A Novel Approach for the Screening of Megaloblastic Anaemia

Feb. 2012, Volume 9, No. 2 (Serial No. 87), pp. 71 79 Journal of US-China Medical Science, ISSN 1548-6648, USA D DAVID PUBLISHING A Novel Approach for the Screening of Megaloblastic Anaemia R. Simon-Lopez 1, M. Egorova 2, N. Tsvetaeva 2, Elena Sukhacheva 1, S. Kolenkin 3 and T. Achildieva 4 1. Scientific Support, Beckman Coulter Eurocenter, Nyon, Switzerland 2. Hematological Scientific Center, Russian Academy of Medical Sciences, Moscow, Russia 3. Laboratory, Medical Center for Central Bank of Russia, Moscow, Russia 4. Product Manager Immunochemistry, Closed Joint-Stock Company Galen, Moscow, Russia Abstract: There is a high prevalence of B12 and folate deficiency in elderly individuals and a deficiency of either causes megaloblastic anaemia. The screening methods for these deficiencies are haemoglobin concentration and mean cell volume; however they are often present without macrocytosis. The study aimed to determine the sensitivity and specificity of parameters relating to neutrophil and monocyte volume in detecting megaloblastic changes, thus detection of B12 and folate deficiencies even before anaemia is apparent. Mean neutrophil and monocyte volumes were measured on Beckman Coulter LH750 haematology analyser using volume, conductivity and scatter. 57 non-anaemic and 109 anaemic patients were screened using these parameters. In the non-anaemic group 5.2% had B12 deficiency, 3.5% had intermediate values and 3.5% had low folate. For anaemic patients prevalence of B12 deficiency was 12.8%, intermediate levels 3.7%, and folate deficiency was 15.6%. Neutrophil and monocyte volume demonstrate utility in the detection of megaloblastic changes that occur due to B12 and folate deficiency. Providing the correct sample is available B12 and folate assays could be automatic reflex tests. This could reduce the number of patients being recalled for further testing, causing inconvenience and adding additional cost to the health service. Key words: Beckman Coulter LH700 haematology instruments, folate, monocyte volume, neutrophil volume, vitamin B12. 1. Introduction Vitamin B12 is a coenzyme involved in two metabolic functions vital to normal cell growth and DNA synthesis, the synthesis of methionine and the conversion of methylmalonyl coenzyme A to succinyl coenzyme A. Vitamin B12 and folate are linked by the reaction pathway for methionine synthesis. A deficiency in either leads to disruption of this metabolic pathway and similar clinical symptoms [1, 2] Vitamin B12 and folate deficiency can lead to neurological and haematological changes and to hyperhomocysteimemia. Symptoms include fatigue, weakness, anorexia, paraesthasia and dizziness, but initial presentations are vague [3]. It is estimated that 5 20% Corresponding author: Elena Sukhacheva, PhD, research field: cellular analysis EMEAI. E-mail: esukhacheva@beckman.com. of individuals over the age of 60 years are B12 deficient [4]. The main causes of B12 deficiency include lack of intrinsic factor and other intestinal factors (e.g., malabsorption), rare genetic factors and inadequate intake [5 7]. Elderly people with low vitamin B12 status frequently lack the classical clinical signs and symptoms of vitamin B12 deficiency, e.g., megaloblastic anaemia. The most common cause of folate deficiency is due to poor dietary intake, although increased folate utilisation, malabsorption, drugs and alcohol may have effects on folate metabolism. A deficiency of either of these nutrients will eventually cause megaloblastic anaemia, characterised by the enlargement and reduction in the number of all rapidly proliferating cells, including bone marrow cells, and is primarily a result of the decreased capacity for DNA synthesis. Because vitamin B12 and folate are linked by the reaction pathway for methionine synthesis, a

