Single-arm trials supporting the approval of anticancer medicinal products in the European Union: contextualization of trial results and observed clinical benefit.

BACKGROUND: Single-arm trials (SATs) can sometimes be used to support marketing authorization of anticancer medicinal products in the European Union. The level and durability of antitumor activity of the product as well as context are important aspects to determine the relevance of trial results. The aim of this study is to provide details on the contextualization of trial results and to evaluate the magnitude of benefit of medicinal products approved based on SATs. MATERIALS AND METHODS: We focused on anticancer medicinal products for solid tumors approved on the basis of SAT results (2012-2021). Data were retrieved from European public assessment reports and/or published literature. The benefit of these medicinal products was evaluated via the European Society for Medical Oncology (ESMO)-Magnitude of Clinical Benefit Scale (MCBS). RESULTS: Eighteen medicinal products were approved based on 21 SATs-few medicinal products were supported by >1 SAT. For the majority of clinical trials, a clinically relevant treatment effect was (pre)specified (71.4%) and most often an accompanying sample size calculation was provided. For 10 studies, each testing a different medicinal product, a justification for the threshold for a clinically relevant treatment effect could be identified. At least 12 out of 18 applications included information to facilitate the contextualization of trial results, including six supportive studies. Of the pivotal SATs analyzed (n = 21), three were assigned an ESMO-MCBS score of 4, which corresponds to 'substantial' benefit. CONCLUSIONS: The clinical relevance of the treatment effects shown by medicinal products for solid tumors tested in SATs is dependent on the effect size and context. To better facilitate regulatory decision making, prespecifying and motivating a clinically relevant effect and aligning the sample size to that effect is important. External controls may facilitate in the contextualization process, but the associated limitations must be addressed.

Revision for coronal malalignment will improve functional outcome up to 5 years postoperatively.

PURPOSE: Revision of a total knee arthroplasty (TKA) for the diagnosis of malalignment is widely performed. However, very little is known about the functional outcome in revision TKA surgery for malalignment. The aim of this study was to assess the functional outcome and to identify factors influencing the functional outcome of patients who have had a revision of a TKA for the diagnosis of malalignment at 5 years follow-up. METHODS: All patients with a revision of a TKA for malalignment as the primary reason were selected from a prospective database. The diagnosis of symptomatic malalignment was made by the surgeon and quantified by radiologic examination. Functional outcome was scored by the functional score of the Knee Society Clinical Rating System (fKSS) at 0, 12, 24 and 60 months. Multiple imputation for missing data and multivariable analysis were performed to identify factors influencing functional outcome. RESULTS: After selection, 105 patients (age: 65.1 ± 9.1 years, gender M:F 30:75) were eligible for outcome analysis. Functional outcome significantly improved from the preoperative (fKSS: 44.1 ± 22.0) to 5 years postoperative (64.7 ± 24.0, p < 0.001) time frames. Higher degree of coronal deviation, younger age and lower preoperative KSS were found to be strongest positive influencing factors for the change in fKSS. CONCLUSION: Revision of TKA for malalignment appears to be an effective treatment to improve functional outcome up to 5 years postoperatively. Higher degree of coronal deviation, younger age and lower preoperative KSS are the strongest contributing factors for functional improvement. LEVEL OF EVIDENCE: Level III; Therapeutic prospective cohort study.

High body mass index and pre-existing autoimmune disease are associated with an increased risk of immune-related adverse events in cancer patients treated with PD-(L)1 inhibitors across different solid tumors.

