Quantitative comparison of immunohistochemical HER2-low detection in an interlaboratory study.

AIMS: Recently, human epidermal growth factor 2 (HER2)-low (i.e. HER2 score 1+ or 2+ without amplification) breast cancer patients became eligible for trastuzumab-deruxtecan treatment. To improve assay standardisation and detection of HER2-low in a quantitative manner, we conducted an external quality assessment-like study in the Netherlands. Dynamic range cell lines and immunohistochemistry (IHC) calibrators were used to quantify HER2 expression and to assess interlaboratory variability. METHODS AND RESULTS: Three blank slides with a dynamic range cell line and an IHC calibrator were stained with routine HER2 assays by 35 laboratories. Four different antibody clones were used: 19 (54.3%) 4B5, six (17.1%) A0485, five (14.3%) DG44 (HercepTest) and five (14.3%) SP3. Laboratories used two different detection kits for 4B5 assays: 14 (73.7%) ultraView and five (26.3%) OptiView. Variability of HER2 expression in cell lines, measured with artificial intelligence software, was median (min-max) = negative core 0.5% (0.0-57.0), 1+ core 4.3% (1.6-71.3), 2+ core 42.8% (30.4-92.6) and 3+ core 96.2% (91.8-98.8). The calibrators DG44 and 4B5 OptiView had the highest analytical sensitivity, closely followed by 4B5 ultraView. SP3 was the least sensitive. Calibrators of A0485 assays were not analysable due to background staining. CONCLUSIONS: As assays were validated for detecting HER2-amplified tumours, not all assays and antibodies proved suitable for HER2-low detection. Some tests showed distinct expression in the negative cell line. Dynamic range cell line controls and quantitative analysis using calibrators demonstrated more interlaboratory variability than commonly appreciated. Revalidation of HER2 tests by laboratories is needed to ensure clinical applicable HER2-low assays.

Quantitative comparison of PD-L1 IHC assays against NIST standard reference material 1934.

Companion diagnostic immunohistochemistry (IHC) tests are developed and performed without incorporating the tools and principles of laboratory metrology. Basic analytic assay parameters such as lower limit of detection (LOD) and dynamic range are unknown to both assay developers and end users. We solved this problem by developing completely new tools for IHC-calibrators with units of measure traceable to National Institute of Standards & Technology (NIST) Standard Reference Material (SRM) 1934. In this study, we demonstrate the clinical impact and opportunity for incorporating these changes into PD-L1 testing. Forty-one laboratories in North America and Europe were surveyed with newly-developed PD-L1 calibrators. The survey sampled a broad representation of commercial and laboratory-developed tests (LDTs). Using the PD-L1 calibrators, we quantified analytic test parameters that were previously only inferred indirectly after large clinical studies. The data show that the four FDA-cleared PD-L1 assays represent three different levels of analytic sensitivity. The new analytic sensitivity data explain why some patients' tissue samples were positive by one assay and negative by another. The outcome depends on the assay's lower LOD. Also, why previous attempts to harmonize certain PD-L1 assays were unsuccessful; the assays' dynamic ranges were too disparate and did not overlap. PD-L1 assay calibration also clarifies the exact performance characteristics of LDTs relative to FDA-cleared commercial assays. Some LDTs' analytic response curves are indistinguishable from their predicate FDA-cleared assay. IHC assay calibration represents an important transition for companion diagnostic testing. The new tools will improve patient treatment stratification, test harmonization, and foster accuracy as tests transition from clinical trials to broad clinical use.

Value of 3D mapping-guided endomyocardial biopsy in cardiac sarcoidosis: Case series and narrative review on the value of electro-anatomic mapping-guided endomyocardial biopsies.

