Digital Imaging and AI Pre-classification in Hematology.

A leukocyte differential of peripheral blood can be performed using digital imaging coupled with cellular pre-classification by artificial neural networks. Platelet and erythrocyte morphology can be assessed and counts estimated. Systems from a single vendor have been used in clinical practice for several years, with other vendors' systems, in a development. These systems perform comparably to traditional manual optical microscopy, however, it is important to note that they are designed and intended to be operated by a trained morphologist. These systems have several benefits including increased standardization, efficiency, and remote-review capability.

Significant Variability in the Identification and Reporting of Band Neutrophils by Participants Enrolled in the College of American Pathologists Proficiency Testing Program: Time for a Change.

CONTEXT.­: Increased band neutrophils in blood smear differential counts ("bandemia") are entrenched in medicine as a flag for sepsis. However, laboratory hematology experts have long advocated for discontinuation of reporting bands separately from segmented neutrophils because of poor sensitivity and specificity, poor interobserver agreement, and availability of alternative biomarkers for sepsis. OBJECTIVE.­: To describe band neutrophil reporting practices and reproducibility of band classification among laboratories participating in the College of American Pathologists (CAP) proficiency testing (PT) program. DESIGN.­: A survey questionnaire was distributed to hematology PT participants. A subsequent morphologic challenge included 12 preselected cell identifications of segmented neutrophils, bands, and metamyelocytes, and a 100-cell manual differential count of a digitally scanned blood smear. RESULTS.­: Among laboratories that reported manual differentials, most respondents reported bands (4554 of 5268; 86.4%). Only 3222 of 4412 respondents (73.0%) provided band reference ranges. Though participants classified "easy" band neutrophils well (78.0%-98.3%), categorization of cell identifications for "moderate" and "difficult" bands was poor (3.1%-39.0% of laboratories), with classification instead as segmented neutrophils. This pattern was seen regardless of laboratory demographic characteristics. Marked variability in band counts was observed on the 100-cell differential count for both CAP PT participants and CAP Hematology and Clinical Microscopy Committee (HCMC) members (coefficients of variation, 55.8% and 32.9%, respectively). Variability was significantly improved when segmented and band neutrophils were grouped together (coefficients of variation, 6.2% and 5.0%, respectively). CONCLUSIONS.­: Most CAP PT-participating laboratories report band counts, many without reference ranges. The survey confirms significant interlaboratory variability of band enumeration when bands are separately identified from segmented neutrophils. This study reaffirms the CAP Hematology and Clinical Microscopy Committee's strong recommendation to group segmented and band neutrophils together in manual differential counts.

Formalin Fixation Followed by Paraffin Embedding Allows Long-Term Storage of Proteins for Liquid Chromatography-Tandem Mass Spectrometry Analysis.

In an anatomical pathology laboratory, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to characterize amyloid deposits identified in formalin-fixed paraffin-embedded tissue (FFPET). However, the development of additional tests is partially limited by the lack of information the passage of time has on the proteins in FFPET. To investigate the reliability of LC-MS/MS in the analysis of old FFPET specimens, 1 bone marrow aspirate clot was analyzed by LC-MS/MS yearly from 2014 to 2018, in 3 consecutive months. Peptide-spectrum match, number of peptides identified, and percentage of the proteins covered were the parameters collected for the hemoglobin subunits alpha (HbA), beta (HbB), delta (HbD), and gamma (HbG). These proteins are constant components of the peripheral blood and are present in high and low abundance, allowing the monitorization of the performance of the test across varying protein concentrations. The hemoglobin subunits were stable over the years studied; 71% to 74% of HbA, 77% to 80% of HbB, 69% to 77% of HbD, and 57% to 63% of HbG were covered, with no statistical difference between 2014 and 2018. The number of peptides identified was also constant, 11 to 13 for HbA, 13 to 15 for HbB, 11 to 14 for HbD, and 7 to 9 for HbG. Peptide spectrum match was only slightly more variable: 209 to 327 for HbA, 569 to 1052 for HbB, 286 to 533 HbD, and 142 to 292 for HbG. In conclusion, high abundance hemoglobins, HbA and HbB, and relatively low abundance ones, HbD and HbG, are preserved in FFPET and confidently identified by LC-MS/MS for at least 5 years.

IgM monoclonal gammopathy of undetermined significance: clinicopathologic features with and without IgM-related disorders.