BACKGROUND: Treatment with anti-PD-(L)1 antibodies, approved for several oncology indications, can lead to immune-related adverse events (irAEs). We aimed to investigate risk factors associated with an increased reporting of irAEs in patients treated with PD-(L)1 inhibitors approved for solid tumor indications. PATIENTS AND METHODS: A retrospective review was performed of individual data from patients in phase II/III registrational studies for PD-(L)1 inhibitors in solid tumors. Data on baseline characteristics and adverse events were extracted. Univariate and multivariable logistic regression models were used to identify risk factors. RESULTS: In total, 5123 patients were included from 15 studies reporting on the use of four PD-(L)1 inhibitors for five solid tumor indications. Univariate analysis suggested that type of study drug (P < 0.001), indication (P = 0.003), body mass index (BMI) (P = 0.001), and baseline autoimmune disease (P < 0.001) were associated with an increased occurrence of any irAE. Using logistic regression analyses, three factors were identified as increasing the risk of irAE: BMI ≥ 30 kg/m2 [odds ratio (OR) 1.5, 95% confidence interval (CI) 1.2-1.8] in comparison to normal BMI, having an autoimmune disease at baseline (OR 1.8, 95% CI 1.1-2.7), and use of a PD-L1 inhibitor (OR 1.6, 95% CI 1.2-2.0). The latter finding is probably biased due to the selection of the studies in the dataset with complete information on baseline characteristics. CONCLUSION: This study was conducted using a large dataset of individual patient data from clinical trials comprising multiple solid tumor indications. We demonstrated that patients with obesity and concurrent autoimmune disease were at increased risk of developing irAEs.

A narrative review of estimands in drug development and regulatory evaluation: old wine in new barrels?

BACKGROUND: An estimand defines the target of estimation for a clinical trial through specification of the treatment, target population, variable, population-level summary and of the strategies for intercurrent events. A carefully defined estimand aligns the clinical trial design and analysis with the scientific question of interest and adequately accounts for so-called intercurrent events. The ICH E9(R1) addendum suggests five estimand strategies. We evaluated to what extent current practice in drug development and regulatory assessment fits in the estimand framework. METHODS: We systematically evaluated what estimands, especially what strategies for intercurrent events are advised in European Medicines Agency disease guidelines, used in sponsors' trials and additionally requested by the European Medicines Agency in assessment dossiers. We selected four therapeutic areas: nervous system, oncology, cardiovascular diseases and respiratory diseases. For each, we evaluated all disease guidelines with approved drugs, the dossiers of the most recently approved drugs matching the guidelines and corresponding regulatory questions. RESULTS: Strategies for intercurrent events were present in 18 (53%) of 34 guidelines, in all 34 sponsor documentations and in 15 (44%) of 34 sets of regulatory questions. Treatment policy was advised in 13 (38%) guidelines and was applied in 9 corresponding sponsor documentations. Of these 9, it was the sole strategy in 4 cases and accompanied by another strategy in 5 cases. Hypothetical strategy was not advised in guidelines. However, it was the leading strategy applied in 25 (74%) sponsor documentations. Composite strategy was advised in 3 (9%) guidelines and applied accompanied by another strategy in 2 corresponding sponsor documentations. While on treatment strategy was not advised in guidelines, but was applied in 2 sponsor documentations. Principal stratum strategy was advised in 2 guidelines but not applied in corresponding sponsor documentations. Of the regulatory questions, treatment policy was present in 2 cases (6%), hypothetical in 6 cases (18%), composite in 6 cases (18%) and while on treatment in 1 case (3%). CONCLUSIONS: Estimand attributes are present in guidelines, sponsor documentations and regulatory questions, but not described as estimands. Treatment policy was most often advised in guidelines, but hypothetical was the leading strategy applied in sponsor documentations. Thus, results indicate not a full concordance between the regulatory target of estimation and what is actually estimated. The lack of concordance was mostly due to limitations in collection of intercurrent events data to enable a treatment policy strategy. There is, therefore, a need to better define estimands at the design stage and throughout the applications dossiers and assessment reports.

Physical therapy aimed at self-management versus usual care physical therapy after hip arthroscopy for femoroacetabular impingement: study protocol for a randomized controlled trial.

