Expansion microscopy of apicomplexan parasites.

Apicomplexan parasites comprise significant pathogens of humans, livestock and wildlife, but also represent a diverse group of eukaryotes with interesting and unique cell biology. The study of cell biology in apicomplexan parasites is complicated by their small size, and historically this has required the application of cutting-edge microscopy techniques to investigate fundamental processes like mitosis or cell division in these organisms. Recently, a technique called expansion microscopy has been developed, which rather than increasing instrument resolution like most imaging modalities, physically expands a biological sample. In only a few years since its development, a derivative of expansion microscopy known as ultrastructure-expansion microscopy (U-ExM) has been widely adopted and proven extremely useful for studying cell biology of Apicomplexa. Here, we review the insights into apicomplexan cell biology that have been enabled through the use of U-ExM, with a specific focus on Plasmodium, Toxoplasma and Cryptosporidium. Further, we summarize emerging expansion microscopy modifications and modalities and forecast how these may influence the field of parasite cell biology in future.

Ultrastructure expansion microscopy reveals the cellular architecture of budding and fission yeast.

The budding and fission yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as invaluable model organisms to study conserved fundamental cellular processes. Although super-resolution microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeasts has remained limited due to the specific know-how and instrumentation required, contrasted with the relative ease of endogenous tagging and live-cell fluorescence microscopy. To facilitate super-resolution microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling a 4-fold isotropic expansion. We demonstrate that U-ExM allows imaging of the microtubule cytoskeleton and its associated spindle pole body, notably unveiling the Sfi1p-Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by monitoring the homeostatic regulation of nuclear pore complex number through the cell cycle. Combined with NHS-ester pan-labelling, which provides a global cellular context, U-ExM reveals the subcellular organization of these two yeast models and provides a powerful new method to augment the already extensive yeast toolbox. This article has an associated First Person interview with Kerstin Hinterndorfer and Felix Mikus, two of the joint first authors of the paper.

Expansion Microscopy-based imaging for visualization of mitochondria in Drosophila ovarian germline stem cells.

Recent studies have shown that mitochondrial morphology can modulate organelle function and greatly affect stem cell behavior, thus affecting tissue homeostasis. As such, we previously showed that the accumulation of fragmented mitochondria in aged Drosophila ovarian germline stem cells (GSCs) contributes to age-dependent GSC loss. However, standard immunofluorescence methods to examine mitochondrial morphology yield images with insufficient resolution for rigorous analysis, while 3-dimensional electron microscopy examination of mitochondrial morphology is labor intensive and allows only limited sampling of mitochondria. To overcome these issues, we utilized the expansion microscopy technique to expand GSC samples by 4-fold in combination with mitochondrial immunofluorescence labeling. Here, we present a simple, inexpensive method for nanoscale optical imaging of mitochondria in the germline. This protocol may be beneficial for studies that require visualization of mitochondria or other fine subcellular structures in the Drosophila ovary.

Improved fluorescent signal in expansion microscopy using fluorescent Fab fragment secondary antibodies.

Expansion microscopy (ExM) is a microscopic imaging approach that can achieve super-resolution visualization of fluorescently labeled biological samples using conventional fluorescence microscopy. The method is based on embedding of a fluorescently labeled biological sample in a hydrogel matrix followed by the physical expansion of the specimen, which is then viewed using a conventional fluorescent microscope. Variations of the method can be used to visualize endogenously expressed fluorescent proteins, such as GFP, fluorescently tagged antibodies, nucleic acids, or other fluorescently tagged molecules. A significant challenge of the method is that the physical expansion of the specimen produces a concommitant reduction in fluorescence intensity, which can make imaging difficult. We describe an approach for amplifying fluorescence signal following expansion of immunolabeled tissue sections by applying fluorescently labeled Fab fragment secondary antibodies to intensify fluorescent signal and enhance detection of labeling using conventional fluorescent microscopy. A method to increase immunofluorescence signal intensity of Expansion Microscopy specimens is described. Method utilizes commercially available reagents. Enhances ability to acquire useful images in expanded tissue samples.

Comparison of absorbed dose extrapolation methods for mouse-to-human translation of radiolabelled macromolecules.

