Triple-Negative Primary Myelofibrosis: A Bone Marrow Pathology Group Study.

Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm driven by canonical gene mutations in JAK2, CALR, or MPL in >80% of the cases. PMF that lacks these canonical alterations is termed triple-negative PMF (TN-PMF). The pathologic and genetic characteristics of TN-PMF compared with those of conventional PMF with canonical driver mutations (DM-PMF) have not been well studied. We aimed to identify clinicopathologic and molecular genetic differences between patients with TN-PMF (n = 56) and DM-PMF (n = 89), all of whom fulfilled the 2016 World Health Organization diagnostic criteria for PMF. Compared with the control group, patients in the TN-PMF group were more likely to have thrombocytopenia and less likely to have organomegaly. The bone marrow in patients with TN-PMF showed fewer granulocytic elements and more frequent dyserythropoiesis. Cytogenetic analysis showed a higher incidence of trisomy 8. Targeted next-generation sequencing revealed a lower frequency of ASXL1 mutations but enrichment of ASXL1/SRSF2 comutations. Our findings demonstrated several clinicopathologic and molecular differences between TN-PMF and DM-PMF. These findings, particularly the observed mutation profile characterized by a higher frequency of ASXL1 and SRSF2 comutation, suggest that at least a subset of TN-PMF may be pathogenetically different from DM-PMF, with potential prognostic implications.

Clinical utility of targeted next-generation sequencing panel in routine diagnosis of hereditary hemolytic anemia: A national reference laboratory experience.

INTRODUCTION: Hereditary hemolytic anemias (HHA) comprise a heterogeneous group of disorders resulting from defective red blood cell (RBC) cytoskeleton, RBC enzyme deficiencies, and hemoglobin (Hb) synthesis disorders such as thalassemia or sideroblastic anemia. MATERIALS AND METHODS: Our hemolytic anemia diagnostic next-generation sequencing (NGS) panel includes 28 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The panel covers the complete coding region of these genes, splice junctions, and, wherever appropriate, deep intronic or regulatory regions are also included. Four hundred fifty-six patients with unexplained hemolytic anemia were evaluated using our NGS panel between 2015 and 2019. RESULTS: We identified pathogenic/likely pathogenic variants in 111/456 (24%) patients that were responsible for the disease phenotype (e.g., moderate to severe hemolytic anemia and hyperbilirubinemia). Approximately 40% of the mutations were novel. As expected, 45/456 (10%) patients were homozygous for the promoter polymorphism in the UGT1A1 gene, A(TA)7 TAA (UGT1A1*28). 8/45 homozygous UGT1A1*28 cases were associated with additional pathogenic mutations causing hemolytic anemia, likely exacerbating hyperbilirubinemia. The most common mutated genes were membrane cytoskeleton genes SPTA1, and SPTB, followed by PKLR. Complex interactions between SPTA1 low expression alleles, alpha-LELY and alpha-LEPRA alleles, and intragenic SPTA1 variants were associated with hereditary pyropoikilocytosis and autosomal recessive hereditary spherocytosis in 23/111 patients. CONCLUSIONS: Our results demonstrate that hemolytic anemia is underscored by complex molecular interactions of previously known and novel mutations in RBC cytoskeleton/enzyme genes, and therefore, NGS should be considered in all patients with clinically unexplained hemolytic anemia and in neonates with hyperbilirubinemia. Moreover, low expression alleles alpha-LELY and alpha-LEPRA should be included in all targeted HHA panels.

δ-Globin Chain Variants Associated with Decreased Hb A2 Levels: A National Reference Laboratory Experience.

