No abstract

CorrespondenceFormic acid decalcification of bone marrow trephines degrades DNA: alternative use of EDTA allows the amplification and sequencing of relatively long PCR products We recently reported a new method for the extraction of DNA from paraYn wax embedded bone marrow trephine biopsies. 1 The DNA extracted from EDTA decalcified bone marrow trephine biopsies using this method was suYciently intact to allow the amplification and sequencing of relatively long polymerase chain reaction (PCR) products, including the 600 bp t(11;14) MTCA PCR product. A shorter 294 bp PCR product could only be amplified from six of 10 formic acid decalcified bone marrow trephine biopsies reported in a previous study by Provan et al. 2 These findings suggested a correlation between DNA degradation and formic acid decalcification, but required a comparative study for confirmation.We have subsequently extracted DNA from 11 formic acid decalcified bone marrow trephine biopsies using our method and determined the quality of DNA using agarose gel electrophoresis and PCR analysis, as in our initial study.The mean DNA yield from the formic acid decalcified blocks was twice that of the EDTA decalcified samples: 9.4 µg and 4.3 µg, respectively. This reflected the fact that the formic acid blocks contained approximately twice as much bone marrow trephine biopsy material as a result of the diVerences in practice between the two centres involved in the study (Exeter, EDTA decalcification; Southampton, formic acid decalcification). However, when the formic acid decalcified DNA samples were analysed by agarose gel electrophoresis, no high molecular weight DNA was detected; only a smear of degraded DNA was seen. In contrast, analysis of the EDTA decalcified bone marrow trephine biopsy DNA samples showed DNA ranging from 5 to 21 kb in length (fig 1).

Seventeen patients with hairy-cell leukaemia (HCL) and peripheral cytopenias were given human lymphoblastoid interferon (Wellferon), 3 megaunits daily or 6 megaunits on alternate days intramuscularly, for 4-24 weeks. Twelve of the patients had undergone splenectomy, three had no palpable spleen and had therefore not been offered surgery, and two patients with substantial splenomegaly were given interferon (IFN) as treatment of first choice. Toxic effects were minor except in one patient who experienced a severe form of somnolence syndrome. In all patients hairy cells (HCs) were cleared from the blood and platelet and Hb levels improved in 2-14 weeks. Neutrophils were improved in 14/17 of the patients. In the two patients with splenomegaly, the spleen became impalpable after 5-8 weeks therapy, and haematological improvement occurred at 12-14 weeks. HC infiltration of the marrow was reduced in all patients, but was complete (less than 5%) in only two, both of whom had impalpable spleens. Immunological surface-marker studies confirmed that light-chain-restricted B cells disappeared from the blood in parallel with the clearance of morphological HCs. There was no evidence of HC maturation and no increase in phenotypic NK cells. T cells were moderately reduced and the relatively greater reduction of Leu 2a+ suppressor cells resulted in increased Leu 3a+/2a+ helper/suppressor ratios in 11/17 of the patients. Early experience in the six patients who have stopped IFN suggests that, after an initial further increase in Hb and neutrophil levels, HCs gradually return with slow deterioration of haematological parameters. Interferon is now the treatment of choice for patients becoming cytopenic post-splenectomy or for patients without splenomegaly. IFN is effective first-line therapy in patients with splenomegaly, but further work is needed to establish whether the agent should replace splenectomy in such patients. Some form of maintenance or re-treatment therapy will probably be necessary.

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.

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.