Report of the International Council for Standardization in Haematology working group for standardization of reticulocyte parameters.

INTRODUCTION: The International Council for Standardization in Haematology convened a working group to assess and propose improvements upon the state of standardization and harmonization of reticulocyte parameters among commercial hematology analyzers. METHODS: An international group of laboratory hematologists prospectively collected and analyzed clinical samples using locally available IVD commercial hematology analyzers. Eight hundred and fifty-five total samples were collected at 6 sites using 9 distinct analyzer types. Samples were assessed for reticulocyte percent (RET%), immature reticulocyte fraction (IRF), and reticulocyte hemoglobin content (RHC). Method comparison and regression statistics were calculated. These analyses were used to determine whether statistical recalibration offered a potential avenue for increasing comparability between these methods. RESULTS: While methods producing reticulocyte percent were the most comparable in this study, the state of harmonization for the IRF and RHC was reduced with pearson correlation coefficients ranging from 0.955 to 0.77 and 0.927 and 0.680, respectively. Nevertheless, use of parameters from the Passing Bablok regression substantially improved the comparability of the results. In addition, precision data was derived which also demonstrated substantial differences between analyzer systems. CONCLUSION: While reticulocyte counting is correlated between the automated methods evaluated in this study, the current state of harmonization of other reticulocyte parameters is not as strong. A major challenge in moving this field forward is the need for commutable materials to facilitate comparisons between analyzers not co-located. A potential alternate approach to improve the current state would be instrument re-calibration. However, this is challenging both technically and due to national regulatory frameworks.

Proceedings of the Association for Pathology Informatics Bootcamp 2022.

The Pathology Informatics Bootcamp, held annually at the Pathology Informatics Summit, provides pathology trainees with essential knowledge in the rapidly evolving field of Pathology Informatics. With a focus on data analytics, data science, and data management in 2022, the bootcamp addressed the growing importance of data analysis in pathology and laboratory medicine practice. The expansion of data-related subjects in Pathology Informatics Essentials for Residents (PIER) and the Clinical Informatics fellowship examinations highlights the increasing significance of these skills in pathology practice in particular and medicine in general. The curriculum included lectures on databases, programming, analytics, machine learning basics, and specialized topics like anatomic pathology data analysis and dashboarding.

Digital image analysis of erythroblastic islands in myelodysplastic syndromes.

INTRODUCTION: Myelodysplastic syndromes (MDS) encompass a diverse group of myeloid neoplasms for which the diagnosis of low-grade subtypes remains challenging. Erythroblastic islands (EBIs) are highly organized units of erythroid proliferation, differentiation, and enucleation. EBI disruption is frequently observed and is believed to be one of the early changes in MDS. METHODS: In this study, we digitally analyzed bone marrow biopsies dual stained with alpha-hemoglobin stabilizing protein (AHSP) and CD163 to quantitatively study features of EBIs in MDS, among MDS subtypes, as well as those in normal marrows and marrows with other causes of anemia. RESULTS: EBIs in MDS specimens were smaller in size and higher in density compared to both normal and non-MDS anemia specimens. Increased CD163 expression within the EBIs is observed in both MDS and other causes of anemia. A combination of increased EBI density and CD163 expression is seen in association with MDS with high-risk cytogenetics and multiple adverse mutations. CONCLUSION: As a proof-of-concept study, we show that EBI features can be relatively easily quantified with AHSP/CD163 dual immunohistochemistry and open-source imaging analysis software, highlighting those that are unique to MDS, and which may be prognostically relevant. Further studies of the measurable EBI features may provide valuable and novel tools to aid MDS diagnosis and prognostication in the era of digital pathology.

Hematology and Machine Learning.

BACKGROUND: Substantial improvements in computational power and machine learning (ML) algorithm development have vastly increased the limits of what autonomous machines are capable of. Since its beginnings in the 19th century, laboratory hematology has absorbed waves of progress yielding improvements in both of accuracy and efficiency. The next wave of change in laboratory hematology will be the result of the ML revolution that has already touched many corners of healthcare and society at large. CONTENT: This review will describe the manifestations of ML and artificial intelligence (AI) already utilized in the clinical hematology laboratory. This will be followed by a topical summary of the innovative and investigational applications of this technology in each of the major subdomains within laboratory hematology. SUMMARY: Application of this technology to laboratory hematology will increase standardization and efficiency by reducing laboratory staff involvement in automatable activities. This will unleash time and resources for focus on more meaningful activities such as the complexities of patient care, research and development, and process improvement.

Haematology laboratory parameters to assess efficacy of CD19-, CD22-, CD33-, and CD123-directed chimeric antigen receptor T-cell therapy in haematological malignancies.