72 A Novel Approach for the Screening of Megaloblastic Anaemia deficiency in either will disrupt this metabolic pathway and lead to the same laboratory findings and medical symptoms. The clinical indications of the disease are of prime importance as routine screening tests, such as the full blood count, are not always abnormal. The usual method of screening for a vitamin B12 and/or folate deficiency in a patient comprises analysing haemoglobin (Hb) and mean corpuscular volume (MCV) which are reported as part of a standard complete blood count (CBC). The normal MCV range is typically 80 100 femtolitres (fl). In pernicious anaemia (macrocytic), MCV can be up to up to 150 fl. A high MCV has been the traditional criterion for detecting vitamin B12 and folate deficiencies however vitamin deficiency is not the most common cause of macrocytosis, excessive alcohol ingestion, reticulocytosis, cytotoxic chemotherapy, and myelodysplastic syndromes may also cause macrocytosis. B12 and folate deficiencies may frequently present with normal cell volumes particularly when there is a concomitant iron deficiency or thalassaemia trait present. A sub-clinical deficiency may be present, an asymptomatic state, in which metabolic insufficiency is demonstrable in seemingly healthy patients who do not have clinical signs of B12 or folate deficiency. This condition may be termed latent deficiency. Its frequency is at least 10-fold that of clinically expressed deficiency and it affects millions of people [8]. An unknown number of these cases may eventually progress to overt clinical manifestation. No diagnostic gold standard test is available for the diagnosis of B12 or folate deficiency but accurate and early diagnosis is important because these deficiencies can lead to life-threatening haematological and neurological abnormalities which may be reversible by treatment with vitamin B12 or folic acid, respectively. Thus, there exists a need to develop more efficient methods for detection of these deficiencies. It has been found that the properties of white blood cells of a subject deficient in vitamin B12 and/or folate are altered, particularly neutrophils and monocytes. The volumes of these cells increase due to megaloblastic changes, these changes are detected using cell volume, conductivity and scatter measurements performed routinely on all samples having a CBC and differential on the Beckman Coulter LH700 series of instruments (Beckman Coulter Miami, Fl, USA). The mean volume of neutrophils and monocytes increase to levels where they are statistically different from normal values. Alterations of these parameters of white blood cell subpopulations can be utilised as an indicator for megaloblastic changes associated with vitamin B12 and/or folate deficiency or a latent deficiency of these vitamins. Previously haematology and clinical chemistry laboratories have worked separately and independently, but nowadays many departments have been combined into one integrated automated laboratory processing all routine tests for both pathologies. The measurement of B12 and folate was traditionally performed by the haematology laboratory but now both are usually analysed on highly automated chemistry instruments. A sample with a high MCV, anaemia or abnormal neutrophil or monocyte volume could trigger an automatic request for a B12 and/or folate assay. The aim of this study was to assess and determine the sensitivity and specificity of parameters based on leucocyte volume, those of the neutrophils, mean neutrophil volume (NeMV) and monocytes, mean monocyte volume (MoMV), in screening and diagnosis of vitamin B12 and/or folate deficiencies, even before any anaemia or clinical symptoms may be apparent. 2. Materials & Methods 2.1 Patients. K 2 EDTA anticoagulated residual blood samples from a routine laboratory in a Moscow hospital were analysed. Samples were selected after all testing was complete. 57 individuals attending the hospital as out-patients, but considered to be normal by their physicians were selected. These samples were not

