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How I investigate difficult cells at the optical microscope
Abstract
Blood cell morphological identification on the peripheral blood and bone marrow films remains a cornerstone for the diagnosis of hematological neoplasms to be integrated with immunophenotyping, molecular genetics, and histopathology. Although standardization is still far from being achieved, with high interobserver variability, in recent years, several classification approaches, from the 1976 FAB to the 2016 WHO classification, have provided hematologists with detailed morphological descriptions for a large number of diseases. Counting blasts and detecting dysplastic specimens are two cornerstones of morphological diagnosis. This review deals with identifying difficult cells, with particular reference of those with relevant diagnostic implications.
... Inter-observer agreement in identifying normal or hypercellular pathological samples with clear morphology is generally satisfactory. However, disagreements can arise when interpreting dysplastic features, especially when clonal cells are present at low frequencies and below diagnostic threshold levels, resulting in low reproducibility [38,39]. The absence of standardized criteria for classifying dysplasia presents a challenge in developing a universally accurate AI algorithm for recognizing dysplastic cells [15]. ...
Digital morphology (DM) analyzers have advanced clinical hematology laboratories by enhancing the efficiency and precision of peripheral blood (PB) smear analysis. This review explores the real-world application of DM analyzers with their benefits and challenges by focusing on PB smear analysis and less common analyses, such as bone marrow (BM) aspirates and body fluids (BFs). DM analyzers may automate blood cell classification and assessment, reduce manual effort, and provide consistent results. However, recognizing rare and dysplastic cells remains challenging due to variable algorithmic performances, which affect diagnostic reliability. The quality of blood film as well as staining techniques significantly influence the accuracy of DM analyzers, and poor-quality samples may lead to errors. In spite of reduced inter-observer variability compared with manual counting, an expert’s review is still needed for complex cases with atypical cells. DM analyzers are less effective in BM aspirates and BF examinations because of their higher complexity and inconsistent sample preparation compared with PB smears. This technology relies heavily on artificial intelligence (AI)-based pre-classifications, which require extensive, well-annotated datasets for improved accuracy. The performance variation across platforms in BM aspirates and rare-cell analysis highlights the need for AI algorithm advancements and DM analysis standardization. Future clinical practice integration will likely combine advanced digital platforms with skilled oversight to enhance diagnostic workflow in hematology laboratories. Ongoing research aims to develop robust and validated AI models for broader clinical applications and to overcome the current limitations of DM analyzers. As technology evolves, DM analyzers are set to transform laboratory efficiency and diagnostic precision in hematology.
... BM blast count by light microscopy was also correlated with WT1 levels in AML, while not in MDS. Conversely, blast count by flow cytometry was positively associated with WT1 levels also in MDS patients, confirming flow cytometry as a more sensitive and specific tool for identification of leukemic cells compared to light microscopy that is operator dependent [29][30][31]. ...
Wilm’s tumor 1 (WT1), a zinc-finger transcription factor and an epigenetic modifier, is frequently overexpressed in several hematologic disorders and solid tumors, and it has been proposed as diagnostic and prognostic marker of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, the exact role of WT1 in leukemogenesis and disease progression remains unclear. In this real-world evidence retrospective study, we investigated prognostic role of WT1-mRNA expression levels in AML and MDS patients and correlations with complete blood counts, flow cytometry counts, and molecular features. A total of 71 patients (AML, n = 46; and MDS, n = 25) were included in this study, and WT1 levels were assessed at diagnosis, during treatment and follow-up. We showed that WT1 expression levels were inversely correlated with normal hemopoiesis in both AML and MDS, and positively associated with blast counts. Flow cytometry was more sensitive and specific in distinguishing normal myeloid cells from neoplastic counterpart even just using linear parameters and CD45 expression. Moreover, we showed that a simple integrated approach combining blast counts by flow cytometry, FLT3 mutational status, and WT1 expression levels might be a useful tool for a better prognostic definition in both AML and MDS patients.
