Safety and quality assurance of chemotherapeutic preparations in a hospital production unit: acceptance sampling plan and economic impact.

OBJECTIVE: The opportunity to apply a sampling plan was evaluated. Costs were computed by a microcosting study. SETTING: In 2003, a sampling plan was defined to reduce the number of chemotherapy quality controls while preserving the same level of quality. Recent qualitative and quantitative changes led us to define a second sampling plan supplemented by an economic evaluation to determine the cost and cost-savings of quality control. METHODS: The study considers preparation produced during four semesters classified into three groups. The first one includes drugs produced below 200 batches a semester. Group 2, those for which the lot of preparation lots would have been rejected twice among these four semesters. Group 3, those would have been accepted (≥3 'acceptable lot'). A single sampling plan by attributes was applied to this group with an acceptance quality level of 1.65% and a lot tolerance percent defective below 5%. A micro-costing study was conducted on quality control, from the sampling to the validation of the results. RESULTS: Among 39 cytotoxic drugs, 11 were sampled which enabled to avoid a mean of 17,512 control assays per year. Each batch of the 28 non-sampled drugs was however analyzed. Costs were estimated at 2.98€ and 5.25€ for control assays depending of the analytical method. The savings from the application of the sampling plans was 153,207€ in 6 years. CONCLUSION: The sampling plan allowed maintaining constancy in number of controls and the level of quality with significant costsavings, despite a substantial increase in drugs to assay and in the number of preparations produced.

Assessment of CD4 T-lymphocyte reactivity by the Cylex ImmuKnow assay in patients following allogeneic hematopoietic SCT.

After allogeneic hematopoietic SCT (alloHSCT), immunosuppressed patients are susceptible to opportunistic infections, and uncontrolled function of the graft can result in GVHD. Accurate immune monitoring may help early detection and treatment of these severe complications. Between October 2005 and November 2007, a total of 170 blood samples were collected from 40 patients after alloHSCT in the Hadassah Hebrew University Medical Center and from 13 healthy controls. We utilized the Cylex ImmuKnow assay for CD4 ATP levels to compare known clinically immunocompromised vs immunocompetent patients after alloHSCT. We also compared the reconstitution of WBC count to the ImmuKnow results and clinical status. The patients' clinical course correlated with the stratification of immune response established by the ImmuKnow assay for solid organ transplantation (immunocompetent vs immunocompromised), and this often differed from their WBC count. On the basis of our observations, we conclude that the ImmuKnow assay is a simple and fast immune-monitoring technique for patients undergoing alloHSCT, with potential to predict clinical course and facilitate prompt management of post-HSCT complications. The assay should be evaluated prospectively in clinical trials.

GnRH-Bik/Bax/Bak chimeric proteins target and kill adenocarcinoma cells; the general use of pro-apoptotic proteins of the Bcl-2 family as novel killing components of targeting chimeric proteins.

In recent years chimeric proteins carrying bacterial toxins as their killing moiety, have been developed to selectively recognize and kill cell populations expressing speciific receptors. The involvement of Gonadotropin releasing hormone (GnRH) has been demonstrated in several adenocarcinomas and a GnRH-bacterial toxin chimeric protein (GnRH-PE66) was thus developed and found to specifically target and kill adenocarcinoma cells both in vitro and in vivo. Because of the immunogenicity and the non-specific toxicity of the bacterial toxins, we have developed new chimeric proteins, introducing apoptosis inducing proteins of the Bcl-2 family as novel killing components. Sequences encoding the human Bik, Bak or Bax proteins were fused to the GnRH coding sequence at the DNA level and were expressed in E. coli. GnRH-Bik, GnRH-Bak and GnRH-Bax new chimeric proteins efficiently and specifically inhibited the cell growth of adenocarcinoma cell lines and eventually led to cell death. All three Bcl2-proteins-based chimeric proteins seem to induce apoptosis within the target cells, without any additional cell death stimulus. Apoptosis-inducing-proteins of the Bcl-2 family targeted by the GnRH are novel potential therapeutic reagents for adenocarcinoma treatment in humans. This novel approach could be widely applied, using any molecule that binds a specific cell type, fused to an apoptosis-inducing protein.

The retention of primary hypoblastic cells underneath the developing primitive streak allows for their prolonged inductive influence.

An experimental study was made of the distribution of the primary hypoblastic cells in the lower layer of the avian blastoderm throughout primitive streak formation and until stage 10 (Hamburger & Hamilton, 1951). The primary hypoblast of stage XIII (Eyal-Giladi & Kochav, 1976) chick blastoderms was exchanged for either an [H3]thymidine-labelled similar chick hypoblast, or a quail primary hypoblast. During the entire period of primitive streak formation, the lower layer proved to be a mosaic of labelled hypoblastic and non-labelled entodermal cells (chick cells of epiblastic origin). The persistance of hypoblastic cells underneath the developing primitive streak is regarded by us as a possible way to prolong the inductive influence of the hypoblast upon the forming streak.

