Hematological cytomorphology: Where we are.

The manuscript discusses the historical evolution of observing blood cell morphology under an optical microscope, from the earliest microscopes in the 17th century to the modern digital era, highlighting key advancements and contributions in the field. Blood has historically held symbolic importance in various cultures, with early medical observations dating back to Hippocrates and Galeno. The discovery of cells and subsequent advancements in microscopy by scientists like Hooke and van Leeuwenhoek paved the way for understanding blood cell morphology. Influential figures such as Hewson, Donné, and Ehrlich followed. Diagnostic cytology evolved from manual cell counting to the development of automated hematological systems. Automated complete blood counting came to support microscopic examination in diagnosing hematological disorders. Morphology is crucial in predicting disease outcomes and guiding treatment decisions, particularly hematological neoplasms. The introduction of flow cytometry and its integration with traditional morphological analysis and the new cytogenetic and molecular techniques revolutionized the classification and prognostication of hematologic disorders. Digital microscopy has emerged as a powerful tool in recent years, offering rapid acquisition and sharing of blood cell images. Integrating Artificial Intelligence with digital microscopy has further enhanced morphological analysis, improving diagnostic efficiency. We also discuss the prospects of AI in pre-classifying blood cells in bone marrow aspirate samples, potentially revolutionizing diagnostic pathways for hematologic diseases. Overall, the manuscript provides a comprehensive overview of the historical development, clinical significance and technological advancements in observing blood cell morphology, underscoring its continued relevance in modern hematology practice.

Digital morphology compared to the optical microscope: A validation study on reporting bone marrow aspirates.

INTRODUCTION: This study aims to evaluate the effectiveness and reliability of the utilization for clinical reporting of the evaluation of digital images of bone marrow aspirates by morphologists and their comparability with the classic microscopic morphological evaluation. METHODS: We scanned 180 consecutive bone marrow needle aspirates smears using the "Metafer4 VSlide" whole slide imaging (WSI) digital scanning system. We evaluated the statistical comparability and the risk of bias of the microscopic readings with those performed on the screen on the digitized medullary images. RESULTS: The evaluation of cellularity on the screen was equivalent, with a higher frequency of "normal" than the analysis of digital preparations. The means and medians of the percentage values obtained on the different cell populations with the microscopic and digital reading were comparable as the main categories are concerned, with an average difference equal to 0 for the neutrophilic and eosinophilic granulocytic series, at -0.2% for the total myeloid cells, at 1.2% for the erythroid series, at -0.4% for the lymphocytes and at -0.4% for the blasts. Dysplastic features were consistently identified in 69/71 cell lineages. CONCLUSION: Our study demonstrated that screen evaluation of digitized bone marrow needle aspirates provides quantitative and qualitative results comparable to traditional microscopic analysis of the corresponding slide smears. Digital images offer significant benefits in reducing the workload of experienced operators, reproducibility and sharing of observations, and image preservation. Even in routine diagnostic activities, their use does not alter the quality of the results obtained in evaluating bone marrow needle aspirates.

A phase II, multicentre trial of decitabine in higher-risk chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a complex clonal hematological disorder classified among myelodysplastic (MDS)/myeloproliferative neoplasms. Prognosis is poor and there is a lack of effective treatments. The hypomethylating agent decitabine has shown activity against MDS and elderly acute myeloid leukemia, but there is little data focusing specifically on its efficacy in CMML. In this prospective, phase 2 Italian study, CMML patients received intravenous decitabine 20 mg/m2 per day on Days 1-5 of a 28-day treatment cycle. Response was evaluated after four and six cycles; patients responding at the end of six cycles could continue treatment with decitabine. Forty-three patients were enrolled; >50% were high-risk according to four CMML-specific scoring systems. In the intent-to-treat population (n=42), the overall response rate after six cycles was 47.6%, with seven complete responses (16.6%), eight marrow responses (19%), one partial response (2.4%) and four hematological improvements (9.5%). After a median follow-up of 51.5 months (range: 44.4-57.2), median overall survival was 17 months, with responders having a significantly longer survival than non-responders (P=0.02). Grade 3/4 anemia, neutropenia and thrombocytopenia occurred in 28.6%, 50% and 38% of patients, respectively. Decitabine appears to be an effective and well-tolerated treatment for patients with high-risk CMML.