The hierarchical organization of the leukemic stem cells (LSCs) is identical to that of healthy counterpart cells. It may be split into roughly three stages: a small number of pluripotent stem cells at the top, few lineage-restricted cells in the middle, and several terminally differentiated blood cells at the bottom. Although LSCs can differentiate into the hematopoietic lineage, they can also accumulate as immature progenitor cells, also known as blast cells. Since blast cells are uncommon in healthy bloodstreams, their presence might be a sign of cancer. For instance, a 20% blast cutoff in peripheral blood or bone marrow is formally used to distinguish acute myeloid leukemia from myelodysplastic neoplasms, which is essential to plan the patients’ management. Many techniques may be useful for blast enumeration: one of them is flow cytometry, which can perform analyses on many cells by detecting the expression of cell surface markers. Leukemic and non-leukemic blast cells might indeed be characterized by the same surface markers, but these markers are usually differently expressed. Here we propose to use CD45, in combination with CD34 and other cell surface markers, to identify and immunophenotype blast cells in patient-derived samples.
Overexpression of Phosphatases of Regenerating Liver 2 (PRL2), detected in numerous diverse cancers, is often associated with increased severity and poor patient prognosis. PRL2-catalyzed tyrosine dephosphorylation of the tumor suppressor PTEN results in increased PTEN degradation, and has been identified as a mechanism underlying PRL2 oncogenic activity. Overexpression of PRL2, coincident with reduced PTEN protein, is frequently observed in Acute Myeloid Leukemia (AML) patients. In the current study, a PTEN-knockdown AML animal model was generated to assess the impact of conditional PRL2 inhibition on the level of PTEN protein and the development and progression of AML. Inhibition of PRL2 resulted in a significant increase in median animal survival, from 40 weeks to greater than 60 weeks. The prolonged survival reflected delayed expansion of aberrantly differentiated hematopoietic stem cells into leukemia blasts, resulting in extended time required for clinically relevant leukemia blast accumulation in the bone marrow niche. Leukemia blast suppression following PRL2 inhibition was correlated with an increase in PTEN, and downregulation of AKT/mTOR regulated pathways. These observations directly established, in a disease model, the viability of PRL2 inhibition as a therapeutic strategy for improving clinical outcomes in AML and potentially other PTEN-deficient cancers by slowing cancer progression.
Background: Ponatinib is a 3rd generation tyrosine kinase inhibitor (TKI) very effective in Chronic Myeloid Leukemia (CML);nevertheless, cardiovascular (CV) adverse events represent a major issue during therapy and some groups proposed to reduce the drug dose for avoiding CV complications. It has been established that the therapeutic plasmatic concentration of Ponatinib must be ≥ 20 ng/mL, but little is known about the relationship between drug dose, plasmatic concentration and toxicity or response to treatment.
Aim: We present here a new high resolution mass spectrometry-based method to determine Ponatinib plasma concentration.
Method: We collected 15 peripheral blood samples from 11 CML patients (6 males and 5 female, mean age 44) in treatment with Ponatinib at different doses (from 45 mg daily to 30 mg weekly). The reason for switch to Ponatinib was toxicity in 4 and resistance in 7 cases. In the resistant cohort, Ponatinib allowed to reach major (MMR) and deep molecular response (DMR) in 28.5% and 71.5% of cases, respectively. Samples were analyzed by LC/HRMS (Ultimate 3000 LC system with TurboFlow technology coupled to a Q-Exactive system). After deproteinization with an acetonitrile/methanol solution (75/25), the samples were injected directly into the system, using Tracefinder® software for quantification analysis. The procedure was validated and successfully applied to the blood samples in routine laboratory analyses and can be considered fast and easy.
Results: The method proves to be reliable with both precision and accuracy higher than 85% and showing a very good specificity and sensitivity. The therapeutic Ponatinib concentration of 20.0 ng/mL was reached in 73.3% of tested samples, regardless of the dosage scheme (15, 30 or 45 mg daily), except for 2 samples from the same patient treated with a very-low dose (30 mg weekly) because of a very-high cardiovascular risk.
Conclusions: 1) even if on a small series, our data confirm the high response rate achievable with Ponatinib; 2) the achievement of the minimal therapeutic concentration regardless of the dosing regimen can explain the dose-independent Ponatinib efficacy also reported in the OPTIC Trial; 3) the method resulted to be accurate and easily applicable for a patient-tailored treatment in clinical practice. In the further steps, we’ll investigate if the cytochromeP450 or efflux-pump polymorphisms might condition the Ponatinib plasma levels.
