Large variation in t(11;14)(q13;q32) and t(14;18)(q32;q21) translocation product size is confirmed by sequence analysis of PCR products.

Polymerase chain reaction is commonly used to detect t(11;14)(q13;q32) and t(14;18)(q32;q21) chromosomal translocations associated with mantle cell lymphoma and follicular lymphoma. We tested a total of 482 samples from patients with suspected non-Hodgkin's lymphoma and sequenced unusual-sized t(11;14)(q13;q32) and t(14;18)(q32;q21) products from 33 of these patients. BCL-1 or BCL-2 gene rearrangements were confirmed in 23 of 33 patients (70%). Considerable size variation was observed using t(11;14) primers, with MTCA and MTCB t(11;14) products ranging from 234 to 934 bp and 143 to 560 bp respectively. Less variability was observed for t(14;18) Major Breakpoint Region (MBR) products (100-252 bp) but Minor Cluster Region (MCR) products ranged from 217 to 498 bp. We demonstrate the utility of sequence analysis to confirm unusual-sized translocation products and reduce false-positive results because of nonspecific amplification.

Analysis of haematopoietic chimaerism by quantitative real-time polymerase chain reaction.

Allogeneic bone marrow transplantation (BMT) with marrow ablative conditioning is the treatment of choice for haematopoietic malignancies. The use of nonmyeloablative stem cell transplants has allowed the treatment of patients previously ineligible for BMT because of age or other disease. These reduced conditioning regimes allow the persistence initially of some recipient cells in the blood and bone marrow (haematopoietic chimaerism). Monitoring of the relative proportion of donor and recipient cells is required to assess the success of the procedure, to predict subsequent rejection or impending relapse and to guide the use of donor lymphocyte infusions. We present a quantitative real-time PCR approach for the measurement of haematopoietic chimaerism using the TaqMan. This approach exploits the presence of single-nucleotide polymorphisms (SNPs) to distinguish cells of patient or donor origin. We have designed and validated a panel of seven allele-specific probes to quantify the contribution of patient and donor cells in the haematopoietic population from 12 patient and donor pairs. We have compared the performance of this approach with an existing method and proved it to be superior in both accuracy and sensitivity. The use of more sensitive and accurate techniques permits earlier intervention for improved clinical outcome.

Quantitation of cyclin D1 over-expression using competitive fluorescent reverse transcription polymerase chain reaction: a tool for the differential diagnosis of mantle cell lymphoma.

Mantle cell lymphoma is characterized by the presence of the t(11;14)(q13;q32) translocation that causes over-expression of the BCL-1 gene and consequent overproduction of its gene product cyclin D1. We have developed a competitive fluorescent reverse transcription polymerase chain reaction assay for the detection and semiquantitation of cyclin D1 over-expression. Using this assay a definitive ratio of the expression of cyclin D1 to cyclins D2 and D3 can be determined, provided good quality RNA is available. A single upstream primer derived from a consensus sequence found in cyclins D1, D2, and D3 was labeled at the 5' end using a fluorescent dye. Downstream primers specific to cyclins D1 and D2 were designed and used in conjunction with a previously published D3 specific primer. The fluorescently labeled PCR products were separated by electrophoresis using an ABI 377 DNA sequencer. Fluorescence emitted from each product was used to determine the ratio of expression of cyclin D1 to D2 and D3 by assigning a dosage quotient [D1/(D2+D3)]. The mean dosage quotient recorded from samples representing 29 non-MCL patients was 0.03 (SD +/- 0.03), the maximum value being 0.11. Samples from eight patients with a diagnosis of MCL generated values greater than 2. Calculation of a dosage quotient using this competitive fluorescent reverse transcription polymerase chain reaction assay allows unequivocal identification of patients with over-expression of cyclin D1, providing a new tool for the differential diagnosis of MCL.

Amplification of PCR products in excess of 600 base pairs using DNA extracted from decalcified, paraffin wax embedded bone marrow trephine biopsies.

AIMS: To establish a robust method of extracting DNA from paraffin wax embedded bone marrow trephine (PBMT) biopsies for the amplification of relatively long polymerase chain reaction (PCR) products. METHOD: Xylene and ethanol were used to remove paraffin wax from eight formalin fixed, EDTA decalcified PBMT biopsies and DNA extraction was performed using a Qiagen QIAamp tissue kit. The DNA samples were amplified using nine different PCR primers sets, including those used to detect chromosomal translocations (t(11;14) and t(14;18), and clonal B cell populations. A t(11;14) PCR product of approximately 600 base pairs (bp) was sequenced using dye terminator cycle sequencing. RESULTS: All eight DNA samples extracted from PBMT biopsies were amplified successfully to generate DNA fragments up to 643 bp in length. Chromosomal translocations and immunoglobulin gene rearrangements were detected by PCR in some of the samples. Sequencing of the t(11;14) PCR product demonstrated the presence of chimaeric sequences, which included both bcl-1 and immunoglobulin heavy chain (IgH) gene sequences, consistent with the presence of this translocation. CONCLUSIONS: This method enables PCR analyses of PBMT biopsies that were not previously possible, offering the prospect of improved accuracy of diagnosis and the monitoring of patients with bone marrow disease.

