Apheresis collection of mononuclear cells for chimeric-antigen receptor therapies.

Collections of lymphocytes to be genetically modified to treat hematologic malignancies have seen a dramatic increase over the last few years as commercial products have been approved. Reports of new products in development that can possibly treat solid organ malignancies represent a massive change in the field. Apheresis is at the center of the collection of cells for the manufacture of these chimeric-antigen receptor therapy products. The expansion of these collections represents one of the areas of apheresis procedures growth. This review will summarize concepts important to this type of collection and variables that need to be optimized to obtain desired cell yields while increasing patients' safety.

Anti-CD20 therapeutic options in immune-mediated thrombotic thrombocytopenic purpura.

Immunosuppression with rituximab in immune-mediated thrombotic thrombocytopenic purpura helps decrease production of autoantibody mediating ADAMTS13 clearance from circulation. Failure to respond to rituximab in a satisfactory way or made difficult by adverse events to the medication does not represent a reason to stop considering anti-CD20 therapies to control antibody production. Therefore, both of atumumab and obinutuzumab with specificity to CD20, represent potentially valuable therapeutic tools in patients who are not candidates for rituximab. Commentary on: Doyle et al. The use of obinutuzumab and ofatumumab in the treatment of immune thrombotic thrombocytopenic purpura. Br J Haematol. 2022;198:391-396.1.

Initial assessment of α-synuclein structure in platelets.

Over the last few years data from our group have indicated that α-synuclein is important in development of immune cells as well as potentially erythrocytes and platelets. The latter is important since this protein may work as negative regulator of granule release. Thus, we sought to begin to understand the structure of this protein in platelets. Flow cytometric analysis of this protein using region-specific (N-terminus, central region and C-terminus) monoclonal antibodies was performed. Antibody to the central region gave the strongest shift among all three antibodies, with the C-terminus having intermediate shift and N-terminus minimal shift. Western blotting using the same antibodies showed similar binding of all antibodies to α-synuclein. These results suggest a similar arrangement of this protein in platelets as seen in neurons. Future studies ought to look at the role that each protein region plays in platelets.

Absolute Immature Platelet Counts Suggest Platelet Production Suppression during Complicated Relapsing Thrombotic Thrombocytopenic Purpura.

Absolute immature platelet counts (A-IPC) aid in diagnosis and treatment follow-up in thrombotic thrombocytopenic purpura (TTP). A-IPC was used to follow a patient on mycophenolate mofetil (MMF) maintenance therapy treated with a prolonged therapeutic plasma exchange (TPE) regimen for relapsing TTP. On admission, the platelet (PLT) count was 95 × 109/L declining to 14 × 109/L in 5 days. Daily TPE was initiated for suspected TTP, and MMF was discontinued. A-IPC and PLT count were 1 × 109/L and 14 × 109/L, respectively, prior to first TPE. A-IPC improved to 3.2 × 109/L with 1 TPE, and on day 5, A-IPC and PLT count were 7.5 × 109/L and 218 × 109/L, respectively. On day 6, A-IPC and PLT count decreased to 4.8 × 109/L and 132 × 109/L further worsening to 0.4 × 109/L and 13 × 109/L, respectively. ADAMTS13 activity remained <5% with an inhibitor; counts did not recover. Initial improvement followed by rapidly declining A-IPC despite therapy suggested production suppression. In TTP, A-IPC may aid in establishing early therapy effects over PLT production.

Bacterial contamination and septic transfusion reaction rates associated with platelet components before and after introduction of primary culture: experience at a US Academic Medical Center 1991 through 2017.