A Novel Approach for the Screening of Megaloblastic Anaemia 73 pre-selected by their haematological results and were classified the as the non-anaemic control group. 109 residual blood samples from patients with anaemia, defined according to the WHO anaemia criteria, (Hb < 12 g/dl in women and Hb < 13 g/dl in men) were selected from samples previously analysed in the hospital laboratory. The WHO definition of anaemia was the only criteria used for selection in order to ensure results were unbiased. The total study population consisted of 82 males (49.4%) and 84 females (50.6%). 2.2 Haematological Analysis All samples were analysed for CBC with 5-part leucocyte differential on the Beckman Coulter LH 750 haematology analyser within eight hours of collection. The total white blood count is performed using the impedance method. After lysis of red blood cells the reagent modifies the nucleated cells, shrinking the cytoplasm and so affecting the original cell size. The histogram produced after analysis of the nucleated cells differentiates the leucocytes from the nucleated red blood cells, platelets, and debris. For the white blood cell differential the analyser makes three measurements as each cell passes through a flow cell which is an electro-optical flow cytometer. Volume (V), conductivity (C) and laser light scatter (S) is measured for each cell. The white cell volume is measured using impedance. The conductivity is measured using a radio frequency probe that determines the nuclear shape, lobularity, density and nuclear/cytoplasmic ratio. Laser light technology analyses the median light scatter of each cell in order to quantify the specific granularity of the cells. The instrument provides a two dimensional histogram of volume and scatter showing the four main populations of leucocytes. Numerical data is also reported for the mean and standard deviation (SD) of the volume, conductivity and scatter for the neutrophils, lymphocytes, monocytes and eosinophils, this data relates to cell size and content. These parameters are measured on all samples selected for a CBC with white cell differential and does affect throughput of the instrument. Reference ranges, stability and precision of these measurements have been previously established [9]. 2.3 Biochemical Markers of Anaemia Traditional biochemical markers of iron deficiency, as well as specific measurements concerning B12 and folate deficiency, were carried out to diagnose and classify any anaemia that may be present in all subjects. Analyses of serum iron, serum ferritin, vitamin B12, serum folate, red blood cell folate, serum erythropoietin, transferrin, C-reactive protein and intrinsic factor antibody were measured in all samples. These biochemical markers were measured using Beckman Coulter instruments, Access (Beckman Coulter Miami, Fl, USA). and SYNCRON CX, (Beckman Coulter Miami, Fl, USA), and Beckman Coulter assay kits. Reference ranges provided by the manafactuer were used. 2.4 Statistical Methods The efficiency of the classical hematological parameters (i.e., MCV) in detecting B12 and/or folate deficiency was compared to the efficiency of white blood cell parameters (i.e., NeMV or MoMV). Receiver operator characteristic (ROC) graphs were plotted and the corresponding area under the curve (AUC) was calculated to assess their potential clinical utility. The ROC curve plots sensitivity versus specificity. The area under the curve is indicative of the diagnostic utility of the parameter to distinguish subjects deficient in vitamin B12 and/or folate from normal subjects or subjects with anaemia due to other causes. Data were analysed and statistical significance determined using Mann-Whitney U test and Student s t-test, as appropriate. ROC curves and Box and

