Three Years Experience with Automated Specimen Processing for Clinical Diagnostic TEM Pathology

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ARUP Laboratories, one of the USA’s largest national reference diagnostic labs (CAP, ISO-15189 & CLIA certified), prepares ∼1,300 renal biopsy TEM specimens annually, plus variable numbers of liver, gastrointestinal, cilia, and muscle (skeletal & heart) biopsies. For these clinical specimens, reproducibility, speed, and labor efficiency are critical. Three years ago, ARUP switched from using microwave prep to an mPrep™ ASP-1000 Automated Specimen Processor (ASP, Microscopy Innovations) workflow [1-2]. We describe here how this ASP workflow has improved consistency, cut reagent consumption, and reduced tedious labor. The EM lab typically receives 4-15 patient renal plus other specimens daily. The workflow (Fig. 1) is shown with renal biopsies, which arrive as 18-gauge needle cores (Fig. 1A) overnight fixed in glutaraldehyde-paraformaldehyde (GA-PA). The cores are cut into 1-2 mm long segments (Fig. 1B), with up to 8 segments from one patient placed into a mPrep/s specimen capsule. The capsules are capped with a barcode-labeled capsule to entrap and identify the patient (Fig. 1C-D). On days with 9-16 patients, two patient-containing capsules are stacked to double processing capacity (Fig. 1D). The loaded and labeled capsules are then attached to the ASP. Figure 1E shows 5 capsules since this day there were only 5 patients. On days with 9-16 patients, capsules are double stacked (Fig. 1D). Thus, the ASP can process up to 16 patient specimens, with each comprising up to 8 segments, for a capacity of 128 tissue segments per automated preparation run. At each reagent-protocol step, the ASP draws reagent into the capsules from 12-channel microplates to fully immerse the specimens (Fig. 1E) and agitates using gentle repeated aspirate-dispense cycles while keeping specimens continuously immersed. This rapidly infiltrates reagents through each tissue piece. Each protocol step uses ∼5 ml, thus providing a reagent consumption of ∼40 µl/step when processing 128 tissue pieces (16 patients) and ∼80 µl/step for 64 tissue pieces (8 patients). The conservative ARUP protocol uses 3 buffer GA-PA rinse-outs, OsO4, uranyl acetate, water rinses, graded ethanols and 6 100% ethanols, and 6 100% acetones, resin infiltration with 1:1 and 3:1 epoxy:acetone, then 3 100% 812-epoxy formulation steps. The operator starts the ASP protocol and then adds OsO4, uranyl acetate, and water rinses while the ASP performs the GA-PA buffer rinse-outs. The ASP alerts lab staff for the timely addition of acetone and epoxy resin but otherwise operates without intervention. Protocol duration for takes about 3 hours. A “Protocol Completed” alert signals staff when the tissue is 100% resin infiltrated. The patient-labeled capsules are then transferred from the ASP to dishes containing an embedding mold with the same number of labeled and resin-filled wells as there are biopsy segments for each patient (Fig. 1F). The biopsy segments are then removed from the capsule, orientated in the wells, and polymerized overnight at 70C. Block facing, microtomy, semi-thin and thin sectioning, grid staining (uranyl acetate and lead citrate), and imaging are done the next morning. Every patient block is sampled with 0.4 µm toluidine blue stained sections on bar-coded glass slides (Fig 1G). Three grids are prepared per block with a minimum of 2 sections per grid. Imaging uses a JEOL 1400 Flash TEM, with a Gatan Rio camera. JPG images are stored on a server for pathologists and client access. Time from sample receipt to images on the server is ∼24 hrs. Figure 2 shows TEM biopsy examples. The ARUP ASP specimen prep workflow requires only 1 person-hour of hands-on effort, divided between 2 persons working together for ∼30 minutes each to ensure no mix-ups when patient specimens are moved and when barcode or human-readable labels are changed. This one-person-hour effort is a substantial reduction from ARUP’s prior microwave workflow requiring 6-7 person-hours, and which demanded nearly nonstop manual reagent exchanges and considerable care to ensure specimens were not accidentally pipetted, damaged, or lost. Reproducible process timing was especially difficult to achieve on days with higher numbers of specimens. By contrast, the ASP provides precise reagent timing and reduced handling of toxic reagents while consuming much less reagent; just 5 mls/step for 1-16 patients consisting of up to 128 biopsy segments, compared to 2 mls/step/patient microwave processing. In summary, the ASP workflow in place for 3 years has improved consistency, cut reagent consumption, and reduced tedious labor. Workflow. A) Specimen package. B) Cut 18-G renal cores into 1-2 mm segments. C-D) Insert up to 8 segments per mPrep/s capsule (arrow), and cap with labeled capsule. D) Single and double stack capsule diagram with 8 renal segments/capsule. E) Five single-stack capsules on ASP 8-channel head for processing. Reagents in microplate (plate), reagent level in capsules (arrow). F) Renal segments labeled and flat embedded. G) Toluidine blue renal slide. TEM pathology examples. A) Renal tissue partially effaced foot processes, B) Renal biopsy with dense deposits disease (arrows), focal segmental and global glomerulosclerosis and tubular atrophy. C) Skeletal muscle biopsy with extensive paracrystalline inclusions (arrows).

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