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This report is being filed to provide additional information in h.6 and h.11.Investigation: since this was a journal publication to assess chimeric antigen receptor t-cell production in adult lymphoma patients between april 2019 and december 2021, the lot numbers were not requested; therefore, a disposable lot history search could not be conducted.Lymphapheresis was carried out using a spectra optia cell separator from terumo bct applying the cmnc collection software.Depending on individual assessment of peripheral veins, lymphocyte collection was performed through a hemodialysis catheter or via a peripheral line.Collection flow rate was set to the individually possible maximum allowing a continuous blood flow considering an acd-a flow rate of max.1.3 ml per liter blood volume per minute with a standard acd-a ratio set to 1:12 (allowed range: 1:8¿1:15).Prerequisites for undergoing apheresis included stable vital signs, a hemoglobin (hb) level above 8.0 g/dl and a platelet (plts) count greater than 30 x 109/l before apheresis.Initial lymphapheresis was successful in 22 of 23 patients.One patient had to undergo a second apheresis owing to an insufficient lymphocyte count after the first run.Hence, 24 apheresis runs were available for analysis.A hemodialysis catheter was required for half of the apheresis runs, while the other half was conducted with peripheral venous access (table 2).Neither central venous access nor peripheral venous access was associated with any significant complications, such as vasovagal reactions.The processed blood volume varied from 5.0 to 13.9 l with a median of 9.9 l (table 2).The median total blood volume (tbv) processed amounted to 1.9 tbv (range 1.3¿3.0 tbv) (table 2), taking into account the german regulations (¿richtlinie zur gewinnung von blut und blutbestandteilen und zur anwendung von blutprodukten¿) in their most recent version, which generally limits leukapheresis to 3 tbv.Anticoagulation was performed with acd-a using a median volume of 1012 ml per apheresis (range 518¿1538 ml) (table 2).The evaluation of the cell separators' internal time-tracking protocols yielded a median collection time of 167 min (range 103¿259 min).Possible serious side-effects reported in the context of lymphapheresis including paresthesia, nausea, headache, pain, hypotension, or cardiovascular events were not observed in any of the evaluated patients.Apheresis success, defined as meeting the requirements of the manufacturer (novartis) comprising a target tnc of =2 x 109, a cd3-positive lymphocyte count of =1 x 109 with an overall viability of =70%, was achieved in 21 of 23 patients (table 4).In total, 17 apheresis products obtained from 16 patients (including the two patients formally not achieving apheresis success) were dispatched to novartis for tisagenlecleucel manufacturing.Unfortunately, three patients died from rapidly progressing disease during car t-cell production.Due to death, car t-cell manufacturing was prematurely aborted in all three patients, and no car t-cell products were generated.Post-apheresis analysis of the apheresis products' composition revealed a median lymphocyte percentage of 38.3% (range: 8.4%¿60.3%), a median cd3-positive cell percentage of 35.9 (range: 4.4%¿55.6%), a median granulocyte percentage of 7% (range: 1.7%¿62.5%), and a median monocyte percentage of 50.1% (range: 11.0%¿62.4%) (table 4).Hemoglobin levels well below 1 g/dl in the apheresate demonstrated the successful targeting of the wbc layer during the apheresis process (table 4).Blood counts were closely monitored before and after apheresis.The median starting hematocrit prior to apheresis was 33.1% (range: 25.2%¿42.2%) and the mean post-apheresis hematocrit amounted to 28.6% (range: 21.3%¿38.6%) (table 5).Moreover, a hemoglobin level of greater than 8 g/dl and a platelet count of greater than 30 g/l were prerequisites to begin apheresis, which were fulfilled in all patients without the need to administer blood products.The lowest hemoglobin level and the lowest platelet count at the start of apheresis were 8.4 g/ dl and 63 g/l respectively (table 5).The median hemoglobin level prior to apheresis was 11.3 g/dl (range: 8.4¿ 14.4 g/dl), and the median pre-apheresis platelet count was 234 g/l (range: 63¿318 g/l) (table 5).Post apheresis median hemoglobin levels and platelet counts amounted to 9.8 g/dl (range: 7.1¿13.2 g/dl) and 150 g/l (range: 51¿244 g/l) (table 5).While no patient required thrombocyte transfusion after apheresis, four patients received rbcs post apheresis due to hemoglobin levels dropping below 8 g/dl.In all those four patients, preapheresis hemoglobin levels ranged between 8.4 and 8.7 g/dl.Among others, one reason for the drop in hemoglobin levels may rely on dilution effects owing to acd-a addition during apheresis.Irrespective of symptoms of anemia, which were not