RESUMO
ABSTRACT: ß-thalassemia is a condition characterized by reduced or absent synthesis of ß-globin resulting from genetic mutations, leading to expanded and ineffective erythropoiesis. Mitoxantrone has been widely used clinically as an antitumor agent considering its ability to inhibit cell proliferation. However, its therapeutic effect on expanded and ineffective erythropoiesis in ß-thalassemia is untested. We found that mitoxantrone decreased α-globin precipitates and ameliorated anemia, splenomegaly, and ineffective erythropoiesis in the HbbTh3/+ mouse model of ß-thalassemia intermedia. The partially reversed ineffective erythropoiesis is a consequence of effects on autophagy as mitochondrial retention and protein levels of mTOR, P62, and LC3 in reticulocytes decreased in mitoxantrone-treated HbbTh3/+ mice. These data provide significant preclinical evidence for targeting autophagy as a novel therapeutic approach for ß-thalassemia.
Assuntos
Modelos Animais de Doenças , Eritropoese , Mitoxantrona , Talassemia beta , Animais , Talassemia beta/tratamento farmacológico , Eritropoese/efeitos dos fármacos , Camundongos , Mitoxantrona/farmacologia , Mitoxantrona/uso terapêutico , Autofagia/efeitos dos fármacos , alfa-Globinas/genética , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismoRESUMO
Red blood cell (RBC) transfusion, limited by patient alloimmunization, demands accurate blood group typing. The Rh system requires specific attention due to the limitations of serological phenotyping methods. Although these have been compensated for by molecular biology solutions, some RhCE ambiguities remain unresolved. The RHCE mRNA length is compatible with full-length analysis and haplotype discrimination, but the RHCE mRNA analyses reported so far are based on reticulocyte isolation and molecular biology protocols that are fastidious to implement in a routine context. We aim to present the most efficient reticulocyte isolation method, combined with an RT-PCR sequencing protocol that embraces the phasing of all haplotype configurations and identification of any allele. Two protocols were tested for reticulocyte isolation based either on their size/density properties or on their specific antigenicity. We show that the reticulocyte sorting method by antigen specificity from EDTA blood samples collected up to 48 h before processing is the most efficient and that the combination of an RHCE-specific RT-PCR followed by RHCE allele-specific sequencing enables analysis of cDNA RHCE haplotypes. All samples analyzed show full concordance between RHCE phenotype and haplotype sequencing. Two samples from the immunohematology laboratory with ambiguous results were successfully analyzed and resolved, one of them displaying a novel RHCE allele (RHCE*03 c.340C>T).
Assuntos
Alelos , Haplótipos , Sistema do Grupo Sanguíneo Rh-Hr , Humanos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Reticulócitos/metabolismo , RNA Mensageiro/genética , Transfusão de Sangue/métodos , FenótipoRESUMO
The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen availability. Deregulation of HIF is associated with the pathogenesis of major human diseases including cardiovascular disease and cancer. Under normoxia, the HIFα subunit is hydroxylated on conserved proline residues within the oxygen-dependent degradation domain (ODD) that labels HIFα for proteasome-mediated degradation. Despite similar oxygen-dependent degradation machinery acting on HIF1α and HIF2α, these two paralogs have been shown to exhibit unique kinetics under hypoxia, which suggests that other regulatory processes may be at play. Here, we characterize the protease activity found in rabbit reticulocytes that specifically cleaves the ODD of HIF1α but not HIF2α. Notably, the cleavage product is observed irrespective of the oxygen-dependent prolyl-hydroxylation potential of HIF1α, suggesting independence from oxygen. HIF1α M561T substitution, which mimics an evolutionary substitution that occurred during the duplication and divergence of HIF1α and HIF2α, diminished the cleavage of HIF1α. Protease inhibitor screening suggests that cysteine proteases cathepsins L and B preferentially cleave HIF1αODD, thereby revealing an additional layer of differential HIF regulation.
