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.

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).

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.

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.

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.

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.

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.

Implementation of Novel Molecular Biomarkers for Non-small Cell Lung Cancer in the Netherlands: How to Deal With Increasing Complexity.

The diagnostic landscape of non-small cell lung cancer (NSCLC) is changing rapidly with the availability of novel treatments. Despite high-level healthcare in the Netherlands, not all patients with NSCLC are tested with the currently relevant predictive tumor markers that are necessary for optimal decision-making for today's available targeted or immunotherapy. An expert workshop on the molecular diagnosis of NSCLC involving pulmonary oncologists, clinical chemists, pathologists, and clinical scientists in molecular pathology was held in the Netherlands on December 10, 2018. The aims of the workshop were to facilitate cross-disciplinary discussions regarding standards of practice, and address recent developments and associated challenges that impact future practice. This paper presents a summary of the discussions and consensus opinions of the workshop participants on the initial challenges of harmonization of the detection and clinical use of predictive markers of NSCLC. A key theme identified was the need for broader and active participation of all stakeholders involved in molecular diagnostic services for NSCLC, including healthcare professionals across all disciplines, the hospitals and clinics involved in service delivery, healthcare insurers, and industry groups involved in diagnostic and treatment innovations. Such collaboration is essential to integrate different technologies into molecular diagnostics practice, to increase nationwide patient access to novel technologies, and to ensure consensus-preferred biomarkers are tested.

Hypothermic Oxygenated Machine Perfusion of the Human Donor Pancreas.

BACKGROUND: Transplantation of beta cells by pancreas or islet transplantation is the treatment of choice for a selected group of patients suffering from type 1 diabetes mellitus. Pancreata are frequently not accepted for transplantation, because of the relatively high vulnerability of these organs to ischemic injury. In this study, we evaluated the effects of hypothermic machine perfusion (HMP) on the quality of human pancreas grafts. METHODS: Five pancreata derived from donation after circulatory death (DCD) and 5 from donation after brain death (DBD) donors were preserved by oxygenated HMP. Hypothermic machine perfusion was performed for 6 hours at 25 mm Hg by separate perfusion of the mesenteric superior artery and the splenic artery. Results were compared with those of 10 pancreata preserved by static cold storage. RESULTS: During HMP, homogeneous perfusion of the pancreas could be achieved. Adenosine 5'-triphosphate concentration increased 6,8-fold in DCD and 2,6-fold in DBD pancreata. No signs of cellular injury, edema or formation of reactive oxygen species were observed. Islets of Langerhans with good viability and in vitro function could be isolated after HMP. CONCLUSIONS: Oxygenated HMP is a feasible and safe preservation method for the human pancreas that increases tissue viability.

Response to HER2 Inhibition in a Patient With Brain Metastasis With EGFR TKI Acquired Resistance and an HER2 Amplification.

A 62-year-old man was referred to our university hospital for treatment of advanced adenocarcinoma of the lung after disease progression on two lines of EGFR TKI and one line of chemotherapy. Fluorescent in situ hybridization analysis upon progression showed an HER2 amplification. At our weekly Molecular Tumor Board (MTB), a decision was made to treat this patient with afatinib, which resulted in a partial response. However, progression was observed with a facial nerve paresis due to a metastasis in the skull. A biopsy of a location in the thorax revealed the presence of an EGFR-T790M mutation associated with acquired resistance, after which treatment with osimertinib was started. After 6 months, disease progression was observed, and a new biopsy was taken from the pelvic bone, which revealed the original amplification of HER2 together with the EGFR-L858R mutation, the EGFR-T790M mutation was not detected. The MTB decided to treat the patient with trastuzumab/paclitaxel. A partial response was observed in different bone lesions, while the skull metastasis with ingrowth in the brain remained stable for 6 months. Because of progression of the bone metastases after 6 months, a biopsy of a lesion in the thorax wall was taken. In this lesion, the EGFR-T790M mutation could be detected again. The MTB advised to start treatment with a combination of osimertinib and afatinib. This resulted in an impressive clinical improvement and a partial response of the bone metastases on the most recent 18-fluorodeoxyglucose positron emission tomography and computer tomography-scan. In conclusion, adjusting treatment to the mutational make-up of the tumor is a great challenge. For optimal treatment response multiple biopsies and re-biopsy upon progression are imperative. As more genes are investigated, treatment decision becomes increasingly difficult, therefore, expert opinions from an MTB is essential.

Improved kidney graft function after preservation using a novel hypothermic machine perfusion device.

OBJECTIVE: To study graft function and ischemia/reperfusion injury of porcine kidneys after preservation with the new Groningen Machine Perfusion (GMP) system versus static cold storage (CS). INTRODUCTION: The increasing proportion of marginal and nonheart beating donors necessitates better preservation methods to maintain adequate graft viability. Hypothermic machine preservation (HMP) is a promising alternative to static CS. We have therefore developed and tested an HMP device, which is portable and actively oxygenates the perfusate via an oxygenator. The aim of the present study was to examine the efficacy of the GMP system in a transplantation experiment. MATERIALS AND METHODS: In a porcine autotransplantation model, kidneys were retrieved and either cold stored in University of Wisconsin CS for 20 hours at 4 degrees C or subjected to HMP using University of Wisconsin machine perfusion at 4 degrees C with 2 different pressure settings: 30/20 mm Hg or 60/40 mm Hg. RESULTS: HMP at 30/20 mm Hg was found to better preserve the viability of kidneys reflected by improved cortical microcirculation, less damage to the proximal tubule, less damage mediated by reactive oxygen species, less proinflammatory cytokine expression, and better functional recovery after transplantation. However, high perfusion pressures (60/40 mm Hg) resulted in higher expression of von Willebrand factor and monocyte chemotactic peptide-1 in postpreservation biopsies and subsequent graft thrombosis in 2 kidneys. CONCLUSIONS: It is concluded that the GMP system improves kidney graft viability and perfusion pressures are critically important for outcome.

