Abnormal cytogenetic clones in patients with aplastic anaemia: response to immunosuppressive therapy.

We report the response to immunosuppressive therapy with antithymocyte globulin (ATG) and cyclosporin or oxymetholone in 13 cases of aplastic anaemia (AA) with an abnormal cytogenetic clone detected at or sometime after diagnosis. Blood and bone marrow examination showed no distinctive morphological features of myelodysplasia (MDS) at diagnosis. Haematological response occurred promptly in eight cases; the remainder responded after additional immunosuppression with or without oxymetholone. Three patients had a late relapse of AA, treated successfully by allogeneic bone marrow transplantation in one; the others responded to oxymetholone. Transformation to MDS or acute leukaemia was not observed after a median follow-up of 4.1 years (range 1.2-11.2). In four patients the cytogenetic clone disappeared after treatment.

Immunostaining of whole agar cultures by APAAP.

We have adapted the alkaline phosphatase-anti alkaline phosphatase (APAAP) technique to demonstrate cell antigen distributions in intact agar culture. The method facilitates batch processing and is no less convenient to perform than standard APAAP procedures. Myeloid and lymphoid antigens generally demonstrated strong staining intensity. However, staining at day 0 consistently produced no antigen expression for two monoclonals (CD11c and CD34) in contrast to positivity in parallel cytospins. CD11c showed rapidly increasing antigen expression over subsequent days of culture whereas the expression of CD34 could not be shown in conventional agar culture at any time from day 0 to day 14. Positivity was only restored in CD34-positive leukaemic cells using a modified culture technique in which cells were cultured as pre-formed small aggregates. Assessment of these aggregates extended to cell cycle analysis using anti-bromodeoxyuridine. CD71 positivity in normal culture samples correlated with colony configuration (whether clones were 'spread' or 'tight' in appearance). CD38 staining of normal bone marrow culture at day 7 showed asymmetrical staining of cells in a small number of micro-groups. The clonal detection of aberrant antigens (CD7, CD2) for assessment of minimal residual disease in AML was a disappointment due to the relative frequency of positive clones in normal culture.

Long-term follow-up of aplastic anaemia.

Of 61 patients with aplastic anaemia (AA) diagnosed in our hospitals, 37 survived more than 2 years; actuarial survival of this latter group was 58%, with a median follow-up of living patients of 10.2 years. Laboratory and clinical data pertaining to these long-term survivors was scrutinized to determine the incidence of clonal disorders, which was identified in 43%. Morphological evidence of the myelodysplastic syndrome (MDS) was found in 13 (35%), including four cases of RAEB; four (11%) developed PNH. Of 23 patients studied, four showed karyotypic abnormalities, but these did not always coincide with morphological features of MDS. Although four patients now have completely normal blood and marrow morphology, and another had normal blood and marrow morphology at the time of death due to unrelated disease, the study confirms the high incidence of cytopenia and morphological abnormality, sufficient to justify a diagnosis of MDS, in patients with a history of AA. No definite survival plateau was identified. However, the natural history of MDS secondary to AA seems to be different to that of MDS arising de novo; the clinical course is relatively indolent, possibly implying a different biology.

Influence of azathioprine on the ferrokinetics of patients with renal failure before and after treatment with erythropoietin.

In 16 patients (9 on azathioprine, 7 not) the ineffective iron turnover (IIT) was much higher in the azathioprine group (62.7 +/- 6.7 vs. 23.5 +/- 3.5 mumol/l blood/day, p < 0.0001, 2-tailed t test), though the red cell iron turnover (RCIT) was similar (42.8 +/- 2.9 vs. 41 +/- 4.8). Erythropoietin improved the anaemia in all patients and raised the RCIT (4 still on azathioprine to 72.2 +/- 9.8, p < 0.003; 7 non-azathioprine patients to 62.7 +/- 5.3, p < 0.01); the IIT remained higher in the azathioprine-treated (85.5 +/- 19.3 vs. 37.1 +/- 5.4; p < 0.013). In 2 patients who discontinued azathioprine, the IIT declined markedly to normal. In summary, azathioprine exacerbates the anaemia of renal failure by augmenting ineffective erythropoiesis, while erythropoietin benefits those on azathioprine as much as other renal patients by stimulating both effective and ineffective erythropoiesis.

Three cases of the myelodysplastic syndrome with pericentric inversion of chromosome 16.

