Clotting factors in cryoprecipitate and cryo-supernatant prepared from MB-treated fresh plasma.
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Blood Coagulation Factors↗
Biomedical subjects
S Bonanad
Publications and source records attributed to S Bonanad.
Influence of methylene blue photoinactivation treatment on coagulation factors from fresh frozen plasma, cryoprecipitates and cryosupernatants.
OBJECTIVE: To study the influence of virus photoinactivation with methylene blue (MB) on the coagulation factors of fresh frozen plasma (FFP) and the corresponding cryoprecipitates and cryosupernatants derived from it. MATERIALS AND METHODS: The photoinactivation procedure of the German Red Cross (Springe) was applied using Biomat (Grifols, Spain). Twenty isogroup pools of three plasma units were made from 60 U of FFP. The pools were split into three bags. One of them was photoinactivated, and pre- and postinactivation samples (MB-plasma) were obtained. The second bag was treated in the same way, followed by the preparation of MB-cryoprecipitate and MB-cryosupernatant. The third bag was not photoinactivated, and was processed in the same way to obtain control cryoprecipitate and cryosupernatant. The prothrombin time and activated partial thromboplastin time were analysed, as well as fibrinogen, factors (F) II, V, VII, VIII, IX, XI and XIII, antithrombin III, von Willebrand (vW) F:RCo, vWF:Ag and the multimeric structure of vWF. RESULTS: In plasma, the proteins most sensitive to photoinactivation were fibrinogen, FV, FVIII, FIX and FXI (24, 32, 28, 23 and 27% loss, respectively). In the MB-cryoprecipitate, the losses were higher for FVIII (23%), moderate for fibrinogen, FXIII and vWF:RCo (18, 14 and 13%, respectively) and minimal (only 3%) for vWF:Ag. In MB-cryosupernatants, the losses were higher for FV (26%) and moderate for fibrinogen (16%), FIX (18%) and FXI (19%), as well as for FII and FXIII (15%). The multimeric structure of vWF was not modified in MB-plasma or in MB-cryoprecipitates. The supernatants (both MB treated as well as controls) showed an absence of multimers of very high and high molecular weight. CONCLUSIONS: The quantitative and qualitative conservation of coagulation factors achieved in MB-plasma-derived products suggest that they are useful for the global replacement of coagulation factors and for deficiencies in FV and FXI. In countries lacking the economic resources to obtain virally inactivated concentrates, MB-cryoprecipitates could be useful in von Willebrand's disease and fibrinogen and FXIII deficiencies. MB-cryosupernatants could be employed in thrombotic thrombocytopenic purpura, in the correction of total or partial deficiencies of prothrombin complex factors and in specific deficiencies of FV and FXI.
Antiviral Agents↗
Quality analysis of blood components obtained by automated buffy-coat layer removal with a top & bottom system (Optipress (R)II).
