Background Transplant patients may have circulating non-ABO red blood cell (RBC) antibodies, but the influence of RBC alloimmunization on solid-organ transplantation outside of the ABO blood group system is not well known. Though RBC antigens have been identified in or on cells other than RBCs, the incidence of non-ABO RBC antibodies in transplant patients has had limited investigation. Identification of non-ABO RBC antibodies in transplant patients may help clarify cases of suspected antibody-mediated rejection (AMR) when human leukocyte antigen (HLA) donor-specific antibodies (DSA) cannot be detected. Study design and methods This investigation tested for the presence and specificity of commonly encountered non-ABO RBC antibodies in the serum of transplant patients (heart, lung, and kidney), using standard hemagglutination antibody screening and antibody panel identification methods. Results Of 135 patients, six (4%) tested positive for clinically significant non-ABO RBC antibodies. The antibodies identified were anti-K and anti-Fya. Discussion This study provides new evidence for the transfusion and transplant community by documenting the frequency of non-ABO RBC antibodies in heart, lung, and kidney transplant patients, and is an important first step in determining the clinical significance of these antibodies in cases of suspected AMR when HLA DSAs are absent.

BACKGROUND:Lipofuscin-like cytoplasmic inclusions have been reported in human blood neutrophils and monocytes but have not been described in dogs. In people, these "green granules of death" have been associated with moderate to severe hepatocellular injury and high mortality.OBJECTIVES:To describe clinicopathologic abnormalities, diagnoses, and outcomes of dogs with greenish inclusions in blood neutrophils or monocytes, and to determine if the inclusions have features of lipofuscin.METHODS:Clinical cases were identified prospectively through routine evaluation of CBC samples. Leukocyte inclusions were characterized with routine staining and assessed for iron and autofluorescence. Additional cases were identified by examination of archived blood smears from dogs meeting search criteria for hepatocellular injury, and clinicopathologic findings were recorded.RESULTS:All 7 prospectively identified dogs with inclusions had inflammation and moderate to marked increases in serum alanine aminotransferase (ALT) activity, as did the 4 dogs identified from the 97 meeting retrospective search criteria. The inclusions were Prussian blue-negative (5/5) with broad-spectrum autofluorescence (5/5) and the appearance of lipofuscin with and without Wright staining. Most clinical diagnoses involved hepatic disorders (5/7 prospective and 3/4 retrospective cases) or pancreatitis (3/7 prospective and 2/4 retrospective cases), and some involved both; 8 of 11 dogs died within 7 days of admission.CONCLUSIONS:Blue-green cytoplasmic inclusions uncommonly found in blood neutrophils ± monocytes of routine canine blood smears have stained and unstained properties of lipofuscin and suggest the presence of hepatocellular injury, often severe. Reporting these inclusions is recommended to guide clinical management.