BACKGROUND: Extrapolation of human absorbed doses (ADs) from biodistribution experiments on laboratory animals is used to predict the efficacy and toxicity profiles of new radiopharmaceuticals. Comparative studies between available animal-to-human dosimetry extrapolation methods are missing. We compared five computational methods for mice-to-human AD extrapolations, using two different radiopharmaceuticals, namely [111In]CHX-DTPA-scFv78-Fc and [68Ga]NODAGA-RGDyK. Human organ-specific time-integrated activity coefficients (TIACs) were derived from biodistribution studies previously conducted in our centre. The five computational methods adopted are based on simple direct application of mice TIACs to human organs (M1), relative mass scaling (M2), metabolic time scaling (M3), combined mass and time scaling (M4), and organ-specific allometric scaling (M5), respectively. For [68Ga]NODAGA-RGDyK, these methods for mice-to-human extrapolations were tested against the ADs obtained on patients, previously published by our group. Lastly, an average [68Ga]NODAGA-RGDyK-specific allometric parameter αnew was calculated from the organ-specific biological half-lives in mouse and humans and retrospectively applied to M3 and M4 to assess differences in human AD predictions with the α = 0.25 recommended by previous studies. RESULTS: For both radiopharmaceuticals, the five extrapolation methods showed significantly different AD results (p < 0.0001). In general, organ ADs obtained with M3 were higher than those obtained with the other methods. For [68Ga]NODAGA-RGDyK, no significant differences were found between ADs calculated with M3 and those obtained directly on human subjects (H) (p = 0.99; average M3/H AD ratio = 1.03). All other methods for dose extrapolations resulted in ADs significantly different from those calculated directly on humans (all p ≤ 0.0001). Organ-specific allometric parameters calculated using combined experimental [68Ga]NODAGA-RGDyK mice and human biodistribution data varied significantly. ADs calculated with M3 and M4 after the application of αnew = 0.17 were significantly different from those obtained by the application of α = 0.25 (both p < 0.001). CONCLUSIONS: Available methods for mouse-to-human dosimetry extrapolations provided significantly different results in two different experimental models. For [68Ga]NODAGA-RGDyK, the best approximation of human dosimetry was shown by M3, applying a metabolic scaling to the mouse organ TIACs. The accuracy of more refined extrapolation algorithms adopting model-specific metabolic scaling parameters should be further investigated.

Coupling Auxin-Inducible Degron System with Ultrastructure Expansion Microscopy to Accelerate the Discovery of Gene Function in Toxoplasma gondii.

Ultrastructure expansion microscopy (U-ExM) is an emerging technique allowing the localization of proteins and cellular structures, at a level of resolution only distinguishable previously via immunoelectron microscopy. U-ExM, as its name indicates, is based on the physical expansion of the sample in the three dimensions without altering its internal features. The proteins of interest are later immunostained for their detection. To accelerate the discovery of gene function in the protozoan parasite Toxoplasma gondii, U-ExM can be coupled to the auxin-inducible degron system (mAiD system). This pipeline led to the subcellular localization of the gene product at unprecedented resolution and simultaneously assessed the consequences of conditional gene disruption. In this chapter, we explain the specific U-ExM protocol used for T. gondii tachyzoite samples and provide non-trivial advice and tips to successfully perform the experiments.

Influence of dosimetry method on bone lesion absorbed dose estimates in PSMA therapy: application to mCRPC patients receiving Lu-177-PSMA-I&T.

