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S F Leitman

Publications and source records attributed to S F Leitman.

69 records · Page 4Linked to original sources
PubMed1988

A simplified automated procedure for generation of human lymphokine-activated killer cells for use in clinical trials.

The administration of lymphokine-activated killer (LAK) cells along with interleukin-2 (IL-2) can mediate regression of tumors in selected patients. A closed automated system utilizing commercial blood cell separators has been developed for washing and Ficoll-Hypaque (FH) separation of lymphocytes, for lymphocyte culture in polyolefin bags, and for concentration of LAK cells out of culture prior to infusion. We now demonstrate that preparation of LAK cells can be simplified by elimination of the FH sedimentation step. Patient leukapheresis was performed using Fenwal CS-3000 blood cell separators, with a mean cellular yield per procedure of 54 X 10(9) WBC (95% lymphocytes), 184 X 10(9) RBC, and 306 X 10(9) platelets (n = 22). These cells were then washed in the same apheresis kit with a counter-current flow of saline, thereby eliminating 85% of platelets while retaining 88% of WBC. Aliquots of the washed cells were separated on FH gradients in 50 ml centrifuge tubes, and both FH-separated and washed-only cells were cultured at 3 X 10(6)/ml with 1500 U/ml IL-2 in polyolefin bags. Cytotoxicities of 22 preparations of LAK cells from 14 patients were evaluated in 4 h 51Cr release assays. Cells that were washed-only averaged 47, 35, and 9 lytic units/10(6) cells against K562, Daudi, and fresh tumor, while FH separated cells averaged 46, 33, and 6 lytic units/10(6) cells respectively. Cellular recoveries using the wash-only technique were 25% greater than when using FH sedimentation. Omission of FH separation saves time and expense in preparation and provides greater numbers of LAK cells for use in adoptive immunotherapy.

Autoanalysis↗
PubMed1988

Technical aspects of lymphokine-activated killer cell production.

Adoptive immunotherapy is a novel approach to treating patients with cancer, utilizing as therapy a patient's own peripheral blood lymphocytes that have been activated by incubation with interleukin-2 (IL-2). These cells develop the ability to mediate tumor regression in vivo and are referred to as lymphokine-activated killer (LAK) cells. The production of LAK cells is a complex and labor-intensive process. Lymphocytes are collected by continuous-flow centrifugation, purified on Ficoll-Hypaque (FH) density gradients, incubated in vitro with IL-2, and then harvested for infusion into the patient. An automated approach to LAK cell generation has been developed using the Fenwal CS-3000 cell separator and polyolefin PL-732 blood storage bags. Lymphocyte concentrates (LC) containing 6.5 x 10(9) mononuclear cells per pack were obtained using standard leukapheresis techniques. Disposable apheresis kits were then modified to allow the LC to be pumped into the separation chamber along with a counter-centrifugal flow of saline, removing the platelets and plasma by elutriation. The remaining cells were underlaid with FH, displacing the lymphocytes into a collection bag, where they were washed and concentrated. Mean leukocyte recovery was 59.2% (99.9% lymphocytes, n = 14). The final product contained 6.7% of the initial platelets and had a hematocrit of less than 1%.(ABSTRACT TRUNCATED AT 250 WORDS)

Automation↗
PubMed1987

Development of an automated closed system for generation of human lymphokine-activated killer (LAK) cells for use in adoptive immunotherapy.

Immunotherapy utilizing the adoptive transfer of lymphokine-activated killer (LAK) cells in conjunction with recombinant interleukin-2 (IL-2) can mediate tumor regression in some patients with advanced cancer. The activation of large numbers of LAK cells was performed in roller bottles in a research laboratory setting and required meticulous aseptic technique, at least one skilled technician per patient and one laminar flow hood per patient. To reduce the complexity and expense of LAK cell generation for human immunotherapy trials we have developed a closed-system automated procedure using a continuous flow blood cell separator. PBL were obtained by standard apheresis techniques. Platelets and plasma were elutriated using countercentrifugal flow of saline in the cell separator machine. The washed PBL were underlaid with Ficoll-Hypaque (FH) in the original separation bag. Lymphocytes were then flushed into a collection bag where they were concentrated and washed with 2 liters of saline. Mean recovery from the automated FH technique was 54.6 +/- 4.3% compared to 62.3 +/- 4.0% using manual methods in 50 ml tubes (P greater than 0.05). Cells were diluted in the collection bag with RPMI 1640 +/- 2% human AB serum and could be dispensed in an automated fashion to polyolefin bags via a sample port with 1000-1500 U/ml IL-2. After 3-4 days of culture in 5% CO2 at 37 degrees C, activated cells from the bags were harvested and washed in a closed system using the continuous flow cell separator. Cell yield from the harvest was 79.2 +/- 5.4% in the automated system compared to 64.9 +/- 5.0% in the standard procedure using manual harvest of roller bottles (P less than 0.01). Lytic capacity of the cells against fresh human tumor in a 4 h 51Cr release assay was equivalent in cells processed either by the automated or the conventional manual method. The advantages of a closed system include decreased potential for microbial contamination and reduced labor and capital equipment costs. This technique may be easily adapted for use with other cell collection and culture systems.