Several risk factors are involved in the pathogenesis of venous thromboembolism
(VTE) in patients affected by cancer, these include cancer type, central venous catheters placement, chemotherapy, radiotherapy, immunomodulatory drugs. The management of VTE may be quite complex in patients with cancer, this is mainly due to an increased risk of recurrent VTE, haemorrhages, morbidity, and hospital admissions. With reference
to haematological malignancies, a high intrinsic risk of VTE is related to several disease-specific factors (high blood viscosity, JAK-2 hyper activation, nephrotic syndrome and elevated cytokines levels) and treatment related factors (immunomodulatory drugs and asparaginase). VTE occurrence may negatively affect treatment of the underlying blood cancer and expose patients to a higher risk of death. Furthermore, patients affected
by haematological malignancies have a particularly higher risk of bleeding because of thrombocytopenia and hyperfibrinolysis. Defining the clinical indication to prophylaxis of VTE is quite challenging, based on the varying risks of VTE and bleeding complications across different haematological malignancies and their specific treatments. Options for the treatment and prevention of cancer associated VTE, previously mainly based on low-molecular- weight heparin (LMWH), have recently been enriched by direct oral anticoagulants (DOACs). These agents have shown a good risk/benefit profile, however they require an accurate patient-based evaluation for their potential interactions with other treatments and bleeding risks. Up-to-date, studies on the treatment of VTE in patients affected by acute leukaemia are scant and specific guidelines on prophylaxis of VTE in patients affected by leukaemia and lymphoma are not available, while single consensus documents have been developed for patients affected by multiple myeloma
and chronic myeloproliferative neoplasm (MPN) with several limits; guidelines on the prevention of VTE in patients with cancer have been recently updated by several scientific societies, and one National Society (AIOM 2020). These recommendations, however, have not been specifically conceived for patients with haematological malignancies, thus the Società Italiana di Ematologia (SIE) has recently developed the first national evidence-based clinical practice guidelines on the administration of pharmacological primary prophylaxis of VTE in patients affected by haematological malignancies. The SIE guidelines have used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology and adopted as reference international guideline, the last updated guidelines from ITAC. The primary aim of these guidelines was to define the primary prophylaxis of VTE schedule in patients affected by haematological malignancies, managed in national structures, including available options, approved for this indication in Italy and published evidences. The target population of these guidelines includes patients affected by acute leukemia, lymphoma and multiple myeloma, not under chronic anticoagulant treatments. The SIE guideline working group (WG) involved Italian haematologists expert in this field (AF, VdS ,MM, AV), three methodology experts (MM,MP, PB) one coordinator (MN), volunteer representatives from scientific societies (MdN, AT,PT,GL) and patients (AP). Guidelines were peer reviewed by 3 external academic clinicians (SS, FR, RS). During the first WG teleconference encounter, recommendations from ITAC guidelines were selected for their further analysis. The following benchmarks were selected: Patients were deemed at high risk of VTE for an estimated VTE risk of more than 5% at 6 months; A pharmacological prophylaxis of VTE was judged acceptable for a ratio of the number needed to treat (NNT) for symptomatic VTE prevention and the number needed to harm (NNH) for major bleeding complications of more than 3; The availability of oral pharmacological prophylaxis of VTE was deemed relevant for patients’ quality of life.PICO questions and recommendations are summarised in Table 1 and 2. Further studies in this field will allow to improve the intricate management of VTE in haematological malignancies.
Newly diagnosed multiple myeloma (MM) who are transplant ineligible (NTE NDMM) are usually treated with multiple-drug combinations including proteasome inhibitors, immunomodulatory drugs and alkylating agents. Recently approved combo therapies including anti-CD38 monoclonal agents and/or lenalidomide improve progression-free survival (PFS) as compared with one of the standard treatments. We thus aimed at assessing the relative efficacy of novel daratumumab-based and lenalidomide based triplets/quadruplets as compared with overall standard treatments for NTE NDMM, namely Rd and VMP. Network metaanalyses (NMA) are accepted evidence-based tools for conducting indirect comparisons among treatments, however, the scientific community is still skeptical regarding their robustness. We, therefore conducted
an umbrella review: fully published NMAs were retrieved by standard searches (EBMASE, Cochrane Library, MEDLINE/PubMed) and appraised by AMSTAR-2 and ROBIS tools. Three indirect comparisons of PFS were targeted: 1) VRD versus VMP, 2) DaraRd versus VMP, 3) DaraVMP versus Rd. Overall 17 NMA addressing NDMM were published
since Jan 2017: 6 fully published ones including both daratumumab- and lenalidomide-based novel treatments were appraised. The overall quality of the NMAs was poor to moderate according to AMSTAR-2 and ROBIS. Each NMA analyzed 6 to 27 trials and 2 ones were company sponsored.