An 8-year-old spayed female Labrador retriever that was presented for a wellness examination at a referring practice had mild anemia with a hematocrit that progressed from 32% to 25% over the next 5 weeks. The dog's reticulocyte concentration was 26 400/μL on day 23, supporting nonregenerative anemia, and no cause was apparent from clinical and laboratory findings. This prompted the submission of bone marrow core and aspirate samples to our laboratory on day 38. These revealed ineffective erythroid hyperplasia with complete maturation but a predominance of rubricytes, rubriphagocytosis, mild lymphoplasmacytosis, and mild to regionally severe collagen myelofibrosis, all supportive of precursor-targeted immune-mediated anemia (PIMA).1 Hemosiderin was markedly increased in aspirate smears and in most regions of the core. No blood transfusions had been given. Immunosuppressive treatment with prednisone was started, but the dog was euthanized 10 days later due to poor clinical progression. Plasma cells in the bone marrow commonly contained coarse crystalline cytoplasmic inclusions that were green to brown in aspirate smears (Wright-Giemsa stain) and golden-brown in tissue sections (H&E stain) (Figure 1); the plasma cells otherwise lacked atypia. The granules were suggestive of iron, and Prussian blue positivity confirmed iron-rich inclusions. Histologic sections of liver collected at necropsy showed no increase in iron within hepatocytes or Kupffer cells. Bone marrow siderotic plasma cells are rarely reported in nonalcoholic human patients, and the authors are unaware of reports in domestic mammals. In people, they have been associated mostly with conditions involving altered iron homeostasis and/or ineffective erythropoiesis: (1) systemic or marrow hemosiderosis from iron-loading anemias (e.g., megaloblastic anemia, sideroblastic anemias, refractory anemia with multilineage dysplasia, and pyruvate kinase-deficiency), recurrent transfusions, or hereditary hemochromatosis,2 but not with iron accumulation from chronic inflammation,2, 3 and (2) marrow disease secondary to chronic alcoholism, which is associated with nutritional deficiencies and megaloblastic or sideroblastic anemias with ineffective erythropoiesis and low to high amounts of hemosiderin.2, 4 More rarely, plasma cell iron inclusions have been reported in people without clear evidence of altered iron homeostasis or ineffective erythropoiesis, for example, a minority of people with malignancies, including plasma cell myeloma.3, 5 Similar to most affected people, the dog in this report had ineffective erythropoiesis and increased marrow hemosiderin that suggests altered iron homeostasis, perhaps related to increased erythroferrone production.6 In people, transmission electron microscopy of siderotic plasma cells has shown increased cytosolic ferritin and iron accumulation in siderosomes,2 perhaps from iron uptake mediated by cell membrane transferrin receptors.2 Siderotic plasma cells reflect disease, but they are currently of little diagnostic value in people and are of unknown diagnostic value in dogs. Recognizing the presence of these cells is the first step in determining if they have any clinical significance. The authors declare they have no conflicts of interest.

Trapped neutrophil syndrome is a rare congenital disease recognized in Border Collies and is characterized by persistent neutropenia with myeloid hyperplasia. The mechanism of neutropenia has not been described. We document the case of a young Border Collie diagnosed with trapped neutrophil syndrome based on clinical features, blood and bone marrow evaluation, and presence of the associated homozygous mutation. Results from flow cytometric and storage studies suggested lower neutrophil survival time. The dog had substantial neutrophilic inflammation in multiple organs, indicating that neutrophils could leave the marrow and enter tissues, making the term "trapped" neutrophil syndrome a misnomer.

BACKGROUND:Cytopenias have been reported in dogs treated with phenobarbital, but detailed descriptions of bone marrow findings and response to treatment are lacking.OBJECTIVES:We aimed to characterize the hematologic findings and clinical outcomes of dogs that had been receiving phenobarbital at the time of marrow evaluation.METHODS:Archived bone marrow slides and clinicopathologic data were reviewed in dogs undergoing marrow evaluation for any hematologic problems that developed while receiving phenobarbital (2008-2020). Dogs were excluded if marrow samples lacked diagnostic value, phenobarbital was withdrawn >1 day before marrow collection, a same-day complete blood count (CBC) was lacking, or dogs had concurrent illness or therapy known to cause cytopenias.RESULTS:Thirteen dogs met inclusion criteria: eight pancytopenic, three anemic/thrombocytopenic, one neutropenic/thrombocytopenic, and one nearly neutropenic. Neutropenia was marked (<700/µL) in eight dogs; all neutrophil concentrations were low or low-normal. Of the 11 anemic dogs (Hct = 12%-42%, median = 29%), three had mild reticulocytosis (eight were tested). One dog had erythroid dysplasia in blood and marrow. All nine neutropenic dogs had evidence of ineffective neutropoiesis: neutrophilic hyperplasia with left shift (9) ± neutrophagocytosis (5). Eight of the 11 anemic dogs had evidence of ineffective erythropoiesis: erythroid hyperplasia (7), left shift (3), and/or rubriphagocytosis (6). No thrombocytopenic dog had megakaryocytic hypoplasia; seven dogs had megakaryocytic hyperplasia. One anemic/thrombocytopenic dog had marked collagen myelofibrosis. The noncytopenic dog had equivocal myeloid hypoplasia with neutrophagocytosis. Median maximal responses and resolution times for neutropenia (n = 6) were 14 days.CONCLUSIONS:Phenobarbital-induced cytopenias should be considered in dogs with multilineage ineffective hematopoiesis, particularly when neutropenia and myeloid hyperplasia are present. However, findings in dogs with immune-mediated neutropenia or precursor-targeted immune-mediated anemia might be indistinguishable.