High prevalence of hemoglobin (Hb) disorders mandates national programs for screening and genetic counseling in many countries. Increased Hb A2 levels are commonly associated with ß-thalassemias, however, various disorders including alteration of δ chains may result in decreased production of Hb A2, thus hindering the diagnosis of ß-thalassemias. The reported data reflect the experience of a large reference laboratory in the United States. In the current study, we have attempted to assess the prevalence and also tried to characterize the identified mutations in the HBD gene resulting in decreased Hb A2 levels. In our cohort, 1.6% of 6486 patients were found to have Hb A2 values of <1.9%. Bidirectional sequencing of the HBD gene demonstrated mutations in 20 cases (19.0% of the individuals with decreased Hb A2). In addition to the previously reported variants, one novel mutation (Hb A2-Utah or HBD: c.46T>C).

Reproducibility of Serum Potassium Values in Serum From Blood Samples Stored for Increasing Times Prior to Centrifugation and Analysis.

GOALS: The goal of this work was to determine if immediate versus postponed centrifugation of samples affects the levels of serum potassium. METHODS: Twenty participants donated normal venous blood that was collected in four serum separator tubes per donor, each of which was analyzed at 0, 1, 2, or 4 hr on the Siemens Advia 1800 autoanalyzer. RESULTS: Coefficients of variation (CVs) for potassium levels ranged from 0% to 7.6% with a mean of 3 ± 2%. ANOVA testing of the means for all 20 samples showed a P-value of 0.72 (>0.05) indicating that there was no statistically significant difference between the means of the samples at the four time points. Sixteen samples were found to have CVs that were ≤5%. Two samples showed increases of potassium from the reference range to levels higher than the upper reference limit, one of which had a 4-hr value that was within the reference or normal range (3.5-5 mEq/l). Overall, most samples were found to have reproducible levels of serum potassium. CONCLUSIONS: Serum potassium levels from stored whole blood collected in serum separator tubes are, for the most part, stable at room temperature for at least 4 hr prior to analysis. However, some samples can exhibit significant fluctuations of values.

Advances in Hemoglobinopathies and Thalassemia Evaluation.

Hemoglobin (Hb) disorders are among the most prevalent inherited diseases. Despite a limited number of involved genes, these conditions represent a broad clinical and prognostic spectrum. The menu of laboratory tests is extensive. From widely available modalities, for example, complete blood count to rather sophisticated molecular technologies, the investigation of Hb disorders recapitulates an increasing complexity of laboratory workup in other medical fields. This review highlights a current state of biochemical and molecular investigation of Hb disorders and offers a glimpse on technologies that are yet to be fully embraced in clinical practice.

How I diagnose systemic mastocytosis.

OBJECTIVES: Systemic mastocytosis (SM) is a neoplasm of mast cells (MCs) characterized by their proliferation in extracutaneous organs. Systemic mastocytosis includes several entities with different clinical courses and prognoses. The rarity of this disease and the diversity of clinical and morphologic presentation make the diagnosis of SM very challenging. The aim of this review is to share our approach to the diagnosis of SM. METHODS: We present 4 cases that highlight the spectrum of clinical and laboratory features of SM and outline the diagnostic process with an emphasis on morphology. RESULTS: Pathology and laboratory medicine play a key role in investigation of SM, as correct diagnosis requires integration of morphologic, molecular, and serologic findings. In addition to awareness of microscopic findings in SM, a pathologist must keep abreast with an expanding menu of ancillary studies, particularly molecular testing. CONCLUSIONS: Systemic mastocytosis is a challenging diagnosis that requires not only a demonstration of a clonal proliferation of MCs but also a correct subclassification based on the recently updated criteria.

Advances in Hematology Analyzers Technology.

The evolution of complete blood count (CBC) methodology from manual calculations to sophisticated high throughput hematology analyzers is the focus of this article. In recent years, hematology testing has greatly benefitted from the combination of various technologies with automated neural networks. In addition to an increasing complexity of the laboratory instrumentation, there is a demand on point of care CBC testing with its benefits and drawbacks. This article highlights exciting advancements of hematology testing from the past to the present and into the future.

Chronic Myelomonocytic Leukemia and Atypical Chronic Myeloid Leukemia: A National Analysis.

