Health technology assessment in clinical biochemistry. Methylmalonic acid: a Danish showcase.

In 1992 plasma methylmalonic acid (MMA) was introduced in Denmark for diagnosing vitamin B-12 deficiency. Now, 10 years later, we report on a health technology assessment (HTA) suggesting that the clinical usefulness of MMA is uncertain. MMA is an obvious component for measurement in the diagnosis of vitamin B-12 deficiency because MMA accumulates when there is a lack of vitamin B-12, and technologically the analysis is of high quality. The diagnostic sensitivity of MMA is high, whereas the diagnostic specificity is debatable, and our results suggest it to be relatively low. The organizational aspect implies that both MMA and P-cobalamins have been increasingly employed, though no consensus on the use of the analyses has emerged. The benefit to the patient is not obvious. An increased level of MMA does not predict further increases over time, and vitamin B-12 treatment shows limited clinical benefit in individuals with a moderately increased MMA. The economic consequences of introducing MMA were an increase in the costs of MMA and P-cobalamins of 12% per year during 1992-2000 and an increase in the turnover of vitamin B-12 preparations of 9% per year. In conclusion, MMA was introduced on sound grounds for both pathophysiological considerations and analytical quality. Our HTA shows that the resources employed to diagnose and to treat vitamin B-12 deficiency have increased considerably, but yet we have no evidence to suggest the clinical benefit.

Vitamin B-12 treatment has limited effect on health-related quality of life among individuals with elevated plasma methylmalonic acid: a randomized placebo-controlled study.

OBJECTIVE: To examine the hypothesis that treatment with vitamin B-12 improves health-related quality of life (HRQOL) in individuals with biochemical signs of vitamin B-12 deficiency. DESIGN: A randomized placebo-controlled study. SETTING: Municipality of Aarhus, Denmark. SUBJECTS: Nonhospitalized individuals (n = 140) with a modest increase in plasma methylmalonic acid (0.40-2.00 micromol L-1) not previously treated with vitamin B-12. INTERVENTION: The participants were randomized to vitamin B-12 injection treatment or placebo weekly for 4 weeks and re-examined 3 months later. The investigator and the participants were blinded to the intervention. MAIN OUTCOME MEASURE: Change in HRQOL assessed by the SF-36 questionnaire from baseline to follow-up examination 3 months later. RESULTS: The participants reported a significantly worser HRQOL than the age- and sex-matched Danish general population (P < 0.001). However, no change was observed after treatment with vitamin B-12 for seven of eight health dimensions. A significant improvement was found only in general health when compared with the placebo group (P = 0.03). CONCLUSIONS: Vitamin B-12 treatment influenced only one of eight dimensions of HRQOL amongst participants with biochemical signs of vitamin B-12 deficiency. We therefore question the benefit of vitamin B-12 treatment amongst elderly with a modestly increased plasma methylmalonic acid as the only sign of vitamin B-12 deficiency.

Vitamin B12 treatment normalizes metabolic markers but has limited clinical effect: a randomized placebo-controlled study.

BACKGROUND: The clinical significance of increased plasma methylmalonic acid (P-MMA) is unclear. We assessed the efficacy of vitamin B12 treatment in reducing P-MMA and plasma total homocysteine compared with the clinical benefits of treatment. METHODS: We studied 140 individuals with mildly to modestly increased P-MMA (0.40-2.00 micromol/L), not previously treated with vitamin B12, in a randomized, placebo-controlled study. A detailed medical history was obtained, and laboratory tests as well as an objective neurologic disability score were performed at baseline and 3 months after the start of intervention. RESULTS: P-MMA (P <0.001) or plasma total homocysteine (P <0.001) decreased in the treatment group vs the placebo group, but no significant difference was found in the change of blood hemoglobin (P = 0.18) and mean cell volume (P = 0.71). Changes in symptom scores did not differ between the groups for symptoms of anemia (P = 0.63), neurologic symptoms (P = 0.21), gastroenterologic symptoms (P = 0.32), or the Neurological Disability Score (P = 0.85). CONCLUSIONS: Treatment with vitamin B12 reduces P-MMA and plasma total homocysteine, but individuals with a mild to modest increase in P-MMA may have only limited clinical benefit from vitamin B12 treatment, at least in the short term.