Assuntos
Catepsina L , Subunidade alfa do Fator 1 Induzível por Hipóxia , Oxigênio , Proteólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Animais , Catepsina L/metabolismo , Catepsina L/genética , Coelhos , Oxigênio/metabolismo , Humanos , Reticulócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , HidroxilaçãoRESUMO
BACKGROUND/AIM: ClFdA is a second-generation antineoplastic agent that has demonstrated significant anticancer activity, particularly against acute lymphoblastic leukemia and has been shown to have radiosensitizing activity. The aim of the study was to explore the genotoxic, cytotoxic and radiosensitizing effects of clofarabine (ClFdA) on bone marrow cells (BMCs), normoblasts and leukocytes of mice in vivo. MATERIALS AND METHODS: Cytotoxicity was determined by the reduction in reticulocytes (RET), and genotoxicity was determined by the induction of micronucleated reticulocytes (MN-RET) in the peripheral blood and by DNA break induction in leukocytes determined by single-cell gel electrophoresis (SCGE). The radiosensitizing capacity of ClFdA was determined in leukocytes and BMCs by SCGE. RESULTS: Two mechanisms of MN-RET induction were identified according to the antecedents, that could be due to inhibition of DNA synthesis and demethylation of G-C regions, and subsequent chromosome fragility. ClFdA cytotoxicity causes two contiguous peaks, an early peak that seems to inhibit MN-RET induction and a second peak that seems to be caused by ribonucleotide reductase (RR) and/or DNA synthesis inhibitions. ClFdA induced early DNA damage in noncycling leukocytes, and also radiosensitizes leukocytes immediately after treatment. ClFdA-ionizing radiation (IR) causes two time-dependent episodes of DNA damage, the latest after 80 min triggers a major breakage of DNA. In terms of the number of damaged cells, leukocytes and BMCs are similarly sensitive to ionizing radiation; BMCs are slightly more sensitive than leukocytes to ClFdA, but BMCs are doubly sensitive to combined treatment. CONCLUSION: ClFdA causes early DNA damage and radiosensitivity in non-proliferating leukocytes, which rules out the most favored hypotheses of the participation of RR and DNA polymerase inhibition.
Assuntos
Clofarabina , Dano ao DNA , Leucócitos , Radiossensibilizantes , Animais , Clofarabina/farmacologia , Camundongos , Radiossensibilizantes/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Arabinonucleosídeos/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/efeitos da radiação , Células da Medula Óssea/metabolismo , Nucleotídeos de Adenina/farmacologia , Masculino , Reticulócitos/efeitos dos fármacos , Reticulócitos/efeitos da radiação , Antineoplásicos/farmacologia , Testes para MicronúcleosRESUMO
BACKGROUND: Myxomatous mitral valve disease (MMVD) is the most common acquired cardiovascular disease in small breed dogs. In contrast to human patients with heart failure (HF), iron deficiency (ID) prevalence in dogs with MMVD is weakly known. The study aimed to assess the usability of ID markers in serum and reticulocyte parameters from whole blood of dogs with MMVD to evaluate early ID symptoms. RESULTS: Sixty-eight dogs (43 male and 25 female) were included in the study. MMVD dogs were assigned according to the 2019 ACVIM guidelines for groups B1 (n = 9), B2 (n = 10), C (n = 27) and D (n = 10). Groups were also combined into B1 and B2 as non-symptomatic HF and C with D as symptomatic HF. Healthy controls were 12 dogs. Serum iron concentration below the reference range in dogs with MMVD was 12.5%. Other ID indices, such as %SAT, UIBC, and TIBC were similar in the MMVD groups and healthy controls (p > 0.05 for all parameters). Statistical comparison between control group and 4 groups of different stages of MMVD showed that significant differences occur only in serum transferrin. The assessment of ferritin and soluble transferrin receptors using Western Blotting did not show differences between control (n = 7) and MMVD (n = 33) dogs. Study has shown positive correlation between ID parameters and echocardiographic indices such as LA/Ao and LVIDdN, and some biochemical parameters. A significant increase in reticulocytes percentage, assessed manually, was observed in the HF group of animals (p = 0.027) compared to the control group. CONCLUSIONS: Studies have shown that ID parameters in serum are not significantly different in dogs with MMVD compared to healthy dogs. However, there is a clear correlation between atrial size and normalised left ventricular size to body size and some biochemical parameters, including ID parameters and therefore the severity of MMVD.
