500-cell Count Research Articles

S100B Is a Useful Marker for the Diagnosis of Haemophagocytic Lymphohistiocytosis

Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic inflammatory clinical syndrome that can be primary/familial or secondary to a variety of underlying conditions. The 2004 diagnostic criteria for HLH require 5 out of 8 of the following clinical and pathological variables to be present: fever, splenomegaly, cytopenia in at least 2 lineages, hypertriglyceridemia/hypofibrinogenemia, haemophagocytosis on pathology examination, low/absent NK cell activity, ferritin greater than 500µg/L or soluble IL-2 receptor (sCD25) greater than 2400 U/mL. At least 5 of these criteria must be met, as each one lacks specificity. With regard to the pathology detection of haemophagocytosis, the sensitivity and specificity reported in the literature were 83% and 60% respectively. Indeed, a degree of haemophagocytosis can be seen outside the context of HLH. Furthermore, some of the diagnostic features of HLH may not be useful when applied to single cases because they may be intrinsic to the underlying condition. Therefore, we wondered whether the histopathological diagnosis might be improved. We investigated whether testing for S100B expression in macrophages might render the detection of haemophagocytosis more specific and sensitive. S100B is a marker of macrophage activation, and can routinely be detected by immunohistochemistry. S100B has pro-inflammatory properties and has been shown to affect macrophage function. It is typically expressed in macrophages of Rosai-Dorfman disease apart of having also a tissue-specific expression pattern, marking melanocytes as well as Langerhans cells and other subsets of dendritic cells.A natural language search of the pathology database at our institution identified 32 patients with bone marrow samples reporting haemophagocytosis as evaluated on bone marrow smear preparations, between January 2002 and July 2015. Cases that did not have available paraffin-embedded bone marrow trephine biopsy and clinical data were excluded. The bone marrow samples of three patients without haemophagocytosis and without any clinical features of HLH were used as controls: a new diagnosis of acute leukemia, a follow-up of acute leukemia post-transplant and a known lymphoma patient with unexplained pancytopenia. Clinical parameters relevant to the diagnosis of HLH were evaluated. Cases were clinically categorized as diagnostic for HLH (≥5 criteria), clinically likely (<5 criteria, but clinical presentation consistent) or clinically non suspicious (<5 criteria and clinical presentation not consistent with HLH). Stains for S100B and CD68 were performed. The percentage of S100B positive cells of all bone marrow cells as well as the percentage of CD68 positive macrophages was calculated from 500-cell counts using a 40x objective lens.The median patient age was 57 years (range 18-80). Underlying clinical diagnoses are reported in Table 1. Expression of S100B ranged from 0-76.4%. Cases without morphological evidence of haemophagocytosis had less than 1% S100B-positive cells. Cases with presence of haemophagocytic cells showed various levels of S100B-positive cells. Of interest, >10% S100B-positive cells (of all cells) was 100% specific for cases meeting ≥4 HLH criteria, 91% specific for cases clinically likely HLH or confirmed HLH. The sensitivity was 60% for cases meeting ≥4 HLH criteria, 52% for cases being likely HLH or confirmed HLH. Percentage of CD68-positive cells or S100B/CD68 ratio did not add relevant information.The high specificity of S100B expression by immunohistochemistry indicates it may be an additional diagnostic feature of HLH. Of note, the test is available in routine immunohistochemistry laboratories. Since not all cases of HLH show increased numbers of S100B-expressing macrophages, the test does not obviate the need for the 2004 criteria. Serological immunoassays for S100B have been developed for use in the context of brain disease/injuries and malignant melanoma. Based on our data, we intend to further investigate the sensitivity and specificity of S100B as a serological marker of HLH. [Display omitted] DisclosuresKuruvilla:BMS: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Abbvie: Consultancy; Amgen: Honoraria; Seattle Genetics: Consultancy, Honoraria; Leukemia and Lymphoma Society Canada: Research Funding; Merck: Consultancy, Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Princess Margaret Cancer Foundation: Research Funding; Lundbeck: Honoraria.