Increased plasma methylmalonic acid level does not predict clinical manifestations of vitamin B12 deficiency.

BACKGROUND: The prevalence of vitamin B(12) deficiency, defined as an elevated concentration of plasma methylmalonic acid (P-MMA), has been estimated to be 15% to 44% in the elderly. However, we do not know whether an increased P-MMA level actually indicates or predicts a clinical condition in need of treatment. PARTICIPANTS AND METHODS: In a follow-up study, 432 individuals not treated with vitamin B(12) were examined 1.0 to 3.9 years after initial observation of an increased P-MMA concentration (>0.28 micromol/L). The examination included laboratory tests, a structured interview to disclose symptoms, a food frequency questionnaire, and a clinical examination including a Neurological Disability Score. RESULTS: Variation in P-MMA levels over time was high (coefficient of variation, 34%). In only 16% of participants, P-MMA levels increased substantially, whereas 44% showed a decrease. Level of P-MMA was significantly but not strongly associated with levels of plasma cobalamins (r = -0.22, P<.001) and plasma total homocysteine (r = 0.37, P<.001). After adjustment for age and sex, we found no associations between P-MMA concentration and the total symptom score (P =.61), the total Neurological Disability Score (P =.64), or other clinical manifestations related to vitamin B(12) deficiency. CONCLUSIONS: An increased level of P-MMA did not predict a further increase with time and clinical manifestations related to vitamin B(12) deficiency. We therefore challenge the use of an increased P-MMA concentration as the only marker for diagnosis of vitamin B(12) deficiency.

Indirect evidence for the potential ability of magnetic resonance imaging to evaluate the myocardial iron content in patients with transfusional iron overload.

The purpose of this study was to evaluate the potential ability of magnetic resonance imaging (MRI) for evaluation of myocardial iron deposits. The applied MRI technique has earlier been validated for quantitative determination of the liver iron concentration. The method involves cardiac gating and may, therefore, also be used for simultaneous evaluation of myocardial iron. The tissue signal intensities were measured from spin echo images and the myocardium/muscle signal intensity ratio was determined. The SI ratio was converted to tissue iron concentration values based on a modified calibration curve from the liver model. The crucial steps of the method were optimized; i.e. recognition and selection of the myocardial slice for analysis and positioning of the regions of interest (ROIs) within the myocardium and the skeletal muscle. This made the myocardial MRI measurements sufficiently reproducible. We applied this method in 41 multiply transfused patients. Our data demonstrate significant positive linear relationships between different iron store parameters and the MRI-derived myocardial iron concentration, which was significantly related to the serum ferritin concentration (rho=0.62, P<0.0001) and to the MRI-determined liver iron concentration (rho=0.36, P=0.02). The myocardial MRI iron concentrations demonstrated also a significant positive correlation with the number of blood units given (rho=0.45, P=0.005) and the aminotransferase serum concentration (rho=0.54, P=0.0008). Our data represents indirect evidence for the ability of MRI techniques based on myocardium/muscle signal intensity ratio measurements to evaluate myocardial iron overload.

Expression of CD2 and activation markers on blood T-helper cell subsets in patients with transfusional iron overload.

The aim of the present study was to investigate the relationship between different measures of iron status, and the expression of CD2, and the activation markers CD25, CD71, CD45RO, HLADR CD38 within the Th-cell subset in patients with progressive transfusional iron overload. We estimated the expression of the activation surface markers on the Th cells of peripheral blood by flow cytometry from 22 multiply transfused patients. The number of CD2 binding sites (BS) on Th cells was significantly higher in the patients (82 917 +/- 30 801) than in age-matched normal controls (41 145 +/- 6989, P < 0.0001). When investigating whether this difference could be due to the iron overload we found the number of CD2 BS closely related to the iron saturation of serum transferrin (TfS) (R2 = 0.78, P < 0.001). The relationship to the serum ferritin concentration and to the number of blood units given was weaker, but also significant (R2 = 0.22, P < 0.027, respectively, R2 = 0.21, P < 0.032). Also the fraction of mature memory Th cells which express CD45RO at a high level was directly related to the TfS (R2 = 0.57, P < 0.0001), while the expression of CD38 within the Th cell fraction was inversely related to the TfS (R2 = - 0.43, P = 0.009). The expression of HLA-DR (but not of CD25 and CD71) was also directly related to the TfS (R2 = 0.29, P = 0.01). Our results show a clear, statistical relationship between the iron status and the expression of surface markers within Th cells in multiply transfused patients.