present in any of those four patients, blood transfusions will be administered as soon as hemoglobin levels drop below 8 g/dl as stipulated by institutional guidelines.In sum, lymphapheresis expectedly caused drops in hematocrit, hemoglobin, and platelet counts, and close monitoring ensured appropriate rbc substitution before patients became symptomatic.Calcium and potassium levels were also regularly checked during the apheresis process to avoid hypocalcemia and hypokalemia, which may both result in potentially relevant arrhythmias or even cardiogenic shock.The median total calcium level measured before apheresis was 2.32 mm (range: 2.08¿2.45 mm).All patients, including those with normal calcium levels, received prophylactic calcium substitution during apheresis by calcium gluconate infusions (usually 2¿6 g calcium).Hence, total serum calcium levels post-apheresis were significantly higher than pre-apheresis with a median calcium level at the end of apheresis of 2.47 mm (range: 2.27¿2.73 mm).Potassium was also prophylactically substituted during apheresis (usually 20¿40 mm potassium chloride were administered) ensuring similar median potassium levels pre- (3.75 mm, range: 3.2¿4.5 mm) and post-apheresis (3.56 mm, range: 3.0¿4.56 mm).In sum, close monitoring and prophylactic substitution prevented severe electrolyte dysbalances during apheresis.Moreover, no side-effects associated with electrolyte dysbalances were observed.In this retrospective study, the authors analyzed the lymphapheresis process of 23 patients diagnosed with aggressive non-hodgkin's lymphoma progressing after first-line chemotherapy, who were considered for in-label car t-cell therapy with cd19-targeting car t cells (tisagenlecleucel).Sufficient (=1 x 10e9 cd3-positive lymphocytes) t-cell collection could be achieved in the vast majority of patients.Additionally, apheresis material from 16 patients was submitted for car t-cell manufacturing, and 13 patients were eventually treated with car t cells with three patients dropping out due to death from rapidly progressing disease resulting in premature abortion of car t-cell manufacturing.Finally, no serious side-effects associated with lymphapheresis were observed.Overall, authors concluded that lymphapheresis for car t-cell manufacturing did not cause any serious side effects in this study.Moreover, a sufficient quantity of cd3 positive lymphocytes for car t-cell production was collected in the majority of patients despite heavy pretreatment and advanced age and disease.Additionally, preemptive lymphocyte collection at an early time point in lymphoma therapy might improve the quantity and quality of collected t cells, especially in patients with dlbcl-nos, to enhance the chances of successful tisagenlecleucel manufacturing.However, for car t-cell products directly generated from fresh aphereses, this approach cannot be applied to.According to therapeutic apheresis: a physician's handbook, as large volumes of donor or patient blood circulate through an apheresis device, blood cells are intentionally or incidentally removed.The small amount of red cells lost in the apheresis circuit may be more apparent in an anemic patient who has meager production capacity and who is receiving multiple procedures.Although generally well tolerated, the large volume leukocytapheresis for stem cell collections in patients often results in a decline in hematocrit and platelet count, particularly because some red cells and platelets are incidentally removed with the stem cells.Since this was a journal publication to assess chimeric antigen receptor t-cell production in adult lymphoma patients between april 2019 and december 2021, the lot numbers were not requested; therefore, a dhr search could not be conducted for this specific incident.All lots must meet acceptance criteria for release.Root cause: based on the available information within the journal article, the authors attributed the drop in hemolglobin levels to dilution effects owing to acd-a addition during apheresis.The authors also indicated that lymphapheresis expectedly caused drops in hematocrit, hemoglobin, and platelet counts.Close monitoring ensured appropriate rbc substitution before patients became symptomatic.A root cause assessment was performed for the anemia.Based on the available information a definitive root cause could not be determined but it is likely due to one or a combination of the possible causes listed below: *patient¿s low initial hemoglobin prior to apheresis *dilution effects owing to acd-a addition during apheresis *patients¿ underlying disease state *incorrect setting of collection preference resulting in too many rbcs in the collected products article citation: harrer dc, heidenreich m, fante ma, et al.Apheresis for chimeric antigen receptor t-cell production in adult lymphoma patients.Transfusion.2022.
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