1) VRD was compared to VMP by 3 moderate-quality NMAs, which consistently reported a significant amelioration of PFS or higher SUCRA of VRD, while OS-HR was not conclusive.
2) DaraRd was compared to VMP by 4 NMAs and the pooled PFSHR ranged from 0.39 to 0.61. A significant amelioration of OS was also reported by the unique NMA assessing this endpoint.
3) DaraVMP was compared versus Rd by 4 NMAs. Pooled HR ranged from 0.35 to 0.71, which was statistically significant in two ones. DaraVMP achieved the highest SUCRA (0.960) in the latest and largest
NMA (Giri et al 2020). Only one NMA compared OS of the two regimens and did not report a significant advantage of DaraVMP.
In conclusion, Dara-VMP, VRD and Dara-Rd show mostly a favorable PFS profile as both directly and indirectly compared with standard frontline treatments for NTE NDMM. NMAs are valuable evidence-based tools, however, their quality needs to be appraised before using their result to support clinical recommendations. Future NMAs are expected to
incorporate also safety endpoints in order to allow benefit to risk assessments.
Blood cancer patients face a relative risk of VTE 28fold higher than the general population. Nevertheless, they also face a significantly higher risk of bleeding than overall cancer patients, thus prevention of VTE is not systematically implemented in this population. A more detailed estimation of VTE risk might support personalized primary prevention decisions. We therefore aimed at reviewing the VTE risk score that have been validated in blood cancer patients. According to GRADE-18 guidelines, we conducted at Nov 2020 a systematic search of literature databases (Cochrane Library, EMBASE, PubMed/MEDLINE) including the studies reported at meetings since Jan 2010. The search was limited to the following blood cancers: multiple myeloma (MM), lymphoma, acute leukemia (AL). Six-month VTE rates for risk categories, sensitivity, specificity, AUC or C-statistics and predictive values of the retrieved scores were recorded. Moreover, PROBASt score was applied for quality appriasal. We retrieved 82 records which resulted in 9 VTE RAMs: 3 ones were devloped in patients with MM, 5 ones in patients with lymphoma and only one was retrieved for AL. Sensitivity of lymphoma RAMs ranged from <50% to 93% and specificity from 54% to 90%. AUC ranged from 0.579 to 0.783. The negative predictive vale of the 5 RAMs was consistently higher than 95% except for the Khorana score (88%). The positive predictive value, however, was very low, ranging from 20% to 35%. Also C-statistics was higher for RAMs specifically developed in lymphoma patients (0.76-0.88) rather than Khorana score (0.60). The expected rate of VTE in high-risk patients ranged from 16% to 25% in lymphoma-specific RAMs, while it was lower than 15% for Khorana score. The expected rate of VTE in low-risk patients was lower than 3.8% in lymphoma specific RAMs. As a result, prophylaxis decisions based on lymphoma specific RAMw achieved a number need to treat lower than 10, namely less than 10 high-VTE-risk lymphoma patients need to receive primary prophylaxis in order to avert 1 VTE. Throly score reported the highest quality. In MM patients, four RAMS were validated: sensitivity ranged from 40% to 85% and specificity from 43% to 72%. C-statistics of the RAMs was poor, ranging from 0.51 to 0.66. Only the IMPEDE-VTE score allowed to discriminate risk categories differing by >10% VTE risk at 6 months.
In conclusion, disease-specific VTE RAMs outperform general RAMs, however they still have suboptimal positive predictive values. Specific bleeding RAMs are also awaited.