BACKGROUND: Myelodysplastic/myeloproliferative overlap syndromes (MDS/MPN) are rare blood cancers characterized by concomitant myeloid hyperplasia and dysplasia. These heterogenous disorders include chronic myelomonocytic leukemia (CMML) and atypical chronic myeloid leukemia (aCML). METHODS: Using two large national cancer databases to examine a total of 15,704 CMML and 702 aCML patients, we report the largest study to date on the incidence, survival and demographic characteristics of CMML and aCML in the United States. RESULTS: Overall age-adjusted incidence of CMML and aCML was 0.63 per 100,000 Americans per year and 0.03 per 100,000 per year, respectively. CMML incidence in the U.S. was noted to rise steadily in the years between 2001 and 2019. Median patient age was 75 and 72 years for CMML and aCML, and the majority of CMML and aCML patients were male (62.9% and 62.0%) and White (90.1% and 86.3%). Median OS was 17.4 months for CMML, and 15.2 months for aCML. Multivariate Cox regression demonstrated features associated with reduced survival, including increasing age, comorbidities, Medicaid insurance status, and low-income residential zip code, highlighting survival disparities in underinsured and socioeconomically disadvantaged patients. In CMML, Black race was associated with inferior survival, while female sex, management at an academic center, and later calendar-year of diagnosis were associated with improved OS. CONCLUSION: These findings underscore the need to better understand the biological basis for such differences in survival and reflect the importance of access to specialized care for patients with these rare disorders.

Applications of Flow Cytometry in Diagnosis and Evaluation of Red Blood Cell Disorders.

Clinical flow cytometry plays a vital role in the diagnosis and monitoring of various red blood cell disorders. The high throughput, precision, and automation potential of this technique allows for cost-effective and timely analysis compared to older and more manual test methods. Flow cytometric analysis serves as the gold standard diagnostic method for multiple hematological disorders, especially in clinical scenarios where an assay needs to have high sensitivity, high specificity, and a short turnaround time. In this review, we discuss the role of flow cytometric analysis in paroxysmal nocturnal hemoglobinuria, fetal-maternal hemorrhage, and hereditary spherocytosis.

Stability of BUN and creatinine determinations on the Siemens Advia 1800 analyzer.

BACKGROUND: Serum creatinine values of patients tend to change as a result of the use of different blanks used for creatinine determinations on the Advia 1650. After upgrading the analyzer to the Advia 1800, creatinine values tended to be more reproducible. As part of a quality assurance investigation to test the reproducibilities of creatinine values, we determined serial creatinine values in the sera of 13 patients whose initial values were either in the reference range or elevated (range 0.58-7.8 mg/dl). These values were determined concurrently with serum blood urea nitrogen (BUN) determinations (range 6.0-84.4 mg/dl) as these two analytes are used together in evaluation of renal function. METHODS: We determined BUN and creatinine values, using the glutamate dehydrogenase lined enzyme assay system and the Jaffe method, respectively. RESULTS: We find that all values for creatinine on samples stored at 4 °C were reproducible as were the corresponding BUN values, which is revealed by low values for the coefficients of variation (CVs), that is, mean CV of 4.55% for creatinine and 2.52% for BUN. One sample with relatively high CV (10.6%) for creatinine was found to have an initial value of 1.1 mg/dl, in the reference range; but, on repeat determinations, the obtained levels were as high as 1.5 mg/dl, above the reference range. BUN values for this sample remained in the reference range, suggesting that no renal disease was present. CONCLUSION: We conclude that creatinine and BUN determinations are stable, but occasional spurious creatinine values can occur on the Advia 1800 analyzer.

Molecular diagnosis of hereditary hemolytic anemias: Recent updates.