BACKGROUND: Patients with metastatic, castration-resistant prostate cancer (mCRPC) present with an increased tumor burden in the skeleton. For these patients, Lutetium-177 (Lu-177) radioligand therapy targeting the prostate-specific membrane antigen (PSMA) has gained increasing interest with promising outcome data. Patient-individualized dosimetry enables improvement of therapy success with the aim of minimizing absorbed dose to organs at risk while maximizing absorbed dose to tumors. Different dosimetric approaches with varying complexity and accuracy exist for this purpose. The Medical Internal Radiation Dose (MIRD) formalism applied to tumors assumes a homogeneous activity distribution in a sphere with unit density for derivation of tumor S values (TSV). Voxel S value (VSV) approaches can account for heterogeneous activities but are simulated for a specific tissue. Full patient-individual Monte Carlo (MC) absorbed dose simulation addresses both, heterogeneous activity and density distributions. Subsequent CT-based density weighting has the potential to overcome the assumption of homogeneous density in the MIRD formalism with TSV and VSV methods, which could be a major limitation for the application in bone metastases with heterogeneous density. The aim of this investigation is a comparison of these methods for bone lesion dosimetry in mCRPC patients receiving Lu-177-PSMA therapy. RESULTS: In total, 289 bone lesions in 15 mCRPC patients were analyzed. Percentage difference (PD) of average absorbed dose per lesion compared to MC, averaged over all lesions, was + 14 ± 10% (min: - 21%; max: + 56%) for TSVs. With lesion-individual density weighting using Hounsfield Unit (HU)-to-density conversion on the patient's CT image, PD was reduced to - 8 ± 1% (min: - 10%; max: - 3%). PD on a voxel level for three-dimensional (3D) voxel-wise dosimetry methods, averaged per lesion, revealed large PDs of + 18 ± 11% (min: - 27%; max: + 58%) for a soft tissue VSV approach compared to MC; after voxel-wise density correction, this was reduced to - 5 ± 1% (min: - 12%; max: - 2%). CONCLUSION: Patient-individual MC absorbed dose simulation is capable to account for heterogeneous densities in bone lesions. Since the computational effort prevents its routine clinical application, TSV or VSV dosimetry approaches are used. This study showed the necessity of lesion-individual density weighting for TSV or VSV in Lu-177-PSMA therapy bone lesion dosimetry.

Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei.

Proper mitochondrial genome inheritance is important for eukaryotic cell survival. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome, the kinetoplast (k)DNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described; however, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC-associated protein TAP110. We find that both depletion and overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA-associated proteins that changed in abundance, including a TEX-like protein that dually localizes to the nucleus and the kDNA, potentially linking replication and segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself; however, once TAP110 has been assembled, it also interacts with the kDNA. Finally, we use ultrastructure expansion microscopy in trypanosomes for the first time, and reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.This article has an associated First Person interview with the first author of the paper.

Improving the resolution of fluorescence nanoscopy using post-expansion labeling microscopy.

The visualization of the cell ultrastructure and molecular complexes has long been reserved for electron microscopy owing to its nanometric resolution. In recent years, this monopoly has been challenged by super-resolution (SR) fluorescence microscopy, which allows the visualization of cell structures with high spatial resolution, approaching virtually molecular dimensions. However, the resolution of current SR microscopy does not systematically reach the level of the ultrastructural information provided by electron microscopy. In this review, we are discussing the potential of revealing cell ultrastructure using the recent method of expansion microscopy (ExM). In particular, we are discussing the limitations that exist in SR and ExM methods that prevent the visualization of nanometric molecular assemblies and how post-labeling expansion could help alleviate them to reveal the molecular cartography of cells with unprecedented details.

Expansion microscopy imaging of various neuronal structures.

Visualization of the spatial distribution of biomolecules with nanoscale precision is essential to understanding the molecular mechanisms of biological phenomena and diseases. Among several state-of-the-art visualization techniques, expansion microscopy (ExM) is an attractive tool, as it can achieve sub-20-nm resolution imaging of biological specimens, even with conventional diffraction-limited microscopy. This chapter first introduces the concept of ExM and its variants and then provides practical guidelines for implementing expansion microscopy and related techniques.

New technical approaches for 3D morphological imaging and quantification of measurements.

3D imaging is becoming more and more popular, as it allows us to identify interactions between structures in organs. Furthermore, it gives the possibility to quantify and size these structures. To allow 3D imaging, the tissue sample has to be transparent. This is usually achieved by using optical tissue clearing protocols. Although using optical tissue clearing often results in perfect 3D images, these protocols have some pitfalls, like long duration of sample preparation (up to several weeks), use of toxic substances, damage to antibody staining, fluorescent proteins or dyes, high refractive indices, and high costs of sample processing.Recently we described [Huang et al., Scientific Reports 9(1): 521 (2019)] a fast, safe, and inexpensive ethyl cinnamate (ECi) based optical tissue clearing protocol. Here, we present extensions of our protocol with respect to the deparaffinization of old paraffin-embedded samples allowing 3D imaging of the blocks. In addition, we learned to remove ECi from the samples allowing the use of routine immunolabeling protocols. Furthermore, we demonstrate new pictures of lungs after expansion microscopy and adaptation of already existing protocols. The aim of our work is, in summary, to describe the advances in these methodologies, focusing on the morphological imaging of kidneys and lungs.

