The Association between Acute Lead Exposure from Indoor Firing Ranges and Iron Metabolism.

Abstract Objective: To assess the impact of short term indoor firing ranges lead exposure and its relationship to iron, ferritin, lead, zinc protoporphyrin and hemoglobin concentration among young adults. Methods: We report of a clinical observation that was carried out in 30 young and healthy soldiers serving in the Israel Defense Forces (IDF) Counter Terrorist Unit (CTU). Blood samples were drawn for Lead (Pb), Zinc Protoporphyrin (ZPP),Iron, Hemoglobin (Hb) and ferritin prior to and after a 6 weeks period of intensive shooting practice in indoor firing ranges. Results: A mean Blood lead level increase (p&lt; 0.0001) with a mean Iron (p&lt;0.0005) and mean ferritin (p&lt;0.0625) decrease simultaneously after 6 weeks period of Lead dusts exposure were demonstrated. We found a trend for inverse correlation between pre-exposure low ferritin levels and post exposure high blood lead levels. Conclusion: We found decreased iron and ferritin levels after short term lead exposure among young adults. This can be explained by competition of iron and lead absorption viatransporters like DMT1 suggesting that lead poisoning can cause iron depletion and that iron depletion can aggravate lead poisoning. This synergistic effect should come to every physicians mind especially when treating patients with a potential risk for each problem separately. Lead (Pb), Zinc Protoporphryn (ZPP), Hemoglobin (Hb), Iron and Ferritin among the CTU soldiers before and after the indoor firing ranges lead exposer variable N Pre-exposure N Post-exposure Difference *P value Pb (mcg/dl) 29 10.3±2.3 30 18.9±3.6 8.8±2.6 0.0001 ZPP(mcg/dl) 29 42±7.5 30 42.9±8.1 0.03±6.5 NS Hb (g/dl) 30 15±0.7 30 14.8±0.9 0.2±0.2 NS MCV (fl) 30 88.7±2.5 30 89±2.7 0.3±0.2 NS Iron (mcg/dl) 29 108.5±43.6 30 77.4±24.4 −30.4±41.2 0.0005 Ferritin (mcg/l) 27 58.1±27.7 16 51±19.9 −6.1±10.7 0.0625 Correlation between Pre-exposure Ferritin levels and Post exposure Lead level.(Person correlation coefficient = −0.28 r=0.14 number of observations = 27) Correlation between Pre-exposure Ferritin levels and Post exposure Lead level.(Person correlation coefficient = −0.28 r=0.14 number of observations = 27)</jats:p

Assessment of Nuclear Homogenicity in Chronic Lymphocytic Leukemia by Image Analysis.

Abstract Maturation of progenitor cells involves gradual chromatin condensation. We have shown that B-CLL cells represnet specific specrtral patterns by multipixel spectral imaging (J. Histocem. Cytohem. 46:113,1998). We further used image analysis techniques to quantify the alteration of chromatin homogenicity in lymphocytes from healthy subjects and B-CLL patients, as well as during maturation of myeloid and erythroid series, using light microscopic digital images of May-Grünwald Giemsa-stained (MGG) cells (B-CLL n=60, control n=22) and transmission electron microscopy (TEM). Nuclei were outlined by mouse cursor. Light microscopy images were converted to grayscale and designated as vector of the red, green and blue channels (sqrt(R*R+G*G+B*B)). Average color intensity (0–255), entropy (0–8), standard deviation, area diversity (0–100) and positive percent area (PPA) measurements were obtained from each nucleus. The entropy and area diversity of B-CLL lymphocyte nuclei was significantly lower than control lymphocytes (entropy 5.6±0.19 vs. 5.44±0.2, respectively, p&amp;lt;0.0001 and area diversity 33.2±4.3 vs. 30.5±2, respectively, p=0.006). In addition, we analyzed MGG-stained and TEM myeloid and erythroid cells at various stages of differentiation (n=155). Both the myeloid and erythroid cell lines showed gradual reduced color intensity, increased entropy and area diversity. Furthermore, MGG-stained megaloblastic proerythroblasts showed significantly higher levels of entropy and area diversity as compared with control proerythroblasts (n=73). Thus, assessment of nuclear entropy and area diversity may help to in the differentiation between normal lymphocytes and B-CLL cells.</jats:p

Combination Of Fluorescence In SITU Hybridization (FISH) and Cytogenetic Techniques Optimize The Diagnostic Process Of Patients With Myelodysplastic Syndrome (MDS)