BACKGROUND: The high incidence of septic transfusion reactions (STRs) led to testing being mandated by AABB from 2004. This was implemented by primary culture of single-donor apheresis platelets (APs) from 2004 and prestorage pooled platelets (PSPPs) from 2007. STUDY DESIGN/METHODS: Platelet (PLT) aliquots were cultured at issue and transfusion reactions evaluated at our hospital. Bacterial contamination and STR rates (shown as rates per million transfusions in Results) were evaluated before and after introduction of primary culture by blood centers that used a microbial detection system (BacT/ALERT, bioMerieux) or enhanced bacterial detection system (eBDS, Haemonetics). RESULTS: A total of 28,457 PLTs were cultured during pre-primary culture periods (44.7% APs; 55.3% at-issue pooled PLTs [AIPPs]) and 97,595 during post-primary culture periods (79.3% APs; 20.7% PSPPs). Forty-three contaminated units were identified in preculture and 34 in postculture periods (rates, 1511 vs. 348; p < 0.0001). Contamination rates of APs were significantly lower than AIPPs in the preculture (393 vs. 2415; p < 0.0001) but not postculture period compared to PSPPs (387 vs. 198; p = 0.9). STR rates (79 vs. 90; p = 0.98) were unchanged with APs but decreased considerably with pooled PLTs (826 vs. 50; p = 0.0006). Contamination (299 vs. 324; p = 0.84) and STR rates (25 vs. 116; p = 0.22) were similar for PLTs tested by BacT/ALERT and eBDS primary culture methods. A change in donor skin preparation method in 2012 was associated with decreased contamination and STR rates. CONCLUSION: Primary culture significantly reduced bacterial contamination and STR associated with pooled but not AP PLTs. Measures such as secondary testing near time of use or pathogen reduction are needed to further reduce STRs.

Use of a whole-cell ELISA to detect additional antibodies in setting of suspected heparin-induced thrombocytopenia.

OBJECTIVES: Type II heparin-induced thrombocytopenia (HIT) is mediated by formation of antibodies to platelet factor 4 (PF4)-heparin complexes. We evaluated anti-PF4-heparin-negative samples for the presence of additional anti-platelet and anti-red blood cell (RBC) antibodies using whole-cell platelet/ RBC ELISAs we developed. METHODS: Seventy-three samples tested for anti-PF4-heparin by ELISA were included: 62 tested negative, 9 tested positive, and 2 had equivocal results. Plasma specimens from healthy donors were used as controls. RESULTS: 100% (9/9) anti-PF4-positive samples had anti-platelet antibodies detected by whole-cell platelet ELISA. 42.2% (27/64) anti-PF4-heparin-negative samples were negative for anti-platelet and anti-RBC antibodies. 32.8% (21/64) negative samples showed reactivity to both platelets and RBC; 12.5% (8/64) negative samples were each reactive with either platelet or RBC ELISA, respectively. Additionally, two samples that tested equivocal by anti-PF4-heparin ELISA had antibodies to both platelets and RBC by whole-cell ELISA. CONCLUSIONS: Our study suggests that patients with thrombocytopenia testing negative for anti-PF4-heparin may still harbor antibodies to platelets. However, additional research is needed to determine the significance of these antibodies. Nevertheless, these findings may encourage clinicians to further investigate patients with possible immune-mediated etiologies of thrombocytopenia and anemia.

Adverse events during apheresis: A 10-year experience at a tertiary academic medical center.

BACKGROUND: Apheresis can be associated with adverse events (AEs). Available studies published on apheresis-associated AEs lack uniformity of data. Unfortunately, there is no common database in the United States (US) to report apheresis-associated AEs. We evaluated our institutional incidence of apheresis-associated AEs and compared it with published literature. STUDY DESIGN AND METHODS: We conducted a 10-year retrospective study of apheresis procedures and associated AEs at our facility, a tertiary academic medical center, from 2007 to 2016. Concurrently, a literature search was conducted on AEs associated with apheresis procedures. Twenty-eight studies including data from US and other countries' facilities were analyzed. RESULTS: The overall AE incidence was 6.9% (396/5684 procedures). Frequency of AEs associated with therapeutic plasma exchange (TPE) was higher (8.5%, P < .0001) compared to other apheresis procedures. Significant correlation between number of TPE and AEs (Spearman rho [rs ] = 0.7, P = .002) was encountered. Furthermore, there was a significant decrease over time of moderate and severe AEs (rs = -0.64, P = .04 and rs = -0.83, P = .003 respectively). Comparison of our institutional AEs (6.9%) to data from other countries (9.8%) and US (22.6%) indicated a significant difference (P < .0001). CONCLUSION: Overall our incidence of AEs was significantly lower than current published literature. Incidence of AEs published in other countries is significantly lower than US rates. Differences in incidence of AEs emphasize need for uniform reporting and stratification of AEs and development of a common database to report AEs. Therefore, we propose a grading rationale in order to standardize reporting of AEs.