A subset of patients with immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) develop IgM-related disorders (IgM-RD) including peripheral neuropathy, cryoglobulinemia and/or cold agglutinin disease (CAD). We examined the clinical and bone marrow pathologic findings in 191 IgM MGUS patients (2016 World Health Oragnization criteria). Clonal plasma cells were identified in 41 of 171 (24%) cases by immunohistochemistry (IHC) and clonal B cells in 43 of 157 (27%). IgM-RD was identified in 82 (43%) cases, including peripheral neuropathy (n=67, 35%), cryoglobulinemia (n=21, 11%), and CAD (n=10, 5%). Cases of CAD showed distinctive features including lack of MYD88 mutations (P=0.048), supporting the concept of primary CAD as a distinct clinicopathologic disorder. Following exclusion of CAD, comparison of the remaining cases with (n=72) or without (n=109) IgM-RD showed IgM-RD to be more frequent in men than women (P=0.02) and to be more highly associated with MYD88 L265P (P=0.011). Cases with and without IgM-RD otherwise showed similar features including serum IgM concentrations, presence of lymphoid aggregates, clonal B cells by flow cytometry or clonal plasma cells by IHC. No differences were observed in overall survival between cases with and without IgM-RD. No cases in this series met criteria for plasma cell type IgM MGUS as defined in the 2022 International Consensus Classification of lymphoid neoplasms. These results show IgM-RD to be common in patients with IgM MGUS. While CAD shows distinctive features, the remaining cases of IgM-RD largely show pathologic findings similar to IgM MGUS without IgM-RD.

Pathology Residents as Testing Personnel in the Hematology Laboratory.

CONTEXT.­: Clinical laboratories and the training of pathology residents are tightly regulated environments. Compliance with regulatory requirements must be addressed when developing entrustable professional activities (EPAs) for pathology residents. OBJECTIVE.­: To describe the development of EPAs for peripheral blood and body fluid review in compliance with Clinical Laboratory Improvement Amendments and College of American Pathologists personnel and testing requirements. To examine the impact of EPA implementation on the workflow in a busy hematology laboratory. DESIGN.­: A training program was designed to prepare pathology residents to function as independent testing personnel in compliance with Clinical Laboratory Improvement Amendments. After a series of lectures, hands-on microscopy sessions, self-assessment quizzes, and achievement of a passing score on a training assessment exam, residents were deemed competent to release certain results independently. The volume and the turnaround time of hematology tests were compared before and after residents were integrated into the laboratory workflow. Faculty and residents were surveyed to assess satisfaction with the training. RESULTS.­: Empowering residents to independently release noncritical results from peripheral blood and body fluid reviews had no adverse impact on test turnaround time. The resident contribution to workflow resulted in a corresponding decrease in the number of cases that required attending pathologist review. Faculty and residents viewed the EPAs as beneficial to service and education. CONCLUSIONS.­: The implementation of the EPAs had a beneficial effect on the laboratory, the trainees, and faculty. Our experience may be helpful to other training programs as EPAs become more widely implemented in residency training.

Risk Assessment of Hospitalized Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Infected Patients Using Laboratory Data and Immune Cell Morphologic Assessment.

CONTEXT.­: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious agent, with the propensity to cause severe illness. While vaccine uptake has been increasing in recent months, many regions remain at risk of significant coronavirus disease 19 (COVID-19)-related health care burden. Health systems will continue to benefit from the availability of a variety of clinical and laboratory models when other triaging models are equivocal. OBJECTIVE.­: To validate previously reported clinical laboratory abnormalities seen in COVID-19 patients and identify what laboratory parameters might be outcome predictive. DESIGN.­: We undertook an observational study of hospital-admitted COVID-19 patients (n = 113), looking at a broad selection of clinical, laboratory, peripheral blood smear, and outcome data during discrete discovery and validation periods from March 2020 to November 2020. RESULTS.­: We confirmed the findings of previous studies noting derangement of a variety of laboratory parameters in COVID-19 patients, including peripheral blood morphologic changes. We also devised a simple-to-use decision tree by which patients could be risk stratified on the basis of red blood cell count, creatinine, urea, and atypical plasmacytoid lymphocyte ("covidocyte") count. This outcome classifier performed comparably to the World Health Organization clinical classifier and the neutrophil-lymphocyte ratio. CONCLUSIONS.­: Our data add to the increasing number of studies cataloguing laboratory changes in COVID-19 and support the clinical utility of incorporating blood morphologic assessment in the workup of hospitalized COVID-19 patients.

IgM Plasma Cell Myeloma.