Smoldering Multiple Myeloma: Impact of the Evolving Pattern on Early Progression

Introduction: Smoldering multiple myeloma (SMM) is a plasma cell dyscrasia defined by the presence of a monoclonal protein (MP) (≥ 30 g/L in serum or >1g/24-hours in urine) and/or plasma cell bone marrow involvement (BMPC) ≥ 10%, in the absence of symptoms due to the gammopathy. The risk of progression to symptomatic disease in patients with SMM is highly variable. Several biomarkers and prognostic index associated with risk of early progression based on tumoral load (M-protein size and percentage of BMPC), M-protein behaviour (evolving vs. non-evolving) and/or immunological status (heavy chain isotype, isotype suppression of uninvolved immunoglobulins and serum free light-chain (FLC) κ/λ ratio) have been recently identified. The identification of patients at risk for early progression is crucial when considering the current possibility of prompt therapeutic intervention. The aim of this study was to analyze the factors associated with early progression to multiple myeloma (MM) in patients diagnosed with SMM and long follow-up in a single institution.Methods: Medical records of the 207 patients (76M/131M; median age 65 years, range 33 to 92) diagnosed with SMM (International Myeloma Working Group criteria, 2003) at our institution between January 1973 and December 2012 were systematically reviewed. Progressive increase in the value of MP was defined as "evolving" when at least 10% increase was observed within the first 6 months from diagnosis when MP was ≥ 30 g/L (Rosiñol et al, Br J Haematol. 2003) or progressive increase in MP in each of the annual consecutive measurements during a period of 3 years in patients with an initial MP < 30 g/L (Rosiñol et al, Mayo Clin Proc. 2007). Immunoparesis was defined as any value below normal in not involved immunoglobulins. Bone marrow aspirates obtained at diagnosis were reviewed independently by 2 observers. Plasma cell percentages were estimated from a 500-cell count by each examiner and the mean values were considered.Results: Sixty-seven patients (33%) accomplished both SMM criteria (MC and BMPCs), while the remaining 140 patients only had one of them. With a follow up of 1,692 years-person, 105 patients had progressed (50.7%) to MM and one case to AL amyloidosis (0.5%). The estimated probability of progression at 2 and 5 years was 19.9% and 44.9% respectively, with a median time to progression (TTP) of 7.3 years (95% CI 3.9 to 10.6).At the time of progression, clinical manifestations were mainly anemia (52%) and skeletal lytic lesions (40%). The presence of renal insufficiency, extramedullary plasmacytomas or hypercalcemia was only identified in 12 patients (5.8%). The median survival after progression was 5 years (95% CI 3.8 to 6.2).Evolving type was recognized in 25% of the patients, and was associated with a probability of progression of 45% and 78.1% at 2 and 5 years and was higher than those with stable MP (HR 4.5; 95% CI 3 to 6.9; p<0.001) (Figure 1). Evolving pattern was more frequently associated with IgA isotype (41.2% vs. 23.8%; p=0.02). Negative impact in median TTP of evolving type was significant in patients either with both diagnostic criteria (MP and BMPCs; 1.3 vs. 6.3 years, p<0.001) and in those with only one of them (3.7 vs. not reached; p<0.001).At the univariate analysis the MP size (< vs. ≥ 30 g/L, median 13.4 vs. 3.1 years, p<0.001), the proportion of BMPCs (< vs. ≥ 20%, 17.2 vs. 2.9 years, p<0.001), the presence or absence of immunoparesis (3.9 vs. 16.9 years, p=0.001) and the evolving pattern (19.4 vs. 3 years, p<0.001) were significantly associated with a higher risk of progression. At the multivariate analysis only the evolving type (HR 4.9, 95% CI 2.8 to 8.7, p<0.001), the proportion of BMPCs (HR 2.5, 95% CI 1.3 to 4.6, p=0.004) and the presence of immunoparesis (HR 1.9, 95% CI 1.03-3.6, p=0.042) retained their statistical significance. Considering these last three variables, a model of risk stratification (Figure 2) was built, ranging from a probability of progression at 2 years of 81.8% for patients with the 3 factors present to only 4% for patients in group 4 (no risk factors).Conclusion: In this series of patients with SMM with long follow up, evolving pattern, proportion of BMPCs and the presence of immunoparesis can accurately predict accurately the risk of early progression to symptomatic disease. Evolving type should be routinely monitored during the follow up of patients with SMM, since it is the most significant predictor for early progression. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of Immunohistochemical Markers in Bone Marrow Sections to Evaluate for Myelodysplastic Syndromes and Acute Myeloid Leukemias

Accurate bone marrow (BM) blast counts (BCs) are essential for diagnosis (dx) of myelodysplasia (MDS), MDS/myeloproliferative (MDS/MPD) disease, or acute myeloid leukemia (AML), and may be difficult in hemodiluted bone marrow aspirates (BMAs). Erythroid precursors (EPs) may be indistinguishable from myeloblasts in BM sections (aspirate clots/cores). We compare the usefulness of immunohistochemistry (IHC) [ie, CD34, CD117, myeloperoxidase (MPO), Hemoglobin A1 (HbA1), and terminal deoxynucleotidyl transferase (TdT)] of BM sections (IHC-BM) with BMA, bone marrow touch preparation (BMTP), and flow cytometry (FC) BCs. The initial BC (48), percentage (%) of Eps (38) (both based on initial 100 to 600-cell counts), and FC expressions of CD34, CD117, Glycophorin A(GLY A), and TdT (44) were tabulated from 50 BMs (MDS, MDS/MPD, or AML). BMAs (48) and BMTPs (25) subsequently received 500-cell counts. IHC-BM was performed (45:formalin, 5:B5-fixed) [CD34 (46), CD117 (45), HbA1 (45), TdT (42), and MPO (45)]. Retrospective BMA BCs revealed a 31% (15/48) discrepant rate between the original/retrospective BMA BCs; 80% revealed an underestimated initial BC. There was a 28% discordance rate between the retrospective BMA and BMTP reviews; 77% showed a higher BMTP BC. IHC showed significantly higher BCs in 19% (9/47), resulting in a different dx (5). However, CD34 and CD117 IHCS revealed lower BCs in 38% and 48%, respectively. The CD34 IHC results were primarily due to CD34-negative blasts by FC. The CD117 IHC results were largely unexplained. EPs were CD34 and CD117-negative. (1) Evaluation for MDS/AML requires 500-cell counts of BMAs and/or BMTPs. (2) CD34 and/or CD117 blasts by FC indicate IHC-BM may increase BC accuracy. (3) CD34 is more reliable than CD117 by IHC; however, in combination, they are most reliable and should be performed on BM clots/cores due to variable reactivity.