AIM: Integration of endomyocardial biopsy (EMB) in the diagnostic workup of cardiac sarcoidosis (CS) is under-recognized in current clinical practice, since capturing focal granulomas is challenging. Our aim was to describe our experience with electro-anatomic mapping (EAM)-guided EMB and provide a comprehensive review of the literature. METHODS AND RESULTS: Five patients (age 49.4 ± 11.4) with suspected CS underwent EAM-guided EMB in Isala Heart Center (Zwolle, the Netherlands) between 2017 and 2019. In all patients, a 3D bipolar voltage map (<0.5-1.5 mV) and unipolar voltage map (LV < 8.3 mV, RV < 5.5 mV) was created using a high-density mapping catheter. The bioptome was connected to the mapping system to guide targeted EMB. Biopsy samples (2-9 samples) were taken from both LV and RV sites, guided by EAM and areas with abnormal electrograms, without complications. CS diagnosis was based on EMB in 2/5 patients. A granuloma was captured in one patient at the LV basal septum with normal bipolar and abnormal unipolar voltage. All patients with delayed enhancement on cardiac magnetic resonance, revealed fibrosis in the biopsy sample. In one patient with suspected isolated cardiac sarcoidosis, diagnosis could not be confirmed by histopathology analysis, while unipolar voltage mapping was abnormal and diastolic potentials were present. Literature search revealed 7 reports (18 patients) describing EAM-guided EMB in CS patients, with 100% of the EMB taken form the RV. CONCLUSION: Unipolar voltage mapping may be superior to target active inflamed tissue and should be evaluated in future research regarding EAM-guided EMB in CS.

Sensitive detection methods are key to identify secondary EGFR c.2369C>T p.(Thr790Met) in non-small cell lung cancer tissue samples.

BACKGROUND: Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. METHODS: Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for which an outcome was available and correct. Additional data on the used test method were collected from the participants. Chi-squared tests on contingency tables and a biserial rank correlation were applied by IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). RESULTS: In 26 of the 1190 tests (2.2%) a technical failure occurred. For the remaining 1164 results, 1008 (86.6%) were correct, 151 (12.9%) were false-negative and 5 (0.4%) included incorrect mutations. Correct p.(Thr790Met) detection improved over time and for repeated scheme participations. In-house non-next-generation sequencing (NGS) techniques performed worse (81.1%, n = 293) compared to non-NGS commercial kits (85.2%, n = 656) and NGS (97.0%, n = 239). Over time there was an increase in the users of NGS. Resection specimens performed worse (82.6%, n = 610 tests) compared to cell line material (90.9%, n = 578 tests), except for NGS (96.3%, n = 344 for resections and 98.6%, n = 312 for cell lines). Samples with multiple mutations were more difficult compared to samples with the single p.(Thr790Met) variant. A change of the test method was shown beneficial to reduce errors but introduced additional analysis failures. CONCLUSIONS: A significant number of laboratories that offer p.(Thr790Met) testing did not detect this relevant mutation compared to the other EQA participants. However, correct identification of this variant is improving over time and was higher for NGS users. Revising the methodology might be useful to resolve errors, especially for resection specimens with low frequency or multiple variants. EQA providers should include challenging resections in the scheme.

Infusing Mesenchymal Stromal Cells into Porcine Kidneys during Normothermic Machine Perfusion: Intact MSCs Can Be Traced and Localised to Glomeruli.

Normothermic machine perfusion (NMP) of kidneys offers the opportunity to perform active interventions, such as the addition of mesenchymal stromal cells (MSCs), to an isolated organ prior to transplantation. The purpose of this study was to determine whether administering MSCs to kidneys during NMP is feasible, what the effect of NMP is on MSCs and whether intact MSCs are retained in the kidney and to which structures they home. Viable porcine kidneys were obtained from a slaughterhouse. Kidneys were machine perfused during 7 h at 37 °C. After 1 h of perfusion either 0, 105, 106 or 107 human adipose tissue derived MSCs were added. Additional ex vivo perfusions were conducted with fluorescent pre-labelled bone-marrow derived MSCs to assess localisation and survival of MSCs during NMP. After NMP, intact MSCs were detected by immunohistochemistry in the lumen of glomerular capillaries, but only in the 107 MSC group. The experiments with fluorescent pre-labelled MSCs showed that only a minority of glomeruli were positive for infused MSCs and most of these glomeruli contained multiple MSCs. Flow cytometry showed that the number of infused MSCs in the perfusion circuit steeply declined during NMP to approximately 10%. In conclusion, the number of circulating MSCs in the perfusate decreases rapidly in time and after NMP only a small portion of the MSCs are intact and these appear to be clustered in a minority of glomeruli.

Validation of a whole-slide image-based teleconsultation network.