Assuntos
Doenças do Cão , Ferro , Cães , Animais , Doenças do Cão/sangue , Feminino , Masculino , Ferro/sangue , Biomarcadores/sangue , Ferritinas/sangue , Insuficiência da Valva Mitral/veterinária , Insuficiência da Valva Mitral/sangue , Deficiências de Ferro/sangue , Doenças das Valvas Cardíacas/veterinária , Doenças das Valvas Cardíacas/sangue , Valva Mitral , Anemia Ferropriva/veterinária , Anemia Ferropriva/sangue , Transferrina/análise , Transferrina/metabolismo , ReticulócitosRESUMO
BACKGROUND: N-(-9 acridinyl)-b-alanine hydrochloride (S-300) is the main byproduct of red blood cell (RBC) amustaline/glutathione(GSH) pathogen reduction, currently undergoing phase III US clinical trials following successful European studies(1-3). Phosphatidylinositol glycan, class A (Pig-a) X-linked gene mutagenesis is a validated mammalian in vivo mutation assay for genotoxicity, assessed as clonal loss of glycosylphosphatidylinositol-linked CD59 cell-surface molecules on reticulocytes (RETs) and RBCs. METHODS: Male Sprague-Dawley rats received continuous infusion of S-300 up to the maximum feasible dose (240 mg/kg/day-limited by solubility and volume) for 28 days. Positive controls received a known mutagen by oral gavage on Days 1-3. Plasma levels of S-300 were assessed by HPLC before, during and after infusion. CD59-negative RBCs and RETs were enumerated in pre-dose and Day 28 samples, using a flow cytometric method. Outcome was evaluated by predetermined criteria using concurrent and historical controls. Toxicity was assessed by laboratory measures and necropsy. RESULTS: S-300 reached maximum, dose-dependent levels (3-15 µmol/L) within 2-8 h that were sustained for 672 h and undetectable 2 h after infusion. Circulating RET levels indicated a lack of hematopoietic toxicity. Necropsy revealed minimal-mild observations related to poor S-300 solubility at high concentrations. Pig-a assessment met the preset acceptability criteria and revealed no increase in mutant RBCs or RETs. CONCLUSIONS: Maximum feasible S-300 exposure of rats by continuous infusion for 28 days was not genotoxic as assessed by an Organization for Economic Cooperation and Development-compliant, mammalian, in vivo Pig-a gene mutation assay that meets the requirements of International Conference on Harmonization (ICH) S2(R1) and FDA guidances on genotoxicity testing.