Abstract Background Cytogenetic analysis in myelodysplastic syndromes (MDS) is the “gold standard” method for identification of clonal chromosomal abnormalities, dependent on culturing the bone marrow cells and analyzing at least 20 metaphases. According to the International System for Human Cytogenetic Nomenclature (ISCN 2009) definition of a clone requires at least two metaphases with the same structural abnormality or the same extra chromosome, or alternatively, the presence of three or more metaphases missing the same chromosome. The minimum number of metaphases to detect a clone is calculated according to the formula presented in Fig.1. To avoid missing a clone present in 14% of cells, 20 metaphases must be analyzed, and to avoid missing a clone present in only 10% of cell 30 metaphases must be analyzed, with 95% confidence. Fluorescence in situ hybridization (FISH) on cultured cells is used as a complementary test, permitting analysis of large numbers of cells. The protocol in our lab includes analyzing, independently, both cytogenetic and FISH, on uncultured BM cells. Aims The aim of our study is to determine the minimal amount of mitoses that must be analyzed by cytogenetic combined with FISH to avoid false-negative results thus optimizing the diagnosis process of MDS patients. Methods Retrospective study was performed on cytogenetic and FISH results of a group of 325 suspected MDS patients analyzed at the Chaim Sheba Medical Center in the years 2009-2011. Results FISH was normal, for the most common MDS abnormalities, (-5,del(5q); -7,del(7q); +8; del(20q), as well as -Y for male patients), in 258 (79.4%) of patients 95.3% of them with normal cytogenetic. One or two abnormal clones, from the intermediate risk group in IPSS, were found in 10 patients (3.9%), at least in 30% of the mitoses. Complex karyotypes with 3 abnormalities, defined as poor risk, were found in two (0.8%) patients in 87.5% of the mitoses. Importantly, when FISH is normal, 9 metaphases are enough to detect minimal size of an abnormal clone with 95 % confidence and 14 metaphases with 99% confidence (Fig.1). Abnormal FISH was detected in 67 of patients. The same abnormalities were found by cytogenetic in 42 (62.6%), at least in 20%of the mitoses. In 25 patients, cytogenetic yielded additional chromosomal aberrations, not detected by FISH, at least in 20% of the mitoses, of them 20 patients (29.9%) with more than one aberration. Notably, in patients with abnormal FISH, analyses of 14 metaphases are enough to detect minimal size of CCA clone present in 20% of mitoses with 95 % confidence and 21 metaphases with 99% confidence (Fig.1). In addition cryptic deletions were detected by FISH, and not by cytogenetic, in 5 patients: del(7q) (n=3),del(5q) and del(20q), one each. Furthermore in 17 of 20 patients (85%) with complex karyotypes del(5q) was involved. Finally, in patients in whom -Y was detected by FISH, cytogenetic did not detect any other chromosomal abnormality implying to the benign nature of this lesion. Summary / Conclusion The combination of FISH, for the five most common abnormalities, and cytogenetic may improve the accuracy and speed of the diagnostic process, and may permit significant reduction of the number of mitoses to be analyzed in suspected MDS patients. The minimal size of mitoses to be analyzed to detect an abnormal clone has to be defined in each laboratory. We suggest that search for cryptic del(5q), del(7q) and del(20q) by FISH be added to the routine BM assessment in MDS patients. Disclosures: No relevant conflicts of interest to declare. </jats:sec

The Prevalence of Iron Deficient Anemia among New Infantry Recruits and Its Effect on Physical Fitness.