Routine blood smear findings in two of four 11-day-old mixed-breed dog littermates were suggestive of a lysosomal storage disease (LSD) that was documented to be mucopolysaccharidosis type VII (MPS VII) by molecular testing. In this condition, a functional β-glucuronidase deficiency results in the accumulation of glycosaminoglycans (GAGs) in cells and tissues where β-glucuronidase is important in GAG degradation. Most neutrophils and a moderate number of lymphocytes within the blood had atypical cytoplasmic magenta inclusions. The bone marrow assessment from one of the two affected pups at 24 days of age revealed similar magenta granulation in myeloid precursor cells that was most prominent in promyelocytes and myelocytes. Moreover, atypical magenta material was present within vacuoles as well as extracellularly in some osteoblasts and macrophages. Histologic bone marrow sections revealed prominent vacuolation of osteoblasts, and some osteoclasts appeared separated from the bone by layers of osteoblasts or hematopoietic cells. At 2 months of age, the second affected dog showed moderate growth retardation and had similar but more prominent hematologic findings that extended to monocytes, eosinophils, and eosinophil precursors. It had an increased number of bone marrow macrophages with many vacuoles that could be seen cytologically to contain magenta material, and there was mild nonselective phagocytosis of hemic cells. Of the hematologic cells, inclusions were most prominent in promyelocytes, myelocytes, and macrophages, cells with relatively high β-glucuronidase activity, and GAG exposure within lysosomes or lysosome-like primary granules of granulocyte precursors.

AbstractBackgroundAlthough precursor‐targeted immune‐mediated anemia (PIMA) is thought to be caused by immune targeting of erythroid precursors (nucleated RBCs, nRBCs), its pathogenesis is unknown. Immunoglobulin G (IgG) or phosphatidylserine (PS) may promote nRBC destruction in PIMA.HypothesisDogs with PIMA have increased nRBC IgG and PS, and dogs with immune‐mediated hemolytic anemia (IMHA) have increased RBC PS compared to healthy dogs.AnimalsBlood from 20 healthy dogs and from dogs with IMHA (11) or other (non‐IMHA) conditions (9), and marrow aspirates with or without blood from 10 healthy dogs and from dogs with PIMA (17) or other (non‐IMHA, non‐PIMA) conditions (7).MethodsMarrow nRBC stages were separated by density gradient. Flow cytometry was used to assess the percentage of RBCs or nRBCs with increased IgG or PS.ResultsRed blood cell (RBC) IgG positivity was increased in 9/11 IMHA dogs and 0/9 non‐IMHA dogs. Red blood cell PS positivity was increased in 10/11 IMHA dogs and 2/9 non‐IMHA dogs. Five of 17 PIMA dogs had increased nRBC IgG positivity in mid‐ or late‐stage fractions, whereas all 7 non‐PIMA dogs were negative. Mid‐ and late‐stage erythroid precursor PS was significantly higher in PIMA dogs compared to healthy dogs. Five of 14 PIMA dogs had increased RBC IgG positivity.ConclusionsImmunoglobulin G and PS may promote destruction of nRBCs in PIMA dogs; PS may promote destruction of RBCs in IMHA dogs.