Cell Separation↗
PubMed1986

Allergic reactions in healthy plateletpheresis donors caused by sensitization to ethylene oxide gas.

We observed immediate-type hypersensitivity reactions in 6 of 600 donors who underwent automated plateletpheresis procedures. Ethylene oxide gas, which was used to sterilize plastic components in the disposable apheresis kits, represented a possible source of sensitization. In skin-prick testing, 4 of the 6 donors who had had a hypersensitivity reaction and none of 40 controls who had not had such a reaction had positive tests when an ethylene oxide-human serum albumin reagent was used (P less than 0.001). Radioallergosorbent testing revealed that serum from 4 of the 6 donors who had reactions, but from only 1 of 145 controls, contained IgE antibodies to ethylene oxide-albumin (P less than 0.001). All six of the donors who had reactions had specific ethylene oxide-induced basophil histamine release (mean release [+/- SE], 34.2 +/- 5.6 percent), whereas none of four controls had specific histamine release in response to ethylene oxide (mean release, 5.7 +/- 1.2 percent; P less than 0.005). Repeat plateletpheresis in five of the donors who reacted was accompanied by a recurrence of mild allergic symptoms. These studies demonstrate that sensitization to ethylene oxide gas can occur in healthy plateletpheresis donors and that it may result in immediate hypersensitivity reactions during the donation. The prevalence of such reactions was 1.0 percent in our apheresis donor population.

Blood Component Removal↗
PubMed1983

Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody.

Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. To gain further insight into the role of fibrinogen in platelet aggregation and to identify the platelet fibrinogen receptor, we developed a monoclonal anti-platelet antibody that inhibited platelet aggregation. The purified antibody, designated A2A9, inhibited platelet aggregation stimulated by 10 microM ADP, 10 microM epinephrine, and thrombin at 1 unit/ml without inhibiting platelet shape change or platelet secretion. A2A9 was also a competitive inhibitor of fibrinogen binding to ADP-stimulated platelets. Fifty percent inhibition of fibrinogen binding occurred at 65 nM A2A9. Direct binding studies using radiolabeled A2A9 demonstrated 47,000 A2A9 binding sites on unstimulated platelets, with a dissociation constant of 60 nM. Platelets from two individuals with Glanzmann thrombasthenia bound essentially no A2A9. Therefore, these data support the hypothesis that receptor-bound fibrinogen mediates platelet aggregation. In order to identify the platelet fibrinogen receptor, A2A9 immobilized on agarose was used for affinity chromatography. Two platelet polypeptides with Mr = 140,000 and 93,000 were recovered from the immobilized A2A9. After disulfide reduction, these Mr values were altered to 125,000 and 116,000. The smaller polypeptide was also found to contain the PlA1 antigen. These data localize the epitope recognized by A2A9 to the platelet membrane glycoprotein IIb-IIIa complex and suggest that this complex forms the physiologic platelet fibrinogen receptor.

Adenosine Diphosphate↗
PubMedDate not supplied

Use of a continuous-flow cell separator in density gradient isolation of lymphocytes.

A continuous-flow technique was developed to isolate and concentrate lymphocytes over Ficoll-Hypaque (FH) density gradients using an automated cell separator (Model CS-3000, Fenwal). Lymphocyte concentrates (LC) containing 6.51 X 10(9) mononuclear cells were obtained by standard leukapheresis techniques. Disposable apheresis kits were modified to allow the LC to be pumped into a separation chamber along with a counter-centrifugal flow of saline, removing the platelets and plasma by elutriation. The remaining cells were underlaid with 300 ml of FH, displacing the lymphocytes into a collection bag, where they were washed and concentrated. Mean leukocyte recovery was 59.2 percent (99.9% lymphocytes, n = 14). The final product contained 6.7 percent of the initial platelets and had a hematocrit of less than 1 percent. In paired studies using split leukocyte concentrates (n = 15), lymphocyte recovery obtained by the automated apheresis technique compared favorably with that obtained by standard manual FH gradients (59.8 +/- 3.4% versus 67.3 +/- 4.2%, p greater than 0.05) and platelet contamination was significantly reduced (2.7 +/- 0.5% versus 26.6 +/- 5.7% residual platelets, p less than 0.001). Equivalent lymphokine-activated killer (LAK) activity was generated from cells isolated by both manual and automated techniques. An automated continuous-flow cell separator can be used for rapid FH isolation of large numbers of lymphocytes, providing a sterile product suitable for human use.