AIMS: Most validation studies on digital pathology diagnostics have been performed in single institutes. Because rapid consultation on cases with extramural experts is one of the most important uses for digital pathology laboratory networks, the aim of this study was to validate a whole-slide image-based teleconsultation network between three independent laboratories. METHODS AND RESULTS: Each laboratory contributed 30 biopsies and/or excisions, totalling 90 specimens (776 slides) of varying difficulty and covering a wide variety of organs and subspecialties. All slides were scanned centrally at ×40 scanning magnification and uploaded, and subsequently assessed digitally by 16 pathologists using the same image management system and viewer. Each laboratory was excluded from digital assessment of their own cases. Concordance rates between the two diagnostic modalities (light microscopic versus digital) were compared. Loading speed of the images, zooming latency and focus quality were scored. Leaving out eight minor discrepancies without any clinical significance, the concordance rate between remote digital and original microscopic diagnoses was 97.8%. The two cases with a major discordance (for which the light microscopic diagnoses were deemed to be the better ones) resulted from a different interpretation of diagnostic criteria in one case and an image quality issue in the other case. Average scores for loading speed of the images, zooming latency and focus quality were 2.37 (on a scale up to 3), 2.39 (scale up to 3) and 3.06 (scale up to 4), respectively. CONCLUSIONS: This validation study demonstrates the suitability of a teleconsultation network for remote digital consultation using whole-slide images. Such networks may contribute to faster revision and consultation in pathology while maintaining diagnostic standards.

Chemotherapy resistant Burkitt lymphoma: Possible novel therapies including CAR-T cell infusion.

Key Clinical Message: The prognosis of patients with relapsed or refractory (R/R) BL is poor with limited response to salvage therapy, especially for patients with early relapse (<6 months) or refractory disease. Novel therapies may be promising but need refinement. Abstract: The prognosis of patients with relapsed or refractory (R/R) BL is poor with a limited response to salvage therapy, especially for patients with early relapse (<6 months) or refractory disease. We present three cases of patients with R/R BL receiving novel therapies followed by a literature review. Amongst others, the patients received inotuzumab ozogamicin, idelalisib, ibrutinib, and CAR-T cell therapy, however, with limited response. In literature, response to several novel agents is described; however, most promising results are seen with CAR-T cell therapy. Concluding from case series, sequential CAR-T cell therapy, targeting multiple B-cell antigens, seems most promising.

A 20-Year-Old Woman With Thoracic Pain and Progressive Dyspnea on Exertion.

CASE PRESENTATION: A 20-year-old woman presented with dry cough, right-sided thoracic pain, and gradually progressive dyspnea on exertion. She had no hemoptysis or fever. There was no relevant medical history. She was a never smoker and used no medication besides oral contraceptives. There were no other risk factors for a pulmonary embolism. There was a family history of ovarian and breast cancer. Physical examination showed a mildly ill-looking woman, with shallow breathing and normal blood oxygen saturation. Auscultation revealed normal breath sounds without crackles or wheezing. Laboratory testing showed a significantly increased D-dimer (4,560 µg/L [normal, < 500 µg/L]), elevated C-reactive protein (131 mg/L [normal, < 5 mg/L]), normal leucocytes, and elevated lactate dehydrogenase (825 units/L [normal, 50 to 250 units/L).

A Consortium for Analytic Standardization in Immunohistochemistry.

CONTEXT.­: The authors announce the launch of the Consortium for Analytic Standardization in Immunohistochemistry, funded with a grant from the National Cancer Institute. As with other laboratory testing, analytic standards are important for many different stakeholders: commercial vendors of instruments and reagents, biopharmaceutical firms, pathologists, scientists, clinical laboratories, external quality assurance organizations, and regulatory bodies. Analytic standards are customarily central to assay development, validation, and method transfer into routine assays and are critical quality assurance tools. OBJECTIVE.­: To improve immunohistochemistry (IHC) test accuracy and reproducibility by integrating analytic standards into routine practice. To accomplish this mission, the consortium has 2 mandates: (1) to experimentally determine analytic sensitivity thresholds (lower and upper limits of detection) for selected IHC assays, and (2) to inform IHC stakeholders of what analytic standards are, why they are important, and how and for what purpose they are used. The consortium will then publish the data and offer analytic sensitivity recommendations where appropriate. These mandates will be conducted in collaboration and coordination with clinical laboratories, external quality assurance programs, and pathology organizations. DATA SOURCES.­: Literature review and published external quality assurance data. CONCLUSIONS.­: Integration of analytic standards is expected to (1) harmonize and standardize IHC assays; (2) improve IHC test accuracy and reproducibility, both within and between laboratories; and (3) dramatically simplify and improve methodology transfer for new IHC protocols from published literature or clinical trials to clinical IHC laboratories.