Although precursor-targeted immune-mediated anemia (PIMA) is thought to be caused by immune targeting of erythroid precursors (nucleated RBCs, nRBCs), its pathogenesis is unknown. Immunoglobulin G (IgG) or phosphatidylserine (PS) may promote nRBC destruction in PIMA. Dogs with PIMA have increased nRBC IgG and PS, and dogs with immune-mediated hemolytic anemia (IMHA) have increased RBC PS compared to healthy dogs. Blood from 20 healthy dogs and from dogs with IMHA (11) or other (non-IMHA) conditions (9), and marrow aspirates with or without blood from 10 healthy dogs and from dogs with PIMA (17) or other (non-IMHA, non-PIMA) conditions (7). Marrow nRBC stages were separated by density gradient. Flow cytometry was used to assess the percentage of RBCs or nRBCs with increased IgG or PS. Red blood cell (RBC) IgG positivity was increased in 9/11 IMHA dogs and 0/9 non-IMHA dogs. Red blood cell PS positivity was increased in 10/11 IMHA dogs and 2/9 non-IMHA dogs. Five of 17 PIMA dogs had increased nRBC IgG positivity in mid- or late-stage fractions, whereas all 7 non-PIMA dogs were negative. Mid- and late-stage erythroid precursor PS was significantly higher in PIMA dogs compared to healthy dogs. Five of 14 PIMA dogs had increased RBC IgG positivity. Immunoglobulin G and PS may promote destruction of nRBCs in PIMA dogs; PS may promote destruction of RBCs in IMHA dogs.

OBJECTIVE To characterize the clinical features of dogs with precursor-targeted immune-mediated anemia (PIMA). ANIMALS 66 dogs with PIMA. PROCEDURES Electronic record databases of a teaching hospital were searched to identify dogs with a diagnosis of nonregenerative anemia between 2004 and 2013. Inclusion criteria included persistent nonregenerative anemia (Hct ≤ 30% and reticulocyte count < 76,000 reticulocytes/μL), cytologic findings supportive of ineffective bone marrow erythropoiesis, and absence of underlying disease. Information regarding clinical signs, clinicopathologic findings, treatment, and outcome was extracted from records of eligible dogs. A regenerative response was defined as a reticulocyte count > 76,000 reticulocytes/μL or sustained increase in Hct of > 5%. Remission was defined as a stable Hct ≥ 35%. RESULTS The median Hct was 13%, and reticulocyte count was 17,900 reticulocytes/μL. Rubriphagocytosis was identified in bone marrow aspirate samples from 61 of 66 dogs. Collagen myelofibrosis was detected in bone marrow biopsy specimens obtained from 31 of 63 dogs. Immune-mediated targeting of mature erythrocytes was uncommon. All dogs received immunosuppressive therapy. Fifty-five dogs developed a regenerative response at a median of 29 days, and 40 of those dogs went into remission at a median of 59 days after PIMA diagnosis. Thromboembolic events were confirmed for 9 dogs and were associated with a decreased survival time. Median survival time was 913 days for all dogs. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that most dogs with PIMA responded to prolonged immunosuppressive therapy. Studies to determine optimal immunosuppressive and thromboprophylactic protocols for dogs with PIMA are warranted.

OBJECTIVE To develop and characterize flow cytometric assays for detecting IgG bound to canine erythrocytes and bone marrow erythroid precursors. SAMPLE Blood samples from 20 healthy and 61 sick dogs with (n = 33) or without (28) immune-mediated hemolytic anemia (IMHA) and bone marrow samples from 14 healthy dogs. PROCEDURES A flow cytometric assay for measurement of IgG on RBCs was developed, and appropriate positive control cells were generated. Analytic and diagnostic performance were characterized. The RBC IgG assay was then combined with density-gradient fractionation of aspirated bone marrow cells and a 2-color process to yield an assay for detecting IgG on nucleated RBCs (nRBCs). Cell sorting and cytologic examination confirmed target cell populations, and anti-dog erythrocyte antigen I (DEAI) blood-typing serum was used to generate IgG-positive nRBCs. RESULTS Within- and between-run coefficients of variation for the RBC IgG assay were 0.1% to 13.9%, and > 90% of spiked IgG-positive RBCs were detected. Diagnostic sensitivity and specificity of the assay for detection of IMHA were 88% and 93%, respectively. Cytologic findings for sorted bone marrow fractions rich in early-, mid-, and late-stage nRBCs from 3 healthy dogs indicated 89% to 98% nRBC purity. After IgG coating with anti-DEAI blood-typing serum, IgG was detected on nRBCs from DEAI-positive, but not DEAI-negative, healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE The developed RBC IgG assay had favorable analytic and diagnostic performance for detection of IM HA in dogs and was successfully adapted to detect IgG on canine nRBCs of various maturation stages. The findings supported the presence of DEAI on canine nRBCs.