Radiation dosimetry of 18F-AzaFol: A first in-human use of a folate receptor PET tracer.

BACKGROUND: The folate receptor alpha (FRα) is an interesting target for imaging and therapy of different cancers. We present the first in-human radiation dosimetry and radiation safety results acquired within a prospective, multicentric trial (NCT03242993) evaluating the 18F-AzaFol (3'-aza-2'-[18F]fluorofolic acid) as the first clinically assessed PET tracer targeting the FRα. MATERIAL AND METHODS: Six eligible patients presented a histologically confirmed adenocarcinoma of the lung with measurable lesions (≥ 10 mm according to RECIST 1.1). TOF-PET images were acquired at 3, 11, 18, 30, 40, 50, and 60 min after the intravenous injection of 327 MBq (range 299-399 MBq) of 18F-AzaFol to establish dosimetry. Organ absorbed doses (AD), tumor AD, and patient effective doses (E) were assessed using the OLINDA/EXM v.2.0 software and compared with pre-clinical results. RESULTS: No serious related adverse events were observed. The highest AD were in the liver, the kidneys, the urinary bladder, and the spleen (51.9, 45.8, 39.1, and 35.4 µGy/MBq, respectively). Estimated patient and gender-averaged E were 18.0 ± 2.6 and 19.7 ± 1.4 µSv/MBq, respectively. E in-human exceeded the value of 14.0 µSv/MBq extrapolated from pre-clinical data. Average tumor AD was 34.8 µGy/MBq (range 13.6-60.5 µGy/MBq). CONCLUSIONS: 18F-Azafol is a PET agent with favorable dosimetric properties and a reasonable radiation dose burden for patients which merits further evaluation to assess its performance. TRIAL REGISTRATION: ClinicalTrial.gov, NCT03242993, posted on August 8, 2017.

Internal radiation dosimetry of a 152Tb-labeled antibody in tumor-bearing mice.

BACKGROUND: Biodistribution studies based on organ harvesting represent the gold standard pre-clinical technique for dose extrapolations. However, sequential imaging is becoming increasingly popular as it allows the extraction of longitudinal data from single animals, and a direct correlation with deterministic radiation effects. We assessed the feasibility of mouse-specific, microPET-based dosimetry of an antibody fragment labeled with the positron emitter 152Tb [(T1/2 = 17.5 h, Eß+mean = 1140 keV (20.3%)]. Image-based absorbed dose estimates were compared with those obtained from the extrapolation to 152Tb of a classical biodistribution experiment using the same antibody fragment labeled with 111In. 152Tb was produced by proton-induced spallation in a tantalum target, followed by mass separation and cation exchange chromatography. The endosialin-targeting scFv78-Fc fusion protein was conjugated with the chelator p-SCN-Bn-CHX-A"-DTPA, followed by labeling with either 152Tb or 111In. Micro-PET images of four immunodeficient female mice bearing RD-ES tumor xenografts were acquired 4, 24, and 48 h after the i.v. injection of 152Tb-CHX-DTPA-scFv78-Fc. After count/activity camera calibration, time-integrated activity coefficients (TIACs) were obtained for the following compartments: heart, lungs, liver, kidneys, intestines, tumor, and whole body, manually segmented on CT. For comparison, radiation dose estimates of 152Tb-CHX-DTPA-scFv78-Fc were extrapolated from mice dissected 4, 24, 48, and 96 h after the injection of 111In-CHX-DTPA-scFv78-Fc (3-5 mice per group). Imaging-derived and biodistribution-derived organ TIACs were used as input in the 25 g mouse model of OLINDA/EXM® 2.0, after appropriate mass rescaling. Tumor absorbed doses were obtained using the OLINDA2 sphere model. Finally, the relative percent difference (RD%) between absorbed doses obtained from imaging and biodistribution were calculated. RESULTS: RD% between microPET-based dosimetry and biodistribution-based dose extrapolations were + 12, - 14, and + 17 for the liver, the kidneys, and the tumors, respectively. Compared to biodistribution, the imaging method significantly overestimates the absorbed doses to the heart and the lungs (+ 89 and + 117% dose difference, respectively). CONCLUSIONS: MicroPET-based dosimetry of 152Tb is feasible, and the comparison with organ harvesting resulted in acceptable dose discrepancies for body districts that can be segmented on CT. These encouraging results warrant additional validation using radiolabeled biomolecules with a different biodistribution pattern.