BACKGROUND: Femoroacetabular impingement has been recognized as a common cause of hip pain and dysfunction, especially in athletes. Femoroacetabular impingement can now be better treated by hip arthroscopy but it is unclear what postoperative rehabilitation of hip arthroscopy should look like. Several rehabilitation protocols have been described, but none presented clinical outcome data. These protocols also differ in frequency, duration and level of supervision. We developed a rehabilitation protocol with supervised physical therapy which showed good clinical results and is considered usual care in our treatment center. However, it is unknown whether, due to the relatively young age and low complication rate of hip arthroscopy patients, rehabilitation based on self-management might lead to similar results. The aims of this pilot study are (1) to determine feasibility and acceptability of the self-management intervention, (2) to obtain a preliminary estimate of the difference in effect between physical therapy aimed at self-management versus usual care physical therapy in patients who undergo hip arthroscopy for femoroacetabular impingement. METHODS/DESIGN: Thirty participants (aged 18-50 years) scheduled for hip arthroscopy will be included and randomized (after surgery) to either self-management or usual care physical therapy in this assessor-blinded randomized controlled trial. After surgery, the self-management group will perform a home-based exercise program three times a week and will receive physical therapy treatment once every 2 weeks for 14 weeks. The usual care group will receive physical therapy treatment twice a week for 14 weeks and will perform an additional home-based exercise program once a week. Assessment will occur preoperatively and at 6, 14, 26 and 52 weeks after surgery. Primary outcomes are feasibility, acceptability and preliminary effectiveness. Feasibility and acceptability will be determined by the willingness to enroll, recruitment rate, adherence to treatment, patient satisfaction, drop-out rate and adverse events. Preliminary effectiveness will be determined using the following outcomes: the International Hip Outcome Tool 33 and hip functional performance as measured with the Single Leg Squat Test 14 weeks after surgery. DISCUSSION: The results of this study will be used to help decide on the need, feasibility and acceptability of a large-scale randomized controlled trial. TRIAL REGISTRATION: This protocol was registered with the Dutch Trial Registry (NTR5168) on 8 May 2015.

Meta-analysis of ultrasound-guided biopsy of suspicious axillary lymph nodes in the selection of patients with extensive axillary tumour burden in breast cancer.

BACKGROUND: Recent studies show that not all patients with breast cancer and positive axillary lymph nodes need additional axillary surgery. A systematic review and meta-analysis of the literature was performed to test the hypothesis that ultrasound-guided biopsy of suspicious nodes can be a useful tool to identify patients with extensive axillary tumour burden. METHODS: PubMed and Embase were searched to identify articles reporting on ultrasound-guided techniques to stage the axilla of patients with breast cancer. The emphasis was to study the number of positive nodes found after axillary lymph node dissection (ALND) following a positive ultrasound-guided biopsy or a positive sentinel lymph node biopsy (SLNB). Information regarding the number of positive nodes thus had to be available. Results were tested for heterogeneity and a meta-analysis was performed. RESULTS: A total of 894 articles were identified, and 115 were selected based on title and abstract information by two independent reviewers. After extensive review, 18 articles were eligible for analysis. Eight studies reported sufficient data to perform a meta-analysis comparing 532 patients with a positive ultrasound-guided biopsy with 248 patients with a negative ultrasound-guided biopsy but a positive SLNB. The number of involved nodes was significantly higher in patients in whom axillary metastasis was detected by ultrasound-guided biopsy (P < 0·001). No heterogeneity in the observed effect was found (I(2) = 22 per cent, P = 0·26). CONCLUSION: Patients with breast cancer in whom axillary metastases are detected by ultrasound-guided biopsy have significantly more involved nodes than SLNB-positive patients. This finding enables further preoperative tailoring of axillary treatment in breast cancer.

Outcomes of colorectal cancer patients with peritoneal carcinomatosis treated with chemotherapy with and without targeted therapy.