BACKGROUND:Precursor-targeted immune-mediated anemia (PIMA) has been suspected in dogs with nonregenerative anemia and bone marrow findings varying from erythroid hyperplasia to pure red cell aplasia. Phagocytosis of erythroid precursors/rubriphagocytosis (RP) reported in some affected dogs suggests a destructive component to the pathogenesis of PIMA.OBJECTIVES:The purpose of the study was to characterize laboratory and clinical findings in dogs with suspected PIMA and RP, with emphasis on cytologic and histologic bone marrow findings.METHODS:Dogs with PIMA and RP were identified by review of paired bone marrow aspirate and core biopsy slides collected over a 4-year period. Samples were systematically assessed and characterized along with other pertinent laboratory data and clinical findings.RESULTS:Twenty-five dogs met criteria for PIMA and had RP that was relatively stage-selective. Erythropoiesis was expanded to the stage of erythroid precursors undergoing most prominent phagocytosis, yielding patterns characterized by a hypo-, normo-, or hypercellular erythroid lineage. A 4th pattern involved severe collagen myelofibrosis, and there was a spectrum of mild to severe collagen myelofibrosis overall. Evidence of immune-mediated hemolysis was rare. Immunosuppressive therapy was associated with remission in 77% of dogs treated for at least the median response time of 2 months.CONCLUSIONS:Bone marrow patterns in dogs fulfilling criteria for PIMA were aligned with stage-selective phagocytosis of erythroid precursors and the development of collagen myelofibrosis, common in dogs with PIMA. Recognition of these patterns and detection of RP facilitates diagnosis of PIMA, and slow response to immunosuppressive therapy warrants further investigation into its pathogenesis.

Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

A juvenile Australian shepherd dog exhibited failure to grow, inappetence, weakness, nonregenerative anemia, neutropenia, and cobalamin deficiency. DNA testing confirmed homozygosity of an amnionless mutation (AMN c.3G > A). Clinical signs resolved with supportive care and parenteral cobalamin supplementation. Inherited selective intestinal cobalamin malabsorption requiring lifelong parenteral supplementation should be considered in Australian shepherds, giant schnauzers, border collies, and beagles that fail to thrive.

Acetaminophen (APAP) hepatotoxicity in mice resembles in most ways that which occurs in humans. Primary mouse hepatic parenchymal cells (HPCs) are the most frequently used cell type in vitro to study mechanisms of APAP hepatotoxicity. It is widely accepted that APAP hepatotoxicity requires bioactivation by cytochromes P450 (CYPs), but this remains unproven in HPCs over the wide range of concentrations that have been used in published reports. Accordingly, we tested the hypothesis that APAP‐induced HPC death in vitro depends solely on CYPs. We evaluated APAP cytotoxicity and bioactivation (formation of APAP‐protein adducts) in the presence and absence of the CYP inhibitor 1‐aminobenzotriazole (1‐ABT). 1‐ABT abolished formation of APAP‐protein adducts at all concentrations tested (0.1‐14mM) but only eliminated cytotoxicity at small APAP concentrations (0.1‐5mM), indicating the presence of a CYP‐independent mechanism at larger concentrations. The APAP deacetylation product, p‐aminophenol (PAP), was detected in HPCs exposed to a large APAP concentration, and a deacetylase inhibitor significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a CYP‐dependent mechanism that operates at smaller APAP concentrations and a CYP‐independent mechanism that predominates at larger concentrations and is later in onset. PAP likely contributes to the latter mechanism. (Supported by NIH grant R01 DK087886)