74 A Novel Approach for the Screening of Megaloblastic Anaemia Whisker plots were determined using MedCalc software (MedCalc Software, Mariakerke, Belgium). 3. Results Cases of B12 and/or folate deficiencies were found in both non-anaemic controls and anaemic subjects. Results indicated that 3/57 (5.2%) of the non-anaemic group had a vitamin B12 deficiency (vitamin B12 < 145 pg/ml); 2/57 (3.5%) of the non-anaemic group with had intermediate values of vitamin B12 (vitamin B12 > 145 <180 pg/ml); and 2/57 (3.5%) of the non-anaemic group had low serum folate. Only 29/57 subjects in the non-anaemic group were found to have normal values for all parameters measured and are termed normal for the purpose of this study. These 29 patient samples were used to establish the normal range for NeMV and MoMV for this study. The ranges are 126.4 149.3 for NeMV and 148.3 173.6 for MoMV From the group of anaemic patients, results indicated that the prevalence of vitamin B12 deficiency was 14/109 (12.8%), the prevalence of intermediate vitamin B12 levels was 4/109 (3.7%), the presence of serum folate deficiency was 17/109 (15.6%), results are shown in Table 1. In addition one patient showed a deficiency of both vitamin B12 and serum folate deficiency and one other an intermediate vitamin B12 level and serum folate deficiency. Results for NeMV and MoMV were statistically different in subjects with normal values for B12 and folate and those with a B12 and/or folate deficiency. 10 patients with low or intermediate levels of B12 had normal CRP values and 13 had raised levels indicating the presence of inflammation or infection; however this did not affect the sensitivity or specificity of NeMV or MoMV in detecting the B12 deficiency. Results for MCV between subjects with normal values for B12 and folate and those with a B12 and/or folate deficiency were not different, Table 2. Fig. 1 (A to D) shows Box and Whisker plots comparing NeMV and MoMV in the 29 normal subjects with patients with either B12 or folate deficiency. Fig. 2 (A to D) shows Box and Whisker plots comparing NeMV and MoMV in patients with anaemia due to other causes with patients with either B12 or folate deficiency. The NeMV and MoMV are higher in patients with B12 or folate deficiency than normal subjects or patients with anaemia due to other causes. Table 1 Descriptive statistics for the different groups, normal subjects, anaemic and non-anaemic patients. The values are the mean of the parameters for the group of patients in each particular group (N = number, MCV (mean cell volume) and NeMV (mean neutrophil volume) and MoMV (Mean monocyte volume), Interm = intermediate). B12/Folate Category N MCV NeMV MoMV B12 (pg/ml) Folate ng/ml Non anaemic group B12 Hb>12 Low B12 3 105.9 150.3 180 B12 90 B12 Hb>12 Interm B12 2 86.7 141.2 168.4 B12 164 B12 Hb>12 Normal B12 48 91.4 138.4 163.3 B12 372.6 Folate Hb>12 Low Folate 2 105.5 147.9 170.2 Folate<2.33 Folate Hb>12 Normal Folate 48 91.4 138.4 163.3 Folate 5.8 Hb>12 All measured parameters normal 29 92.1 138.5 162.1 Normal Anaemic patients B12 Hb<12 Low B12 14 94.9 152.9 178.8 B12 95.9 B12 Hb<12 Interm B12 4 87.1 144.1 177.8 B12 164.8 B12 Hb<12 Normal B12 62 84.6 142.5 169 B12 355.1 Folate Hb<12 Low Folate or RBC Folate 17 93.5 151.7 178.1 Folate 1.9 Folate Hb<12 Normal Folate 62 84.6 142.5 169 Folate 355.1 B12 and Folate Hb<12 Anaemia due to other causes 62 84.6 142.5 169 Normal

A Novel Approach for the Screening of Megaloblastic Anaemia 75 Table 2 Comparative statistics between the different groups, normals, those with and without anaemia and patients with or without folate or B12 deficiencies. The cut-off values for MCV, NeMV and MoMV obtained from the ROC analysis are used as the criterion for indication of a vitamin B12 and/or folate deficiency (p < 0.05 was considered significant, ROC = Receiver operator Characteristic, AUC = area under the curve, Sens = sensitivity, Specif = specificity). N Parameter T-Test ROC AUC Sens % Specif % Cut-Off 23 vs 29 B12 Def & Interim vs Normals Significance ROC MCV P = 0.477 0.475 31.10 100.00 > 99.5 P = 0.8145 NeMV P < 0.0001 0.826 82.60 75.90 > 140.32 P = 0.0001 MoMV P < 0.0001 0.895 87.00 