Hereditary hemolytic anemia (HHA) is a heterogeneous group of disorders due to genetically caused defects in red blood cell membrane structure, enzymes, heme and globin synthesis, erythroid proliferation, and differentiation. Traditionally, the diagnostic process is complex and includes a plethora of tests from routine to highly specialized ones. The inclusion of molecular testing has significantly improved the diagnostic yield. The value of molecular testing is broader than just rendering the correct diagnosis, as it may also guide therapeutic decisions. As more molecular modalities become available for clinical use, it is imperative to understand their benefits and disadvantages pertaining to the HHA diagnostics. Re-evaluation of the traditional diagnostic workflow may also bring forth additional benefits. This review focuses on the current state of molecular testing for HHA.

Granulomas in bone marrow biopsies: clinicopathological significance and new perspectives.

Bone marrow granulomas in trephine biopsies are a rare and usually incidental finding. Possible causes include infectious (especially tuberculous and rarer non-tuberculous mycobacteria, but also many other bacterial, viral, fungal and parasitic agents) and non-infectious causes (especially medications, autoimmune disease, sarcoidosis, haematological and non-haematological malignancy). Necrotising granulomas are generally suggestive of an infectious aetiology (tuberculosis being the most common), whereas fibrin ring granulomas are associated with Q-fever and Epstein Barr Virus, although exceptions are possible. Every case suspicious for infectious aetiology should undergo further analysis like special staining (Ziehl-Neelsen for acid-fast rods) or molecular studies. The histomorphology should always be clinically correlated. In cases in which no infectious cause can be identified, untargeted metagenomics may represent a valid diagnostic tool that may become standard in the near future for bone marrow diagnostics. In this review, we have analysed the published data from 1956 up to today, and we report aspects of epidemiology, aetiology, diagnostic algorithms, differential diagnosis and the role of metagenomics in bone marrow biopsies with granulomas.

Validation of MYC and BCL6 rapid break apart digital fluorescence in situ hybridization assays for clinical use.

OBJECTIVE: Detection of gene rearrangements in MYC (a family of regulator genes and proto-oncogenes) and human B-cell lymphoma 6 (BCL6) using fluorescence in situ hybridization (FISH) are important in the evaluation of lymphomas, in particular diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma. Our current clinical MYC and BCL6 FISH workflow involves an overnight hybridization of probes with digital analysis using the GenASIs Scan and Analysis instrument (Applied Spectral Imaging). In order to improve assay turnaround time SureFISH probes were validated to reduce the hybridization time from 16 hours down to 1.5 hours. METHODS: Validation was a four-phase process involving initial development of the assays by testing new probes in a manual protocol, and cytogenetic studies to confirm the probe specificity, sensitivity, and localization. In the next phase, the assays were validated as a manual assay. The third phase involved development of the digital FISH assays by testing and optimizing the GenASIs Scan and Analysis instrument. In the final phase, the digital FISH assays were validated. RESULTS: Cytogenetic studies confirmed 100% probe sensitivity/specificity, and localization patterns. Negative reference range cutoffs calculated from 20 normal lymph nodes using the inverse of the beta cumulative probability density function (Excel BETAINV calculation) were 11% inclusive for both manual and digital MYC and BCL6 assays. There was 100% concordance between the manual and digital methods. The shortened hybridization time decreased the overall workflow time by 14.5 hours. CONCLUSIONS: This study validates the use of the SureFISH MYC and BCL6 probes on formalin fixed paraffin embedded (FFPE) tissue sections using a hybridization time of 1.5 hours that shortened the overall workflow by 14.5 hours. The process described also provides a standardized framework for validating digital FISH assays in the future.

Myeloid sarcoma with NPM1 mutation may be clinically and genetically distinct from AML with NPM1 mutation: a study from the Bone Marrow Pathology Group.