Abstract Background: Oxygen carrying capacity is clearly an important factor in physical fitness. There is limited information regarding the optimal hemoglobin level and iron stores status for physical activity and most studies followed relatively few participants. This information is usually on people participating in different sports of variable intensities, and it is difficult to predict how hemoglobin level might affect sports level in the general population. Object: To assess hemoglobin and iron stores levels at induction and their impact on physical fitness. Methods: 363 new infantry recruits were asked to participate in the study. Prior to recruitment all subjects were provided with a written, itemized protocol for a self-administered training regimen. Blood samples for hemoglobin, iron, ferritin, transferrin, folic acid and soluble transferrin receptor were taken from recruits that consented. A detailed medical and nutritional history was noted. Recruits performed the Wingate field fitness test including a 2Km. run. Recruits were grouped by the hemoglobin levels as pronounced anemia (&lt;12 g/dl), intermediate anemia (&lt;13g/dl), mild anemia (&lt;14 g/dl) or normal (&gt;14 g/dl). Results: Laboratory information was available for 358 recruits, 333 of which also completed the Wingate test. Mean hemoglobin was 13.8 ± 1.0 g/dl (SD). 11.7% 37% and 4.5% of the recruits had lower levels that 14,13 and 12 g/dl, respectively. Mean ferritin was 57 ± 34 ng/ml (SD) with 15% of the recruits under the 25ng/ml norm. Mean folic acid level was 6.1 ±2.1 ng/ml (SD) with 7.9% of recruits under 3.7ng/ml. On multivariate analysis, pre-induction training improved running scores (P&lt;0.001). Intermediate pre-induction hemoglobin level (12–14 g/dl) was associated with significantly better 2 km. running time (530sec ± 69) than both the lower hemoglobin group (570sec ± 77) and even the higher hemoglobin group (552sec ± 86, P&lt;0.05). Smoking and poultry consumption were not associated with running scores or anemia. Discussion: The subjects in this study are normal healthy young men, not sportsmen by acceptable criteria. The only pre-selection is that they volunteered and were found fit for the infantry. The high rate of abnormal hemoglobin and ferritin values in this population is most likely the result of “sports anemia” due to the intense physical training regimen adopted prior to their recruitment. The improvement of running results with higher hemoglobin than 12 g/dl is certainly in line with previous work. The anemia may be partly encountered to physiologic dilutional pseudoanemia caused by plasma volume expansion reactive to physical activity. The decline of running result with lower hemoglobin, less than 12g/dl, can be explained with depleted iron stores. Depletion of iron stores could be also a result of strenuous exercise with recurrent events of bowel ischemia during exercise and occult blood loss, or poor diet as this group had also lower levels of folic acid. The decrease in running ability with increased hemoglobin above 14 g/dl is surprising and will need further evaluation.</jats:p

Somatic Expansion of the Frataxin Gene GAA Repeats in MDS Patients.

Abstract The mitochondria play an important role in both apoptosis and heme synthesis. In patients with Myelodysplastic syndrome (MDS) the marrow is characterized by defective hematopoiesis, increased apoptosis and the presence of iron laden mitochondria. The molecular mechanisms responsible for increased apoptosis remain incompletely understood. Frederic’s ataxia (FRDA), the most common inherited ataxia, is a severe autosomal-recessive disease characterized by neurodegeneration, cardiomyopathy and diabetes, resulting from reduced synthesis of the mitochondrial protein frataxin which is involved in mitochondrial energy production and other cellular functions by providing iron for heme synthesis and iron–sulfur cluster (ISC) assembly and repair, serving as a Fe (II) donor for ferrochelatase. The underlying mutation consists of an unstable expansion of GAA repeats in the first intron of the frataxin gene. Long expansions of a GAA tri-nucleotide in FRDA patients range from 66 to more than 1,700 repeats, whereas the normal range of repeats varies from 7 to 36. Abnormal expansion results in reduced frataxin mRNA levels, leading to reduced function of the respiratory chain. The aim of the present study was to determine if frataxin gene mutations occurred in MDS patients. We analyzed DNA from peripheral blood (PB) of 29 MDS patients and from 22 healthy marrow (BM) donors using repeat-Primed PCR. We also sampled genomic DNA products from buccal smears of the MDS patients. In MDS patients PCR of PB in 9 out of 24 patients (37%) showed short length (2–8 repeats), whereas PB of the remaining 15 patients (62.5%) showed longer PCR products (10–43 repeats, still in the “normal” range for FRDA). The PCR products of the buccal smears from all 14 patient samples were short (2–7 repeats), including those from 9 patients who had longer repeats in PB. In healthy BM donors, PCR of PB detected short length repeats (4–5 repeats) in17 of 20 individuals (85%), whereas 3 samples (15%) had longer PCR products (11–26 repeats). This was statistically significantly different from patients with MDS (P= 0.0014). The results indicate that MDS patients exhibit longer frataxin gene products than healthy individuals in PB, but not in buccal DNA. These data suggest a somatic mutation in the frataxin gene in hematopoetic cells of patients with MDS. Further studies will explore the impact of this mutation on mitochondrial function and on the pathophysiology of MDS.</jats:p

Population-Based Prevalence of Anemia, Iron, Folic Acid and Vitamin B12 Deficiency among Young Adults in Israel.