The impact of a pathologist's personality on the interobserver variability and diagnostic accuracy of predictive PD-L1 immunohistochemistry in lung cancer.

OBJECTIVES: Programmed death-ligand 1 (PD-L1) is the only approved predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). However, predictive PD-L1 immunohistochemistry is subject to interobserver variability. We hypothesized that a pathologist's personality influences the interobserver variability and diagnostic accuracy of PD-L1 immunoscoring. MATERIALS AND METHODS: Seventeen pathologists performed PD-L1 immunoscoring on 50 resected NSCLC tumors in three categories (<1%;1-49%;≥50%). Also, the pathologists completed a certified personality test (NEO-PI-r), assessing five personality traits: neuroticism, extraversion, openness, altruism and conscientiousness. RESULTS: The overall agreement among pathologists for a series of 47 tumors was substantial (kappa = 0.63). Of these, 23/47 (49%) tumors were entirely negative or largely positive, resulting in a kappa value of 0.93. The remaining 24/47 (51%) tumors had a PD-L1 score around the cutoff value, generating a kappa value of 0.32. Pathologists with high scores for conscientiousness (careful, diligent) had the least interobserver variability (r = 0.6, p = 0.009). Also, they showed a trend towards higher sensitivity (74% vs. 68%, p = 0.4), specificity (86% vs. 82%, p = 0.3) and percent agreement (83% vs. 79%, p = 0.3), although not significant. In contrast, pathologists with high scores for neuroticism (sensitive, anxious) had significantly lower specificity (80% vs. 87%, p = 0.03) and percent agreement (78% vs. 85%, p = 0.03). Also, a trend towards high interobserver variability (r = -0.3, p = 0.2) and lower sensitivity (68% vs. 74%, p = 0.3) was observed, although not significant. Pathologists with relatively high scores for conscientiousness scored fewer tumors PD-L1 positive at the ≥ 1% cut-off (r = -0.5, p = 0.03). In contrast, pathologists with relatively high scores for neuroticism score more tumors PD-L1 positive at ≥ 1% (r = 0.6, p = 0.017) and ≥ 50% cut-offs (r = 0.6, p = 0.009). CONCLUSIONS: This study is the first to demonstrate the impact of a pathologist's personality on the interobserver variability and diagnostic accuracy of immunostaining, in the context of PD-L1 in NSCLC. Larger studies are needed for validation of these findings.

PD-L1 immunohistochemistry in non-small-cell lung cancer: unraveling differences in staining concordance and interpretation.

Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is accepted as a predictive biomarker for the selection of immune checkpoint inhibitors. We evaluated the staining quality and estimation of the tumor proportion score (TPS) in non-small-cell lung cancer during two external quality assessment (EQA) schemes by the European Society of Pathology. Participants received two tissue micro-arrays with three (2017) and four (2018) cases for PD-L1 IHC and a positive tonsil control, for staining by their routine protocol. After the participants returned stained slides to the EQA coordination center, three pathologists assessed each slide and awarded an expert staining score from 1 to 5 points based on the staining concordance. Expert scores significantly (p < 0.01) improved between EQA schemes from 3.8 (n = 67) to 4.3 (n = 74) on 5 points. Participants used 32 different protocols: the majority applied the 22C3 (56.7%) (Dako), SP263 (19.1%) (Ventana), and E1L3N (Cell Signaling) (7.1%) clones. Staining artifacts consisted mainly of very weak or weak antigen demonstration (63.0%) or excessive background staining (19.8%). Participants using CE-IVD kits reached a higher score compared with those using laboratory-developed tests (LDTs) (p < 0.05), mainly attributed to a better concordance of SP263. The TPS was under- and over-estimated in 20/423 (4.7%) and 24/423 (5.7%) cases, respectively, correlating to a lower expert score. Additional research is needed on the concordance of less common protocols, and on reasons for lower LDT concordance. Laboratories should carefully validate all test methods and regularly verify their performance. EQA participation should focus on both staining concordance and interpretation of PD-L1 IHC.

Formalin fixation for optimal concordance of programmed death-ligand 1 immunostaining between cytologic and histologic specimens from patients with non-small cell lung cancer.