Cell Separation↗
PubMedDate not supplied

Viability of platelets following storage in the irradiated state. A pair-controlled study.

Gamma irradiation of blood products is a standard practice recommended for the prevention of posttransfusion graft-versus-host disease in susceptible hosts. We studied the effects of irradiation on stored platelet concentrates and evaluated whether platelets could be stored for 5 days in the irradiated state without adverse effects on their viability. Using a pair-controlled design in which each of six normal subjects acted as his or her own control, we compared in vitro storage characteristics and in vivo kinetics of platelet concentrates exposed to 30 Gy and stored for 5 days with those of platelet concentrates simply stored for 5 days without irradiation. Irradiation had no significant effects on in vitro storage characteristics (platelet count, mean platelet volume, pH, and white cell count) or on in vivo kinetics, including initial recovery and mean platelet survival. Using the multiple-hit model, initial recovery was 49.6 +/- 10.8 percent, and mean platelet survival was 5.6 +/- 1.05 days for irradiated concentrates, compared with 51.3 +/- 13.0 percent and 5.9 +/- 0.50 days, respectively, for the unirradiated control concentrates. We conclude that irradiation of platelet concentrates with up to 30 Gy has no effect on their in vivo recovery or survival, and that irradiation administered before storage of platelet concentrates does not interfere with their clinical efficacy.

Blood Platelets↗
PubMedDate not supplied

Efficacy of various methods of confidential unit exclusion in identifying potentially infectious blood donations.

To determine the efficacy of various methods of confidential unit exclusion (CUE) among donors at increased risk of HIV exposure, we surveyed AABB institutional members on their experience with 3 CUE methods: ballot or barcode, completed at the time of donation, and call-back, performed by the donor after leaving the donor center. From June 1985 to December 1987, 5,049,883 donations at 48 donor centers were evaluable for analysis. The results of this survey suggest that ballot and barcode methods of CUE are important adjuncts to other donor screening procedures in identifying potentially infectious units, and that both of these methods are superior to the call-back system of unit exclusion.

Blood Banking↗
PubMedDate not supplied

Detection of cytomegalovirus antibody in platelet concentrates by fluorescence immunoassay and latex agglutination.

A passive latex agglutination (PLA) test for cytomegalovirus (CMV) antibody detection has been shown to be an acceptable method of screening both donor sera and plasma from units of red cells and platelets stored in CPDA-1. However, most plateletpheresis concentrates are collected in ACD, and CMV antibody testing of ACD-stored products has not been systematically evaluated by PLA. Sera and ACD-stored platelet concentrate bag segments from 104 donors were tested by PLA and by a solid-phase fluorescence immunoassay (FIAX) as a reference standard for CMV-IgM and CMV-IgG antibodies. Sera were stored at both 4 and 22 degrees C and were tested on Days 1 and 5 of storage; segments were tested daily for 5 days. Of 63 donor samples (61%) that tested negative for CMV-IgG by FIAX, there were two false-positive results in bag segments by PLA testing, one on Day 1 and the other on Day 2 of storage. PLA testing was consistently positive in sera and segments in the 40 donors (38%) who tested positive for CMV-IgG by FIAX. Potential false-negative PLA results occurred in five bag segments derived from one donor whose serum gave equivocal CMV-IgG results on FIAX. The sensitivity and specificity of the PLA assay were 100 percent for donor sera tested at both 4 and 22 degrees C and 91.5 and 98.4 percent, respectively, for platelet bag segment tests. Although no donors positive for CMV-IgM were identified, 15 (14.4%) had equivocal IgM anti-CMV test results.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Viral↗
PubMedDate not supplied

The effect of prestorage irradiation on posttransfusion red cell survival.

Transfusion-associated graft-versus-host disease (TA-GVHD) may occur whenever immunologically competent allogeneic lymphocytes are transfused to an immunocompromised recipient. Irradiation of blood components eliminates the risk of TA-GVHD but may damage the cellular elements in the transfused component, particularly if the cells are stored for prolonged periods in the irradiated state. To study the effect of irradiation on long-term storage of red cells, AS-1 red cells from eight normal subjects were prepared on two occasions. On one occasion, the units were stored as standard AS-1 red cells for 42 days at 4 degrees C; on the other, they were exposed to 3000 cGy radiation within 4 hours of collection and then were stored as AS-1 red cells for 42 days at 4 degrees C. The donations were at least 12 weeks apart. Irradiated units demonstrated significant elevations in poststorage plasma hemoglobin (Hb) (623 +/- 206 vs. 429 +/- 194 g/dL [6230 +/- 2060 vs. 4290 +/- 1940 g/L], p less than 0.02) and plasma potassium (78 +/- 4 vs. 43 +/- 9 mEq/L [78 +/- 4 vs. 43 +/- 9 mmol/L], p less than 0.01) and significant decreases in red cell ATP (1.9 +/- 0.2 vs. 2.1 +/- 0.3 microM/g Hb, p less than 0.04) and 24-hour posttransfusion red cell recovery (68.5 vs. 78.4%, p less than 0.02), as compared to nonirradiated units. It can be concluded that irradiation with 3000 cGy damages red cells and that long-term storage in the irradiated state may enhance this damage. Red cells should not be stored for 42 days after irradiation with 3000 cGy.