OBJECTIVES: Immunoglobulin M plasma cell myeloma (IgMPCM) is a rare entity that is difficult to distinguish from other IgM-related neoplasms. The study aims to characterize the clinicopathologic features of IgMPCM, including MYD88 L265P and CXCR4 mutations. METHODS: From our institutional archives, bone marrow biopsy specimens from January 1, 2008, to December 1, 2018, with monotypic plasma cells (PCs) expressing IgM that met current International Myeloma Working Group/World Health Organization criteria for PCM were included. Sanger sequencing was used to test for MYD88 L265P and WHIM-like CXCR4 mutations. RESULTS: Nine cases of IgMPCM were identified. Serum IgM paraproteins were detected in eight cases. CD138-positive PC burden averaged 41.9% (5%-80%). In four cases, PCs had lymphoplasmacytic morphology with cyclin D1 expression by immunohistochemistry. Three of four tested cases were positive for t(11;14) by fluorescence in situ hybridization, one with monosomy 13. The remaining case was positive for del13q14. All were negative for MYD88 L265P and WHIM-like CXCR4 mutations. Eight patients received immunochemotherapy, with four receiving autologous hematopoietic stem cell transplant. Median follow-up was 61 months (range, 11-120). All patients were alive except one. CONCLUSIONS: Distinguishing IgMPCM from other IgM-related disorders requires correlation with clinical, laboratory, and radiologic findings. Exclusion of MYD88 L265P and WHIM-like CXCR4 mutations may be useful to diagnose IgMPCM.

Characterisation and prognostic impact of immunoparesis in relapsed multiple myeloma.

Characterisation and prognostic impact of immunoparesis in relapsed multiple myeloma (MM) is lacking in the current literature. We evaluated 258 patients with relapsed MM, diagnosed from 2008 to 2015, to investigate the prognostic impact of deep immunoparesis on post-relapse survival. On qualitative immunoparesis assessment, no, partial and full immunoparesis was present in 9%, 30% and 61% of patients, respectively. Quantitative immunoparesis was assessed by computing the average relative difference (ARD) between polyclonal immunoglobulin(s) and corresponding lower normal limit(s), with greater negative values indicating deeper immunoparesis. The median ARD was -39%, with an optimal cut-off of -50% for overall survival (OS) by recursive partitioning analysis. Deep immunoparesis (ARD ≤-50%) was associated with a higher tumour burden at first relapse compared to none/shallow [ARD >-50%] immunoparesis. The OS (P = 0·007) and progression-free survival (PFS; P < 0·001) differed significantly between the deep and none/shallow immunoparesis groups. Kaplan-Meier estimates for 3-year OS were 36% and 46%, and for 2-year PFS were 17% and 27%, respectively. On multivariable analysis (MVA) for PFS, both qualitative and quantitative immunoparesis retained negative prognostic impact independently. However, only quantitative immunoparesis was independently prognostic for OS on MVA. Depth of immunoparesis in relapsed MM is an important prognostic factor for post-relapse survival in the era of novel agents and continuous therapy.

IgA plasma cell neoplasms are characterized by poorer long-term survival and increased genomic complexity compared to IgG neoplasms.

The characteristics of the IgA plasma cell neoplasms are not clearly reported in the literature. The main goal of this study is to examine IgA plasma cell neoplasms (PCN) and to compare them to IgG lesions. After at least 5 years from the identification of an M protein, 98 cases were selected, the presentation and clinical evolution of 45 IgA neoplasms were compared to 43 cases of IgG gammopathies. The classification at presentation as monoclonal gammopathy of undermined significance (MGUS)-22 of 45 IgA and 20 of 43 IgG (49 vs 46%), plasma cell myeloma (PCM)-22 of 45 IgA and 22 of 43 IgG (49 vs 51%) and smoldering PCM (SPCM)-1 each (2% for both) was essentially identical. No solitary plasmacytomas were identified. At presentation, IgA patients were younger (66.5 ± 11.3 vs. 69.2 ± 10.7 years), less likely to have bone lesions (12/45 vs 18/43, p < 0.14) or immunoparesis (51% vs. 63%), differences statistically insignificant. Cases with normal fluorescence in-situ hybridization (FISH) results, 27% for IgA vs 61% for IgG (p < 0.037) were statistically different. The IgA patients had worse survival (80 vs 108 months median IgA vs IgG, p < 0.013), difference not detectable in the first 5 years, but substantial after 10. In conclusion, poorer long-term survival and increased genomic complexity by FISH are characteristics of IgA PCNs.

