Cts Sibinga

Autologous stem cell transplantation in multiple myeloma after VAD and EDAP courses : a high incidence of oligoclonal serum Igs post transplantation

Thirty-seven patients with multiple myeloma (stage II and III, 65% increased β2-microglobulin level) were prospectively treated with a median of 3.7 VAD courses (range 2–8) followed by cyclophosphamide (6 g/m2) in conjunction with G-CSF (5 μg/kg filgrastrim (n = 14), or 3.5 μg/kg lenograstrim (n = 22)), and peripheral stem cell (PSC) isolation. After regeneration this was followed by one EDAP course and high-dose melphalan (HDM, 200 mg/m2) in combination with re-infusion of PSC. Adequate stem cell mobilization was obtained with both G-CSF regimens. A median of 41 × 106 CD34+ cells/kg (range 4.5–161) was collected in a median of 1.6 leukapheresis procedures following filgrastrim (n = 14) and 24 × 106 CD34+ cells/kg (range 2.3–80) in a median of 1.7 leukapheresis procedures following lenograstrim (n = 22) which indicated no significant difference (P = 0.24) between both G-CSF regimens. A rapid hematological recovery was obtained after HDM with reinfusion of a median of 9.3 × 106 CD34+ cells/kg. After the total courses the overall response was 84% with a complete remission rate of 30%. Currently the median overall survival is 44.0 months (95% CI 38.9–49.1) with a median follow-up of 33 months (range 3–51) and a median event-free survival of 29.0 months (95% CI 25.3–32.7) (n = 33). Post transplantation a high incidence of oligloclonal serum immunoglobulins (Igs) was observed. In 73% of the patients new oligoclonal or monoclonal serum bands were noticed 3 months post transplantation. IgG-λ and IgG-κ bands predominated. In 48% of the cases the oligoclonal Igs disappeared after a median follow-up of 22 months (range 8–36), whereas in 52% of the cases the oligoclonal Igs persisted with a median follow-up of 31 months (range 21–45), which did not correlate with a significant difference in overall, and event-free survival between both subgroups. Bone Marrow Transplantation (2000) 25 , 723–728.

Validation of the determination of Delta F508 mutations of the cystic fibrosis gene in over 11000 mouthwashes

Mouthwashes can be used as a DNA resource for mutation detection and, because collection and DNA isolation is simple and cheap, they could in particular, be used for large numbers of samples. To determine the failure rate (the proportion of mouth samples in which no PCR product was obtained) and the specificity of buccal epithelial cell mutation detection in large numbers of samples, we collected mouthwashes and blood samples from 11413 blood donors and tested the mouthwashes for the ΔF508 mutation, which has an estimated frequency of 75% among cystic fibrosis chromosomes in The Netherlands. Blood samples were tested for the ΔF508 mutations only if the mutation was identified in the mouthwash or in the case of a failure to obtain PCR products. The sensitivity of the test was determined in mouthwashes of 75 ΔF508 carriers known from earlier family studies. These samples were offered blindly between the mouthwashes of the blood donors. Both specificity and sensitivity of the mouthwash procedure were 100%. The overall failure rate was 5.6%. This large figure was caused mainly by insufficient rinsing of the mouth in one particular blood bank. Exclusion of the results of this blood bank reduced the failure rate to 1.8%. Our results also confirm that for a large number of samples the mouthwash procedure is suitable for mutation detection and, with proper instructions, can be used in community screening.

Prevalence of Delta F508 cystic fibrosis carriers in The Netherlands: Logistic regression on sex, age, region of residence and number of offspring

Abstract An average cystic fibrosis (CF) carrier frequency of 1 in 25 in Europe is cited in numerous reports, although a great variability in estimated prevalences has been found in different European populations. The estimates of these frequencies were based on numbers of CF patients before identification of the gene in 1989. Here we report the results of a study to determine the carrier frequency of the ΔF508 mutation in The Netherlands by analyzing mouthwashes and matched blood samples from 11 654 blood donors all over the country. We analyzed possible relationships between a number of theoretically explanatory variables and the ΔF508 carrier frequency by means of univariate and multivariate logistic regression. These variables were: distance of the blood banks from the northeastern part of the country (distance); whether the blood donors knew that we were looking for a CF mutation; sex and age of the donor; and number of children of the donor (family size). We detected a ΔF508 carrier frequency of 1 in 42 (95% CI 1/37–1/47) in The Netherlands. If we assume that the relative frequency of the ΔF508 mutation among carriers and patients is comparable in The Netherlands, this gives an estimated overall CF carrier frequency of 1 in 32 (95% CI 1/28–1/36), significantly less than 1 in 25. The univariate logistic regression analysis of the effects of the explanatory variables on the carrier frequency revealed no significant relationships, except for an increase in carrier frequency with increasing distance from the northeastern region. In the multivariate analysis with all five independent variables, distance, age and family size were significantly related to the carrier frequency, but sex and CF information were not. There was a significant interaction between age and family size. In our final model, distance, age and family size were positively related to the carrier frequency, while the interaction of age with family size showed a negative relation. These results confirm that there is a gradient in gene frequency with low frequencies in the northeastern part of the country and high frequencies in the southern part. They also suggest a relation of age and family size with carrier frequency. This relation, however, is too complex to be explained by heterozygote advantage.