Blood Preservation↗
PubMedDate not supplied

Alloimmunization after granulocyte transfusions.

BACKGROUND: Although granulocyte transfusions are recommended for neutropenic patients with bacterial infections that are unresponsive to antibiotic therapy, the presence of white cell (WBC) antibodies in the recipient can render these transfusions ineffective. STUDY DESIGN AND METHODS: A 25-year-old man with chronic granulomatous disease experienced a pulmonary transfusion reaction while receiving granulocyte transfusions, and he was found to be immunized to neutrophil antigen NA2. A retrospective study of alloimmunization to HLA and neutrophil antigens in 18 patients with chronic granulomatous disease who had also received repeated granulocyte transfusions was then performed. Sera were tested in lymphocytotoxicity, granulocyte agglutination, granulocyte immunofluorescence, monoclonal antibody immonobilization of granulocyte antigen, and immunoprecipitation assays. RESULTS: After the granulocyte transfusions, sera from 14 of the 18 patients contained WBC antibodies. Seven sera samples reacted in the lymphocytotoxicity, granulocyte immunofluorescence, and granulocyte agglutination assays; seven reacted in the lymphocytotoxicity and granulocyte immunofluorescence assays but not the granulocyte agglutination assay, and four did not react. When the monoclonal antibody immobilization of granulocyte antigen assay was used, three sera samples reacted with Fc gamma receptor III, three with the 58- to 64-kDa protein carrying the neutrophil antigen NB1, one with CD11a, and one with CD18. Antibodies from three patients immunoprecipitated a neutrophil protein of 60 kDa. Overall, antibodies to neutrophil antigens other than HLA could be detected in sera from eight patients. Transfusion reactions occurred in 11 of the 14 individuals with WBC antibodies and in none of the 4 without antibodies. Seven pulmonary reactions occurred in patients with WBC antibodies. The patients with WBC antibodies were given significantly more granulocyte concentrates (78 +/- 65 vs. 29 +/- 15 units, p < 0.05). CONCLUSION: Recipients of granulocyte transfusions often become alloimmunized. Screening for WBC antibodies periodically during transfusions, after adverse reactions, or before subsequent transfusions is indicated. If WBC antibodies are present, no further granulocyte transfusions should be given unless the granulocytes are collected from HLA- and/or neutrophil antigen-compatible donors.

Adolescent↗
PubMedDate not supplied

Adoptive transfer of cloned melanoma-reactive T lymphocytes for the treatment of patients with metastatic melanoma.

This report describes a phase I study of the adoptive transfer of cloned melanoma antigen-specific T lymphocytes for therapy of patients with advanced melanoma. Clones were derived from peripheral blood lymphocytes or tumor-infiltrating lymphocytes of patients who had received prior immunization with the melanoma-associated antigen, gpl00. In response to its cognate antigen, each clone used for treatment secreted large amounts of interferon-gamma and granulocyte-macrophage colony-stimulating factor, lesser amounts of interleukin (IL)-2 and tumor necrosis factor-alpha, and little or no IL-4 and IL-10. Clones also demonstrated recognition of human leukocyte antigen-matched melanomas using cytokine secretion and lysis assays. Twelve patients received 2 cycles of cells alone; 11 patients received additional cycles of cells and were randomized between two schedules of IL-2 (125,000 IU/kg subcutaneously daily for 12 days versus 720,000 IU/kg intravenously every 8 h for 4 days). A total of 51 cycles of cells were administered, with an average of 1 x 10(10) cells per cycle. Peripheral blood samples were analyzed for persistence of transferred cells by T-cell receptor-specific polymerase chain reaction. Transferred cells reached a maximum level at 1 h after transfer but rapidly declined to undetectable levels by 2 weeks. One minor response and one mixed response were observed (both in the high-dose IL-2 arm). This report demonstrates the safety and feasibility of cloned T-cell transfer as a therapy for patients with cancer. The lack of clinical effectiveness of this protocol suggests that transfer of different or additional cell types or that modulation of the recipient host environment is required for successful therapy.

Adult↗