Background
Dysplastic neutrophils commonly show at least 2/3 reduction of the content of cytoplasmic granules by morphologic examination. Recognition of less granulated dysplastic neutrophils by human eyes is difficult and prone to inter-observer variability. To tackle this problem, we proposed a new deep learning model (DysplasiaNet) able to automatically recognize the presence of hypogranulated dysplastic neutrophils in peripheral blood.
Methods
Eight models were generated by varying convolutional blocks, number of layer nodes and fully connected layers. Each model was trained for 20 epochs. The five most accurate models were selected for a second stage, being trained again from scratch for 100 epochs. After training, cut-off values were calculated for a granularity score that discerns between normal and dysplastic neutrophils. Furthermore, a threshold value was obtained to quantify the minimum proportion of dysplastic neutrophils in the smear to consider that the patient might have MDS. The final selected model was the one with the highest accuracy (95.5 %).
Results
We performed a final proof of concept with new patients not involved in previous steps. We reported 95.5% sensitivity, 94.3% specificity, 94% precision, and a global accuracy of 94.85%.
Conclusions
The primary contribution of this work is a predictive model for the automatic recognition in an objective way of hypogranulated neutrophils in peripheral blood smears. We envision the utility of the model implemented as an evaluation tool for MDS diagnosis integrated in the clinical laboratory workflow.
The World Health Organization (WHO) Classification of Tumours of the Haematopoietic and Lymphoid Tissues was last updated in 2008. Since then there have been numerous advances in the identification of unique biomarkers associated with some myeloid neoplasms and acute leukemias - largely derived from gene expression analysis and next generation sequencing (NGS) that can significantly improve the diagnostic criteria as well as the prognostic relevance of entities currently included in the WHO classification and that also suggest new entities that should be added. Therefore, there is a clear need for a revision to the current classification. The revisions to the categories of myeloid neoplasms and acute leukemia will be published in a monograph in 2016 and reflect a consensus of opinion of hematopathologists, hematologists, oncologists and geneticists. The 2016 edition represents a revision of the prior classification rather than an entirely new classification and attempts to incorporate new clinical, prognostic, morphologic, immunophenotypic, and genetic data that has have emerged since the last edition. The major changes in the classification and their rationale are presented here.
Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders characterized by dysplastic, ineffective, clonal and neoplastic hematopoiesis. MDS represent a complex hematological problem: differences in disease presentation, progression and outcome have necessitated the use of classification systems to improve diagnosis, prognostication, and treatment selection. However, since a single biological or genetic reliable diagnostic marker has not yet been discovered for MDS, quantitative and qualitative dysplastic morphological alterations of bone marrow precursors and peripheral blood cells are still fundamental for diagnostic classification. In this paper, World Health Organization (WHO) classification refinements and current minimal diagnostic criteria proposed by expert panels are highlighted, and related problematic issues are discussed. The recommendations should facilitate diagnostic and prognostic evaluations in MDS and selection of patients for new effective targeted therapies. Although, in the future, morphology should be supplemented with new molecular techniques, the morphological approach, at least for the moment, is still the cornerstone for the diagnosis and classification of these disorders.
According to WHO 2008 guidelines, the required percentage of cells manifesting dysplasia in the bone marrow to qualify as significant is 10% or over in one or more hematopoietic cell lineages, but this threshold is controversial. No 'normal' values have been established. Therefore, we investigated dyshematopoiesis in bone marrow aspirate squash preparations of 120 healthy bone marrow donors.
Bone marrow squash slides of 120 healthy unrelated bone marrow donors were examined independently by 4 experienced morphologists. Samples were taken from the first aspiration during the harvest. Bone marrow preparation and assessment were performed according to WHO recommendations and ICSH guidelines.
More than 10% dysmyelopoiesis could be detected in 46% of bone marrow aspirate squash preparations with 26% in 2 or more cell lineages and 7% in 3 cell lineages in healthy bone marrow donors. Donors under the age of 30 years exhibited more dysgranulopoietic changes and dysmegakaryopoietic changes (P<0.001) compared to the older donors. Female donors showed more dysgranulopoietic changes than male donors (P = 0.025). The concordance rate between the 4 investigators was modest in dysgranulopoiesis but poor in dyserythropoiesis and dysmegakaryopoiesis.
