Validation of an automated iron stain process for use with bone marrow aspirate smear slides.

The assessment of bone marrow iron stores is typically performed on an aspirate smear slide that has been manually stained by a technologist using a commercially available kit. This approach can contribute to inconsistent results and limit the broad use of iron staining in bone marrow specimens, particularly when laboratories have low staffing and/or high specimen volumes. Here, we describe the adaptation and validation of the Ventana Benchmark automated stainer and iron stain kit for routine clinical use of staining iron in bone marrow aspirate smear slides. We assessed accuracy and precision of the Ventana automated iron staining protocol compared to the Perls Prussian blue manual iron staining index method. Hematopathologists assigned Gale scores and enumerated the percentages of erythroid sideroblasts on paired patient bone marrow aspirate smear slides stained by the automated method and the manual iron staining method. We found a similar level of performance of the Ventana automated iron stain relative to the index manual method (as assessed by Pearson correlation and Bland-Altman analyses). In addition, there was low imprecision between replicates performed via the automated iron stain protocol. We also report superior qualitative findings of the automated method in ease of localization of iron storage, visualization of sideroblasts, and counterstain consistency. Automated iron staining of bone marrow aspirate smear slides performed similarly to the manual method and may allow for accurate routine evaluation of bone marrow iron stores as part of bone marrow analysis.

Diagnostic performance of automated red cell parameters in predicting bone marrow iron stores.

OBJECTIVES: The aim of the study was to determine the diagnostic performance of novel automated red cell parameters for estimating bone marrow iron stores. METHODS: The study was a retrospective single-centre study based on data from an automated haematology analyser and results of bone marrow iron staining. Red cell parameters were measured on a Sysmex XN-series haematology analyser. Bone marrow iron stores were assessed semiquantitatively by cytochemical reaction according to Perls. RESULTS: The analysis included 429 bone marrow aspirate smears from 393 patients. Median age of patients was 67 years, 52 % of them were female. The most common indication for bone marrow examination was a plasma cell dyscrasia (n=104; 24 %). Median values of percentage of hypochromic and hyperchromic red blood cells (%HYPO-He, %HYPER-He), reticulocyte haemoglobin equivalent (RET-He) and microcytic red blood cells (MicroR) were statistically significantly different between cases with iron deplete and iron replete bone marrow. In a logistic regression model, ferritin was the best predictor of bone marrow iron stores (AUC=0.891), outperforming RET-He and %HYPER-He (AUC=0.736 and AUC=0.722, respectively). In a combined model, ferritin/MicroR index achieved the highest diagnostic accuracy (AUC=0.915), outperforming sTfR/log ferritin index (AUC=0.855). CONCLUSIONS: While single automated red cell parameters did not show improved diagnostic accuracy when compared to traditional iron biomarkers, a novel index ferritin/MicroR has the potential to outperform ferritin and sTfR/log ferritin index for predicting bone marrow iron stores. Further research is needed for interpretation and implementation of novel parameters and indices, especially in the context of unexplained anaemia and myelodysplastic syndromes.

Bone marrow macrophage iron content and sideroblast count in iron- and ESA-naïve patients with CKD-related anemia.

BACKGROUND: Since in chronic kidney disease (CKD) iron deficiency anemia (IDA) can coexist with inflammation-induced immobilization of iron in macrophages (anemia of chronic disorders - ACD), we assessed the utility of ferritin, transferrin saturation (TSAT), and hepcidin for differentiation of mixed IDA-ACD from ACD, using bone marrow (BM) examination as reference. METHODS: This cross-sectional, single-center study analyzed 162 non-dialysis iron and epoietin-naïve CKD patients (52% males, median age 67 years, eGFR 14.2 mL/min 1.73 m2, hemoglobin 9.4 g/dL). BM aspiration, serum hepcidin (ELISA), ferritin, TSAT, and C-Reactive protein (CRP) were the main studied parameters. RESULTS: ACD was seen in 51%, IDA-ACD in 40%, while "pure" IDA in only 9%. In univariate and binomial analyses, IDA-ACD differed from ACD by lower ferritin and TSAT, but not by hepcidin or CRP. Correspondingly, in receiver operating curve analysis, ferritin and TSAT differentiated IDA-ACD from ACD, at cutoffs of 165 ng/mL and 14%, but with moderate precision (sensitivity and specificity of 72%, and 61%, respectively). CONCLUSION: The mixed pattern IDA-ACD could be more prevalent than estimated in non-dialysis CKD. Ferritin and, to a lesser degree, TSAT are useful in the diagnosis of IDA superimposed on ACD, while hepcidin, although reflecting BM macrophages iron, seems to have limited utility.