BACKGROUND AND OBJECTIVE: There are Council of Europe recommendations for the quality of blood components. We analyzed the quality of blood components processed by a top & bottom system (Optipress((R)) II), the routine method used in our blood bank, to test whether the components reached the recommended quality. DESIGN AND METHODS: Blood was collected in triple CPD-SAGM bags (Optipac((R)) Baxter). Whole blood (WB) was centrifuged at 4,158 g for 14 min before separation by an automated top & bottom system (Optipress((R) )II). Platelet concentrate (PC) was prepared by pooling four isogroup buffy-coat (BC) units before low-speed centrifugation, and transferring the supernatant (4 BC-PC) to a 5-day storage bag (PL732, Baxter). An alternative approach involved PC preparation from a single BC unit by adding approximately 70 mL of plasma before centrifugation, followed by transfer of the platelet concentrate (1BC-PC) to a 300 mL Teruflex((R)) transfer bag. Both 4 BC-PC and 1 BC-PC were stored in a flat agitator at 22 degrees C for up to 5 days after collection. Cell counts were determined, along with hemoglobin and hematocrit in a Sysmex K-800 cell counter. The pH was determined on day 5 at 22 degrees C. Weights were measured and volumes were calculated based on specific gravity. Statistical analyses were carried out using the Kolmogorov-Smirnov test as a normality distribution test, the t-test for parametric values and Wilcoxon's test as a non-parametric test. Statistical significance between samples was considered to have been reached when p<0.05. RESULTS: The best parameters for configuring the system were: strength 25; BC volume 33-55; level of BC 5.5. Red blood cell (n = 1,434) volume was 279+/-20 mL, with 54.92+/-7.16 g of hemoglobin. More than 96% of units had fewer than 1.2x10(9) white blood cells. Fresh plasma volume (n = 803) averaged 279+/-19 mL, with a white blood cell contamination of fewer than 0.1x10(9)/L in all samples examined (n = 23). Platelet recovery in BC was 92+/-9% of platelets present in WB; the percentage of removed leukocytes was 74+/-10%, and between 13 and 15% of RBCs were lost in the BC (95% confidence interval). The BC volume (n = 1,037) fitted the target volume of 60 mL, except for some devices, when Optipress II((R)) lost the configuration for this parameter. Of 4 BC-PCs 80.3% yielded more than 0.6x10(11) platelets per unit, whereas this criterion was only met by 59.7% of 1 BC-PCs, and a greater proportion of 1 BC-PCs (58.8%) showed pH values within the range of 6.5-7.4 after 5 days of storage in comparison with 4 BC-PCs (44.25%). INTERPRETATION AND CONCLUSIONS: Optipress II((R)) provides standardized, leukocyte-poor blood components. Council of Europe requirements were met in a large percentage of red-cell concentrates, with less than 92 and 74% of the original platelets and leukocytes, respectively, and a small hemoglobin loss per unit. The system gave an optimal yield in terms of plasma volume. The top & bottom technique allowed us to reduce the number of blood units per platelet concentrate from 6 to 4 units, with similar platelet yields compared with traditional procedures. Nevertheless, the storage conditions must be improved to satisfy all Council of Europe requirements for platelet concentrates.
Blood Cell Count↗
A new method for phenotyping red blood cells using microplates.
BACKGROUND AND OBJECTIVES: The supply of phenotyped red blood cells (RBC) for patients with several RBC antibodies presents a difficult task to hospital blood banks and regional blood centers. The aim of this study was to establish a low-cost typing system to allow extensive phenotyping of regular blood donors for clinically significant RBC antigens. MATERIALS AND METHODS: We developed a new buffer that greatly intensifies the antigen-antibody reaction and thus reduces the quantity of serum needed for phenotyping. The procedure was carried out on microplates. RESULTS: A total of 20,435 regular blood donors have been typed to date. For 752 units required for transfusion, 3,584 phenotyping tests were performed, validating the results by tube or gel typing methods; agreement was achieved in all cases. CONCLUSION: This technique seems adequate for phenotyping a large number of RBC units at very low cost, thus facilitating the availability of phenotyped blood.
Antibodies↗
[Analysis of the quality of hemoderivatives obtained using a buffy-coat extraction system with a top-and-bottom technique (Optipress II)].