Ultrastructural changes in peripheral blood leukocytes in α-synuclein knockout mice.

Effects of α-synuclein deficiency on cellular blood components have not been extensively investigated. This study evaluated ultrastructural changes of leukocytes in α-synuclein knockout (KO) mice using electron microscopy (EM). The following ultrastructural characteristics were quantified in leukocytes: mitochondria, primary granules, specific granules (SG), Golgi apparatus (GA), inclusions, rough-endoplasmic reticulum (RER), smooth-endoplasmic reticulum (SER), and cellular projections (CP). EM showed increased numbers or amounts of SG, inclusions, and SER in KO group (5.3 ±â€¯4.5 in WT vs. 14.1 ±â€¯10.3 in KO, p = 0.02; 0.4 ±â€¯0.9 in WT vs. 3.2 ±â€¯2.8 in KO, p = 0.007; and 7.7 ±â€¯6.7 in WT vs. 17.7 ±â€¯12.2 in KO, p = 0.03, respectively). Although CP number was not significantly different between the two groups (13.4 ±â€¯5.3 in WT vs. 16.3 ±â€¯7.5 in KO, p = 0.32), their size and shapes were altered in KO mice. Notably, findings occurred in the setting of significant lymphopenia. α-Synuclein deficiency leads to changes in size and shape of secretory particles and increases in SER, SG, and inclusions, indicating a potential role for α-synuclein in vesicular trafficking in leukocytes. Further studies are needed to elucidate functions mediated by α-synuclein.

Detection of septic transfusion reactions to platelet transfusions by active and passive surveillance.

Septic transfusion reactions (STRs) resulting from transfusion of bacterially contaminated platelets are a major hazard of platelet transfusion despite recent interventions. Active and passive surveillance for bacterially contaminated platelets was performed over 7 years (2007-2013) by culture of platelet aliquots at time of transfusion and review of reported transfusion reactions. All platelet units had been cultured 24 hours after collection and released as negative. Five sets of STR criteria were evaluated, including recent AABB criteria; sensitivity and specificity of these criteria, as well as detection by active and passive surveillance, were determined. Twenty of 51,440 platelet units transfused (0.004%; 389 per million) were bacterially contaminated by active surveillance and resulted in 5 STRs occurring 9 to 24 hours posttransfusion; none of these STRs had been reported by passive surveillance. STR occurred only in neutropenic patients transfused with high bacterial loads. A total of 284 transfusion reactions (0.55%) were reported by passive surveillance. None of these patients had received contaminated platelets. However, 6 to 93 (2.1%-32.7%) of these 284 reactions met 1 or more STR criteria, and sensitivity of STR criteria varied from 5.1% to 45.5%. These results document the continued occurrence of bacterial contamination of platelets resulting in STR in neutropenic patients, failure of passive surveillance to detect STR, and lack of specificity of STR criteria. These findings highlight the limitations of reported national STR data based on passive surveillance and the need to implement further measures to address this problem such as secondary testing or use of pathogen reduction technologies.

α-Synuclein concentration increases over time in plasma supernatant of single donor platelets.

OBJECTIVES: In platelets, α-synuclein is important in calcium-dependent granule release. Notably, cells release α-synuclein in setting of cell damage or death. Therefore, we investigated α-synuclein levels in plasma of single donor platelet (SDP) units during storage. METHODS: Aliquots were obtained from same SDP units for 7 days from day of donation. Additionally, randomly sampled SDP units at same storage time points were also assayed by enzyme-linked immunosorbent assay. RESULTS: α-Synuclein in SDP plasma increased continuously over time at each assayed time point. Significant increases were measured on day 3 (11.7 ± 9.6 ng/mL, P = 0.025), day 5 (15.3 ± 5.9 ng/mL, P = 0.002), and highest on day 7 (23.7 ± 5.6 ng/mL, P < 0.0001) compared to day 0 (1.1 ± 0.8 ng/mL). Similar significant results were obtained in randomly sampled SDP units at same corresponding time points. Flow cytometry showed that platelets had strong expression of α-synuclein and lacked expression of other synucleins. CONCLUSIONS: Increases of α-synuclein during SDP storage is a steady and continuous process that increases with time. Our findings indicate that α-synuclein may represent a biomarker of platelet biological state during storage. Further research will be needed to determine how α-synuclein increases correlate with platelets' function.