INTRODUCTION: Chimeric antigen receptor (CAR) T cell products are available to treat relapsed/refractory B-lymphoblastic leukaemia/lymphoma (B-ALL), diffuse large B-cell lymphoma, mantle-cell lymphoma, and myeloma. CAR products vary by their target epitope and constituent molecules. Hence, there are no common laboratory assays to assess CAR T cell expansion in the clinical setting. We investigated the utility of common haematology laboratory parameters to measure CAR T cell expansion and response. METHODS: Archived CellaVision images, absolute lymphocyte counts, and Sysmex CPD parameters spanning 1 month after CD19-CAR, UCAR19, CD22-CAR, CD33-CAR, and UCAR123 therapy were compared against donor lymphocyte infused control patients. Additionally, CellaVision images gathered during acute EBV infection were analysed. RESULTS: CellaVision images revealed a distinct sequence of three lymphocyte morphologies, common among CD19-CAR, CD22-CAR and UCAR19. This lymphocyte sequence was notably absent in CAR T cell non-responders and stem-cell transplantation controls, but shared some features seen during acute EBV infection. CD19-CAR engraftment kinetics monitored by quantitative PCR show an expansion and persistence phase and mirror CD19-CAR ALC kinetics. We show other novel CAR T cell therapies (UCAR19, CD22-CAR, CD33-CAR and UCAR123) display similar ALC expansion in responders and diminished ALC expansion in non-responders. Furthermore, the CPD parameter LY_WY fluorescence increased within the first week after CD19-CAR infusion, preceding the peak absolute lymphocyte count (ALC) by 3.7 days. CONCLUSION: Autologous and allogeneic CAR T cell therapy produce unique changes in common haematology laboratory parameters and could be a useful surrogate to follow CAR T-cell expansion after infusion.

Hemolysis After Medication Exposure in Pediatric Patients With G6PD Deficiency.

Hemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficiency varies by mutation status and the oxidative stressor. Although classified by percent of enzymatic deficiency, variability in normal G6PD values clouds assessment of hemolysis risk by level. This was a retrospective, single institution, cohort study assessing risk of postexposure medication-induced hemolysis in G6PD deficient patients. Exposures occurred in 87 of 1415 deficient patients. Only 2 of 87 medication-exposed patients had hemolytic episodes and both had very low enzymatic activity. No hemolytic events occurred with G6PD levels >7 units/g hemoglobin. Correlation of levels with mutation may improve predictive capacity for hemolysis in G6PD deficiency.

Thromboelastography Changes of Whole Blood Compared to Blood Component Transfusion in Infant Craniosynostosis Surgery.

ABSTRACT: Surgical treatment of craniosynostosis with cranial vault reconstruction in infants is associated with significant blood loss. The optimal blood management approach is an area of active investigation. Thromboelastography (TEG) was used to examine changes in coagulation after surgical blood loss that was managed by transfusion with either whole blood or blood components. Transfusion type was determined by availability of whole blood from the blood bank.This retrospective study examined differences in posttransfusion TEG maximum amplitude (MA), a measure of the maximum clot strength, for patients transfused with whole blood or blood components. We included all patients less than 24 months old who underwent cranial vault remodeling, received intraoperative transfusions with whole blood or blood components, and had baseline and posttransfusion TEG measured. Whole blood was requested for all patients and was preferentially used when it was available from the American Red Cross.Of 48 eligible patients, 30 received whole blood and 18 received blood components. All patients received an intraoperative antifibrinolytic agent. The posttransfusion MA in the whole blood group was 61.8 mm (IQR 59.1, 64.1) compared to 57.9 mm (IQR 50.5, 60.9) in the blood components group (P = 0.010). There was a greater posttransfusion decrease in MA for patients transfused with blood components (median decrease of 7.7 mm [IQR -3.4, 6.3]) compared with whole blood (median decrease of 2.1 mm [IQR -9.6, 7.5] P < 0.001).Transfusion with blood components was associated with a greater decrease in MA that was likely related to decreased postoperative fibrinogen in this group. Patients who received whole blood had higher postoperative fibrinogen levels.

Implementation of an Extraction-Free COVID Real-Time PCR Workflow in a Pediatric Hospital Setting.

BACKGROUND: This study outlines the development, implementation, and impact of a laboratory-developed, extraction-free real-time PCR assay as the primary diagnostic test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a pediatric hospital. METHODS: Clinical specimens from both upper and lower respiratory tract sources were validated, including nasopharyngeal aspirates, nasopharyngeal swabs, anterior nares swabs, and tracheal aspirates (n = 333 clinical samples). Testing volumes and laboratory turnaround times were then compared before and after implementation to investigate effects of the workflow changes. RESULTS: Compared to magnetic-bead extraction platforms, extraction-free real-time PCR demonstrated ≥95% positive agreement and ≥97% negative agreement across all tested sources. Implementation of this workflow reduced laboratory turnaround time from an average of 8.8 (+/-5.5) h to 3.6 (+/-1.3) h despite increasing testing volumes (from 1515 to 4884 tests per week over the reported period of testing). CONCLUSIONS: The extraction-free workflow reduced extraction reagent cost for SARS-CoV-2 testing by 97%, shortened sample handling time, and significantly alleviated supply chain scarcities due to the elimination of specialized extraction reagents for routine testing. Overall, this assay is a viable option for laboratories to increase efficiency and navigate reagent shortages for SARS-CoV-2 diagnostic testing.