PURPOSE: The aim of the present study is to know the results of the quality analysis of blood components processed with a Top & Bottom system (Optipress II) as a routine method in our blood bank, and compare it with the CE recommendations for quality of blood components. MATERIAL AND METHODS: Blood was collected in triple CPD-SAGM bags (Optipac, Baxter) and whole blood (WB) were centrifuged at 4,158 g, 14 min. Blood separation was performed by an automated Top & Bottom system (Optipress II), in which parameters were individually configured in preliminary trials. The buffy-coat (BC) layer was maintained within the configured levels during the separation process and remained into the original bag, whereas red cells (RBC) were collected into the bottom satellite bag (with 100 mL of SAGM) and fresh plasma (FP) was sent to the top satellite bag. Platelet concentrate (PC) was prepared by two different ways: 4 isogroup buffy-coats units were pooled by means of a sterile connector device (TSCD-201, Terumo) before a low centrifugation (1,040 g, 9 min) and the supernatant (4BC-PC) was transferred into a PL732 bag (Fenwal, Baxter); the other PC was prepared from one unit of BC by additioning approximately 70 mL of FP before centrifugation (321 g, 6 min) and following transference of the platelet concentrate (1BC-CP) into a 300 mL (Teruflex, Terumo) transfer bag. Both, 4BC-PC and 1BC-PC, were stored in a flat agitator at 22 degrees C to up five days after collection. We determined cell counts, haemoglobin, and hematocrit in a Sysmex K-800 cell counter in WB and blood components. Nageotte chamber was used when low white blood cells (WBC) counts were obtained. We also determined pH values on day five at 22 degrees C in a Crison 2000. Weights were measured and volumes were calculated using specificity gravity. Statistical analysis were carried out by Kolmogorov-Smirnov test as a normality distribution test, t-test for parametrical values and Wilcoxon-test as a no parametrical test (p < 0.05 was considered as Wilcoxon a significant value between different samples). RESULTS: The best parameters to configure the system were: strength: 25; BC volume: 33-35; level of BC: 5.5. RBCs (n: 1434) volume was 279 +/- 20 mL with 54.92 +/- 7.16 g of haemoglobin. More than 96% units had less than 1.2 x 10(9) WBC. FP volume (n: 803) averaged 279 +/- 19 mL with a WBC contamination less than 0.1 x 10(9)/L in all examined samples (n: 23). Platelet recovery in BC 92 +/- 9 percent of platelets present in WB, the percentage of removed leukocytes was 74 +/- 10 and between 13 and 15% of RBCs were lost in the BC (CI 95%). The BC volume (n: 1037) fitted the target volume of 60 mL (59-61 mL, CI 95%) except in some devices, where Optipress II lost the configuration for this parameter. 4BC-CPs (n: 325) showed a platelet yield per unit greater than 1BC-CPs (226). In addition, 80.3% of 4BC-CPs yielded more than 0.6 x 10(11) platelets per unit, whereas this criteria was only met in 59.7% of 1BC-CPs (p < 0.001). The ratio volume oper 10(9) platelets in 1 BC-CPs was significantly higher (1.57 mL) than 4BC-CPs (1.31 mL), and a greater level of 1BC-CPs (58.8%) showed pH values within 6.5-7.4 after 5 days of storage in comparison with 4BC-CPs (44.25%) (p < 0.001). CONCLUSIONS: Optipress II provides standardized and poor leukocytes blood components. CE requirements were met in a great percentage of red-cell concentrates with less than 92 and 74 percent of original platelets and leukocytes, respectively and a low loss of haemoglobin per unit. Plasma volume obtained with this system represents an optimal yield. Top and Bottom technique allowed us to reduce the number of blood units per platelet concentrate, from six to four units with similar platelet yield compared to traditional procedures. Nevertheless, we must improve the storage conditions, in orter to satisfy all the CE requirements for platelet concentrates.
Adult↗
Prolonged molecular remission after PML/RAR alpha-positive autologous peripheral blood stem cell transplantation in acute promyelocytic leukemia: is relevant pretransplant minimal residual disease in the graft?
The contribution of residual malignant cells contaminating the autologous graft with the occurrence of post-transplant relapse in acute myeloid leukemia (AML) is still unclear. The presence of a specific molecular marker (the PML/RAR alpha rearrangement) in acute promyelocytic leukemia (APL) offers the opportunity to investigate better the pathogenesis of disease recurrence after transplant. We report an APL patient who received high-dose chemotherapy and peripheral blood stem cell (PBSC) autograft in second hematologic remission. Two leukaphereses that tested PML/RAR alpha positive by RT-PCR were obtained during the post-reinduction hematopoietic recovery, while the patient also tested PCR positive in the BM, and was reinfused after myeloablative chemotherapy (BUCY4), when the patient had spontaneously converted to PCR negative in the marrow. At present, he remains in continuous molecular and hematologic remission 22 months after PBSC transplantation. This is the second report of an APL patient who was transplanted in molecular remission with a PML/RAR alpha-positive PBSC autograft. As in the previous report, the prolonged clinical and molecular remission experienced post-transplant suggests that autologous PBSC infusion is still worthy of consideration for patients with APL in spite of the detection of PML/RAR alpha-positive cells in the PBSC collections. Possible underlying mechanisms and the potential role of molecular monitoring of the graft, as well as the host, before and after transplant, in patients with APL undergoing autologous HSCT are also discussed.