Current state of apheresis technology and its applications.

Apheresis is at the forefront of therapeutic approaches for an increasing number of indications caused by formation of pathologic antibodies treated by therapeutic plasma exchange, or through the use of red cell exchanges to overcome complications secondary to sickle cell crises. Likewise, the number of hematopoietic stem cell transplants has continued to grow annually and this is the direct result of the expansion of apheresis collections. Over the years a number of apheresis platforms have been utilized, but as one of the oldest and most widely used systems, the COBE Spectra, has ceased to be used therapeutically and at blood centers for donations there is an active search to find suitable systems that will replace it and have the versatility to perform as many procedures as possible. Computer innovations have made it possible with current apheresis technology to obtain more real-time information of the procedure which permits the operator to adjust parameters not only to optimize the specific procedure but also to safeguard against potential adverse events. The focus of this review is to go over available clinical data describing the operation, outcomes and applications of apheresis platforms, and discuss those systems that are likely to meet clinical demands and those of blood donation centers.

Absolute immature platelet counts in the setting of suspected heparin-induced thrombocytopenia may predict anti-PF4-heparin immunoassay testing results.

BACKGROUND: Heparin-induced-thrombocytopenia (HIT) is a disease mediated by antibodies to platelet factor 4 (PF4)-heparin complexes. Immature platelet fraction (%-IPF) and absolute immature platelet count (A-IPC) measure newly-released platelets into circulation and can prove useful in differentiating patients with thrombocytopenic presentations due to consumptive or hypoproduction processes. Therefore, we evaluated utility of A-IPC in a cohort of thrombocytopenic patients suspected of HIT. PATIENTS AND METHODS: Twenty-six thrombocytopenic patients (<150 × 109/L) tested for anti-PF4-heparin and 36 non-thrombocytopenic controls were included. Platelet count, %-IPF, and A-IPC were determined at time of anti-PF4-heparin testing. RESULTS: Sixteen patients tested anti-PF4-heparin negative and 10 tested positive. Patients with positive anti-PF4-heparin did not differ in A-IPC from normal range (7.2 ±â€¯2.9 × 109/L vs. 7.1 ±â€¯3.2 × 109/L respectively; p = 0.97). However, there was a significant A-IPC decrease in patients negative for anti-PF4-heparin compared to normal range and those testing anti-PF4-heparin positive (4.2 ±â€¯3.1 × 109/L vs. 7.1 ±â€¯3.2 × 109/L vs. 7.2 ±â€¯2.9 × 109/L respectively, p < 0.01). An A-IPC of greater than 5 × 109/L characterized 80% of anti-PF4-heparin positive cases. CONCLUSION: A-IPC measurements can complement anti-PF4-heparin testing of patients suspected of HIT while potentially predicting anti-PF4-heparin immunoassay results.

Dynamic changes in absolute immature platelet count suggest the presence of a coexisting immune process in the setting of thrombotic thrombocytopenic purpura.

BACKGROUND: Previous studies have indicated the usefulness of absolute immature platelet counts (A-IPCs) in the management and diagnostic algorithm of thrombotic thrombocytopenic purpura (TTP). Specifically a threefold increase in A-IPC from baseline may be diagnostic of TTP. Here, A-IPC was used to understand a coexisting immune dysregulation complicating TTP treatment. CASE REPORT: A 17-year-old previously healthy female was admitted with altered mental status, petechiae, anemia, thrombocytopenia, and schistocytes on peripheral smear. Daily therapeutic plasma exchange (TPE) and corticosteroids were started for suspected TTP supported by ADAMTS13 activity of less than 5%, inhibitor more than 8, and more than threefold A-IPC increase from baseline post-TPE initiation. Despite daily TPE, the patient had significant and unexpected decreases in platelet (PLT) counts and A-IPCs during her hospital course. After each PLT count decline, response to TPE and immunosuppression led to increasingly prolonged count recovery with subsequent episodes. Decreases in both PLTs and A-IPCs indicated that both mature and immature PLTs were being cleared from circulation. Recovery occurred once A-IPC dynamics indicated restored negative feedback in relation to PLT count. CONCLUSION: Serial monitoring of A-IPC dynamics was indicative of coexisting processes in the setting of ADAMTS13 deficiency. Uncoupling of the expected A-IPC and PLT count seen in TTP suggested the presence of such an immune process in addition to TTP with high ADAMTS13 inhibitor. Monitoring of A-IPC is a clinically valuable, rapid, and noninvasive thrombopoietic measurement when TTP is suspected.