The poor reliability of the 10% cut off was partly related to the proximity of the current criteria to the observed cut-off mean values of the normal population. These findings question the current WHO threshold of the 10% or over necessary for the percentage of cells manifesting dysplasia to be considered significant, and suggest that either a higher threshold would be more appropriate or different thresholds should be set for each lineage.
This paper describes the methodology used to develop a consensual glossary for haematopoietic cells within Diagnostics-WP10 of European-LeukemiaNet EU-project. This highly interactive work was made possible through the use of the net, requiring only a single two-day meeting of actual confrontation and debate. It resulted in the production of a freely accessible tool that could be useful for training as well as harmonization of morphological reports in onco-haematology especially, without geographic limitation, not limited to European countries. Moreover, this collective work resulted in the production of a consensus statement, taking into account individual practices, collegial agreement and literature data.
The monocyte is still the most difficult cell to identify with confidence in the peripheral blood or in the bone marrow in healthy individuals as well as in patients with infections, and in those with leukemic proliferations. The goal of this study was to establish morphological definitions so that monocytes, including immature monocytes, could be separated from the spectrum of monocyte precursors. Cells from peripheral blood or bone marrow were selected to provide a large panel of normal and leukemic cells at different maturational stages and were submitted to 5 experts, who had previously reached a consensus, on the basis of microscopy, in defining 4 subtypes: monoblast, promonocyte, immature monocyte, mature, monocyte. They achieved a good concordance rate of 76.6% and a high kappa rate confirming that the criteria for defining the 4 subtypes could be applied consistently. It has now to be established whether these monocyte subtypes correlate with immunological or molecular markers and are clinically relevant.
Three patients with acute leukemia, disseminated intravascular coagulation, and a specific acquired chromosome abnormality [t (15;17)] were found by transmission electron microscopy to have the typical distribution of granules seen in promyelocytes. However, the average granule sizes were 120, 170, and 180 nm, respectively, for the three patients, significantly less than the 250-nm resolution of light microscopy. We regard the leukemia in these three patients as comprising a distinct clinical, ultrastructural, and cytogenetic entity that we have chosen to call "microgranular" acute promyelocytic leukemia.
The CBC count and leukocyte differential count (LDC) are among the most frequently requested clinical laboratory tests. These analyses are highly automated, and the correct interpretation of results requires extensive knowledge of the analytic performance of the instruments and the clinical significance of the results they provide. In this review, we analyze the state of the art regarding traditional and new parameters with emphasis on clinical applications and analytic quality. The problems of some traditional parameters of the CBC count, such as platelet counts, some components of the LDC such as monocyte and basophil counts, and other commonly used indices such as red cell volume distribution width and platelet indices such as mean platelet volume and platelet distribution width are considered. The new parameters, evaluated from analytic and clinical viewpoints, are the available components of the extended differential count (hematopoietic progenitor cells, immature granulocytes, and erythroblasts), the immature reticulocyte fraction, the reticulocyte indices, the fragmented RBCs, and the immature platelet fraction.
Three patients with acute leukemia, disseminated intravascular coagulation, and a specific acquired chromosome abnormality [t (15;17)] were found by transmission electron microscopy to have the typical distribution of granules seen in promyelocytes. However, the average granule sizes were 120, 170, and 180 nm, respectively, for the three patients, significantly less than the 250-nm resolution of light microscopy. We regard the leukemia in these three patients as comprising a distinct clinical, ultrastructural, and cytogenetic entity that we have chosen to call “microgranular” acute promyelocytic leukemia.
In this era of genome medicine, the sub-classification of myeloid neoplasms, including myelodysplastic syndrome (MDS), is now supported by genetic testing in selected cases. However, as the initial suspicion and primary diagnosis of the disease still largely relies on morphological features and numbers of hematopoietic cells, the establishment of a uniform diagnostic basis, especially for cell morphology, is essential. In this study, we collected nearly 100,000 hematopoietic cell images from 499 peripheral blood smear specimens from patients with MDS and used these to evaluate the standardization of morphological classification by medical technologists. The observers in this study ranged between two to eleven for each image, and the images were classified according to MDS criteria through a web-based system. We found considerable inter-observer variance in the assessment of dysplastic features. Observers did not recognize cytoplasmic hypo-granularity unless almost all granules in neutrophils were absent. Pseudo Pelger-Huët anomalies were also often overlooked, except for cells with a very typical "pince-nez" appearance. Taken together, this study suggests a requirement for further standardization in terms of morphological cell classification, and a need for the development of automatic cell classification-supporting devices for the accurate diagnosis of MDS.