Assuntos
Testes de Mutagenicidade , Ratos Sprague-Dawley , Animais , Masculino , Ratos , Testes de Mutagenicidade/métodos , Antígenos CD59/genética , Reticulócitos/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Proteínas de Membrana/genética , Mutagênese/efeitos dos fármacos , Mutagênicos/toxicidadeRESUMO
The parasite Plasmodium vivax preferentially invades human reticulocytes. Its merozoite surface protein 1 paralog (PvMSP1P), particularly the 19-kDa C-terminal region (PvMSP1P-19), has been shown to bind to reticulocytes, and this binding can be inhibited by antisera obtained by PvMSP1P-19 immunization. The molecular mechanism of interactions between PvMSP1P-19 and reticulocytes during P. vivax invasion, however, remains unclear. In this study, we analyzed the ability of MSP1P-19 to bind to different concentrations of reticulocytes and confirmed its reticulocyte preference. LC-MS analysis was used to identify two potential reticulocyte receptors, band3 and CD71, that interact with MSP1P-19. Both PvMSP1P-19 and its sister taxon Plasmodium cynomolgi MSP1P-19 were found to bind to the extracellular loop (loop 5) of band3, where the interaction of MSP1P-19 with band3 was chymotrypsin sensitive. Antibodies against band3-P5, CD71, and MSP1P-19 reduced the binding activity of PvMSP1P-19 and Plasmodium cynomolgi MSP1P-19 to reticulocytes, while MSP1P-19 proteins inhibited Plasmodium falciparum invasion in vitro in a concentration-dependent manner. To sum up, identification and characterization of the reticulocyte receptor is important for understanding the binding of reticulocytes by MSP1P-19.
Assuntos
Antígenos CD , Plasmodium vivax , Proteínas de Protozoários , Receptores da Transferrina , Reticulócitos , Plasmodium vivax/metabolismo , Plasmodium vivax/genética , Reticulócitos/metabolismo , Reticulócitos/parasitologia , Humanos , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Receptores da Transferrina/metabolismo , Receptores da Transferrina/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/genética , Ligação Proteica , Proteína 1 de Superfície de Merozoito/metabolismo , Proteína 1 de Superfície de Merozoito/genética , Malária Vivax/parasitologia , Malária Vivax/metabolismo , AnimaisRESUMO
Background: Cancer-related anemia (CRA) is a functional iron deficient anemia, and the early diagnosis will improve the prognosis of the patients. This prospective study aimed to investigate the utility of mean reticulocyte volume (MRV) in the early diagnosis of CRA. Methods: A total of 284 first-diagnosed cancer patients were enrolled, and the subjects were assigned anemia and non-anemia groups by hemoglobin (Hb) concentrations. The mature RBC and reticulocyte indices were detected with BC-7500 blood analyzer, and the MRV, reticulocyte hemoglobin (RHE) content, and reticulocyte production index (RPI) were obtained. ROC curves were constructed in identifying anemia diagnosed by the combination of RHE and RPI. An adjusted multivariate analyse and quartiles were used to assess the associations of MRV with early CRA diagnosed by combining RBC indices (MCV, MCH and MCHC), respectively. Results: No statistical differences were observed in MCV, RHE and MRV levels between anemia and non-anemia subjects (p > 0.05). MRV exhibited a complete or high correlation with the RHE levels (r = 1.000, p < 0.001), or MCV, MCH, and MCHC in anemia patients (R: 0.575-0.820, p < 0.001). ROC curves analyse indicated a highest area under curve of 0.829 (95% CI [0.762-0.895]) and 0.884 (95% CI [0.831-0.936]) for MRV in identifying anemia in male and female patients, respectively (p < 0.001). When the optimal cutoff values of MRV were set at 100.95 fl in males and 98.35 fl in females, the sensitivity and specificity were 1.00 and 0.68, and 1.00 and 0.73, respectively. The regression analyse showed that, when being as a categorical variable, MRV showed an odds ratio of 19.111 (95% CI [6.985-52.288]; p < 0.001) for the incidence of CRA. The incidence of overall anemia demonstrated a more significant decrease trend along with the increase of MRV quartiles (p-trend < 0.001). Conclusion: This study revealed that the MRV can be used as a convenient and sensitive index in early diagnosis of cancer-related anemia, and decreased MRV level may be the powerful predictor of overt anemia in cancer patients.
Assuntos
Anemia , Neoplasias , Humanos , Feminino , Masculino , Reticulócitos , Detecção Precoce de Câncer , Estudos Prospectivos , Anemia/diagnóstico , Hemoglobinas , Neoplasias/complicaçõesRESUMO
The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.