Comparison of two apheresis systems during hematopoietic progenitor stem cell collections at a tertiary medical center.

BACKGROUND: The Spectra Optia is a newer apheresis system developed based on the COBE Spectra platform. COBE Spectra requires more manual control, while Spectra Optia offers greater automation. The purpose of this study was to compare the two systems during hematopoietic progenitor stem cell (HPSC) collections. STUDY DESIGN AND METHODS: A retrospective review of 41 collections performed in 26 subjects at a tertiary medical center between June 1, 2013, and December 31, 2013, was conducted, 11 with the Spectra Optia and 30 with the COBE Spectra. Six patients underwent two consecutive daily collections, first on the Spectra Optia followed by the COBE Spectra. RESULTS: Procedure run time with the Spectra Optia was considerably longer than with the COBE Spectra (283 ± 11 min vs. 217 ± 2 min, respectively; p < 0.01). Mean CD34+ cell yields with the Spectra Optia were comparable with those of the COBE Spectra. Products collected with the Spectra Optia had less red blood cell contamination. However, platelet (PLT) attrition was greater with the Spectra Optia. Similar results were obtained in patients who were collected on consecutive days in both systems. CONCLUSION: Collections with the Spectra Optia take longer and lead to greater PLT losses during HPSC collections.

Absolute immature platelet count dynamics in diagnosing and monitoring the clinical course of thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening diagnosis requiring prompt initiation of therapeutic plasma exchange (TPE). Measurement of immature platelet (PLT) fraction (%-IPF) differentiates PLT consumption or destruction from hypoproduction. STUDY DESIGN AND METHOD: Our study evaluated %-IPF changes over the course of TTP treated with TPE and as a measure of treatment efficacy. Eleven idiopathic TTP patients, two human immunodeficiency virus (HIV)-associated TTP patients, and five non-TTP patients with thrombocytopenia were enrolled into our study. All patients were treated with TPE and had ADAMTS13 activity measured. RESULTS: All idiopathic TTP patients had a significantly increased %-IPF and decreased absolute immature PLT count (A-IPC) and PLT count at presentation. An A-IPC value of less than 5 × 10(9) /L at presentation has 84.6% sensitivity, 80% specificity, and 91.7% positive predictive value for diagnosing TTP. A concurrent steady decline in %-IPF and increased PLT counts toward normal was observed in TTP patients undergoing TPE. The A-IPC, however, showed an increase and decrease curve that was not seen in the two HIV-associated TTP patients with no response to TPE and the five non-TTP patients. More importantly, reaching an A-IPC ratio of 3 compared to baseline value during TPE can readily differentiate idiopathic TTP from the other two groups and is correlated with good clinical responses to TPE. An abrupt increase of A-IPC during TPE was also noted in a TTP patient who relapsed 3 days before PLT count decrease. A-IPC is positively correlated with ADAMTS13 activity at presentation but negatively correlated with ADAMTS13 activity during recovery. CONCLUSION: A-IPC should be routinely analyzed for diagnosing and monitoring TTP patients.

Absolute immature platelet count helps differentiate thrombotic thrombocytopenic purpura from hypertension-induced thrombotic microangiopathy.

ADAMTS13 activity measurement is used in the diagnostic algorithm of thrombotic thrombocytopenic purpura (TTP), but results may not be available before initiation of therapeutic plasma exchange (TPE). The immature platelet fraction (%-IPF) and the calculated absolute immature platelet count (A-IPC) represent a test of real-time thrombopoiesis, and can be performed in most laboratories using automated analyzers. Here we report on using A-IPC kinetics to exclude idiopathic TTP in a patient with severe hypertension, thrombocytopenia, and acute renal failure, which was confirmed by a normal ADAMTS13. The complete resolution of thrombocytopenia occurred once blood pressure was controlled favoring a diagnosis of hypertension-induced thrombotic microangiopathy.