Acetaminophen (APAP)-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation participates in APAP hepatotoxicity. Here, we found that APAP overdose in mice caused liver damage accompanied by significant thrombocytopenia and accumulation of platelets in the liver. These changes were attenuated by administration of the direct thrombin inhibitor lepirudin. Platelet depletion with an anti-CD41 antibody also significantly reduced APAP-mediated liver injury and thrombin generation, indicated by the concentration of thrombin-antithrombin (TAT) complexes in plasma. Compared with APAP-treated wild-type mice, biomarkers of hepatocellular and endothelial damage, plasma TAT concentration, and hepatic platelet accumulation were reduced in mice lacking protease-activated receptor (PAR)-4, which mediates thrombin signaling in mouse platelets. However, selective hematopoietic cell PAR-4 deficiency did not affect APAP-induced liver injury or plasma TAT levels. These results suggest that interconnections between coagulation and hepatic platelet accumulation promote APAP-induced liver injury, independent of platelet PAR-4 signaling. Moreover, the results highlight a potential contribution of nonhematopoietic cell PAR-4 signaling to APAP hepatotoxicity.

BACKGROUND A captive Red Panda developed a regenerative anemia with Heinz bodies after being sprayed by a skunk. A definite cause-and-effect relationship between skunk musk and oxidative erythrocyte damage has not been reported, but it was suspected in one reported case of a dog with Heinz body hemolytic anemia. OBJECTIVE The objective was to determine whether skunk musk induces oxidative HGB damage in vitro. METHODS Plasma and RBC were harvested from heparinized blood of 3 dogs, 3 cats, and a Red Panda. Skunk musk was solubilized in ethanol and mixed with plasma from each species to make stock solutions of 4% musk and 4% ethanol. Aliquots of RBC were resuspended in autologous stock solutions and solvent controls to yield musk concentrations of 0%, 0.04%, and 0.4% (by volume). Aliquots were incubated at 37°C for 4-72 hours and assessed for oxidative damage by visual inspection, optical absorbance spectroscopy, transmission electron microscopy, and light microscopy after Wright and vital New Methylene Blue staining. RESULTS Dose-dependent brown color and absorption changes characteristic of methemoglobin were present by 4 hours and increased over 24 hours (Red Panda) and 72 hours (dog and cat). Similarly, there were time-dependent (all species) and dose-dependent (dog and cat) increases in the number of Heinz bodies, which were present by 4 hours and numerous by 24 hours. CONCLUSIONS In vitro, skunk musk causes Heinz body and methemoglobin formation in canine, feline, and Red Panda RBC, supporting the clinical association between Heinz body hemolytic anemia and skunk spray exposure.

A mixed-methods evaluation was conducted to study learner attitudes and knowledge about clinical pathology across a curricular change that instituted a stand-alone clinical pathology course in place of content within a previously integrated pathology course structure. Groups of pre- and post-change students were assessed three times across the two semesters leading up to graduation. At each time, rank-ordered and open-ended response items probed attitudes, and multiple-choice items assessed knowledge. Data about student clinical pathology performance were also collected from clinical pathology instructors and supervising clinicians. Student rank-ordered items were evaluated by factor analysis; resulting factor-scale scores, multiple-choice scores, and rank responses from study cohorts were statistically assessed between groups and within each group over time. Intraclass correlations were calculated for the coding of student open-ended responses, and all coded responses were compared among groups. Analysis revealed that students in the revised curriculum had greater satisfaction with their training and greater confidence in data interpretation compared to students without exposure to an independent clinical pathology course. Although differences in knowledge of clinical pathology were not detected, it was also apparent that the independent clinical pathology course filled a student-perceived curricular need without raising criticisms related to diminished integration with anatomic pathology. Secondary study outcomes included formative feedback for course improvement, evidence of clerkship efficacy, and baseline data for further studies.