Peripheral Blood and Bone Marrow Findings in Chronic Alcoholics with Special Reference to Acquired Sideroblastic Anemia.

Anemia associated with alcoholism has numerous causes, most common being megaloblastic anemia and acquired sideroblastic anemia (SA). The bone marrow aspirate (BMA) and bone marrow iron (BMIr) findings and their correlation with peripheral blood smear (PBS) have not been extensively described in literature. We aim to study the spectrum of hematological abnormalities in chronic alcoholics. Complete blood count (CBC), PBS, BMA and BMIr of 71 chronic alcoholics were studied retrospectively over a period of 3 years. The slides were reviewed by 2 pathologists. The clinical history, CBC, PBS, BMA and BMIr findings were recorded. Out of 71 patients, 68 (95.77%) had anaemia. Red cell morphology varied from normocytic-normochromic, microcytic-hypochromic, macrocytic, to dimorphic anaemia. Principal findings seen on BMA were erythroid hyperplasia and megaloblastic maturation. BMIr was available in 41 patients; iron stores were decreased in 2 (4.88%), normal in 14 (34.15%), increased in 25 (60.97%). Seven (17.07%) cases showed presence of ring sideroblasts. Chronic alcoholics show a variety of abnormalities in BMA, which closely mimic many haematological disorders. A history of alcoholism should always be taken in these circumstances. SA should be ruled out in all chronic alcoholics with anaemia not responding to vitamin B12/folic acid, even with macrocytic picture on PBS.

Prevalence of Iron Deficiency Anemia in Biochemically Defined Moderate to Severely Anemic patients in a Tertiary Care Centre.

INTRODUCTION: Anemia due to iron deficiency and chronic diseases is common occurrence in developing country like Nepal, the latter seen in patients with various inflammatory, autoimmune, and malignant disorders . The Intensive method of marrow iron examination, which this study has employed, provides clinically useful iron status classification in cases of functional iron deficiency. The aim of the study is to find out the prevalence of iron deficiency anemia in biochemically defined moderate to severe anemic patients in tertiary care center. METHODS: A descriptive cross-sectional study was done in 43 patients who underwent bone marrow aspiration for evaluation of any cause and had moderate to severe anemia at the same time over a period of one year from Nov 2015 to 2016. Ethical clearance was obtained from Institutional Review Committee. The bone marrow iron stores were assessed by"intensive method" apart from the routinely used Gale's method. Data was collected and entry were done in Statistical Package for Social Sciences version 24. Point estimate at 95% Confidence Interval was calculated along with frequency and proportion for binary data. RESULTS: The intensive grading system demonstrated normal marrow iron store in 13 (30.2%), depleted iron stores in 3 (7%), functional iron deficiency in 14 (32.6%), and combined deficiency in 13 (30.2%) patients. Mean log ferritin concentration was lower in patients with depleted iron stores (2.2µg/l) than in those with normal (2.7µg/l), and functional iron deficiency (2.4µg/l). The mean log ferritin in combined deficiency was lower than the mean log ferritin concentration in iron store deficiency (1.9µg/l). CONCLUSIONS: The prevalence of functional iron deficiency anemia was greatest when the intensive method for assessment of bone marrow iron was used, thus differentiating four different iron status categories, including functional iron deficiency, from actual iron store deficiency, avoiding unnecessary iron supplementation in the former group.