BACKGROUND: Although systemic therapies have shown to result in survival benefit in patients with metastatic colorectal cancer (mCRC), outcomes in patients with peritoneal carcinomatosis (PC) are poor. No data are available on outcomes of current chemotherapy schedules plus targeted agents in mCRC patients with PC. METHODS: Previously untreated mCRC patients treated with chemotherapy in the CAIRO study and with chemotherapy and targeted therapy in the CAIRO2 study were included and retrospectively analysed according to presence or absence of PC at randomisation. Patient demographics, primary tumour characteristics, progression-free survival (PFS), overall survival (OS), and occurrence of toxicity were evaluated. RESULTS: Thirty-four patients with PC were identified in the CAIRO study and 47 patients in the CAIRO2 study. Median OS was decreased for patients with PC compared with patients without PC (CAIRO: 10.4 versus 17.3 months, respectively (p ≤ 0.001); CAIRO2: 15.2 versus 20.7 months, respectively (p < 0.001)). Median number of treatment cycles did not differ between patients with or without PC in both studies. Occurrence of major toxicity was more frequent in patients with PC treated with sequential chemotherapy in the CAIRO study as compared to patients without PC. This was not reflected in reasons to discontinue treatment. In the CAIRO2 study, no differences in major toxicity were observed. CONCLUSION: Our data demonstrate decreased efficacy of current standard chemotherapy with and without targeted agents in mCRC patients with PC. This suggests that the poor outcome cannot be explained by undertreatment or increased susceptibility to toxicity, but rather by relative resistance to treatment.

MicroRNA expression in formalin-fixed paraffin embedded tissue using real time quantitative PCR: the strengths and pitfalls.

MicroRNAs (miRNAs) are a group of small non-coding RNAs with a huge impact in a wide range of biological processes, including cancer. The evidence collected to date demonstrates that miRNAs represent valid diagnostic, prognostic and predictive markers in cancer. The identification of these miRNA biomarkers in archived tissues has been facilitated by novel development and refinement of detection methodologies. Quantitative real-time reverse-transcription PCR (qRT-PCR) is one of the most common methods used to detect low levels of miRNAs with high sensitivity and specificity. However, several technical parameters should be identified and optimized in order to obtain meaningful and reproducible results. The purpose of this review is to describe some of these technical parameters and improve the validity and reliability of miRNA expression studies.

KRAS mutation analysis: a comparison between primary tumours and matched liver metastases in 305 colorectal cancer patients.

BACKGROUND: KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor antibody in metastatic colorectal cancer (CRC). KRAS mutation analysis is usually performed on primary tumour tissue because metastatic tissue is often not available. However, controversial data are available on the concordance of test results between primary tumours and corresponding metastases. We assessed the concordance of KRAS mutation status in a study of 305 primary colorectal tumours and their corresponding liver metastases. METHODS: Patients with histologically confirmed CRC who underwent surgical resection of the primary tumour and biopsy or surgical resection of the corresponding liver metastasis were included. KRAS mutation analysis was performed for codons 12 and 13. RESULTS: KRAS mutation was detected in 108 out of 305 primary tumours (35.4%). In 11 cases (3.6%), we found a discordance between primary tumour and metastasis: 5 primary tumours had a KRAS mutation with a wild-type metastasis, 1 primary tumour was wild type with a KRAS mutation in the metastasis, and in 5 cases the primary tumour and the metastasis had a different KRAS mutation. CONCLUSION: We observed a high concordance of KRAS mutation status of 96.4% (95% CI 93.6-98.2%) between primary colorectal tumours and their corresponding liver metastases. In only six patients (2.0%; 95% CI 0.7-4.2%), the discordance was clinically relevant. In this largest and most homogenous study to date, we conclude that both primary tumours and liver metastases can be used for KRAS mutation analysis.

A new, simple and objective prognostic score for phase I cancer patients.