Aiding clinical assessment of neonatal sepsis using hematological analyzer data with machine learning techniques.

INTRODUCTION: Early diagnosis and antibiotic administration are essential for reducing sepsis morbidity and mortality; however, diagnosis remains difficult due to complex pathogenesis and presentation. We created a machine learning model for bacterial sepsis identification in the neonatal intensive care unit (NICU) using hematological analyzer data. METHODS: Hematological analyzer data were gathered from NICU patients up to 48 hours prior to clinical evaluation for bacterial sepsis. Five models, Support Vector Machine, K-nearest-neighbors, Logistic Regression, Random Forest (RF), and Extreme Gradient boosting (XGBoost), were trained on 60 hematological and nine clinical variables for 2357 cases (1692 control, 665 septic). Clinical feature only models (nine variables) were additionally trained and compared with models including hematological variables. Feature importance was used to assess relative contributions of parameters to performance. RESULTS: The three best performing models were RF, Logistic Regression, and XGBoost. RF achieved an average accuracy of 0.74, AUC-ROC of 0.73, Sensitivity of 0.38, and Specificity of 0.88. Logistic Regression achieved an average accuracy of 0.70, AUC-ROC of 0.74, Sensitivity of 0.62, and Specificity of 0.73. XGBoost achieved an average accuracy of 0.72, AUC-ROC of 0.71, Sensitivity of 0.40, and Specificity of 0.85. All models with hematological variables had significantly stronger performance than models trained on only clinical features. Neutrophil parameters had the highest average feature importance. CONCLUSIONS: Machine learning models using hematological analyzer data can classify NICU patients as sepsis positive or negative with stronger performance compared to clinical feature only models. Hematological analyzer variables could augment current sepsis classification machine learning algorithms.

Facilitating Test Status Information Communication in the Time of COVID-19.

The Coronavirus 2019 pandemic has strained nearly every aspect of pathology practice, including preanalytic, analytic, and postanalytic processes. Much of the challenges result from high demand for limited severe acute respiratory syndrome coronavirus 2 testing capacity, a resource required to facilitate patient flow throughout the hospital system and society at large. At our institution, this led to unprecedented increases in inquiries from providers to laboratory staff relating to the expected time to result for their patients. The demand was great enough to require redeployment of staff to handle the laboratory call volume. Although these data are available in our laboratory information system, the data do not interface to our electronic health record system. We developed systems using the R statistical programming language that abstract the necessary data regarding severe acute respiratory syndrome coronavirus 2 polymerase chain reaction testing from our lab system in real time, store it, and present it to clinicians for on demand querying. These data have been accessed over 2500 times by over 100 distinct users. Median length of each user session is approximately 4.9 minutes. Because our lab information system does not persistently store tracking information while our system does, we have been able to iteratively recalculate time to result values for each tracking stop as workflows have changed over time. Facility with informatics and programming concepts coupled with clinical understanding have allowed us to swiftly develop and iterate on applications which provide efficiency gains, allowing laboratory resources to focus on generating test results for our patients.

Clinical impact of genomic characterization of 15 patients with acute megakaryoblastic leukemia-related malignancies.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia but is approximately 500 times more likely to develop in children with Down syndrome (DS) through transformation of transient abnormal myelopoiesis (TAM). This study investigates the clinical significance of genomic heterogeneity of AMKL in children with and without DS and in children with TAM. Genomic evaluation of nine patients with DS-related TAM or AMKL, and six patients with non-DS AMKL, included conventional cytogenetics and a comprehensive next-generation sequencing panel for single-nucleotide variants/indels and copy-number variants in 118 genes and fusions involving 110 genes. Recurrent gene fusions were found in all patients with non-DS, including two individuals with complex genomes and either a NUP98-KDM5A or a KMT2A-MLLT6 fusion, and the remaining harbored a CBFA2T3-GLIS2 fusion, which arose from both typical and atypical cytogenetic mechanisms. These fusions guided treatment protocols and resulted in a change in diagnosis in two patients. The nine patients with DS had constitutional trisomy 21 and somatic GATA1 mutations, and those with DS-AMKL had two to four additional clinically significant somatic mutations. Comprehensive genomic characterization provides critical information for diagnosis, risk stratification, and treatment decisions for patients with AMKL. Continued genetic and clinical characterization of these rare cancers will aid in improving patient management.