Impact of Interventions to Change CBC and Differential Ordering Patterns in the Emergency Department.

Objectives: A CBC with leukocyte differential (CBC-DIFF) is a frequently ordered emergency department (ED) test. The DIFF component often does not add to clinical decision making. Our objective was to evaluate the impact of a performance improvement project on CBC ordering. Methods: ED orders for CBC-DIFF were identified through the laboratory information system. Two interventions were evaluated: an educational intervention regarding CBC-DIFF uses and a reprioritization of ED CBC-DIFF and CBC in the electronic medical record (EMR) orders. Pearson χ2 tests were used to assess for differences in the proportions. Results: There was no difference in the proportion of CBC tests performed after the education intervention (175/6,192, 2.8% [95% CI, 2.39%-3.21%] vs 219/6,270, 3.5% [95% CI, 3.05%-3.95%]). There was a significant increase in CBC samples ordered following the EMR intervention (604/6,044, 9.1% [95% CI, 8.37%-9.83%]; P < .01). Conclusions: Reprioritizing EMR laboratory orders can reduce overutilization of CBC-DIFF testing.

Transient abnormal myelopoiesis of a newborn not associated with chromosome 21 abnormalities or GATA1 mutations.

Transient abnormal myelopoiesis (TAM) is a disorder of Down syndrome newborns characterized by megakaryocytic blasts indistinguishable from acute myeloid leukemia (AML), which undergoes spontaneous remission. Acquired GATA1 mutations are present in blasts of both TAM and the subsequent AML which sometimes develops. We present a unique case of a newborn with leukemic megakaryoblasts indistinguishable from those of TAM who had neither extra material from chromosome 21 in the germline or blasts, nor evidence of GATA1 mutations. These findings suggest there are other genetic abnormalities that can lead to TAM besides GATA1 mutation in the setting of trisomy 21. Pediatr Blood Cancer 2015;62:353-355. © 2014 Wiley Periodicals, Inc.

Lymphoblastic transformation of follicular lymphoma: a clinicopathologic and molecular analysis of 7 patients.

Approximately 30% of patients with follicular lymphoma (FL) transform to a more aggressive malignancy, most commonly diffuse large B cell lymphoma. Rarely, FL transformation results in clinical findings, histology, and immunophenotype reminiscent of B-lymphoblastic leukemia/lymphoma. We report the largest series to date with detailed analysis of 7 such patients. Lymphoblastic transformation occurred on average 2 years after initial diagnosis of FL. Five patients had prior intensive chemotherapy. Two patients developed mature high-grade lymphoma, followed by the lymphoblastic transformation. FL had BCL2 gene rearrangement in 4 of 5 cases. High-grade transformation was accompanied by MYC gene rearrangement (5 of 5). Transformation was characterized by expression of TdT, loss of Bcl6, variable loss of immunoglobulin light chain, and persistence of Pax-5, Bcl2, and CD10. Whole-exome sequencing in 1 case revealed presence of several actionable mutations (CD79B, CCND3, CDK12). FL, aggressive mature B cell lymphoma, and lymphoblastic transformation were clonally related in 6 evaluable cases. After transformation, survival ranged from 1 to 14 months. Four patients died of disease, 2 were in remission after stem cell transplant, and 1 was alive with disease.

Utility and diagnostic pitfalls of SOX11 monoclonal antibodies in mantle cell lymphoma and other lymphoproliferative disorders.

Mantle cell lymphoma (MCL) is classically characterized by t(11;14) leading to cyclin D1 overexpression. Recently the transcription factor SOX11 has been discovered to be expressed in most MCL, including cyclin D1-negative cases. In this study we assess the performance of 2 commercially available monoclonal antibodies, Atlas Antibodies (Stockholm, Sweden) clone CLO142 and Cell Marque (Rocklin, CA) clone MRQ-58, for SOX11 immunohistochemistry in MCL, both cyclin D1 positive and cyclin D1 negative, as well as in cases of other small B-cell lymphoproliferative disorders, diffuse large B-cell lymphomas (DLBCLs), Burkitt lymphomas, and lymphoblastic leukemia/lymphomas. We also performed Western blots to further characterize the antibody specificity. Both antibodies show reliable, clear nuclear staining in MCL with variable specificity. However, the MRQ-58 antibody was more specific for MCL than CLO142, which showed considerably more nonspecific staining, especially in DLBCLs (59% positive vs. 4% positive with MRQ-58). In addition we reconfirmed the utility of SOX11 IHC for identifying cases of cyclin D1-negative blastoid MCL. However, we also identified cases of SOX11-positive DLBCL and splenic marginal zone lymphoma. Although SOX11 IHC is a powerful, and relatively accessible, tool to identify MCLs with variant immunophenotypes and/or morphology, these latter 2 cases highlight the need for strict criteria for interpreting SOX11 staining.