First in-human radiation dosimetry of the gastrin-releasing peptide (GRP) receptor antagonist 68Ga-NODAGA-MJ9.

BACKGROUND: Gastrin-releasing peptide receptor antagonists have promise in theranostics of several highly incident tumours, including prostate and breast. This study presents the first human dosimetry of 68Ga-NODAGA-MJ9 in the first five consecutive patients with recurrent prostate cancer included in a dual-tracer positron emission tomography (PET) protocol. Five male patients with biochemical relapse of prostate adenocarcinoma underwent four whole-body time-of-flight PET/CT scans within 2 h after tracer injection. To be used as input in OLINDA/EXM 2.0, time-integrated activity coefficients were derived from manually drawn regions of interest over the following body regions: brain, thyroid, lungs, heart, liver, gallbladder, spleen, stomach, kidneys, adrenals, red marrow, pancreas, intestines, urinary bladder and whole body. Organ absorbed doses and effective dose (ED) were calculated with OLINDA/EXM 2.0 using the NURBS voxelized phantoms adjusted to the ICRP-89 organ masses and ICRP103 tissue-weighting factors. Additional absorbed dose estimations were performed with OLINDA/EXM 1.1 to be comparable with similar previous publications. RESULTS: The body regions receiving the highest absorbed doses were the pancreas, the urinary bladder wall, the small intestine and the kidneys (260, 69.8, 38.8 and 34.8 µGy/MBq respectively). The ED considering a 30-min urinary voiding cycle was 17.6 µSv/MBq in male patients. The increment of voiding time interval produced a significant increase of absorbed doses in bladder, prostate and testes, as well as an increase of ED. ED also increased if calculated with OLINDA/EXM 1.1. These results have been discussed in view of similar publications on bombesin analogues or on other commonly used theranostic peptides. CONCLUSIONS: The pancreas is the most irradiated organ after the injection of 68Ga-NODAGA-MJ9, followed by the urinary bladder wall, the small intestine and the kidneys. ED is in the same range of other common 68Ga-labelled peptides. Differences with similarly published studies on bombesin analogues exist, and are mainly dependent on the methodology used for absorbed dose calculations. TRIAL REGISTRATION: Clinicaltrial.Gov identifier: NCT02111954 , posted on 11/042014.

18F-FPYBF-2, a new F-18 labelled amyloid imaging PET tracer: biodistribution and radiation dosimetry assessment of first-in-man 18F-FPYBF-2 PET imaging.

OBJECTIVE: Recently, a benzofuran derivative for the imaging of ß-amyloid plaques, 5-(5-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)benzofuran-2-yl)- N-methylpyridin-2-amine (18F-FPYBF-2) has been validated as a tracer for amyloid imaging and it was found that 18F-FPYBF-2 PET/CT is a useful and reliable diagnostic tool for the evaluation of AD (Higashi et al. Ann Nucl Med, https://doi.org/10.1007/s12149-018-1236-1 , 2018). The aim of this study was to assess the biodistribution and radiation dosimetry of diagnostic dosages of 18F-FPYBF-2 in normal healthy volunteers as a first-in-man study. METHODS: Four normal healthy volunteers (male: 3, female: 1; mean age: 40 ± 17; age range 25-56) were included and underwent 18F-FPYBF-2 PET/CT study for the evaluation of radiation exposure and pharmacokinetics. A 10-min dynamic PET/CT scan of the body (chest and abdomen) was performed at 0-10 min and a 15-min whole-body static scan was performed six times after the injection of 18F-FPYBF-2. After reconstructing PET and CT image data, individual organ time-activity curves were estimated by fitting volume of interest data from the dynamic scan and whole-body scans. The OLINDA/EXM version 2.0 software was used to determine the whole-body effective doses. RESULTS: Dynamic PET imaging demonstrated that the hepatobiliary and renal systems were the principal pathways of clearance of 18F-FPYBF-2. High uptake in the liver and the gall bladder, the stomach, and the kidneys were demonstrated, followed by the intestines and the urinary bladder. The ED for the adult dosimetric model was estimated to be 8.48 ± 1.25 µSv/MBq. The higher absorbed doses were estimated for the liver (28.98 ± 12.49 and 36.21 ± 15.64 µGy/MBq), the brain (20.93 ± 4.56 and 23.05 ± 5.03µ Gy/MBq), the osteogenic cells (9.67 ± 1.67 and 10.29 ± 1.70 µGy/MBq), the small intestines (9.12 ± 2.61 and 11.12 ± 3.15 µGy/MBq), and the kidneys (7.81 ± 2.62 and 8.71 ± 2.90 µGy/MBq) for male and female, respectively. CONCLUSIONS: The ED for the adult dosimetric model was similar to those of other agents used for amyloid PET imaging. The diagnostic dosage of 185-370 MBq of 18F-FPYBF-2 was considered to be acceptable for administration in patients as a diagnostic tool for the evaluation of AD.