AIM: To ensure safety and optimise efficacy, careful patient selection for participation in oncologic phase I trials is warranted. Therefore, we did a validation study on existing phase I prognostic scores, and subsequently aimed to make an even more simple prognostic score. PATIENTS AND METHODS: We retrospectively analysed characteristics and clinical outcome of 122 patients who participated in eight different phase I studies in our centre. A literature search was performed for existing prognostic scores which were validated in our dataset. Additionally, a simple prognostic score able to predict overall survival (OS), progression free survival (PFS), 90-d mortality and duration of participation was developed. RESULTS: In our population the median OS was 31 (range 4-241) weeks, median PFS was 10 (range 3-119) weeks. Thirteen patients (11%) died within 90-d and median participation duration was 11 (range 1-119) weeks. Two out of five existing prognostic scores could be validated in this dataset for OS. Based on multivariate analyses a new, objective and simple score, consisting of LDH, sodium and haemoglobin, was tested. High score (2-3 points) compared to low score (0-1) significantly predicted OS (HR 1.878, p = 0.003), PFS (HR 1.156, p = 0.038), participation duration (HR 1.858, p = 0.004) and 90-d mortality (OR 4.200, p = 0.022). CONCLUSION: We propose a new prognostic model, using LDH, sodium and haemoglobin, helpful to predict OS, PFS, participation duration and 90-d mortality. Larger multicentre studies are needed to validate this score.

Systematic review of the effect of external beam radiation therapy to the breast on axillary recurrence after negative sentinel lymph node biopsy.

BACKGROUND: Axillary recurrence after negative sentinel lymph node biopsy (SLNB) in patients with invasive breast carcinoma remains a concern. Previous investigations to identify prognostic factors for axillary recurrence identified that a disproportionate number of patients with an axillary recurrence after negative SLNB were not treated with external beam radiation therapy (EBRT) of the breast as part of initial treatment. This finding prompted a systematic review to test the hypothesis that EBRT to the breast reduces the risk of axillary recurrence after negative SLNB. METHODS: A literature search was performed in PubMed, the Cochrane Library and the Spanish-language database LILACS to identify articles publishing data regarding follow-up of sentinel lymph node (SLN)-negative patients. Reports and articles lacking information on the initial treatment were excluded. RESULTS: Forty-five articles were accepted for review. A total of 23,357 SLN-negative patients were identified with median follow-up ranging from 15 to 102 months. Some 18,878 patients were treated with EBRT to the breast as part of their initial treatment. One hundred and twenty-seven patients with an axillary recurrence were identified, of whom 73 had EBRT as part of their initial treatment. Meta-analysis showed that EBRT was associated with a lower rate of axillary recurrence (P < 0·001), but this finding was subject to heterogeneity. CONCLUSION: This review and meta-analysis showed that EBRT is associated with a significantly lower axillary recurrence rate after negative SLNB.

The effect of prophylactic calcium and magnesium infusions on the incidence of neurotoxicity and clinical outcome of oxaliplatin-based systemic treatment in advanced colorectal cancer patients.

BACKGROUND: Peripheral sensory neurotoxicity is a frequent and potentially debilitating side effect of oxaliplatin treatment. Calcium and magnesium (Ca/Mg) infusions are frequently used to prevent this toxicity. However, concerns about a negative impact of Ca/Mg infusions on outcome have been raised. We retrospectively assessed the effect of Ca/Mg infusions on the incidence of neurotoxicity and on clinical outcome in advanced colorectal cancer (ACC) patients treated in the phase III CAIRO2 study. MATERIALS AND METHODS: Seven hundred and fifty five previously untreated ACC patients were randomised between treatment with capecitabine, oxaliplatin and bevacizumab or the same combination with the addition of cetuximab. Patients were retrospectively divided into two groups: patients in the Ca/Mg(+) group received Ca/Mg at least during their first treatment cycle, and patients in the Ca/Mg(-) group did not. RESULTS: Seven hundred and thirty two patients were evaluable for this analysis. The Ca/Mg(+) group consisted of 551 patients, the Ca/Mg(-) group consisted of 181 patients. The incidence of all grade neurotoxicity in the Ca/Mg(+) group and the Ca/Mg(-) group was 85% and 92%, respectively (p = 0.02), and the incidence of grade ≥ 2 neurotoxicity was 40% and 45%, respectively (p = 0.22). The median PFS in the Ca/Mg(+) versus Ca/Mg(-) group was 10.1 versus 10.7 months (p = 0.92), the median OS was 19.8 versus 20.7 months (p = 0.10), and the response rate was 43.1% versus 50% (p = 0.11), respectively. CONCLUSIONS: In this largest retrospective analysis to date we observed that Ca/Mg infusions significantly reduced all grade oxaliplatin-related neurotoxicity. Ca/Mg infusions did not affect the clinical efficacy of treatment.