Aggressive B-cell lymphomas with translocations involving BCL6 and MYC have distinct clinical-pathologic characteristics.

OBJECTIVES: Recently described, aggressive B-cell lymphomas with genetic abnormalities involving MYC and BCL2 have been shown to have a poor prognosis when treated with regimens for diffuse large B-cell lymphomas. Similar data on cases with concurrent MYC and BCL6 translocation are still scant. Moreover, little is known regarding the morphologic and immunophenotypic characteristics of these cases, which further complicates their identification. This study describes six cases of aggressive B-cell lymphoma with translocations involving MYC and BCL6. METHODS: Six cases of large B-cell lymphoma with translocation involving MYC and BCL6 confirmed by fluorescence in situ were identified. The morphologic, immunophenotypic, and clinical features of the cases were examined. RESULTS: All the patients were older women, and in 50% of cases, the presentation was extranodal. In two cases, the liver was involved at presentation. A starry-sky pattern was a constant feature of the cases in which the morphology could be reliably assessed. Five of six cases had an immunophenotype corresponding to the germinal center B cells, and only one was positive for BCL2, an immunophenotype reminiscent of that of Burkitt lymphoma. CONCLUSIONS: B-cell lymphomas with translocations involving MYC and BCL6 have morphologic and immunophenotypic features suggestive of Burkitt lymphoma or gray zone lymphoma, and they tend to be diagnosed mainly in women, often in extranodal locations.

Hypercoagulable states: an algorithmic approach to laboratory testing and update on monitoring of direct oral anticoagulants.

Hypercoagulability can result from a variety of inherited and, more commonly, acquired conditions. Testing for the underlying cause of thrombosis in a patient is complicated both by the number and variety of clinical conditions that can cause hypercoagulability as well as the many potential assay interferences. Using an algorithmic approach to hypercoagulability testing provides the ability to tailor assay selection to the clinical scenario. It also reduces the number of unnecessary tests performed, saving cost and time, and preventing potential false results. New oral anticoagulants are powerful tools for managing hypercoagulable patients; however, their use introduces new challenges in terms of test interpretation and therapeutic monitoring. The coagulation laboratory plays an essential role in testing for and treating hypercoagulable states. The input of laboratory professionals is necessary to guide appropriate testing and synthesize interpretation of results.

Intensive chemotherapy and immunotherapy in patients with newly diagnosed primary CNS lymphoma: CALGB 50202 (Alliance 50202).

PURPOSE: Concerns regarding neurocognitive toxicity of whole-brain radiotherapy (WBRT) have motivated development of alternative, dose-intensive chemotherapeutic strategies as consolidation in primary CNS lymphoma (PCNSL). We performed a multicenter study of high-dose consolidation, without WBRT, in PCNSL. Objectives were to determine: one, rate of complete response (CR) after remission induction therapy with methotrexate, temozolomide, and rituximab (MT-R); two, feasibility of a two-step approach using high-dose consolidation with etoposide plus cytarabine (EA); three, progression-free survival (PFS); and four, correlation between clinical and molecular prognostic factors and outcome. PATIENTS AND METHODS: Forty-four patients with newly diagnosed PCNSL were treated with induction MT-R, and patients who achieved CR received EA consolidation. We performed a prospective analysis of molecular prognostic biomarkers in PCNSL in the setting of a clinical trial. RESULTS: The rate of CR to MT-R was 66%. The overall 2-year PFS was 0.57, with median follow-up of 4.9 years. The 2-year time to progression was 0.59, and for patients who completed consolidation, it was 0.77. Patients age > 60 years did as well as younger patients, and the most significant clinical prognostic variable was treatment delay. High BCL6 expression correlated with shorter survival. CONCLUSION: CALGB 50202 demonstrates for the first time to our knowledge that dose-intensive consolidation for PCNSL is feasible in the multicenter setting and yields rates of PFS and OS at least comparable to those of regimens involving WBRT. On the basis of these encouraging results, an intergroup study has been activated comparing EA consolidation with myeloablative chemotherapy in this randomized trial in PCNSL, in which neither arm involves WBRT.