This is a translation from the Italian of the second edition of a textbook on the morphology of the blood and bone marrow in blood diseases. Morphological details are supplemented by detailed descriptions of the output and role of automated instruments in disorders of the blood. Despite the enormous advances made in immunophenotyping and in cytogenetic and molecular aspects of haematology, morphology of the blood and marrow remains fundamental in haematological diagnosis. The authors are renowned in this field. I have translated the text into English so that their expertise can be shared with a wider audience.
The European LeukemiaNet(ELN) is composed of several workpackages, four of them being directly involved in the various aspects of diagnosis. On the occasion of the annual ELN meeting of 2015 in Mannheim, these four workpackages collectively examined the current situation and future prospects of cytomorphology, flow cytometry, cytogenetics, next generation sequencing and minimal residual disease detection in the context of leukemia diagnosis and follow-up. This document summarizes the outcome of this compendium. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
These guidelines provide information on how to reliably and consistently report abnormal red blood cells, white blood cells and platelets using manual microscopy. Grading of abnormal cells, nomenclature and a brief description of the cells are provided. It is important that all countries in the world use consistent reporting of blood cells. An international group of morphology experts have decided on these guidelines using consensus opinion. For some red blood cell abnormalities, it was decided that parameters produced by the automated haematology analyser might be more accurate and less subjective than grading using microscopy or automated image analysis and laboratories might like to investigate this further. A link is provided to show examples of many of the cells discussed in this guideline.
© 2015 John Wiley & Sons Ltd.
I read the Editorial by van’t Veer and Haferlach with interest.[1][1] I disagree with their premise that clinical hematologists should abandon their microscopes. Whereas undoubtedly there have been many very useful technological advances which have shed light on the pathogenesis of hematologic
We are very happy with the reaction of Prof. Shaun McCann[1][1] to our recent Editorial[2][2] because it may initiate more thinking and discussion about the role of morphology in the diagnosis of hematologic malignancies. What’s more, we agree with most of his statements. Not, however, that our
Since Dimitri Romanowski, in 1891, invented a staining technique that made intracellular structures visible, and Gustaaf Giemsa, in his studies of malaria detection, described a method to stabilize and standardize this technique, light microscopic examination and description of the cells on films of blood and bone marrow has been the most important tool in the diagnosis and classification of hematologic malignancies.1,2 Subsequently, in 1976, this morphological assessment formed the basis of the French-American-British (FAB) classification.3 To define the lymphatic, myeloid and monocytic character of the cells more specifically than the degree obtained using only morphology, cytochemical reactions for myeloperoxidase and non-specific esterase, also performed on the film, were included. Soon immunological methods were added to this classification for the detection of megakaryoblasts and those blasts that are negative both for myeloperoxydase and B- and T-lymphoid markers.4,5
This revision is intended to update the 1994 ICSH guidelines. It is based on those guidelines but is updated to include new methods, such as digital image analysis for blood cells, a flow cytometric method intended to replace the reference manual 400 cell differential, and numerous new cell indices not identified morphologically are introduced. Haematology analysers are becoming increasingly complex and with technological advancements in instrumentation with more and more quantitative parameters are being reported in the complete blood count. It is imperative therefore that before an instrument is used for testing patient samples, it must undergo an evaluation by an organization or laboratory independent of the manufacturer. The evaluation should demonstrate the performance, advantages and limitations of instruments and methods. These evaluations may be performed by an accredited haematology laboratory where the results are published in a peer-reviewed journal and compared with the validations performed by the manufacturer. A less extensive validation/transference of the equipment or method should be performed by the local laboratory on instruments prior to reporting of results.