Radiation dosimetry of the α4ß2 nicotinic receptor ligand (+)-[18F]flubatine, comparing preclinical PET/MRI and PET/CT to first-in-human PET/CT results.

BACKGROUND: Both enantiomers of [18F]flubatine are new radioligands for neuroimaging of α4ß2 nicotinic acetylcholine receptors with positron emission tomography (PET) exhibiting promising pharmacokinetics which makes them attractive for different clinical questions. In a previous preclinical study, the main advantage of (+)-[18F]flubatine compared to (-)-[18F]flubatine was its higher binding affinity suggesting that (+)-[18F]flubatine might be able to detect also slight reductions of α4ß2 nAChRs and could be more sensitive than (-)-[18F]flubatine in early stages of Alzheimer's disease. To support the clinical translation, we investigated a fully image-based internal dosimetry approach for (+)-[18F]flubatine, comparing mouse data collected on a preclinical PET/MRI system to piglet and first-in-human data acquired on a clinical PET/CT system. Time-activity curves (TACs) were obtained from the three species, the animal data extrapolated to human scale, exponentially fitted and the organ doses (OD), and effective dose (ED) calculated with OLINDA. RESULTS: The excreting organs (urinary bladder, kidneys, and liver) receive the highest organ doses in all species. Hence, a renal/hepatobiliary excretion pathway can be assumed. In addition, the ED conversion factors of 12.1 µSv/MBq (mice), 14.3 µSv/MBq (piglets), and 23.0 µSv/MBq (humans) were calculated which are well within the order of magnitude as known from other 18F-labeled radiotracers. CONCLUSIONS: Although both enantiomers of [18F]flubatine exhibit different binding kinetics in the brain due to the respective affinities, the effective dose revealed no enantiomer-specific differences among the investigated species. The preclinical dosimetry and biodistribution of (+)-[18F]flubatine was shown and the feasibility of a dose assessment based on image data acquired on a small animal PET/MR and a clinical PET/CT was demonstrated. Additionally, the first-in-human study confirmed the tolerability of the radiation risk of (+)-[18F]flubatine imaging which is well within the range as caused by other 18F-labeled tracers. However, as shown in previous studies, the ED in humans is underestimated by up to 50 % using preclinical imaging for internal dosimetry. This fact needs to be considered when applying for first-in-human studies based on preclinical biokinetic data scaled to human anatomy.

Pathology consultation on electronic crossmatch.

OBJECTIVES: The full crossmatch is traditionally the final step in compatibility testing, acting as a serologic double check for ABO compatibility and unexpected RBC antibodies. In this review, we discuss the development of electronic crossmatch (EXM), an approach for determining when EXM can be used, and its strengths and weaknesses. METHODS: Because EXM relies on highly sensitive screening assays, antibodies are frequently encountered whose clinical significance must be investigated and interpreted. Our approach is to obtain further history, perform enhanced tube testing, and consider tests of immune reactivity or RBC survival. RESULTS: For those without clinically significant antibodies, we found two alternatives: immediate-spin crossmatch (IS XM) and EXM. IS XM is prone to error related to serologic interference, whereas EXM depends on the accuracy of the sample label, accurate data entry, and informatics to avoid errors. CONCLUSION: EXM is an alternative to the serologic test in patients who have no clinically significant antibodies.