Assuntos
Eritrócitos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Reticulócitos , Transporte Biológico , Cálcio/metabolismo , Eritrócitos/metabolismo , Reticulócitos/metabolismo , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canais Iônicos/metabolismoRESUMO
2-Methylfuran (2-MF) is an important member of the furan family generated during food thermal processing. An in-vivo multiple endpoint genotoxicity assessment system was applied to explore the genotoxic mode of action and threshold of 2-MF. Male Sprague-Dawley rats received 2-MF by oral gavage at doses of 0.16, 0.625, 2.5, and 10â¯mg/kg.bw/day for 120 days. An additional 15 days were granted for recovery. The Pig-a gene mutation frequency of RET and RBC showed significant increases among the 2-MF groups on day 120. After a 15-day recovery period, the Pig-a gene mutation frequency returned to levels similar to those in the vehicle control. The tail intensity (TI) values of peripheral blood cells at a dose of 10â¯mg/kg.bw/day significantly increased from day 4 and remained at a high level after the recovery period. No statistical difference was found in the micronucleus frequency of peripheral blood between any 2-MF dose group and the corn oil group at any timepoint. 2-MF may not induce the production of micronuclei, but it could cause DNA breakage. It could not be ruled out that 2-MF may accumulate in vivo and cause gene mutations. Hence, DNA, other than the spindle, may be directly targeted. The mode of action of 2-MF may be that it was metabolized by EPHX1 to more DNA-active metabolites, thus leading to oxidative and direct DNA damage. The point of departure (PoD) of 2-MF-induced genotoxicity was derived as 0.506â¯mg/kg bw/day.
Assuntos
Dano ao DNA , Reticulócitos , Ratos , Animais , Masculino , Ratos Sprague-Dawley , Testes para Micronúcleos , Reticulócitos/metabolismo , Furanos/toxicidade , Furanos/metabolismo , DNA/metabolismo , Testes de MutagenicidadeRESUMO
The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and peroxiredoxin 2 (Prx2) are particularly important in erythroid cells. Reticulocytes and other erythroid precursors may adapt their biosynthetic mechanisms to cell defects or to changes in the bone marrow environment. Our aim was to perform a comparative study of the mRNA levels of CAT, GPX1, PRDX2 and SOD1 in reticulocytes from healthy individuals and from patients with hereditary spherocytosis (HS), sickle cell disease (SCD) and ß-thalassemia (ß-thal), and to study the association between their transcript levels and the reticulocyte maturity indices. In controls, the enzyme mRNA levels were significantly correlated with reticulocyte maturity indices for all genes except for SOD1. HS, SCD and ß-thal patients showed younger reticulocytes, with higher transcript levels of all enzymes, although with different patterns. ß-thal and HS showed similar reticulocyte maturity, with different enzyme mRNA levels; SCD and HS, with different reticulocyte maturity, presented similar enzyme mRNA levels. Our data suggest that the transcript profile for these antioxidant enzymes is not entirely related to reticulocyte maturity; it appears to also reflect adaptive mechanisms to abnormal erythropoiesis and/or to altered erythropoietic environments, leading to reticulocytes with distinct antioxidant potential according to each anemia.