Abstract Background: The prevalence of anemia and iron deficiency among adolescents and young adults in Israel has not yet been assessed. Studies performed among specific populations in Israel demonstrated highly variable rates of anemia of 6–58%, with iron deficiency rates of 13–38%. We have set out to investigate the prevalence of anemia, iron, folic acid and vitamin B12 deficiency among young males and females that are requited to the army in Israel (IDF). Methods: A random sample of 366 new recruits to the IDF (270 males and 96 females) participated in the study and undergone blood testing for Hemoglobin, Iron, Transferrin, Ferritin, Vitamin B12, Folic acid on recruitment day. Results: Anemia among females (Hb&lt;12 gr/dl), was 15.1% compared with 11.4% among males (Hb&lt;14 gr/dl). Transferrin saturation values indicating iron deficiency (15%&gt;) were also more prevalent among females compared with males (28.1% and 9.7%, respectively), as were low ferritin rates (37.2% and 14.3%, respectively). Vitamin B-12 deficiency (&lt;180 pg/ml) was found in 6.6–9.6% of the subjects, and folic acid deficiency was detected in 6.4–11.7% of the subjects. Low-level paternal education and immigration were two factors found to be negatively associated with anemia. Smoking was associated with low ferritin levels in males. Anemia and iron levels were not affected significantly by the type of service designed, as combat soldiers or for clerical work. Conclusions: Anemia and iron deficiency occur frequently among young adults requited to the IDF. They were more prevalent among females; but in males the prevalence is higher than expected. Low content of iron in the diet, infection like H Pylori and engaging in strenuous exercise preparing to service, may contribute to depleted iron stores. Low-level paternal education and immigration was surprisingly protecting factor, may be as a result of different eating habits. Anemia and iron deficiency, are known to reduce physical work capacity and mental performance, therefore it is important to perform more research to identify possible etiologies for intervention like education for proper nutrition, and adjustment of the diet in the military to tackle this prevalent problem.</jats:p

Assessment By MRI T2*of Iron Accumulation and Removal in Transfusion Dependent MDS Patients

Introduction: Transfusion dependency in Myelodysplastic syndrome (MDS) patients is associated with shortened over-all survival and leukemia-free survival; however, it is not clear whether this detrimental effect is mediated by transfusional iron overload itself or whether the need for RBC transfusion is a surrogate marker of disease severity. Additionally, despite the negative effects of anemia and transfusion dependence on disease outcomes and patient quality of life, the clinical impact of iron overload in MDS remains controversial. Overall, the most common non-leukemic cause of death in MDS is heart failure. Recent T2* cardiac MRI (CMR) studies have shown that cardiac iron accumulation in MDS patients is variable and infrequent. T2* CMR is noninvasive, robust and the best available modality for assessing iron organ quantification. The current study assessed iron overload in transfusion dependent MDS patients using ferritin levels combined with myocardial and liver iron quantification using T2* CMR. The study monitored iron accumulation dynamics while on iron chelation therapy. Methods: We collected retrospectively clinical data in 18 RBC transfusion dependent MDS patients that had a CMR in our institution; some of the patients have serial scans. All scans were obtained using a 1.5 T scanner (Signa HDx ver 15 GE). Scans interpretation was performed using a dedicated workstation (Medis Medical imaging version 7.6, the Netherlands) for left ventricular ejection fraction measurement and T2* investigation as previously described. T2* values were recorded in milliseconds and converted to mg/gr dry tissue of myocardial liver and pancreatic tissue. Results: Ten of 18 patients were chelated. During the study period 4 patients have died from pneumonia, brain tumor, complications of Allo BMT and transformation to AML. Demographic data and prognostic score of MDS are shown in table 1. Before the first CMR, patients were treated with median of 48 PC (14-145) with median ferritin levels 1691.3ng/ml (269.4-3730ng/ml). In Table 2 we show iron accumulation in the first CMR. Iron accumulation in the heart was seen in 2 patients. The results are shown as severity of accumulation and not absolute numbers, as normal and abnormal range is different in the different organs. Seven patients had yearly serial CMR done, 6 of them were treated with iron chelation. We found iron accumulation in the heart in 4 of them. In table 3 we show example of a patient that was treated with chelation. Another patient, that had decrease ejection fraction with only moderate accumulation in the heart in CMR, improved her heart function with chelation. During the study period all patients continue to be transfusion dependent except 1 patient that became transfusion independent. She was transfused with 182 PC with maximal ferritin of 6926ng/ml. She was chelated with deferiprone and then with desferal. She is now transfusion independent for a year. Conclusion: Blood transfusion dependency in MDS patients, leads to high ferritin levels and evidences in CMR of iron accumulation in parenchymal orangs including the heart. During the study 4 patients had evidence of iron accumulation in the heart with 2 of them with severe accumulation that improved with iron chelation. Liver and pancreas accumulation was seen in 12 and 8 patients accordingly. Prospective studies documenting the correlation between chelation therapy and improvement in organ damage and survival are needed. Disclosures No relevant conflicts of interest to declare. </jats:sec