Adult↗
Comparison of two reverse transcription-polymerase chain reaction methods for detection of AML1/ETO rearrangement in the M2 subtype of acute myeloid leukaemia.
Two reverse transcription-polymerase chain reaction methods to detect the AML1/ETO rearrangement in the M2 subtype of acute myeloid leukaemia those of Downing et al. (Blood 1993; 81: 2860-5) and Satake et al. (Br J Haematol 1995; 91: 892-8) were evaluated. Bone marrow samples, one at diagnosis and two in complete remission from a patient with M2 subtype of acute myeloid leukaemia, with t(8;21), were analysed using both methods. The Kasumi-1 cell line was used as a positive control and a patient with M3 subtype of acute myeloid leukaemia as a negative control. To confirm the feasibility of Satake's method a group of 35 patients with subtypes of acute myeloid leukaemia at diagnosis were studied. The method of Downing requires Southern blotting and hybridization with a specific probe because it often generates non-specific amplification products. By contrast, the method of Satake yields only a single amplification product, using one single round of PCR in samples at diagnosis, or two rounds in complete remission samples. The sensitivity of this method allows the detection of a single Kasumi-1 cell in 10(6) normal cells. The AML1/ETO rearrangement was observed in 5 of the 35 cases of acute myeloid leukaemia at diagnosis (14.3%) and in 3 of the 14 cases of M2 subtype of acute myeloid leukaemia (21.4%). The two remaining positive cases corresponded to the acute myeloid leukaemia subtypes M4 and M6. The results indicate that the method of Satake better meets the requirements of the clinical laboratory due to its greater simplicity, specificity, sensitivity and feasibility, thus making it more appropriate for use in diagnosing and monitoring minimal residual disease.
Base Sequence↗
HLA-DQA, -DQB and -DRB allele contribution to narcolepsy susceptibility.
The association of narcolepsy with HLA class I antigens and HLA class II alleles was studies in a series of Spanish narcoleptic patients. The haplotype DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 was found to be significantly associated with the disease, while the haplotype DRB1*0701-DRB4*01-DQA1*0201-DQB1*02 might confer a slight protective effect against narcolepsy. Gene dose-effect was not seen in any of the involved alleles, and linkage disequilibrium between the positively associated alleles was found to be stronger in patients than in controls. Statistical analysis applied to identify the HLA allele truly responsible for the association did not clearly discriminate between the contribution of DRB1*1501 and that of DQB1*0602, but it proved that the association with DQA1*0102 is secondary to that with DRB1*1501/DQB1*0602. Analysis of the diagnostic value of typing for the narcolepsy-associated alleles demonstrated a very high negative predictive value and revealed that this test can be convenient for exclusion of narcolepsy in cases when the diagnosis is not evident after clinical evaluation and the marker haplotype is absent. Finally, a family study indicated that narcolepsy is a multifactorial disorder that involves HLA genes under an incomplete penetrance model, with possible influences from environmental factors or other genes different to HLA genes.
Adolescent↗
Cytokines and platelet activation in stored pooled buffy-coat-derived platelet concentrates: the issue of transfusional reactions.
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Cytokines↗
[Detection of the PML/RAR alpha rearrangement in acute promyelocytic leukemia using a reverse PCR method].