Interaction of epiblast and hypoblast in the formation of the primitive streak and the embryonic axis in chick, as revealed by hypoblast-rotation experiments.

Three types of experiments were performed to determine the interaction between the epiblast and hypoblast for primitive streak formation: (1) Hypoblasts of blastoderms from stages XIII E.G & K to 3 H & H were separated from the epiblasts and rotated by 90 degrees counterclockwise; (2) hypoblasts from stages XIII E.G & K to 3 H & H blastoderms were rotated by 180 degrees; (3) hypoblasts were exchanged between blastoderms of different developmental stages and placed at 90 degrees counterclockwise to the axis of the recipient epiblast. In all blastoderms studied only a single PS developed. After rotation of the hypoblast by 90 degrees, the direction of the PS was according to the orientation of the hypoblast at stage XIII, whereas at older stages it gradually shifted towards the axis of the epiblast. At stage 3- H & H the PS is already imprinted in the epiblast and cannot be shifted. After rotation of the hypoblast by 180 degrees the PS originated at the point near the marginal zone at which the inductive part of the hypoblast interacted with a component epiblast. Conclusions are drawn about the dynamics of the inductiveness of the hypoblast and the competence of the epiblast for the PS formation and orientation.

In vivo helper activity of enriched populations of antigen-specific T lymphocytes.

Enrichment of murine antigen-specific T cells was achieved by stimulation of primed lymph node cells with macrophages containing the immunizing antigen. After a week in culture, the in vitro-sensitized lymphocytes had an increased helper activity. Inoculation of 10(7) egg albumin (OVA)-enriched T cells together with hapten coupled to OVA, elevated the number of splenic antihapten-producing cells of primed or unprimed mice. Augmentation of the hapten specific B-cell response could be observed as early as 4 days following the injection of the enriched population and peaked at Day 7. The enhancing effect of the enriched population of antigen-specific T cells was carrier specific since it occurred only in the presence of hapten coupled to the T-cell sensitizer [2,4-dinitrophenol (DNP)-OVA]. When given with the hapten conjugated to an irrelevant carrier [DNP-human gamma globulin (HGG)], the enriched lymphocytes caused a depression in the anti-DNP response. The capacity of in vitro enriched lymphocytes to promote a substantial antigen-specific helper activity (up to 1 anti-DNP producing cell per 10(3) spleen cells) upon adoptive transfer to nonirradiated mice, provides an experimental system for studying B-T collaboration in vivo under the normal physiological conditions.

Marginal zone cells--the primitive streak-inducing component of the primary hypoblast in the chick.

(1) Removal of both the area opaca and the marginal zone of the area pellucida from a blastoderm stripped of its hypoblast (type-IV operation) prevents the regeneration of a normally functioning primary hypoblast. (2) Stage-XIII E.G. & K blastoderms (prior to the appearance of PS) after a type-IV operation do not form a primitive streak. (3) In order type-IV operated blastoderms in which the primitive streak has already begun to appear, the regeneration of a non-functional hypoblast did not support the normal maturation of the primitive streak, and in many cases the already existing rudimentary streak was absorbed. (4) Type-IV operated blastoderms from stage 3+ H + H and on developed normally. (5) It is concluded that the cellular contribution of the marginal zone to the primary hypoblast is instrumental in the latter's capacity to induce a PS.

In vivo activity of affinity-purified helper factor from antigen-specific helper clone.

Supernatant from culture of a virally transformed OVA-specific helper T clone (C-41) was examined for the presence of soluble helper factor. Inoculation of helper clone supernatant into DNP-KLH-primed mice enhanced the IgG anti-DNP response when given with DNP-OVA. The C-41 supernatant did not trigger the DNP-primed B cells in mice when injected with hapten (DNP) coupled to an unrelated carrier (BSA). The carrier-dependent helper activity of C-41 supernatant in vivo demonstrates the presence of an antigen-specific T helper factor in the media of the cultured helper clone. Extensive immunization of F1(C57BL X BALB/c) mice with the helper clone resulted in the production of anti C-41 antibodies. Monoclonal antibodies prepared from the immunized mice were screened for specificity of binding to other transformed T lines and clones, some specific to OVA. Monoclonal antibodies that stained the C-41 cells exclusively were considered clone-specific. Supernatants of the helper clone were passed over columns of anti-clone-specific antibodies. The eluates from three antibodies were active as antigen-specific helper factor, i.e., they elevated the IgG anti-DNP response in vivo in a linked recognition fashion in the presence of DNP-OVA. The affinity-purified factor was inactive when injected with DNP-BSA or DNP-BSA + OVA. Thus, we describe the antigen-specific immune function of a clone-produced helper factor in normal mice.

Immortalization of antigen specific, helper T cell lines by transformation with the radiation leukemia virus (RadLV).