Inversion of chromosome 16, inv(16)(p13q22), is characteristic of acute myeloid leukaemia (AML) with eosinophilia and is rarely found in the myelodysplastic syndrome (MDS). We report three cases of MDS in which inv(16) was observed. They were classified to FAB subtypes RA, RARS and RAEBT; eosinophilia or abnormal eosinophils were not observed. The disease appeared to be stable in all three patients. MDS with inv(16) without eosinophilia may be a rare subgroup associated with a good prognosis.

Monitoring of iron requirements in renal patients on erythropoietin.

We studied 38 patients (9 haemodialysis, 18 peritoneal dialysis, 11 advanced renal failure) over the first 12 weeks of erythropoietin therapy. In 14 iron-overloaded patients (ferritin > 500 micrograms/l the haemoglobin (+/- SEM) increased from 6.74 +/- 0.27 to 9.85 +/- 0.36 g/dl (P < 0.0001) entirely by mobilizing iron reserves (reduced from 1,220 +/- 73 to 739 +/- 111 mg, P < 0.0001). In the 24 non-overloaded patients (ferritin < 500 micrograms/l) the haemoglobin rose similarly from 7.04 +/- 0.18 to 10.70 +/- 0.36 g/dl (P < 0.0001), partly from iron reserves (depleted from 200 +/- 74 to -44 +/- 77 mg, P = 0.016) and partly from oral iron supplements (305 +/- 110 mg). In the overloaded patients the ferritin declined from 1057 micrograms/l (geometric mean, range 504-3699) to 317 micrograms/l (42-1505, P < 0.0001). In the non-overloaded patients it declined from 82 micrograms/l (8-461) to 45 micrograms/l (5-379, P = 0.016). The transferrin saturation (TS) in the overloaded patients appeared to decline from 38.3 +/- 7.2% to 24.0 +/- 3.7% but this was not statistically significant. In the non-overloaded the TS was unchanged (23.3 +/- 2.4 before and 28.1 +/- 3.6% after treatment). Considering all 38 patients together, the haemoglobin correlated negatively with the ferritin (r = 0.3731, P < 0.001) but not with the TS. The TS correlated with the serum ferritin initially (r = 0.75, P < 0.001) but not after the first 4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of azathioprine on response of renal anaemia to subcutaneous recombinant human erythropoietin.

AIMS: To determine the effect of concomitant azathioprine treatment on the response of patients with renal failure to treatment with subcutaneous recombinant human erythropoietin (r-HuEPO). METHODS: Two groups of patients with renal failure not receiving haemodialysis were studied. One comprised seven patients receiving erythropoietin alone, the second consisted of nine patients who were also treated with azathioprine. The haematological changes were monitored, and the functional erythropoietic response was studied by two different ferrokinetic models. One analysed the initial, the other the extended plasma iron clearance. Studies were performed before r-HuEPO treatment on all 16 patients, and repeated on 11 of these when the target haemoglobin (10-11 g/dl) was achieved and stabilised. Total erythropoiesis was determined using both techniques. Analysis of the extended plasma iron clearance also permitted calculation of both effective and ineffective erythroid activity. RESULTS: The haematological response to r-HuEPO was the same for both patient groups. Measurement of total erythropoiesis by both ferrokinetic methods showed good correlation. For those receiving long term azathioprine, the percentage ineffective erythropoiesis was high compared with that of the other patients, and remained so for as long as they continued with azathioprine. For those uncomplicated by azathioprine treatment, r-HuEPO increased levels of both effective and ineffective erythropoiesis by the same degree. A substantial reduction in ineffective erythropoiesis was shown only by those patients who either discontinued or reduced their azathioprine once they started r-HuEPO treatment. CONCLUSIONS: Azathioprine increases ineffective erythropoiesis. In this study, the r-HuEPO dose was sufficient to overcome this effect and promoted effective erythropoiesis so that the anaemia lessened. Measurement of total erythropoiesis provided limited information on the functional changes involved, differentiation of effective from ineffective erythropoiesis being necessary to define the changes after azathioprine reduction or withdrawal.

High incidence of type II autoantibodies in pernicious anaemia.

AIMS: To investigate the incidence of type II autoantibodies to intrinsic factor in pernicious anaemia. METHODS: Three hundred and forty four serum samples submitted for intrinsic factor antibody (IFAB) analysis on clinical or laboratory grounds were tested by an established radioassay and a new enzyme linked immunosorbent assay (ELISA) method for type I and total IFAB, respectively. Sixty of these were found to be positive by ELISA; this method was used to test further, 40 samples of adequate volume for types I and II antibodies. RESULTS: Type II antibodies were detected in 39 of the 40 sera tested. A comparative analysis indicated that seven samples contained pure type II antibody, being positive for total and type II by ELISA, but negative for type I by both the ELISA and radioassay technique. CONCLUSIONS: The occurrence of type II antibody, both alone and in combination with type I, seems to be more common than has previously been recognised, and emphasises the advantage of using a technique which will detect both types of antibody.