Cardiac function during iron chelation therapy in adult non-thalassaemic patients with transfusional iron overload.

It is well-documented that iron chelation by desferrioxamine protects/improves the cardiac function in blood transfusion-dependent children suffering from beta-thalassaemia. In patients who do not become dependent upon blood transfusion until adulthood (ANT-patients), iron chelation by desferrioxamine may affect the cardiac function in unknown ways, presumably because age-related changes in the heart may cause iron chelation to affect the cardiac function in different ways. We therefore followed the left ventricular ejection fraction (LVEF) by multigated radionuclide angiography in 16 iron-loaded ANT-patients during iron chelation alone and after increasing the efficacy of chelation by vitamin C supplementation. During 12 months of iron chelation the mean LVEF fell significantly from 63.3% to 58.0% (p=0.04). Individual changes in LVEF did not correlate significantly with age but with the pretreatment liver iron concentration. After initiation of vitamin C supplementation, the mean LVEF increased from 55.9% to 65.3% (p=0.01). Our data suggest that in ANT-patients prolonged desferrioxamine treatment without vitamin C supplementation may be associated with reduced LVEF, whereas vitamin C supplementation seems to benefit the cardiac function. Similar findings have not been described in beta-thalassaemia and may hence be specific for ANT-patients. However, our findings have to be confirmed by controlled studies.

Acyclovir prophylaxis and fever during remission-induction therapy of patients with acute myeloid leukemia: a randomized, double-blind, placebo-controlled trial.

PURPOSE: A randomized, double-blind, placebo-controlled trial was performed to estimate the preventive effect of the antiherpetic drug acyclovir on fever, incidence of bacteremia, use of antibiotics, and presentation of infections in patients with acute myeloid leukemia (AML). PATIENTS AND METHODS: Ninety herpes simplex virus (HSV)-seropositive patients aged 18 to 84 years were included. Forty-five patients received acyclovir (800 mg by mouth daily) and 45 placebo. The patients were examined daily for 28 days from the initiation of remission-induction chemotherapy. RESULTS: Fever developed in all patients in both groups. Acyclovir prophylaxis postponed the development of an oral temperature > or = 38.0 degrees C by 3 days (95% confidence interval [CI], 1 to 4 days; P = .03) and the initiation of antibacterial treatment by 3 days (95% CI, 1 to 5 days; P = .008). The duration of fever, use of antibacterial treatment, incidence of bacteremia, and need for systemic antifungal therapy were not affected by acyclovir prophylaxis. At fever development, acyclovir prophylaxis affected the incidence and localization pattern of oral ulcers. Thus, in the acyclovir group, the number of nonfungal oral infections was reduced (relative risk, 0.45 [95% CI, 0.24 to 0.85]) and mainly located on the soft palate (relative risk, 2.49 [95% CI, 1.19 to 5.22]). CONCLUSION: Acyclovir prophylaxis has an impact on fever development, but not on the duration of fever or the need for antibiotics. It does not reduce the incidence of bacteremia, but the presentation of acute oral infections is changed.

In B-cell chronic lymphocytic leukaemia chromosome 17 abnormalities and not trisomy 12 are the single most important cytogenetic abnormalities for the prognosis: a cytogenetic and immunophenotypic study of 480 unselected newly diagnosed patients.