Within the past decade the World Health Organization (WHO) has published two extensive and well-regarded syllabi on the classification and definitions of the myelodysplastic syndromes (MDS).[1][1],[2][2] Although predominantly based on the French-American-British (FAB) morphological approach to the
Early mortality in acute promyelocytic leukemia has been reported to occur in less than 10% of patients treated in clinical trials. This study reports the incidence and clinical features of acute promyelocytic leukemia patients treated at Stanford Hospital, CA, USA since March 1997, focusing on early mortality. We show that the risk of early death in acute promyelocytic leukemia patients is higher than previously reported. In a cohort of 70 patients who received induction therapy at Stanford Hospital, 19% and 26% died within seven and 30 days of admission, respectively. High early mortality was not limited to our institution as evaluation of the Surveillance, Epidemiology and End Results Database demonstrated that 30-day mortality for acute promyelocytic leukemia averaged 20% from 1977-2007 and did not improve significantly over this interval. Our findings show that early death is now the greatest contributor to treatment failure in this otherwise highly curable form of leukemia.
Recently several parameters have been introduced to the complete blood count such as nucleated red blood cells, immature granulocytes; immature reticulocyte fraction, immature platelet fraction and red cell fragments as well as new parameters for detection of functional iron deficiency. Leucocyte positional parameters, which may diagnose specific diseases (e.g. differentiate between abnormal lymphocytes in leukaemia and viral conditions and may also detect malarial infection) are now available. At this time they are only used for research; however, generally such parameters later become reportable. One manufacturer's routine analyser allows measurement of cells by flow cytometry using monoclonal antibodies. Currently, there are no accredited external quality assessment schemes (EQAS) for these parameters. For a number of parameters, on some instruments, there is no internal quality control, which brings into question whether these parameters should be used for clinical decision making. Other more established parameters, such as mean platelet volume, red cell distribution width and the erythrocyte sedimentation rate do not have EQAS available. The UK National EQAS for General Haematology held a workshop earlier this year in 2008 to discuss these parameters. Participants were asked to provide a consensus opinion on which parameters are the most important for inclusion in future haematology EQAS.
The bone marrow examination is an essential investigation for the diagnosis and management of many disorders of the blood and bone marrow. The aspirate and trephine biopsy specimens are complementary and when both are obtained, they provide a comprehensive evaluation of the bone marrow. The final interpretation requires the integration of peripheral blood, bone marrow aspirate and trephine biopsy findings, together with the results of supplementary tests such as immunophenotyping, cytogenetic analysis and molecular genetic studies as appropriate, in the context of clinical and other diagnostic findings. Methods for the preparation, processing and reporting of bone marrow aspirates and trephine biopsy specimens can vary considerably. These differences may result in inconsistencies in disease diagnosis or classification that may affect treatment and clinical outcomes. In recognition of the need for standardization in this area, an international Working Party for the Standardization of Bone Marrow Specimens and Reports was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines based on preferred best practices. The guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus.
Among AML with maturation, acute promyelocytic leukemia (APL) represents a distinct subtype which accounts for 5-10% of all the FAB variants. APL may be recognized by different cytological pictures: (i) Hypergranular APL, the most typical form, showing promyelocytes with cytoplasm packed with purple granules. Most of the primary granules may be incorporated into Auer rods, sometimes stacked in bundles of faggots. (ii) Microgranular APL, characterized by fine dustlike granulation in the cytoplasm; some promyelocytes may even appear agranular by light microscopy. Most of the cells show bilobed or folded nuclei, a picture which may simulate that of acute myelomonocytic leukemia. (iii) Hyperbasophilic form, characterized by cells with high N/C ratio, and strongly basophilic cytoplasm with either sparse or no granules. Conspicuous cytoplasmatic budding is usually present, recalling the feature of micromegakaryocytes. Strong positivity for myeloperoxidase, Sudan black B and chloroacetate esterase represents the typical cytochemical pattern of M3; usually a weaker reactivity may be observed in M3v. However, sometimes a degree of cytochemical heterogeneity of APL cells may be observed, as suggested by cases displaying a strong sodium fluoride-sensitive non-specific esterase reaction. Recently a distinct entity associated with basophilic differentiation has been described. Differential diagnosis of this form with M2-baso subtype and with cases of MDS or AML with basophilia (M2, M4 with t(6;9) translocation) may be obtained by the use of cytochemistry, cytogenetic investigations, and electron microscopy.
World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues
- E S Jaffe
- N L Harris
- H Stein
- J W Vardiman
Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. World
Health Organization Classification of Tumours. Pathology and
Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon:
IARC; 2001.