BACKGROUND: Immunohistochemical staining of programmed death-ligand 1 (PD-L1) is used to determine which patients with non-small cell lung cancer (NSCLC) may benefit most from immunotherapy. Therapeutic management of many patients with NSCLC is based on cytology instead of histology. In this study, concordance of PD-L1 immunostaining between cytology cell blocks and their histologic counterparts was analyzed. Furthermore, the effect of various fixatives and fixation times on PD-L1 immunoreactivity was studied. METHODS: Paired histologic and cytologic samples from 67 patients with NSCLC were collected by performing fine-needle aspiration on pneumonectomy/lobectomy specimens. Formalin-fixed, agar-based or CytoLyt/PreservCyt-fixed Cellient cell blocks were prepared. Sections from cell blocks and tissue blocks were stained with SP263 (standardized assay) and 22C3 (laboratory-developed test) antibodies. PD-L1 scores were compared between histology and cytology. In addition, immunostaining was compared between PD-L1-expressing human cell lines fixed in various fixatives at increasing increments in fixation duration. RESULTS: Agar cell blocks and tissue blocks showed substantial agreement (κ = 0.70 and κ = 0.67, respectively), whereas fair-to-moderate agreement was found between Cellient cell blocks and histology (κ = 0.28 and κ = 0.49, respectively). Cell lines fixed in various alcohol-based fixatives showed less PD-L1 immunoreactivity compared with those fixed in formalin. In contrast to SP263, additional formalin fixation after alcohol fixation resulted in preserved staining intensity using the 22C3 laboratory-developed test and the 22C3 pharmDx assay. CONCLUSIONS: Performing PD-L1 staining on cytologic specimens fixed in alcohol-based fixatives could result in false-negative immunostaining results, whereas fixation in formalin leads to higher and more histology-concordant PD-L1 immunostaining. The deleterious effect of alcohol fixation could be reversed to some degree by postfixation in formalin.

Methylprednisolone Treatment in Brain Death-Induced Lung Inflammation-A Dose Comparative Study in Rats.

Background: The process of brain death (BD) leads to a pro-inflammatory state of the donor lung, which deteriorates its quality. In an attempt to preserve lung quality, methylprednisolone is widely recommended in donor lung management. However, clinical treatment doses vary and the dose-effect relation of methylprednisolone on BD-induced lung inflammation remains unknown. The aim of this study was to investigate the effect of three different doses methylprednisolone on the BD-induced inflammatory response. Methods: BD was induced in rats by inflation of a Fogarty balloon catheter in the epidural space. After 60 min of BD, saline or methylprednisolone (low dose (5 mg/kg), intermediate dose (12.5 mg/kg) or high dose (22.5 mg/kg)) was administered intravenously. The lungs were procured and processed after 4 h of BD. Inflammatory gene expressions were analyzed by RT-qPCR and influx of neutrophils and macrophages were quantified with immunohistochemical staining. Results: Methylprednisolone treatment reduced neutrophil chemotaxis as demonstrated by lower IL-8-like CINC-1 and E-selectin levels, which was most evident in rats treated with intermediate and high doses methylprednisolone. Macrophage chemotaxis was attenuated in all methylprednisolone treated rats, as corroborated by lower MCP-1 levels compared to saline treated rats. Thereby, all doses methylprednisolone reduced TNF-α, IL-6 and IL-1ß tissue levels. In addition, intermediate and high doses methylprednisolone induced a protective anti-inflammatory response, as reflected by upregulated IL-10 expression when compared to saline treated brain-dead rats. Conclusion: We showed that intermediate and high doses methylprednisolone share most potential to target BD-induced lung inflammation in rats. Considering possible side effects of high doses methylprednisolone, we conclude from this study that an intermediate dose of 12.5 mg/kg methylprednisolone is the optimal treatment dose for BD-induced lung inflammation in rats, which reduces the pro-inflammatory state and additionally promotes a protective, anti-inflammatory response.

Multicentre study on the consistency of PD-L1 immunohistochemistry as predictive test for immunotherapy in non-small cell lung cancer.