Clinicopathological features and outcome in advanced colorectal cancer patients with synchronous vs metachronous metastases.

BACKGROUND: Synchronous metastases of colorectal cancer (CRC) are considered to be of worse prognostic value compared with metachronous metastases, but only few and conflicting data have been reported on this issue. METHODS: We retrospectively investigated patient demographics, primary tumour characteristics and overall survival (OS) in 550 advanced CRC patients with metachronous vs synchronous metastases, who participated in the phase III CAIRO study. For this purpose only patients with a prior resection of the primary tumour were considered. RESULTS: The clinical and pathological characteristics associated with poor prognosis that we observed more often in patients with synchronous metastases (n=280) concerned an abnormal serum lactate dehydrogenase (LDH) concentration (P=0.01), a worse WHO performance status (P=0.02), primary tumour localisation in the colon (P=0.002) and a higher T stage (P=0.0006). No significant difference in median OS was observed between patients with synchronous metastases and metachronous metastases (17.6 vs 18.5 months, respectively, P=0.24). CONCLUSION: Despite unfavourable clinicopathological features in patients with synchronous metastases with a resected primary tumour compared to patients with metachronous metastases, no difference in the median OS was observed. Possible explanations include a (partial) chemoresistance in patients with metachronous disease because of previous adjuvant treatment, whereas differences between the two groups in screening procedures resulting in a lead time bias to diagnosis or in prognostic molecular markers remain speculative.

The effect of hemodilution during normothermic cardiac surgery on renal physiology and function: a review.

Although the definitions of renal dysfunction vary, loss of renal function is a common complication following cardiac surgery using cardiopulmonary bypass (CPB). When postoperative dialysis is required, mortality is approximately 50%. CPB-accompanied hemodilution is a major contributing factor to renal damage as it notably reduces oxygen delivery by reducing the oxygen transport capacity of the blood as well as disturbing the microcirculation. To minimize hypoxemic damage during CPB, lowering of body temperature is applied to reduce the patient's metabolic rate. At present, however, temperature management during elective adult cardiac surgery is shifting from moderate hypothermia to normothermia. To determine whether the currently accepted levels of hemodilution during CPB can suffice the normothermic patient's high oxygen demand, we focused this study on renal physiology and postoperative renal function. Hemodilution reduces the capillary density through a diminished capillary viscosity, thereby, redistributing blood from the renal medulla to the renal cortex. As the physiology of the renal medulla makes it a hypoxic environment, this part of the kidney appears to be especially at risk for hypoxic damage caused by a hemodilution-induced lowered oxygen transport and oxygen delivery. In addition, hemodilution is also likely to disturb the hormonal systems regulating renal blood distribution. Clinical studies, mostly of retrospective or observational nature, show that perioperative nadir hematocrit levels lower than approximately 24% are associated with an increased risk to develop postoperative renal failure. A better comprehension of the cause-and-effect relation between low perioperative hematocrits and loss of postoperative renal function may enable more effective renal protective strategies.

Reliability of point-of-care hematocrit, blood gas, electrolyte, lactate and glucose measurement during cardiopulmonary bypass.