Cytokine profile of autologous platelet-derived eye drops in patients with ocular chronic graft-versus-host disease.

Ocular chronic GVHD is efficaciously treated with autologous platelet-derived eye drops. We investigated the cytokine content of eye drops produced using a non-gelified lysate obtained from autologous platelet-rich plasma in six patients with ocular GVHD. In both the responding (n = 4) and the resistant (n = 2) patients, the eye drops were significantly enriched with various growth factors, in amounts proportional with the platelet counts. In contrast, chemokine ligand and interleukin levels were similar to those of plasma. The non-responding patients showed the highest levels of chemokine (C-X-C motif) ligand (CXCL)10. These findings provide possible explanations for beneficial or detrimental effects of eye drops.

Expanding the Living Donor Pool "Second Act": Laparoscopic Donor Nephrectomy and ABO-Incompatible Kidney Transplantation Improve Donor Recruitment.

BACKGROUND: To safely expand our living donor pool, we recently decided to work on 3 areas: analysis of causes of exclusion of potential donors, the results of which we recently published, introduction of laparoscopic donor nephrectomy (LDN), and ABO-incompatible (ABOi) transplantation. We sought to determine the impact of the new strategy on living donor recruitment and transplantation during over a 10-year period at a single institution. METHODS: From January 2005 to September 2014, we evaluated 131 living donors. Of these, 80 (61%) were genetically related, 51 (39%) unrelated, 119 (91%) ABO compatible (ABOc), 12 ABOi (9%). The analysis was divided into 2 eras: era 1, 2005-2010 (n = 53) included the use of open lumbotomy and acceptance of ABOc only; and era 2, 2011-2014 (n = 78), which saw the introduction of LDN and ABOi transplantation. RESULTS: Forty-five (34%) potential candidates successfully donated, 67 (51%) were excluded, and 19 (15%) were actively undergoing evaluation. Overall, 53 potential donors were evaluated in era 1 (8.8 donors/year), 78 in era 2 (19.5 donors/year). There were fewer excluded donors in era 2 vs era 1 (62% era 1 vs 44% era 2), and living donor kidney transplantation (LDKT) significantly increased in era 2 vs era 1 (3.3/year era 1 vs 7.1/year era 2). The establishment of an ABOi LDKT program led to a 15% increase of evaluations in era 2 (12/78 donors). CONCLUSIONS: LDN along with ABOi LDKT allowed for an improvement in recruitment of living donors and corresponding LDKT.

ICSH recommendations for the standardization of nomenclature and grading of peripheral blood cell morphological features.

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.

ICSH: on board for new projects.

The International Council for Standardization in Hematology (ICSH) is a not-for-profit organization aimed at improving global quality and harmonization of analytical methods, and achieving reliable and reproducible results in diagnostic hematology. ICSH co-ordinates Working Groups of experts to examine laboratory methods and instruments for hematological analyses, and co-operates with different international organizations which have similar scientific goals. Among seven ongoing approved projects, three ICSH projects have been selected and will be presented in the ICSH session at the XXVIIth ISLH International Symposium on Technological Innovations in Laboratory Hematology in The Hague, on May 2014. The project on 'Guideline for flow cytometric evaluation of patients with suspected acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS)' covers different aspects of the application of immunophenotyping by multiparameter flow cytometry (MFC) in the diagnosis of AML and MDS including integration into multimodal diagnostic workflow, quality control, antibody selection, interpretation of findings, reporting, and personnel. Data from the pilot study of the project for 'International Standardization of Hematology Reporting Units' suggest that there is a wide variation in reporting units for the routine blood cell count and highlights the areas of nomenclature and units of measurement where standardization is necessary and feasible, such as units for cell counts, white cell differentials, and hemoglobin concentration. The project on 'Standardization of HbA2 measurement and its implications for clinical practice' starts from the observation that different instruments give different results for hemoglobin A2; it is aimed at producing recommendations as to how instrument manufacturers and laboratories should assess their equipment before using it to analyze patient samples. These projects are examples of how the ICSH represents a great opportunity for scientists involved in hematology laboratory to participate in a process of expert collaboration and discussion all around the world.