Utility of repeat testing in the evaluation for von Willebrand disease in pediatric patients.

BACKGROUND: Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is caused by quantitative and qualitative defects in von Willebrand factor (VWF). The laboratory diagnosis of VWD in pediatric patients is complicated by VWF interassay and intra-assay variability, stress-induced elevations in VWF levels, and a lack of significant bleeding history with which to correlate test results. OBJECTIVE: Guidelines recommend repeat testing in patients with a high suspicion of VWD and unclear laboratory assay results; however, no studies have evaluated the utility of repeat VWF testing in pediatric patients. METHODS: This retrospective single-center cohort study aimed to determine clinical variables associated with requiring more than one test to diagnose VWD and to establish a cutoff VWF value above which further testing is not informative. RESULTS: Of 811 patients evaluated for a suspected bleeding disorder, 22.2% were diagnosed with VWD, with ~70% diagnosed on the first test. Patients with VWD were younger (5.8 vs. 8.5 years, P = .002) and more likely to have a family history of VWD (38% vs. 22%, P < .001) than those without VWD. Univariate analysis failed to identify any clinical variables that correlated with needing multiple tests for a VWD diagnosis. A cutoff of 100 IU/dL for VWF antigen or activity on the first test yielded negative predictive values >95%. CONCLUSIONS: We demonstrate that the majority of pediatric patients had diagnostic VWF values on the first set of testing. Pediatric patients without a family history of VWD and VWF levels >100 IU/dL may not need further testing to rule out the diagnosis of VWD.

Surgical Pathology Resident Rotation Restructuring at a Tertiary Care Academic Center.

Changes in the field of pathology and resident education necessitate ongoing evaluation of residency training. Evolutionary change is particularly important for surgical pathology rotations, which form the core of anatomic pathology training programs. In the past, we organized this rotation based on subjective insight. When faced with the recent need to restructure the rotation, we strove for a more evidence-based process. Our approach involved 2 primary sources of data. We quantified the number of cases and blocks submitted per case type to estimate workload and surveyed residents about the time required to gross specimens in all organ systems. A multidisciplinary committee including faculty, residents, and staff evaluated the results and used the data to model how various changes to the rotation would affect resident workload, turnaround time, and other variables. Finally, we identified rotation structures that equally distributed work and created a point-based system that capped grossing time for residents of different experience. Following implementation, we retrospectively compared turnaround time and duty hour violations before and after these changes and surveyed residents about their experiences with both systems. We evaluated the accuracy of the point-based system by examining grossing times and comparing them to the assigned point values. We found overall improvement in the rotation following the implementation. As there is essentially no literature on the subject of surgical pathology rotation organization, we hope that our experience will provide a road map to improve pathology resident education at other institutions.

Gain of CD26 expression on the malignant T-cells in relapsed erythrodermic leukemic mycosis fungoides.

Loss of CD26 surface expression on the circulating malignant T-cell is the most widely accepted diagnostic marker in patients with leukemic cutaneous T-cell lymphoma (CTCL). CTCL cases with reemergence of CD7 and/or CD26 surface expression are unusual and of uncertain prognosis. We report the case of an erythrodermic leukemic mycosis fungoides patient who had achieved temporary remission after several months on multimodality immunotherapy and extracorporeal photopheresis, but who relapsed with aggressive disease phenotypically characterized by CD4+ T-cells with high CD26 expression. Polymerase chain reaction studies and high-throughput sequencing analyses from peripheral blood mononuclear cells at presentation and relapse consistently showed an identical clonal T-cell receptor suggesting evolution of her original malignant clone which lacked CD26 expression. Interestingly, quantitative expression of the sialomucin, CD164, mirrored her clinical picture, thus favoring its reliability as a novel biomarker in CTCL.

Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy.

The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity.

Pleural mass forming extramedullary hematopoiesis masquerading as a malignant neoplasm.

Extramedullary hematopoiesis (EMH) represents the presence of immature hematopoietic elements and their differentiation into mature blood components outside of the medullary bone and may be seen in a variety of circumstances in the postnatal period, but is most strongly associated with disorders of the hematopoietic system. Postnatally, EMH is typically identified at sites of fetal hematopoiesis, the spleen, and liver, but occasional reports have identified it in nearly every tissue of the body. We report a case of EMH presenting as pleural mass, initially suspected to represent a neoplastic process in a patient with multiple comorbidities, including history of carcinoma, but without co-existing hematologic disorder. On-site evaluation of the fine-needle aspiration specimen was initially suspicious for a malignant neoplasm, but further evaluation revealed the lesion to be a mass forming focus of non-hepatosplenic EMH. In the era of increasing utilization of imaging, mass forming EMH is increasingly detected. When unsuspected, EMH may present a diagnostic challenge for the pathologist and may be confused for a neoplastic process.