86.20 > 167.11 P = 0.0001 17 vs 29 B12 def vs Normals MCV P = 0.1906 0.615 52.90 100.00 > 99.5 P = 0.3223 NeMV P < 0.0001 0.844 70.59 93.1 > 145.4 P = 0.0001 MoMV P < 0.0001 0.905 88.24 86.21 > 167.11 P = 0.0001 19 vs 29 Folate Def (Folate or Red cell folate low) vs Normals MCV P = 0.3322 0.519 52.60 86.20 > 89.5 P = 0.3761 NeMV P < 0.0001 0.844 89.50 75.90 > 140.32 P = 0.0001 MoMV P < 0.0001 0.878 73.70 93.10 > 169.5 P = 0.0001 36 vs 29 B12 or Folate Def (Folate low or Red cell folate low) vs Normals MCV P = 0.8622 0.602 55.60 86.20 > 89.5 P = 0.1803 NeMV P < 0.0001 0.822 83.30 75.90 > 140.32 P = 0.0001 MoMV P < 0.0001 0.875 80.60 86.20 > 167.11 P = 0.0001 14 vs 62 B12 Def and anaemic vs other cause of anaemia MCV P = 0.0067 0.857 50.00 98.40 > 99.7 P = 0.1396 NeMV P < 0.0001 0.735 85.70 55.10 > 143.10 P = 0.0025 MoMV P < 0.0012 0.689 64.30 66.00 > 172.35 P = 0.0204 18 vs 62 B12 Def (Low & Interim) and anaemic vs other cause of anaemia MCV P = 0.0108 0.634 38.90 98.40 > 99.7 P = 0.1242 NeMV P < 0.0005 0.699 66.70 67.20 > 146.05 P = 0.0040 MoMV P < 0.0004 0.723 72.20 66.00 > 172.35 P = 0.0011 17 vs 62 Folate Def (Folate low or Red cell folate low) and anaemic vs other cause of anaemia MCV P = 0.0046 0.662 82.00 46.80 > 85.0 P = 0.0251 NeMV P < 0.0002 0.711 88.20 49.20 > 141.30 P = 0.0020 MoMV P < 0.0009 0.691 76.50 54.10 > 168.70 P = 0.0099 35 vs 62 B12 Def (Low & Interim) or Folate Def and anaemic vs other cause of anaemia MCV P = 0.0102 0.611 30.0 96.80 >96.9 P = 0.894 NeMV P = 0.0018 (Mann 0.702 66.70 49.20 >141.34 P 0.0004 Whitney MoMV P = 0.0005 0.681 83.30 42.60 166.91 P = 0.0023 ROC analysis was used to determine the sensitivity and specificity of MCV, NeMV and MoMV in the diagnosis of vitamin B12 or folate deficiency. MoMV was more accurate in detecting samples with the B12 deficiency followed by the NeMV. When comparing patients with vitamin B12 or folate deficiencies with patients with anaemia due to other causes, the most accurate test was the NeMV, followed by the MoMV;

76 A Novel Approach for the Screening of Megaloblastic Anaemia Table 3 Examples of data for patients with B12 and/or folate deficiencies both with and without anaemia. Grey shaded cells show when B12 or Folate deficiencies are detected by MoMV or NeMV. Figures in bold are abnormal results (IFAb = Intrinsic factor antibody, Fe = serum iron, CRP = C-reactive protein, TRF = transferrin, Hb = haemoglobin, MCV = mean cell volume, NeMV = mean neutrophil volume, MoMV = mean monocyte volume, Interm = intermediate). Patient 62 Patient 113 Patient 159 Patient 154 Patient 137 Patient 125 Test Interm B12, no anaemia Interm B12, anaemia Low B12, no anaemia Low B12, anaemia Low Folate, anaemia Low B12, Low Folate, anaemia Ferritin mg/dl 80.8 77.1 168.4 38.1 160.9 159.3 Folate ng/ml 4.94 6.32 2.6 5.26 1.64 1.64 IFAb miu/ml 5.09 1.21 1.08 1.23 1.1 1.17 B12 pg/ml 161 158 128 102 368 145 RBC Folate 281.1 341.26 284.71 156.98 Fe µg/dl 10.2 2.9 9.1 1.5 3.2 5.3 CRP mg/l 2.9 10.9 16.7 4.4 9.1 1.5 TRF mg/dl 240 178 158 216 150 164 Hgb g/l 12.1 9.53 12.36 8.08 9.53 9.75 MCV fl 88.9 84.7 92.1 73.8 85.3 90.5 NeMV fl 137.93 146.36 150.92 150.71 166.98 141.068 MoMV fl 176.21 178.93 182.58 173.81 188.75 181.66 180 220 170 160 210 200 190 150 180 140 170 160 130 150 120 NeMV Low B12 NeMV Normal B12 (A) 140 MoMV Low B12 MoMV Normal B12 (B) (C) (D) Fig. 1 Box and Whisker Plots comparing normal controls with B12 and folate deficient patients. 1A normal controls (right side of graph) with B12 deficient subjects (left side of graph) using mean neutrophil volume (NeMV). 1B normal controls (right side of graph) with B12 deficient subjects (left side of graph) using mean monocyte volume (MoMV). 1C comparing normal controls (right side of graph) with folate deficient subjects (left side of graph) using mean neutrophil volume (NeMV). 1D comparing normal controls (right side of graph) with folate deficient subjects (left side of graph) using mean monocyte volume (MoMV).