AIMS: This study describes a molecular method of reverse transcription polymerase chain reaction (RT-PCR) to detect the rearrangement PML/RAR alpha in acute promyelocytic leukaemia (APL) in order to assess its specificity and sensibility, and to evaluate its utility in the characterization of APL patients. PATIENTS AND METHODS: Between January and June of 1995, 64 samples of bone marrow and peripheral blood stem cells (PBSC) cytapheresis were studied. There were 58 APL samples (23 patients: 10 samples obtained with disease activity, 43 samples in complete remission (CR) and 5 PBSC samples) and 6 control samples, of non-APL hematological neoplasms (3 other AML, 1 CML, 1 ALL, and 1 MDS). On the RNA obtained from the isolated mononuclear cells of each sample a conserved region of the PML/RAR alpha fusion gene was amplified by using a RT-PCR with specific primers. RESULTS: The sensitivity assays were performed by diluting PML/RAR alpha positive RNA samples into RNA of controls. The RT-PCR assay was capable to detect the PML/RAR alpha until an 1/1000 dilution in negative control RNA. Nine out of 58 APL samples failed in the amplification of the control gene, and were considered non-evaluable. None of the 6 control samples showed PML/RAR alpha rearrangement. Nine out of 10 APL samples with disease activity were positive for the presence of PML/RAR alpha (the non-positive sample was a non-evaluable one). Six out of 43 APL samples in CR showed the rearrangement, 3 of them corresponding to 2 patients who posteriory relapsed 12 and 19 months after 1st CR. The other 3 positive samples came from other 3 APL patients (24 months in 3rd CR, 14 months in 1st CR and early CR), who remained in CR at the end of the study. No relapse could be noted in patients with negative PCR samples. PML/RAR alpha was not found in any of the 5 APL PBSC samples studied. CONCLUSIONS: The RT-PCR method described here seems to be highly specific as it only detects this rearrangement in LPA patients. Furthermore, the presence of PML/RAR alpha in CR patients could be related to relapse. For all these reasons, this molecular method shows great usefulness and can be advocated, not only for assessing diagnosis, but for as monitoring minimal residual disease in the post remission follow up.
Bone Marrow↗
Autologous bone marrow transplantation for patients with acute myeloblastic leukemia in relapse after autologous blood stem cell transplantation.
Leukemic relapse remains the most frequent reason for treatment failure in patients with acute myeloblastic leukemia (AML) treated with autologous blood stem cell transplantation (ABSCT). The aim of this study was to evaluate the possible role of autologous bone marrow transplant (ABMT) in patients with AML who relapse after ABSCT. Eighteen consecutive patients were enrolled in the study. At ABMT, 17 patients were in untreated relapse and one was in third complete remission (CR). The preparative regimen was BAVC, and consisted of BCNU 800 mg/m2 on day -6, M-AMSA 150 mg/m2/day on days -5 to -3, VP-16 150 mg/m2/day on days -5 to -3 and Ara-C 300 mg/m2/day on days -5 to -3. There were two regimen-related deaths (11%). Thirteen out of 17 patients in untreated relapse before ABMT achieved CR (76%). The cumulative risk of relapse was 58 +/- 13% at 3 years. Seven patients are in CR between 7+ and 53+ months, with a disease-free survival (DFS) probability of 36 +/- 12% at 3 years. The probability of DFS after ABMT was clearly higher in those patients relapsing later than 7 months after the first autograft (52%) than in patients relapsing earlier (20%)(P = 0.02). In a significant proportion of patients, remission duration was clearly longer after ABMT than ABSCT. We conclude that BAVC conditioning followed by ABMT is associated with a low treatment-related toxicity and results in prolonged DFS in a substantial number of AML patients who relapse after ABSCT. Until better therapeutic options become available, ABMT in untreated relapse is a useful alternative in this group of very poor-risk patients.
Acute Disease↗
[Levels of cytokeratin CK19 expression in mononuclear blood cells evaluated using a reverse PCR (RT-PCR)].