Oxygenation during hypothermic rat liver preservation: an in vitro slice study to demonstrate beneficial or toxic oxygenation effects.

Hypothermic machine perfusion (HMP) of abdominal organs is shown to be superior compared to cold storage. However, the question remains if oxygenation is required during preservation as oxygen is essential for energy resynthesis but also generates toxic reactive oxygen species (ROS). To determine if oxygenation should be used during HMP, urea-synthesis rate, adenosine triphosphate (ATP), and generation of ROS were studied in an in vitro model, modeling ischemia-reperfusion injury. Furthermore, expression of uncoupling protein-2 (UCP-2) mRNA was assessed since UCP-2 is a potentially protective protein against ROS. Rat liver slices were preserved for 0, 24, and 48 hr in University of Wisconsin machine perfusion solution (UW-MP) with 0%, 21%, or 95% oxygen at 0-4 degrees C and reperfused for 24 hours. In the 0% and 95% groups, an increase of ROS was found after cold storage in UW-MP. After slice reperfusion, only the 0% oxygen group showed higher levels. The 0% group showed a lower urea-synthesis rate as well as lower ATP levels. mRNA upregulation of UCP-2 was, in contrast to kidney mRNA studies, not observed. In conclusion, oxygenation of UW-MP gave better results. This study also shows that ROS formation occurs during hypothermic preservation and the liver is not protected by UCP-2. We conclude that saturation of UW-MP with 21% oxygen allows optimal preservation results.

Initial blood washout during organ procurement determines liver injury and function after preservation and reperfusion.

Organ procurement is the first step toward effective liver preservation and comprises a thorough washout of blood components from the microvasculature. To study the efficacy of optimal blood washout of the liver, three groups were compared including low-pressure perfusion with UW-CSS (12 mmHg, group A), which is the routine method in clinical practice, high-pressure perfusion with UW-CSS (100 mmHg, group B) and low-pressure perfusion with modified UW solution (12 mmHg, group C). After procurement all livers were preserved in original UW-CSS for 0, 24 or 48 h, followed by reperfusion in oxygenated Williams Medium E for 24 h at 37 degrees C. Histology results of livers procured in group A, showed good hepatocyte viability but also remaining erythrocytes. However, injury parameters were high and ATP concentrations were low. No functional differences were found. Group B, high pressure, and group C, modified UW-CSS, both showed better results. High-pressure washout is preferable since the warm ischemia time during procurement is short. We propose to use high-pressure UW-CSS perfusion for the initial blood washout of the donor liver instead of the usually used low-pressure washout.

Effect of University of Wisconsin organ-preservation solution on haemorheology.

In conventional cold-storage organ preservation, the donor organ is flushed with University of Wisconsin (UW) solution at 0-4 degrees C. The initial flush is used to wash out blood from the microcirculation to allow optimal preservation with the UW solution. The component hydroxyethyl starch (HES) of UW is known to cause relatively high viscosity and a possible interaction with blood, i.e. increased red blood cell (RBC) aggregation. The aim of this study was to investigate the influence of the HES component on the viscosity of UW and the aggregation behaviour of blood during washout. Viscosity aspects were measured with a cone-plate rheometer. HES-induced RBC aggregation was studied by means of an optical aggregation measuring device. The experiments were carried out with rat whole blood and mixtures of rat whole blood with UW-solution and UW without HES (UWmod), at 4 degrees C. The viscosity of blood at 4 degrees C is two-times higher than at 37 degrees C; the UW/blood mixture at 4 degrees C is 1.3-times more viscous than blood at 37 degrees C; the 4 degrees C UWmod/blood mixture equals the viscosity of blood at 37 degrees C. The UW/blood mixture shows a ninefold increased aggregation compared with whole blood. These aggregates are larger than the diameter of the sinusoids in the rat liver. A mixture of whole blood and UWmod shows a lower aggregation than blood. Apart from an increased viscosity, HES in UW causes increased RBC aggregation. The aggregates are larger than the diameter of the sinusoids. Initial washout could be optimised by pre-flushing to improve the viability of the liver and to decrease delayed graft function.

Hyperaggregating effect of hydroxyethyl starch components and University of Wisconsin solution on human red blood cells: a risk of impaired graft perfusion in organ procurement?

BACKGROUND: The standard preservation solution used during organ procurement and preservation of most organs is the University of Wisconsin (UW) solution. Despite its superiority over other cold storage solutions, the inclusion of hydroxyethyl starch (HES) as one of the components of the UW solution has been both advocated and denied. This study determined whether HES had any effect on red blood cell (RBC) aggregability and correlated aggregation parameters with HES molecular weight. METHODS: Human RBC aggregability and deformability were investigated in vitro, at 4 degrees C, with a laser-assisted optical rotation cell analyzer. The study of RBC aggregation in a binary HES-HES system gave an indication about the nature of HES-RBCs interactions. Bright field microscopy and atomic force microscopy were used to morphologically characterize the aggregates size and form. RESULTS: High molecular weight HES and UW solution had a potent hyperaggregating effect; low molecular weight HES had a hypoaggregating effect on RBC. RBC aggregates were of large size and their resistance to dissociation by flow-induced shear stress was high. CONCLUSION: The authors' in vitro experiments conclusively showed that the physiologic function of RBCs to form aggregates is significantly affected in the presence of HES. The use of high molecular weight HES in UW solution accounts for extended and accelerated aggregation of erythrocytes that may result in stasis of blood and incomplete washout of donor organs before transplantation.