Myeloid sarcoma (MS) is currently considered equivalent to de novo acute myeloid leukemia (AML); however, the relationship between these entities is poorly understood. This retrospective multi-institutional cohort study compared 43 MS with NPM1 mutation to 106 AML with NPM1 mutation. Compared to AML, MS had more frequent cytogenetic abnormalities including complex karyotype (p = .009 and p = .007, respectively) and was enriched in mutations of genes involved in histone modification, including ASXL1 (p = .007 and p = .008, respectively). AML harbored a higher average number of gene mutations (p = .002) including more frequent PTPN11 mutations (p < .001) and mutations of DNA-methylating genes including DNMT3A and IDH1 (both p < .001). MS had significantly shorter overall survival (OS) than AML (median OS: 44.9 vs. 93.2 months, respectively, p = .037). MS with NPM1 mutation has a unique genetic landscape, and poorer OS, compared to AML with NPM1 mutation.

First study comparing genetic profiles of MS and AML with a common disease-defining lesion.NPM1^Mut MS may be genetically distinct from NPM1^Mut AML.NPM1^Mut MS may have inferior overall survival compared to NPM1^Mut AML.

Lymphoid aggregates in bone marrow: a diagnostic pitfall.

Lymphoid aggregates in bone marrow specimens are a relatively frequent finding that may pose a diagnostic challenge for a pathologist. The distinction between reactive and neoplastic aggregates has significant clinical relevance. Although many testing modalities such as immunohistochemistry, flow cytometry and molecular studies are currently available in clinical laboratories, the appropriate utilisation of these modalities and the awareness of their potential pitfalls are important. When a neoplastic process is ruled out, the significance of benign lymphoid aggregates in bone marrow is often unclear, as they may be associated with a broad spectrum of conditions including infections, autoimmune disorders, medications, or may even be idiopathic.This review focuses on evidence-based criteria that can aid in making the distinction between benign and malignant lymphoid aggregates and discusses the advantages, disadvantages and limits of ancillary tests used for this purpose. Finally, the most common aetiologies of benign lymphoid aggregates and their associations with specific diseases are discussed.

Laboratory approach to investigation of anemia in pregnancy.

Anemia is a global health problem in all age groups. According to World Health Organization (WHO), approximately 40% of pregnant women are anemic. Iron deficiency anemia (IDA) due to nutritional deficiency is the most common cause. The incidence of IDA varies worldwide depending on the socioeconomic status, but it remains the leading cause even in developed countries. Physiologic anemia of pregnancy due to relatively higher expansion of blood volume in comparison with elevated red blood cell mass also occurs frequently. Complete blood count (CBC) in the first trimester is recommended for all pregnant women to screen for anemia. The screening of pregnant women for IDA in absence of anemia is still debatable. If IDA is suspected, ferritin level of <30 ng/ml is diagnostic. Iron supplementation is recommended for all pregnant women to compensate the increased demand.

Laboratory approach to investigation of anemia with a focus on pyruvate kinase deficiency.

Anemia is a major health burden worldwide and affects approximately one-third of world's population. It is not a diagnosis; it is a manifestation of an underlying pathophysiology leading to either decreased hemoglobin (Hb), hematocrit (Hct), or red blood cells (RBCs). Iron deficiency anemia is still the most common cause of anemia worldwide. The symptoms are usually due to the underlying compensatory responses to decrease in oxygen delivery to the tissues. Laboratory investigation should start with complete blood count (CBC), reticulocyte count (RC), and peripheral smear evaluation. Further testing depends on these indices, that is, iron parameters and hemoglobinopathies/thalassemia evaluation in microcytic hypochromic anemia, vitamin B12, and folic acid level in macrocytic anemia. Increased RC denotes adequate bone marrow response and points toward hemolytic process and vice versa. Anemia diagnosis can be complex and confusing for the practicing physician. This review tries to give a practical simplistic approach to the diagnosis, focusing mainly on the basic parameters, that is, CBC, RC, and peripheral smear etc. Moreover, we have also tried to provide an update on the pyruvate kinase deficiency, as there has been recent exciting development in the management of these patients.