AIMS: The sensitivity and specificity of a reverse transcription PCR method (RT-PCR) to detect cytokeratin K19 (CK19) expression was evaluated with the purpose of assessing its capability to detect the presence of breast cancer tumour cells in peripheral blood progenitor cell collection that had to be reinfused to breast cancer patients submitted to intensive chemotherapy as haematopoietic support. PATIENTS AND METHODS: Two breast cancers as positive samples were used and 34 samples of mononucleated blood cells as negative controls: 18 peripheral blood samples from normal subjects, 14 from different types of leukaemias (M3, M4Eo, M2, etc.) and two from two patients with Hodgkin's lymphoma. The method studied is a nested RT-PCR that amplifies the CK19 expression from the sample RNA extracted following the method of phenol-chloroform. RESULTS: The right performance of the method is demonstrated by observing the detection of CK19 transcripts in the breast cancer RNA and by obtaining good blank results both with non transcribed RNA and with DNA. Moreover, the method has an excellent sensitivity such as to allow the detection of CK19 transcripts in a 10(-6) dilution of cDNA reverse transcribed from 1 microgram of breast cancer RNA. The CK19 transcripts were also detected in the 64% of RNA obtained from the mononucleated blood cells controls, although the percentage of positivities was lower (47%) in the RNA from peripheral blood samples. Nevertheless it should be remarked that the levels of CK19 expression in the blood mononucleated cells is almost negligible since it used to extinguish at 1:5 cDNA dilution. CONCLUSIONS: The method studied is specific and has a high sensitivity that explains the detection of CK19 illegitimate expression approximately a half in mononucleated blood cells negative controls. However, the levels of CK19 expression in mononucleated blood cells were almost negligible and it used to extinguish at 1:5 cDNA dilution, therefore it could be concluded that the method might be useful to detect breast cancer occult tumours cells in mononucleated blood cell collection, always provided that a lower amount of cDNA is taken, thus decreasing to nil almost the false positive samples and keeping always a good sensitivity.
Biomarkers, Tumor↗
[Immunohematologic study in 112 patients with myelodysplastic syndromes: 10-year analysis].
PURPOSE: To describe the immunohematological response after multiple red blood cells transfusions in 112 patients with myelodysplastic syndromes (MDS). PATIENTS AND METHODS: From 1982 to 1992 every patient with MDS entered the study. Immunohematological studies consisted of an antibody screening test (AST) and a direct antiglobulin test (DAT) at diagnosis and after every four transfused units of red blood cell concentrates. RESULTS: Twenty-three out of 112 patients (20.5%) presented a positive AST and/or DAT during the study period. Eleven out of the 23 patients (9.8%) developed a positive DAT, six of them with red cell alloantibodies associated. In the remaining 12 patients (10.7%) the immune response observed was the development of red cell alloantibodies without a positive DAT. No patient presented with positive AST or DAT at diagnosis and no differences were observed in the pattern of immunohematological response and the FAB subtype. CONCLUSION: Patients with MDS have disordered immune systems with a high prevalence of autoantibodies against red cells. However, the incidence of red cell alloimmunization is similar to that observed in other multitransfused populations. Finally, despite the high immunization rate, the absence of any hemolytical reaction make the AST and DAT a safe and effective method of pretransfusion testing in this heavily transfused population.
Adolescent↗
Rapid progression of multiple myeloma following G-CSF mobilization.
A 23-year-old man diagnosed as having multiple myeloma was treated with melphalan and prednisone monthly. After six cycles, an autologous peripheral blood stem cell transplantation (ABSCT) was planned. Peripheral blood mononuclear cells were collected after G-CSF mobilization (5 micrograms/kg/day for 5 days). Ten days after the last dose of G-CSF the patient showed a striking progression of multiple myeloma. A 57% infiltration of plasma cells in bone marrow and recurrence of laboratory abnormalities were evident. The patient's clinical course strongly suggests that myeloma progression was triggered by G-CSF and supports the concept of G-CSF mediated autocrine stimulation of myeloma growth.
Adult↗
Carboplatin and etoposide in acute myeloid leukemia.
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Adult↗
[Transfusion policy toward a patient with an anti-Cartwright antibody (anti-Yta)].
We have observed one patient with hepatic cirrhosis showing on her antibody screening an anti-Yta. We studied the 51 Cr-labelled erythrocyte survival using either Yt (a+) and autologous blood red cells. She was transfused two Yt (a+) red blood cell concentrates without any adverse reaction.
Aged↗