Two-colour immunoenzymatic technique using sequential staining by APAAP to evaluate two cell antigens.

AIMS: To extend the alkaline phosphatase-antialkaline phosphatase (APAAP) immunoenzyme single stain method to a more generally applicable double stain technique. This will allow two primary antibodies of the same isotype of IgG and specifically the nuclear antigen bromodeoxyuridine (BRdU) to be evaluated with a cell surface antigen identifier. METHOD: Sequential applications of the APAAP method showed two antigen sites by different dye couplings to a common alkaline phosphatase substrate, producing blue and red reaction products on the same slide. Antigens on different cell populations as well as those in different compartments of the same cell were analysed. The method allowed a surface antigen monoclonal to be revealed first, using an optimal fixative, before alcohol/gluteraldehyde fixation was used to start the second (BRdU) staining sequence. RESULTS: An analysis of double staining of T lymphocyte subsets (CD4 and CD8) showed no significant difference in the order of application of the primaries (n = 10) and no significant difference from their corresponding single stain results (n = 50), confirming the validity of the technique where antigens are exclusively distributed. Other examples, including antigens distributed in different compartments of the same cell, displayed discrete staining which implied validity. CONCLUSION: Double staining by APAAP with this technique seems to be applicable to those cases where antigens are exclusively distributed and includes cases where different compartments of the same cell are stained. It is especially useful in revealing antigens that require different fixation and preparation--that is DNA incorporated BRdU with a surface antigen. But it does seem to have a limited ability to produce a dual colour at a common site.

Comparative ferrokinetic study with initial and extended iron clearance models.

Erythrokinetic studies were performed on 10 patients with chronic myelofibrosis and 11 patients with myelodysplasia (MDS). Values for plasma iron turnover, marrow iron turnover, and erythron transferrin uptake were derived using two ferrokinetic models. One entailed analysis of the extended plasma iron clearance over a number of days, the other comprised analysis of the initial plasma iron clearance during the first few hours of the study. A close correlation was found between the variables quantifying total erythropoiesis (marrow iron turnover and erythron transferrin uptake) in the two methodologies. Functional classifications produced by both models and based on the values for plasma iron turnover, marrow iron turnover, and erythron transferrin uptake were compared. Both models identified functional heterogeneity in the group with myelofibrosis and functional homogeneity within the MDS group. Each method produced comparable data on erythropoiesis. The main reason for analysing the extended plasma iron clearance is to differentiate levels of effective and ineffective erythropoiesis. The short analysis presents the practical advantages associated with a one-day study. This could be further enhanced if the level of effective erythropoiesis could be clearly defined.

Bone marrow necrosis complicating chronic myeloid leukaemia.

Two women with chronic myeloid leukaemia in chronic phase were found to have bone marrow necrosis when severe bone pains and falling blood counts prompted a marrow examination to exclude blast transformation. One patient survived for 12 months following the event without transforming. The second patient died soon after and was found to have widespread extramedullary disease.

Continuous assessment of performance in External Quality Assessment Schemes.

A simple, standardised procedure was used for processing data from External Quality Assessment Schemes and for producing performance charts. A graphic presentation of cumulative performance is generated for each test, using an integrated software package to display results in a standardised format. This permits interpretation of satisfactory and poor performances and facilitates identification of "outlying" results. Although most results are recorded as Deviation Indices, the system is flexible and can be used to present other performance variables, such as Percentage Deviations and relevant textual information relating to interpretative assessments. It is therefore readily adaptable for use by other pathology disciplines.

Failure of mithramycin to control the myeloid blast phase of chronic granulocytic leukemia: a report on nine patients and review of the literature.

After reports of the successful use of mithramycin and hydroxyurea in the myeloid blast phase of chronic granulocytic leukemia, we treated nine patients according to the protocol devised by Koller and Miller (1986). There were no complete responses, but one patient had a partial response with a transient return to the chronic phase. Of the remaining eight patients, two experienced lessening of bone pains, and one a reduction in spleen size, but without hematological improvement. The regimen was associated with significant toxicity, and no overall survival advantage. We present a review of published data regarding the use of mithramycin in chronic granulocytic leukemia which supports the results in our series. The combination of mithramycin and hydroxyurea is largely ineffective in the blast phase of chronic granulocytic leukemia, but may be of value in the accelerated phase.