Concordant expression of class II and class III CD34 epitopes on haemopoietic cells in leukapheresis and cord blood samples with CD45/CD34 dual staining.

We used flow cytometry with CD45/CD34 dual antibody labelling to investigate the relative expression of class II and class III CD34 epitopes on haemopoietic progenitor cells (HPC) in 22 leukapheresis and cord blood samples. There was a close correlation between class II (QBEnd-10) and class III (clone 581) CD34+ cells (R2= 0.975, ratio of class III to class II CD34+ count 1.24 +/- 0.04). The linear relationship between class II and III CD34 epitopes on HPC suggests that the choice of antibody class is not a source of random variation in the quality assurance of CD34+ cell enumeration.

Acute myeloid leukaemia developing in a patient with longstanding untreated chronic lymphocytic leukaemia.

A patient with chronic lymphocytic leukaemia untreated for 5 years subsequently developed acute myeloid leukaemia. It is suggested that reduced immunocompetence is the likely mechanism in this case and may also be a contributory factor in those cases which have been ascribed to the use of alkylating agents or radiation.

Refractory anaemia terminating in a combined lymphoproliferative and myeloproliferative disorder.

We report a case of non-sideroblastic refractory anaemia which evolved to a double lymphomyeloproliferative disorder. At presentation, bone marrow appearances and peripheral blood pancytopenia without myelomonocytosis were consistent with a diagnosis of non-sideroblastic refractory anaemia. Subsequently, the patient developed pronounced myelomonocytosis and lymphocytosis with prolymphocytes. Light and transmission electron microscopy as well as surface marker studies were compatible with a diagnosis of prolymphocytic transformation of chronic lymphocytic leukaemia/prolymphocytic leukaemia associated with myelomonocytic leukaemia. The pathogenesis of such double lympho-myeloproliferative disorders is discussed in the light of the evidence for common lymphoid and myeloid progenitor cells and some recent advances in the immunology of the myelodysplastic syndromes.

Acute thrombocytopenia following ingestion of indomethacin.

A case of acute thrombocytopenia with shortened platelet survival following ingestion of indomethacin is reported. Drug history and absence of infection exclude both the role of other agents and an acute episode of idiopathic thrombocytopenia. The patient exhibited a poor response to immunosuppressive treatment and a slow resolution of the thrombocytopenia.

Detection of platelet-bound and serum antibodies in autoimmune thrombocytopenia by enzyme-linked assay.

Platelet antibodies were looked for in 47 patients with autoimmune thrombocytopenia using a modification of the enzyme-linked assay previously described. Surface-bound antibodies measured as increased platelet-associated IgG were found in 32 (68%) of the patients. After incubation in test sera, the platelet-associated IgG of normal donor platelets was significantly increased in 27 of the 47 patients (57%), thus demonstrating the presence of platelet antibodies free in their sera. 6 patients had antibodies only in the serum without any elevation of their platelet-associated IgG. When both tests are evaluated together no antibody was detected by either the direct or the indirect test in 9 of the 47 patients (29%) studied. The technique used is described and the interpretation of our results discussed.

Sarcoidosis associated with combined immunodeficiency.

The association of multiple non-caseating granulomata and a positive Kviem test is normally considered to be indicative of a diagnosis of sarcoidosis. However, although depressed cell-mediated immunity is commonly described, it is extremely rare to find a humoral immune paresis. A patient is reported who had multiple granulomata, depressed cellular and humoral immunity and a positive Kveim test.

Non-excretory multiple myeloma.

The clinical and laboratory features in five patients with non-excretory myeloma are described including plasma cell immunofluorescence and, in two cases, ultrastructure. Findings are compared with those in similar patients previously reported, and those in excretory disease. Clinical, haematological and biochemical features were similar to those found in excretory myeloma, showing differences only in relation to the absence of serum or urinary monoclonal immunoglobin. Cellular cytological and ultrastructural features allowed no differentiation from excretory cells. Within this non-excretory group distinction between secretory and non-secretory myeloma cells is possible on the basis of immunofluorescence. Differing patterns of intermittent excretion occurred in three of these patients.

The significance of Ph1 mosaicism: a report of six cases of chronic granulocytic leukaemia and two cases of acute myeloid leukaemia.

Six cases of chronic granulocytic leukaemia (CGL) and two cases of acute myeloid leukaemia (AML) with dual populations of karyotypically normal and Philadelphia (Ph1) chromosome-positive cells are described. GTG and QF-banding characterized the Ph1 as resulting from a 9/22 translocation in all eight cases. Four of the patients suffering from CGL presented with 100% Ph1-positive bone marrows, and after receiving intensive chemotherapy, karyotypically normal cells were demonstrated. The other two patients with CGL showed Ph1 mosaicism at presentation. The two patients with AML exhibited Ph1 mosaicism at presentation and throughout the course of the disease. In both of these patients, a marker No. 10 chromosome was found in some of the Ph1-positive cells and in one hyperdiploidy was observed to have developed only in the clone with the additional chromosome anomaly.