Of 560 consecutive, newly diagnosed untreated patients with B CLL submitted for chromosome study, G-banded karyotypes could be obtained in 480 cases (86%). Of these, 345 (72%) had normal karyotypes and 135 (28%) had clonal chromosome abnormalities: trisomy 12 (+12) was found in 40 cases, 20 as +12 alone (+12single), 20 as +12 with additional abnormalities (+12complex). Other frequent findings included abnormalities of 14q, chromosome 17, 13q and 6q. The immunophenotype was typical for CLL in 358 patients (CD5+, Slg(weak), mainly FMC7-) and atypical for CLL in 122 patients (25%) (CD5-, or Slg(strong) or FMC7+). Chromosome abnormalities were found significantly more often in patients with atypical (48%) than in patients with typical CLL phenotype (22%) (P < 0.00005). Also +12complex, 14q+, del6q, and abnormalities of chromosome 17 were significantly more frequent in patients with atypical CLL phenotype, whereas +12single was found equally often in patients with typical and atypical CLL phenotype. The cytomorphology of most of the +12 patients was that of classical CLL irrespective of phenotype. In univariate survival analysis the following cytogenetic findings were significantly correlated to a poor prognosis: chromosome 17 abnormalities, 14q+, an abnormal karyotype, +12complex, more than one cytogenetic event, and the relative number of abnormal mitoses. In multivariate survival analysis chromosome 17 abnormalities were the only cytogenetic findings with independent prognostic value irrespective of immunophenotype. We conclude that in patients with typical CLL immunophenotype, chromosome abnormalities are somewhat less frequent at the time of diagnosis than hitherto believed. +12single is compatible with classical CLL, and has no prognostic influence whereas chromosome 17 abnormalities signify a poor prognosis. In patients with an atypical CLL immunophenotype, chromosome abnormalities including +12complex, 14q+, del 6q and chromosome 17 are found in about 50% of the patients, and in particular chromosome 17 abnormalities suggest a poor prognosis.

Distinct circulation patterns in peripheral blood of leukocyte subpopulations during the first 24 hours following autologous bone marrow transplantation.

We have studied the recirculation patterns of leukocyte subpopulations during the first 24 h at 5 min before and 5, 15, 180, and 1440 min after autologous bone marrow transplantation (ABMT) in 14 patients with cancer (6 with AML, 5 with malignant lymphomas, 2 with ALL, and 1 with Ewing's sarcoma) using multiparameter flow cytometry and measurements of myeloid progenitors (CFU-GM). Although the great majority of the injected cell populations were undetectable 5 min after graft infusion, the number of CD3+ T lymphocytes increased at 5 and 15 min and again at 24 h post-ABMT. In contrast, the number of CD56+ natural killer (NK) cells decreased rapidly after ABMT to remain low throughout the observation period. Mature myeloid and monocytic cells (identified by their expression of CD66 and CD14, respectively) were present before, as well as after ABMT in numbers indicating that they were probably of endogenous origin. Immature myeloid cells were identified in a three-color flow cytometric assay as CD13+ CD14- CD66- and tended to increase during the first 15 min after ABMT. Finally, when CFU-GM were followed longitudinally, they were found to be practically absent before ABMT but were clearly detectable in 12 of 14 patients throughout the observation period. We conclude that leukocyte subsets exhibit different recirculation patterns after ABMT, and in light of the increased knowledge about leukocyte-endothelial interactions, these data could provide a platform for attempts to control leukocyte recirculation during stem cell infusion.

[Diagnosis of minimal residual disease in autologous bone marrow transplantation]. / Diagnostik af minimal residual sygdom ved autolog knoglemarvstransplantation.

ABMT (autologous bone marrow transplantation) is being increasingly used in the treatment of malignant diseases, including the acute leukaemias. Although ABMT seems to be superior to conventional chemotherapy in terms of disease-free survival, an unacceptably high frequency of relapse after ABMT remains a major problem. As such relapse may be due to malignant cells in the graft or residual malignant cells surviving in the patient after preconditioning therapy, it is essential to be able to detect and eradicate residual malignant cells. The article presents a review of available methods for the detection of minimal residual disease in conjunction with ABMT, especially regarding their relative sensitivity and specificity.

The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload.