ICSH guidelines for the evaluation of blood cell analysers including those used for differential leucocyte and reticulocyte counting.

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.

Topical use of phytotherapic cream (Capilen® cream) to prevent radiodermatitis in breast cancer: a prospective historically controlled clinical study.

AIM: The purpose of this study was to evaluate the ability of a topical phytotherapic product (Capilen® cream) to limit acute radiodermitis and delay the use of corticosteroids in patients with breast cancer (BC). METHODS: From January 2012 to August 2012, 30 consecutive patients, undergoing radiotherapy with adjuvant intent, were invited to use Capilen® cream two times daily two weeks before and during radiotherapy. An historical group was used as an external control. Acute skin toxicity was scored weekly according to RTOG/EORTC criteria. Time of occurrence of acute skin toxicity was taken as endpoint. RESULTS: Compliance was good. Overall, no significative statistical difference was observed in rate of acute radiation dermatitis, 46.7% in experimental arm versus 63.3% in the historical control group, although only 3.3% of Capilen® cream treated patients had a G3 acute radiation dermatitis versus 10% of the control group. A delay in the onset of radition dermatitis in patients treated with Capilen® cream (P=0.04) was showed. CONCLUSION: Our findings suggested that Capilen® cream plays a role in reducing acute radiation dermatitis in breast cancer patients treated with adjuvant radiotherapy. Further evidence is needed to confirm these results.

Metabolic responses in non-small cell lung cancer after hypofractionated stereotactic radiotherapy PET and hypofractionated radiotherapy.

BACKGROUND: The aim of our study was to evaluate the pattern of local failure after stereotactic body radiotherapy (SBRT) of non small cell lung cancer (NSCLC) lesions relating to different type of 18F-FDG positron emission tomography (PET) response. METHODS: Thirteen NSCLC patients for a total of 15 lesions (primary early or locally advanced and metastases) underwent PET before and 6 months after SBRT. Maximum standard uptake value (SUVmax) <2.5 was considered as cut off for complete response (CR) while lesion reduction > or =50% with residual value above 2.5 for partial response (PR). RESULTS: With a median follow up of 30 months pre- and post-SBRT mean SUV max values were 8.2 (range 14.2-3.7) and 2.4 (range 12.9-0), respectively. No "in field recurrence" was observed while 3 cases of "out field recurrence" occurred as regional nodes progression at 7.8 and 14 months after treatment. Three years overall survival, local control and distant metastases free survival were respectively 66.7%, 63.3% and 44.4%. Actuarial 75% and 53.3% 3-year local control, 60% and 40% 3-years distant metastases free survival were observed for complete and partial PET response, respectively, after SBRT. Thereafter, 60% and 50% 3-year overall survival were observed for complete and partial response. CONCLUSIONS: Clinical results were significantly better for "responder" than "non responder" and for "complete" than "partial response" group. Moreover, our data seem to confirm that a significant subset of patients maintain a low metabolic activity without developing local relapse on longer follow up.

Can we decrease the acute proctitis in prostate cancer patients using hyaluronic acid during radiation therapy: a prospective historically controlled clinical study.