Autologous cryopreserved platelets and prophylaxis of bleeding in autologous bone marrow transplantation

SummaryAutologous platelets were harvested and cryopreserved in eight consecutive patients elected for ablative chemotherapy and autologous bone marrow transplantation (ABMT) for solid malignancy. There was a 19% loss in platelet count after the freeze thaw and wash procedure; with an in vitro functional loss of 40–60%. No correlation could be found for individual platelet transfusions between in vitro functional tests and in vivo recovery. Six consecutive patients received a total of 16 autologous platelet transfusions in the aplastic phase of ABMT. No bleeding was observed during the study period and there was no CMV infection in the recipients. While improvement in freezing and subsequent handling is desirable, autologous cryopreserved platelets can safely be used for the prophylaxis of bleeding during aplasia in patients treated with ABMT.

Double cryoprecipitated factor VIII concentrate from heparinised plasma and its heat treatment

SummaryIn an attempt to implement the small pool concept in Factor VIII purification, cryoprecipitate derived from heparinised plasma was reprecipitated in the cold providing a factor VIII concentrate for freeze drying and heat treatment. There was considerable purification; only 1% of the original plasma proteins was left in the final product. Factor VIII: C concentration was about 19 IU/ml. Factor VIII related antigen (RAg) appeared heterogeneous, with a broad base and asymmetry on crossed immunoelectrophoresis. Fibrinogen content was 15 g/l. In contrast to high-purity commercial concentrates, fibronectin was considerably concentrated. Immunoglobulin contents were similar to a high-purity commercial product. The amount of other plasma proteins was very small, varying from less than 0.2% for C 3 complement to 2.3% ceruloplasmin. In some respects the preparation may be considered as an intermediate-purity Factor VIII concentrate. Following addition of 2% sucrose before freeze drying, Factor VIII, total protein and fibrinogen remain virtually stable (less than 15% loss) during heating of the material to 60, 64 or 68°C for 24 to 72 h without changes of protein spectrum following heating. The heated product when stored at 4°C remains stable for at least 3 months. In two severe haemophiliacs receiving this heat treated product, in vivo Factor VIII recovery was 100% with a mean half life of 10.2 h.

PLASMA-EXCHANGE IN 5 PATIENTS WITH ACUTE GUILLAIN-BARRE-SYNDROME

During the last 3 years plasma exchanges were undertaken in 5 patients with acute Guillain Barré syndrome (G.B.S.). All the patients were admitted in the intensive respiratory care unit and had received six plasma exchange procedures over two weeks (each procedure consists of 2–3 L exchange). The first patient improved dramatically after the second exchange. Moderate success was obtained in two patients. One patient did not show any effect. The fifth patient received plasma exchange one day after her recovery phase had begun but the course of recovery remained uneffected. The effect of plasma exchange was analysed as the patients’ response to motor activity, and compaired with a historical control group consisting of 50 acute G.B.S. patients admitted in the intensive respiratory care area over the last 25 years. Plasma exchange does not seem to have excerted any significant effect although at any given time the plasma exchange group had higher motor activity than that of the control group. A controlled clinical trial especially in the early phase of the disease is emphasized.

Comparison of apheresis and other methods for separation and purification of hemopoietic stem cells: initial experience with a blood buffy coat model for the use of autologous bone marrow transplantation.

With an increasing number of bone marrow transplantations (BMT) being contemplated in leukemia and cancer patients, it is prudent for blood banks to develop a suitable program within their resources for harvesting, purifying and freezing bone marrow stem cells. In order to do this, initially a prototype has been developed involving buffy coat model (BC) using normal donor blood. Centrifugation, sedimentation and machine apheresis methods were separately evaluated leading to a combined and sequential handling procedure. Blood was passed through a cell separator resulting collection of BC with 90% reduction of the volume showing 80% recovery of total leucocytes and 87% yield of mononuclear cells. Following centrifugation the cells with DMSO were frozen in a controlled freezing system and stored in liquid nitrogen. After thawing 94% cells were recovered with 93% viability. The initial experience gained in the model system could be incorporated in autologous BMT program in patients but requires modifications for improved results; the latter will be described separately.

Alloimmunization by Plasma Infusion

REFERENCES tact red cells were also found in thawed FFP. Although the amount was small, the use of FFP during plasmapheresis in pregnant Rhesus negative women has been suspected of having provided additional atlo-immunization stimuli (3). If Wolfowitz and Shechters (1) claim that red cells or their fragmented products in FFP could be immunogenic, then our results do support the recent plea for the avoidence of infusion of a large amount of Rh positive plasma to Rhesus negative females of child bearing age.