Antigen-specific immune T lymphocytes of male C57BL/6 mice were enriched in vitro on monolayers of antigen-pulsed syngeneic macrophages. The cells were treated in vitro with RadLV and inoculated intrathymically into irradiated female C56BL/6 animals. Thymomas arising in the inoculated recipients were characterized as donor- (male) type according to their karyotype. In vivo and in vitro cell lines were established from the primary lymphomas, two of which (designated ROT/6.1 and ROT/6.2) were capable of providing antigen- (carrier) specific help in normal or preimmunized mice. None of the lymphomas could induce antigen-specific DTH reaction. Five months after their establishment, ROT/6.2 alone retained its carrier specificity. ROT/6.2 consisted mainly of Lyt-1+ cells, whereas the ROT/6.1 population was more heterogeneous and contained Lyt-1+, Lyt-2+, and Lyt-3+ cells. The carrier specificity of the latter may have been lost due to selection against the specific helper cells during prolonged passages.

Inactivation of interleukin-8 by the C5a-inactivating protease from serosal fluid.

The complement fragment C5a and the cytokine interleukin-8 (IL-8) are proinflammatory peptides with potent chemotactic activity toward neutrophils. We have previously shown that C5a can be inactivated by a protease that is found in normal synovial and peritoneal fluids but is absent from serosal fluids obtained from patients with familial Mediterranean fever (FMF). We report here that serosal fluids can also eliminate the chemotactic activity of IL-8. The agent responsible for IL-8 elimination appears to be the C5a-inactivating protease, because the pure protease can inactivate IL-8, inactivation of IL-8 by normal peritoneal fluid is partly prevented by an antibody raised against the purified C5a-inactivating protease, and IL-8 is not inactivated by peritoneal fluids from patients with FMF. The ability of this protease to inactivate both, early (C5a) and late (IL-8) inflammatory mediators identifies it as a potentially significant regulator of inflammation.

Effect of serum amyloid A on selected in vitro functions of isolated human neutrophils.

Serum amyloid A (SAA) is an acute phase reactant whose levels in the blood rise as part of the body's response to stress and inflammation. Previous studies have suggested that SAA may carry an anti-inflammatory potential. We evaluated the effects of SAA on human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. At concentrations higher than 10 microg/mL, SAA inhibited neutrophil myeloperoxidase (MPO) release. This effect was located in the N-terminal--that is, amino acid residues 1-14--of the SAA molecule. Directed neutrophil migration was inhibited at the same SAA concentrations. Several amino acid residues (1-14, 15-104, 83-104) contributed to this effect. Neutrophil O2- production was inhibited at low concentrations of SAA (0.1 to 1 microg/ml) and was stimulated at concentrations higher than 50 microg/mL. Neutrophil O2- production induced by phorbol myristate acetate (PMA) and O2- generated by the xanthine-xanthine oxidase reaction were not affected by SAA. These results add to previous data suggesting that SAA, at concentrations recorded in the serum during inflammation, modulates neutrophil function; thus it may play a role in the down-regulation of the inflammatory process.

Purification and characterization of a C5a-inactivating enzyme from human peritoneal fluid.

Earlier work has suggested that familial Mediterranean fever, an inherited disorder characterized by sporadic episodes of inflammation involving the pleural and peritoneal cavities and the joints, is caused by the lack of a C5a inactivator normally found in serosal fluid. We have purified this inactivator from ascites fluid and obtained a protein of molecular weight 53 to 56 kD with a specific activity 10,000-fold greater than the crude material. On Western blot, an inhibitory antibody recognized a single antigenic species at the same molecular weight. The enzyme had no activity against denatured bovine serum albumin. With recombinant C5a as substrate, the Km and Vm were 3.4 mumol/L and 52 nmol C5a/min/mg protein, respectively.

Expression of the familial Mediterranean fever gene and activity of the C5a inhibitor in human primary fibroblast cultures.

Familial Mediterranean fever (FMF) is an inherited disease whose manifestations are acute but reversible attacks of sterile inflammation affecting synovial and serosal spaces. The FMF gene (MEFV) was recently cloned, and it codes for a protein (pyrin/marenostrin) homologous to known nuclear factors. We previously reported the deficient activity of a C5a/interleukin (IL)-8 inhibitor, a physiologic regulator of inflammatory processes, in FMF serosal and synovial fluids. We now describe the concomitant expression of MEFV and C5a/IL-8-inhibitor activity in primary cultures of human fibroblasts. Fibroblasts grown from synovial and peritoneal tissues displayed C5a/IL-8-inhibitor activity that could be further induced with phorbol myristate acetate (PMA) and IL-1 beta. Very low levels of chemotactic inhibitor were evident in skin fibroblast cultures or in peritoneal and skin fibroblasts obtained from FMF patients. MEFV was expressed in peritoneal and skin fibroblasts at a lower level than in neutrophils and could be further induced by PMA and IL-1 beta. In the FMF cultures, the MEFV transcript carried the M694V mutation, consistent with the genetic defect found in patients with this disease. MEFV was also expressed in other cell lines that do not produce C5a/IL-8 inhibitor. These findings suggest that human primary fibroblast cultures express MEFV and produce C5a/IL-8-inhibitor activity. The interrelationship between pyrin, the MEFV product, and the C5a/IL-8 inhibitor requires further investigation. (Blood. 2000;96:727-731)