BACKGROUND: The purpose of this study was to evaluate the ability of rectal suppository of hyaluronic acid to limit symptoms of acute radiation proctitis in patients with prostate cancer (PC). MATERIALS AND METHODS: From January 2011 to October 2011, 50 consecutive patients, undergoing radiotherapy with radical or adjuvant intent for PC, were invited to use rectal suppository of hyaluronic acid (HA: Cicatridina suppository, Farma-Derma s.r.l., Sala Bolognese, BO, Italy) daily, before radiation delivering. An historical group was used as an external control. Acute rectal toxicity was scored weekly according to RTOG criteria. Time to occurrence of acute rectal toxicity was taken as endpoint. RESULTS: Compliance was good. Only 2% of HA treated patients had a G2 acute proctitis versus 7% of historical group, globally a difference was observed in rate of acute proctitis between the experimental arm and the control group: 32% in experimental arm versus 45% in control group (p = 0.08). A delay in the onset of acute rectal toxicity in patients treated with HA (p = 0.04) was showed. CONCLUSIONS: Our findings suggested the role of HA in reducing acute proctitis in prostate cancer patients treated with radiotherapy. Further trials are needed to confirm these results.

Fragmented red blood cells automated measurement is a useful parameter to exclude schistocytes on the blood film.

INTRODUCTION:   The diagnosis of thrombotic microangiopathies (TMA) or disorders that may mimic their features remains difficult. Mechanical hemolytic anemia with the detection of shistocytes on the blood smear is a cornerstone finding to assess the diagnosis, but microscopic evaluation of shistocytes is still problematic with wide interobserver variations. Some of the latest generation automated blood cell counters (ABCC) propose an original quantitative approach of fragmented red cells (FRC), aiming to be equivalent to the microscopic count. This parameter has been poorly evaluated. METHODS:   To assess the predictive value (PV) of this test, we conducted studies comparing automated and microscopic counts of FRC/schistocytes, based on the analysis of thousands samples in four university hospitals and using the 2 ABCC currently available (Siemens ADVIA series, Sysmex XE-2100). RESULTS: Reference range for FRC was <0.3% for the ADVIA and <0.5% for the XE-2100. The presence of FRC below a threshold determined at 1% (ADVIA and XE-2100) had a negative PV close to 100% to exclude the presence of schistocyte on the blood smear, but in relationship with a poor PV value. CONCLUSIONS: Our study validated the utility of the immediately available FRC parameter on ABCC to exclude schistocytes and the diagnosis of TMA.

ICSH recommendations for identification, diagnostic value, and quantitation of schistocytes.

Schistocytes are fragments of red blood cells (RBCs) produced by extrinsic mechanical damage within the circulation. The detection of schistocytes is an important morphological clue to the diagnosis of thrombotic microangiopathic anemia (TMA). Reporting criteria between different laboratories, however, are not uniform, owing to variability of shape and nature of fragments, as well as subjectivity and heterogeneity in their morphological assessment. Lack of standardization may lead to inconsistency or misdiagnosis, thereby affecting treatment and clinical outcome. The Schistocyte Working Group of the International Council for Standardization in Haematology (ICSH) has prepared specific recommendations to standardize schistocyte identification, enumeration, and reporting. They deal with the type of smear, method of counting, morphological description based on positive criteria (helmet cells, small, irregular triangular, or crescent-shaped cells, pointed projections, and lack of central pallor). A schistocyte count has a definite clinical value for the diagnosis of TMA in the absence of additional severe red cell shape abnormalities, with a confident threshold value of 1%. Automated counting of RBC fragments is also recommended by the ICSH Working Group as a useful complement to the microscope, according to the high predictive value of negative results, but worthy of further research and with limits in quantitation.

Immunophenotyping of acute leukemia and lymphoproliferative disorders: a consensus proposal of the European LeukemiaNet Work Package 10.