Percentage of CD34 + Cells in a Minimum 500-Cell Count in Bone Marrow Trephines of Patients with Chronic Myeloid Leukaemia Provides the Best Correlation with Aspirate Blast Count.

We studied the expression of CD34 in 169 bone marrow trephines of 53 patients with CML in chronic, accelerated and blastic phases. The bone marrow blast count ranged from 0–82% (median: 4.5%). All were receiving imatinib treatment. We aimed to establish which counting method in bone marrow trephines provided the best correlation with the blast count of the corresponding bone marrow aspirate in all phases of the disease. Paraffin embedded sections were stained with the monoclonal antibody QBEND10.CD34 expression in each marrow trephine was evaluated by three counting methods. The first method, established in our department for several years, expressed the percentage of CD34 positive cells vs. all nucleated cells in a minimum 500-cell count (% CD34+/500 cells). The second recorded the total number of CD34 positive cells in ten high power fields (CD34+/10hpf) using x 40 magnification /numerical aperture 0.65. The third method evaluated the highest number of CD34 positive cells present in a single high power field (CD34+/hpf), this value being taken from the previous 10 hpf count. The two latter methods were employed because they may be more applicable for general use by practicing histopathologists. In addition, counting over 10 hpf provides a much larger sample size than a 500-cell count. CD34 expression in endothelial cells served as a positive internal control.All three methods showed significant positive correlation with blast count (% CD34+/500 cells: Pearson's Correlation - 0.723, sig - <0.001; CD34+/10hpf: Pearson's Correlation - 0.434, sig - <0.001; CD34+/hpf: Pearson's Correlation - 0.372, sig - <0.001). In conclusion, our previously established method of recording the percentage of CD 34 positive cells vs. all nucleated cells in a minimum 500-cell count provided the best correlation with the blast count.

Total Number of CD34 + Cells Per Ten High Power Fields in Bone Marrow Trephines of Patients with Chronic Myeloid Leukaemia Correlates with Probability of Complete Cytogenetic Response during the First Year of Treatment with Imatinib.

We studied expression of CD34 positive cells in the bone marrow trephines of two groups of patients with CML treated with imatinib. In the first group (n=17), patients went on to achieve sustained complete cytogenetic response during their disease course. In the second group (n =33) complete cytogenetic response was not achieved and patients later developed either accelerated or blastic phase disease. We studied paraffin embedded bone marrow trephines stained with the monoclonal antibody QBEND10 during an eighteen month period following initiation of imatinib therapy and recorded the highest CD34 positive cell counts and aspirate blast counts in three six-month intervals. CD34 positive cells were evaluated using three counting methods. The first method, established in our department for several years, expressed the percentage of CD34 positive cells vs. all nucleated cells in a minimum 500-cell count (% CD34+/500 cells). The second recorded the total number of CD34 positive cells in ten high power fields (CD34+/10hpf) using x 40 magnification /numerical aperture 0.65 and the third method utilised the highest number of CD34 positive cells expressed in a single high power field (CD34+/hpf), the value being taken from the previous CD34+/10 hpf count.We hypothesised that the overall CD34 positive cell burden and absolute CD34 positive numbers would be more important in predicting the likelihood of complete cytogenetic response and this would be better assessed by volumetric evaluation than percentage calculation relative to other nucleated cells.CD34 expression in endothelial cells was taken as a positive internal control. We aimed to establish which variable: % blast count, % CD34+/500 cells, CD34+/10hpf and CD34+/hpf provided better correlation with subsequent cytogenetic response to treatment. Values for each variable were dichotomised at the following levels; aspirate blast count <5 and > 5, % CD34+/500 cells < 2 and > 2%, CD34+/10hpf < 60 and > 60 cells and CD34+/hpf < 10 and > 10 cells. Univariate analysis was performed on samples within the first six months, seven to twelve months and thirteen to eighteen months. Only CD34+/10hpf showed significant discriminatory ability throughout the entire eighteen month period (Pearson's Chi-square 2-sided sig: 0.031, 0.003 and 0.007 in the first six months, seven to twelve months and thirteen to eighteen months samples respectively). By logistic regression, we confirmed that CD34+/10hpf had the best correlation with cytogenetic response during the first year following initiation of imatinib therapy. Larger studies are required to confirm these initial findings and to establish volumetric assessment of CD34 positive cells as a predictor for cytogenetic response.