BACKGROUND: Recently, the GEM Premier blood gas analyser was upgraded to the GEM Premier 3000. In addition to pH, pCO2, pO2, Na+, K+, Ca2+, and hematocrit measurement, glucose and lactate can be measured on the GEM Premier 3000. In this prospective clinical study, the analytical performance of the GEM Premier 3000 was compared with the Ciba Corning 865 analyser for blood gas/electrolytes/metabolites, and for hematocrit with the Sysmex XE 2100 instrument. METHODS: During a 6-month period, 127 blood samples were analysed on both the GEM Premier 3000 analyser and our laboratory analysers (Ciba Corning 865/Sysmex 2100 instrument), and compared using the agreement analysis for quantitative data. RESULTS: With the exception of K+, the other parameters (pCO2, pO2, Na+, Ca2+, hematocrit, glucose, and lactate) can be described in terms of the mean and standard deviation of the differences. For K+ measurement, a clear linear trend (r=0.79, p<0.001) in the deviation of the GEM Premier 3000 from the Ciba Corning was noticed, ie, in the lower or upper K+ reference range, the GEM Premier 3000 measured systematically too low or too high, respectively. Furthermore, in comparison with the other parameters, a therapeutically unacceptable systematic difference (mean of difference: -2.2%, p=0.05) in hematocrit measurement on the GEM Premier 3000 was observed for hematocrit values below 30%. The variance of the readings for the GEM Premier 3000 measurements was at clinically acceptable levels. CONCLUSION: The GEM Premier 3000 analyser seems to be suitable for point-of-care testing of electrolytes, metabolites, and blood gases during cardiopulmonary bypass. However, its downward bias in hematocrit values below 30% suggests that using the GEM Premier 3000 as a transfusion trigger leads to overtreatment with packed red cells.

Leukocyte removal efficiency of cell-washed and unwashed whole blood: an in vitro study.

Leukocyte filtration of the cardiopulmonary bypass (CPB) perfusate after cardiac surgery has evolved as an important technique to prevent effector functions mediated by activated leukocytes. However, little is known about the filtration efficiency. Therefore, an in vitro study was conducted to define the leukocyte removal rate of a transfusion leukocyte-depletion filter, using cell-washed and unwashed whole porcine blood. In addition, the influence of different cell-washing protocols on the elimination rate of blood cells (leukocytes and platelets) was investigated. Fresh, diluted, pooled, heparinized, porcine blood was processed using either a high-flow (HF, n = 5) or quality-wash (QW, n = 5) protocol on a continuous auto-transfusion system, or was left unprocessed (control n=5). Thereafter, all samples were filtered using a transfusion leukocyte-depletion filter. Blood samples for measurement of hematocrit, white blood cell count, including leukocyte differentiation and platelet count, were taken before and after filtration. To compare the experimental groups, the removal rate was presented as the fraction of leukocytes or platelets removed per plasma volume. Cell washing significantly altered the fraction of leukocytes removed per plasma volume when compared to unprocessed blood (2.07 and 2.36 in the HF and QW groups, respectively, versus 1.34 in the control group, p = 0.008 for both). No statistically significant difference in leukocyte removal rate was observed between the different cell-washing protocols. The leukocyte differential count showed that, during all experiments, the neutrophils were removed most efficiently (99.7%). Overall, significantly more platelets were depleted after cell washing compared to the control group (1.47 and 1.60 in the HF and QW groups, respectively, versus 1.12 in the control group, p =0.008 and 0.032, respectively). Furthermore, the amount of blood that could be filtered using a single pass technique did not significantly differ between the experimental groups. However, a larger variation in the total amount of filtered blood was observed in the unprocessed group (570+/-398 mL) compared to the cell-washed groups (360+/-42 and 430+/-97 mL in the HF and QW groups, respectively). In conclusion, blood processing with an auto-transfusion system significantly enhances the leukocyte and platelet removal efficiency of the transfusion leukocyte-depletion filter that was studied. In particular, neutrophils were efficiently removed.