The European LeukemiaNet (ELN), workpackage 10 (WP10) was designed to deal with diagnosis matters using morphology and immunophenotyping. This group aimed at establishing a consensus on the required reagents for proper immunophenotyping of acute leukemia and lymphoproliferative disorders. Animated discussions within WP10, together with the application of the Delphi method of proposals circulation, quickly led to post-consensual immunophenotyping panels for disorders on the ELN website. In this report, we established a comprehensive description of these panels, both mandatory and complementary, for both types of clinical conditions. The reason for using each marker, sustained by relevant literature information, is provided in detail. With the constant development of immunophenotyping techniques in flow cytometry and related software, this work aims at providing useful guidelines to perform the most pertinent exploration at diagnosis and for follow-up, with the best cost benefit in diseases, the treatment of which has a strong impact on health systems.

Morphological diagnosis by bone marrow aspirate of toxoplasmosis infection in an HIV-positive patient.

An HIV-positive woman receiving antiretroviral therapy developed an opportunistic toxoplasma infection, detected by morphological examination of bone marrow aspirate in the absence of serological positivity. The intracellular presence of Toxoplasma gondii was confirmed by indirect immunofluorescence microscopy, using a polyclonal antitoxoplasma antiserum on marrow smears. This case report confirms the utility of morphological bone marrow examination for the diagnosis of parasitic infections in patients with impaired host defenses.

Blastoid mantle cell lymphoma occurring in a patient in complete remission of chronic myelogenous leukemia.

The development of a de novo lymphoma in patients affected by chronic myelogenous leukemia (CML) is a rare event. The introduction of new molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH), allows a correct differential diagnosis between lymphoid blastic crisis and a blastoid variant of mantle cell lymphoma (MCL), which shows an aggressive behavior and some molecular characteristics detectable by cytogenetics and immunohistochemistry. We report a case of a blastoid variant of MCL that developed in a patient with CML who achieved complete cytogenetic and molecular response to imatinib mesylate treatment.

The risk of thrombosis in patients with acute leukemia: occurrence of thrombosis at diagnosis and during treatment.

BACKGROUND: Thromboembolism can occur during acute leukemia, especially acute lymphoid leukemia (ALL) treated with L-asparaginase. Yet, most reports are anecdotical and scarce data are available on the risk of thrombosis in acute myeloid leukemia (AML). OBJECTIVES: To evaluate the risk of thrombosis in patients with acute leukemia. PATIENTS AND METHODS: Three-hundred and seventy-nine consecutive adult patients with newly diagnosed acute leukemia were recruited in an observational cohort study conducted from January 1994 to December 2003. Diagnosis was ALL in 69 patients, acute promyelocytic leukemia (APL; FAB subtype M3) in 31, and non-M3 AML in 279. All first or recurrent symptomatic thromboembolic events objectively diagnosed were recorded. RESULTS: Twenty-four patients of the overall 379 (6.3%; 95% CI 4.1%-9.2%) had a first thrombosis, venous in 80% of the cases and arterial in 20%. At diagnosis, thrombosis was a presenting manifestation in 13 cases (3.4% of the whole cohort): 1.4% in ALL, 9.6% in APL, and 3.2% in non-M3 AML patients. Follow-up was carried out on 343 patients without thrombosis at diagnosis and further 11 thrombotic events (3.2%) were recorded. At 6 months from diagnosis, the cumulative incidence of thrombosis was 10.6% in ALL, 8.4% in APL, and 1.7% in non-M3 AML patients. The patients who received L-asparaginase had a 4.9-fold increased risk of thrombosis in comparison with those who did not (95% CI 1.5-16.0). The fatality rate due to thrombosis was 0.8%. CONCLUSIONS: In patients with acute leukemia, the risk of thrombosis is not negligible. Thombosis can be a presenting symptom at diagnosis in a significant portion of cases with APL (9.6%) and non-M3 AML (3.2%); a similar rate of thrombosis can occur during the subsequent course of the disease. The incidence of symptomatic thrombosis at diagnosis is relatively low in ALL patients (1.4%), but is significantly increased by further treatment up to 10.6%. Strategies of antithrombotic prophylaxis should be investigated in this setting.