremely heterogeneous, the vast majority being point mutations. This study also demonstrates the efficiency of hydrolink gel electrophoresis as a method of screening for unknown mutations by heteroduplex detection.

Is pure red cell aplasia (PRCA) a clonal disorder?

Pure red cell aplasia (PRCA) is an uncommon disorder, many cases lacking a well defined aetiology. This report describes three cases of PRCA (two idiopathic and one associated with B-CLL) who were investigated to assess the possibility of their PRCA being associated with a clonal proliferation of T-lymphocytes. The results show that one patient had evidence of T-cell receptor (TCR) gamma chain rearrangement, and the other had a TCR delta chain rearrangement. These two cases raise the possibility of PRCA being associated with a clonal proliferation of T-cells and further studies are warranted.