A Novel Approach for the Screening of Megaloblastic Anaemia 77 (A) (B) (C) (D) Fig. 2 Box and Whisker Plots comparing patients with anaemia due to other causes with B12 and folate deficient patients. 2A comparing subjects deficient in B12 (left side of graph) to other subjects with anaemia (right side of graph) using mean neutrophil volume (NEMV). 2B comparing subjects deficient in B12 (left side of graph) to other subjects with anaemia (right side of graph) using mean monocyte volume (MoMV). 2C comparing subjects deficient in folate (left side of graph) to other subjects with anaemia (right side of graph) using mean neutrophil volume (NeMV). 2D comparing subjects deficient in folate (left side of graph) to other subjects with anaemia (right side of graph) using mean monocyte volume (MoMV). results are shown in Table 2. Using ROC analysis several slightly different cut-offs were calculated for the diagnosis of deficiency in different groups of patients but on a routine daily basis, on the instrument used in this study, the cut-off values would be a NeMV> 143 and MoMV > 169. Table 3 shows some example results from patients with B12 or folate deficiencies. The data demonstrates that the NeMV and the MoMV can detect B12 or folate deficiencies in patients presenting whether they are anaemic or not. In all cases presented B12 and/or folate deficiencies are detected by NeMV and/or MoMV in patients with a normal, or in one case low, MCV. 4. Discussion Deficiencies of folate or vitamin B12 are widespread and constitute a major global burden of morbidity affecting all age groups. Prevalence of B12 deficiency among the general population varies from 3 5% [12] and from 5-20% among people older than 65 years [5] In this study 14% of samples were found to be B12 deficient, and 10% folate deficient, this high prevelance is probably because the patient population attending this hospital are mostly older than 60 years. Deficiencies may arise when there is insufficient intake of the nutrients, inadequate absorption, increased loss,

78 A Novel Approach for the Screening of Megaloblastic Anaemia or due to increased need. The most common cause of increased need of folate is pregnancy where it is required for proper fetal development, any deficiency prior to pregnancy will intensify during gestation. Screening for these deficiencies is advocated to prevent anaemia in elderly patients and others at risk [10].The most simple test is a CBC to measure Hb and MCV to detect a macrocytic anaemia; however it has previously been shown that frequently there is no anaemia present with B12 deficiency [11]. Adding to the complications of detecting a possible B12 and/or folate deficiency by a routine CBC alone is that it is frequently associated with iron deficiency and poorly correlated with increased MCV levels [12]. Tests needed for diagnosis of deficiency need to be restricted to avoid unnecessary investigation of unaffected individuals but be applied broadly enough to include all patients with a clinical state or laboratory abnormality that may be associated with a deficiency of B12 or folate. The initial investigation of these patients should always be a CBC and blood film examination. The blood film will show oval macrocytes, in varying numbers, and megaloblastic changes to granulocytes. These show a tendency toward gigantism, hyperpolymorphism and hypersegmention. Using VCS technology the LH700 series of instruments are able to detect these changes in the neutrophils and monocytes before a blood film is examined. The new parameters, NeMV and MoMV are related to the size of neutrophils and monocytes. The volume of these two cell types increase to levels which are statistically different from normal values in the presence of either deficiency and show better sensitivity and specificity for the detection of megaloblastic changes related to a B12 and/or folate deficiency, even in the presence of anaemia due to other causes, than MCV. The NeMV and MoMV in either B12 or folate deficiency show good separation from normal values, and anaemia due to other causes, demonstrated in Figs. 1 2. 18 of the patients selected because of low Hb concentration (the cause for anaemia was unknown at that time) were found to have a low, or interim level, of B12 and 17 a low serum folate or red cell folate and deficiency, or interim levels, of B12 and folate was present in 2 patients. Despite the fact that most of these patients had a normal MCV their condition was detected by a raised NeMV or MoMV, or both. Using these two new leucocyte parameters seven non-anaemic patients enrolled into the study were found to have either a B12 or folate deficiency, two of these also had a normal MCV. These patients were enrolled into the study only if their physicians considered them normal so were therefore showing no clinical signs of either B12 or folate deficiency. This is subclinical deficiency where metabolic insufficiency is demonstrable in seemingly healthy patients who do not have megaloblastic anaemia, neurological symptoms or other clinical signs of B12 or folate deficiency. This condition is termed a latent deficiency and it has been reported that the frequency of latent deficiency is at least 10-fold that of clinically expressed deficiency and affects millions of people [7]. An unknown number of these cases may eventually progress to overt clinical manifestation. Recently these same parameters have been found to predict sepsis and non-systemic bacterial infection [13] which might lead to the supposition that in the presence of infection NeMV or MoMV would loose specificity for the detection of B12 and/or folate deficiency by showing false positive results in these circumstances. In this small group of patients this did not seem to be the case. Further studies are needed to assess the affect of other causes of macrocytosis, such as alcoholism, on the volumes of neutrophils and monocytes. Ultimately, confirming the presence of either a B12 or folate deficiency and distinguishing one from the other depends on laboratory testing of the vitamins in blood, but using the LH700 parameters NeMV and MoMV, automatically produced on all CBC and differential results, appears to allow for efficient screening of all samples tested within the laboratory. This will result in faster and more efficient diagnoses

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