Long-term follow-up data are presented on changes in peripheral blood counts and Hb requirements of 11 patients with myelodysplastic syndromes (MDS) during iron chelation treatment with desferrioxamine for up to 60 months. The erythroid marrow activity was indirectly evaluated by repeated determinations of the serum transferrin receptor concentration. The efficacy of iron chelation was evaluated by repeated quantitative determination of the liver iron concentration by magnetic resonance imaging. Reduction in the Hb requirement ( > or = 50%) was seen in 7/11 (64%) patients. Five patients (46%) became blood transfusion independent. Platelet counts increased in 7/11 (64%) patients and the neutrophil counts in 7/9 (78%) evaluable patients. All patients in whom iron chelation was highly effective showed improvement of erythropoietic output accompanied by an increase in the serum transferrin receptor concentration. It is concluded that reduction in cytopenia in MDS patients may be accomplished by treatment with desferrioxamine, if the iron chelation is efficient and the patients are treated for a sufficiently long period of time. Exactly how treatment with desferrioxamine works remains a challenge for further investigation.

Serum procollagen III peptide concentration in iron overload.

Patients with severe iron overload may develop hepatic fibrosis due to iron toxicity. Unfortunately, the follow-up of the fibrogenic activity during treatment by histological examination of tissue biopsies carries potential side effects, and may therefore not be justified ethically. Recently, the serum concentration of procollagen type III peptide (S-PIIINP) has been shown to be a valid serum marker of the activity of collagen metabolism in conditions with hepatic fibrosis unrelated to iron overload. In order to evaluate the potential usefulness of this test in patients with fibrosis due to iron overload, we investigated the relationship between the PIIINP serum concentration and the size of iron overload in 18 patients with hereditary haemochromatosis (HH) and in 14 patients with transfusional iron overload. A close correlation was found between S-ferritin and S-PIIINP (r = 0.73, p < 0.0001). Follow-up of 6 patients during iron depletion treatment revealed a normalization of the serum aminotransferase concentration before normalization of S-PIIINP was found. This may indicate that excess iron directly induces an increase in fibrogenesis rather than the increased fibrogenesis is secondary to hepatocellular injury caused by iron excess. Thus, serial measurements S-PIIINP may be useful in follow-up of the fibrogenic process due to iron overload.

Myeloid progenitors in remission bone marrow in patients with malignant blood diseases: clues for slow hematopoietic regeneration after ABMT?

Slow hematopoietic recovery is a well-known feature in patients undergoing autologous bone marrow transplantation (ABMT), and here we demonstrate that compared with patients with other malignant blood diseases, the thrombocytopenia in acute myeloid leukemia (AML) patients stands out, with the median time to recovery of > 50 x 10(9) platelets being 101 days for AML patients and 22-37 days for other patient groups. We have consequently evaluated the content and fate of myeloid progenitor cells (BFU-E, CFU-GM, and CFU-GEMM) in bone marrow preparations from cancer patients in an attempt to uncover factors of importance for their slow hematopoietic recovery after ABMT using three experimental setups. First, we analyzed progenitors in marrow samples from 36 patients [18 AML, 5 acute lymphoblastic leukemia (ALL), and 13 non-Hodgkin's lymphoma (NHL)] exposed to multiple doses of severely myelotoxic cytoreductive regimens and observed that AML patients had suppressed numbers of CFU-GEMM and BFU-E (80% that of normal volunteers) but increased levels of CFU-GM. In contrast, the decrease in ALL patients was more pronounced (50% that of normal volunteers) and demonstrable for all progenitors. NHL patients exhibited normal progenitor frequencies of CFU-GM and CFU-GEMM, whereas their BFU-E counts were found to be increased nearly two-fold. Second, we followed the fate of progenitors through the laboratory manipulations of marrow grafts and found no evidence for selective losses in terms of disease (7 AML, 5 NHL, 1 ALL, and 2 testicular germ cell tumor patients) and progenitor type. Third, we retrospectively evaluated the cell yields of grafts from 121 cases [39 AML, 20 ALL, 22 NHL, 9 Burkitt's lymphoma (BL), 18 Hodgkin's disease (HD), and 13 testicular germ cell tumors] and found those of heavily treated AML, HD, and germinal tumors most affected, with a clear negative correlation in AML patients between age on the one hand and mononuclear cell yield and CFU-GM content on the other. We conclude that the amount of previous chemotherapy and age of patients appear to be among the determining factors on the number of myeloid progenitor cells in cancer patients and that thresholds for acceptability of grafts should take these variables into account.