AIMS: Investigate the impact of interlaboratory- and interobserver variability of immunohistochemistry on the assessment of programmed death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC). METHODS: Two tissue microarrays (TMAs) were constructed from 50 (TMA-A) and 51 (TMA-B) resected NSCLC cases, and distributed among eight centres. Immunostaining for PD-L1 was performed using Agilent's 22C3 pharmDx Assay (pharmDx) and/or a 22C3 laboratory developed test (LDT). The interlaboratory variability of staining- and interobserver variability of scoring for PD-L1 were assessed in selected critical samples (samples at the cut-off of positivity) and non-critical samples. Also, PD-L1 epitope deterioration in time in stored unstained slides was analysed. Krippendorff's alpha values (0=maximal, 1=no variability) were calculated as measure for variability. RESULTS: For interlaboratory variability of immunostaining, the percentage of PD-L1 positive cases among centres ranged 40%-51% (1% cut-off) and 23%-30% (50% cut-off). Alpha values at 1% cut-off were 0.88 (pharmDx) and 0.87 (LDT) and at 50% cut-off 0.82 (pharmDx) and 0.95 (LDT). Interobserver variability of scoring resulted in PD-L1 positive cases ranging 29%-55% (1% cut-off) and 14%-30% (50% cut-off) among pathologists. Alpha values were at 1% cut-off 0.83 (TMA-A) and 0.66 (TMA-B), and at 50% cut-off 0.77 (TMA-A) and 0.78 (TMA-B). Interlaboratory variability of staining was higher (p<0.001) in critical samples than in non-critical samples at 50% cut-off. Furthermore, PD-L1 epitope deterioration in unstained slides was observed after 12 weeks. CONCLUSIONS: The results provide insight in factors contributing to variability of immunohistochemical assessment of PD-L1, and contribute to more reliable predictive testing for PD-L1.

Staining Performance of ALK and ROS1 Immunohistochemistry and Influence on Interpretation in Non-Small-Cell Lung Cancer.

Selection of non-small-cell lung cancer patients for treatment relies on the detection of expression of anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1) protein by immunohistochemistry (IHC). We evaluated staining performance for different IHC protocols and laboratory characteristics, and their influence on ALK and ROS1 interpretation during external quality assessment schemes between 2015 and 2018. Participants received five formalin-fixed, paraffin-embedded cases for staining by their routine protocol, whereafter at least two pathologists scored them simultaneously under a multihead microscope and awarded a graded expert staining score (ESS) from 1 to 5 points based on staining quality. European Conformity in Vitro Diagnostic kits (such as D5F3) revealed a better ALK ESS compared with laboratory-developed tests. ESS was indifferent to the applied antibody dilution or a recent protocol change. Lower ESSs were observed for higher antibody incubation times and temperatures. ESS for various ROS1 protocols were largely similar. Overall, for both markers, ESS improved over time and for repeated external quality assessment participation but was independent of laboratory setting or experience. Except for ROS1, ESS positively correlated with laboratory accreditation. IHC stains with lower ESS correlated with increased error rates in ALK and ROS1 interpretation and analysis failures. Laboratory characteristics differently affected staining quality and interpretation, and laboratories should assess both aspects, and less common protocols need improvement in staining performance.

Effects of Oxygen During Long-term Hypothermic Machine Perfusion in a Porcine Model of Kidney Donation After Circulatory Death.

BACKGROUND: Hypothermic machine perfusion (HMP) has become standard care in many center's to preserve kidneys donated after circulatory death (DCD). Despite a significant reduction in metabolism at low temperatures, the remaining cellular activity requires oxygen. Because of the role and safety of oxygen during HMP has not been fully clarified, its supply during HMP is not standard yet. This study investigates the effect of administering oxygen during HMP on renal function in a porcine DCD model. METHODS: After 30 minutes of warm ischemia, porcine slaughterhouse kidneys were preserved for 24 hours by means of cold storage (CS), or HMP with Belzer Machine Perfusion Solution supplemented with no oxygen, 21% or 100% oxygen. Next, kidneys were reperfused for 4 hours in a normothermic machine perfusion setup. RESULTS: HMP resulted in significantly better kidney function during normothermic machine perfusion. Thiobarbituric acid-reactive substances, markers of oxidative stress, were significantly lower in HMP preserved kidneys. HMP preserved kidneys showed significantly lower aspartate aminotransferase and lactate dehydrogenase levels compared with kidneys preserved by CS. No differences were found between the HMP groups subjected to different oxygen concentrations. Adenosine triphosphate levels significantly improved during HMP when active oxygenation was applied. CONCLUSIONS: This study showed that preservation of DCD kidneys with HMP is superior to CS. Although the addition of oxygen to HMP did not result in significantly improved renal function, beneficial effects were found in terms of reduced oxidative stress and energy status. Oxygen addition proofed to be safe and did not show detrimental effects.