Pathological Mechanisms and Novel Testing Methods in Thrombotic Thrombocytopenic Purpura.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon, but potentially disabling or even deadly, thrombotic microangiopathy with a well-studied mechanism of ADAMTS13 deficiency or dysfunction. While established treatments are largely effective, the standard ADAMTS13 testing required to definitively diagnose TTP may cause delays in diagnosis and treatment, highlighting the need for rapid and effective diagnostic methods. Additionally, the heterogeneous presentation and varied inciting events of TTP suggest more variation in its mechanism than previously thought, implying three potential pathways rather than the accepted two. The recent discovery of ADAMTS13 conformation as a potential contributor to TTP in addition to the proposal of using the absolute immature platelet count (A-IPC) as a biomarker, present novel areas for monitoring and treatment. A-IPC in particular may serve as a more rapid and accurate diagnostic test to distinguish TTP from non-TTP TMAs and to monitor treatment response and relapse. These considerations highlight the need to further study TTP in order to improve best practices and patient care.

Immature platelet counts in transfused platelet units given to neonates.

BACKGROUND: Immature platelets, young and large platelets recently released from the bone marrow, have gained interest over the last decade as a clinically informative variable during thrombocytopenic presentations. These immature platelets are found in all donated platelet units, however, the role, if any, that these younger platelets play post transfusion is not known. It has also been reported that the immune response can affect responses to platelet transfusions. Thus, we looked at PLT increments in a cohort of neonates receiving platelet transfusions in our neonatal intensive care unit. METHODS: During a twelve-month period, platelet transfusions received by neonates born and not discharged from our institution at time of transfusion were retrospectively analyzed. In the study period a total of 33 patients received either a single or multiple transfusions during their hospitalization, for a total of 100 transfusion events. RESULTS: The cohort was mostly premature neonates with a mean gestational age of 29.6 weeks. The units transfused appeared to have a broad range of absolute immature platelet counts (A-IPC) but overall, it was similar between those receiving single or multiple transfusions. Considering that platelet count was similar among aliquots transfused, it appeared that count increments were influenced by higher A-IPC content of the aliquot especially among 2nd trimester and 3rd trimester premature neonates. Patients with higher baseline platelet count (PLT) tended to receive a single transfusion aliquot while those receiving multiple transfusions had lower baseline PLT (p = 0.0022). Looking at aliquot dose, regardless if receiving a single or multiple transfusions, younger patients received incrementally higher dose (ml/kg) with each transfusion. CONCLUSIONS: A-IPC in platelet aliquots transfused to neonates may influence post-transfusion PLT. Full effect of A-IPC in platelet aliquots may not be seen since irradiation of units may hamper immature platelets viability and function. Further research is needed to determine if A-IPC plays an active role to limit the need for further transfusions of patients receiving transfusions.

Bacterial Contamination of Platelet Products.

Transfusion of bacterially contaminated platelets, although rare, is still a major cause of mortality and morbidity despite the introduction of many methods to limit this over the past 20 years. The methods used include improved donor skin disinfection, diversion of the first part of donations, use of apheresis platelet units rather than whole-blood derived pools, primary and secondary testing by culture or rapid test, and use of pathogen reduction. Primary culture has been in use the US since 2004, using culture 24 h after collection of volumes of 4-8 mL from apheresis collections and whole-blood derived pools inoculated into aerobic culture bottles, with limited use of secondary testing by culture or rapid test to extend shelf-life from 5 to 7 days. Primary culture was introduced in the UK in 2011 using a "large-volume, delayed sampling" (LVDS) protocol requiring culture 36-48 h after collection of volumes of 16 mL from split apheresis units and whole-blood derived pools, inoculated into aerobic and anaerobic culture bottles (8 mL each), with a shelf-life of 7 days. Pathogen reduction using amotosalen has been in use in Europe since 2002, and was approved for use in the US in 2014. In the US, recent FDA guidance, effective October 2021, recommended several strategies to limit bacterial contamination of platelet products, including pathogen reduction, variants of the UK LVDS method and several two-step strategies, with shelf-life ranging from 3 to 7 days. The issues associated with bacterial contamination and these strategies are discussed in this review.