Assuntos
Anemia Falciforme , Esferocitose Hereditária , Talassemia beta , Humanos , Reticulócitos , Talassemia beta/genética , Antioxidantes , RNA Mensageiro/genética , Superóxido Dismutase-1 , Esferocitose Hereditária/genética , Anemia Falciforme/genéticaRESUMO
BACKGROUND/AIM: Undernutrition is a serious health problem prevalent in poor countries, affecting millions of people worldwide, especially young children, pregnant women, and sick elderly individuals. This condition increases vulnerability to infections, leading to widespread use of antibiotic treatments in undernourished populations. The objective of the present study was to determine the in vivo genotoxic and cytotoxic effects of trimethoprim-sulfamethoxazole (TMP-SMX) treatment according to nutritional conditions. MATERIALS AND METHODS: The effects of TMP-SMX treatment were measured by analyzing the kinetics of micronucleated reticulocytes (MN-RET) induced in the peripheral blood of young, well-nourished (WN) and undernourished (UN) rats. RESULTS: In the WN group, two distinct peaks of MN-RET were observed, while the UN group had a significantly higher basal frequency of MN-RET compared to the WN group and only a later peak. Reticulocyte (RET) frequency slightly decreased in WN, indicating a poor cytotoxic effect. In contrast, in the UN, the treatment caused a significant increase in RET frequency. The results indicate that SMX's aromaticity index decreases when formed with TMP, suggesting potentially fewer toxic effects. CONCLUSION: In vivo TMP-SMX produces two MN-RET induction peaks in WN animals, indicating two DNA damage induction mechanisms and consequent micronucleus production. The UN rats did not display the two peaks, indicating that the first MN induction mechanism did not occur in UN, possibly due to pharmacokinetic effects, decreased metabolism or effects on cell proliferation. TMP-SMX has a slight cytotoxic effect on WN. In contrast, in the UN, the antibiotic treatment seems to favor early erythropoiesis.
Assuntos
Desnutrição , Combinação Trimetoprima e Sulfametoxazol , Humanos , Criança , Ratos , Animais , Feminino , Gravidez , Pré-Escolar , Idoso , Combinação Trimetoprima e Sulfametoxazol/toxicidade , Reticulócitos , Dano ao DNARESUMO
The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.
Assuntos
Malária Falciparum , Plasmodium vivax , Humanos , Receptores de Superfície Celular , Eritrócitos , Reticulócitos , Antígenos CD2 , Moléculas de Adesão CelularRESUMO
INTRODUCTION: Detection of iron deficiency (ID) remains challenging. We aimed to evaluate the performance of reticulocyte hemoglobin equivalent (Ret-He) as a potential diagnostic marker to assess ID and iron deficiency anemia (IDA) in a large pediatric cohort. METHODS: A total of 3158 patients (aged 15 days to 19 years with a median age of 8.5 years; 60.2% female) were retrospectively studied. Statistical analysis was performed (a) to evaluate relationship of Ret-He with other relevant complete blood count and iron panel parameters; (b) to compare the levels of Ret-He in ID and IDA groups to a control group; and (c) to assess sensitivity and specificity of Ret-He in ID, IDA, and anemia without ID groups. RESULTS: Ret-He values were significantly positively correlated to ferritin and transferrin saturation (TSAT). The median Ret-He was significantly lower in ID. A Ret-He cutoff of ≤30.0 pg distinguished cases of ID from the control group with a sensitivity of 90.2%, specificity of 59.5%, and area under curve (AUC) of 0.88. Ret-He showed better diagnostic performance in the IDA group and acceptable performance for ID without anemia. The sensitivity, specificity, and AUC were 90.1%, 80.9%, and 0.93 for IDA at cutoff value of ≤27.4 pg, and 80.8%, 51.1%, and 0.70 for ID without anemia at cutoff value of ≤30.8 pg, respectively. CONCLUSION: Our large pediatric tertiary care hospital study demonstrates that Ret-He is a reliable marker to help confirm IDA in pediatric population. However, further studies are needed for its use to capture the early stages of ID.
Assuntos
Anemia Ferropriva , Anemia , Deficiências de Ferro , Humanos , Criança , Feminino , Masculino , Reticulócitos , Estudos Retrospectivos , Centros de Atenção Terciária , Curva ROC , Anemia Ferropriva/diagnóstico , Hemoglobinas/análiseRESUMO
INTRODUCTION: The International Council for Standardization in Haematology convened a working group to assess and propose improvements upon the state of standardization and harmonization of reticulocyte parameters among commercial hematology analyzers. METHODS: An international group of laboratory hematologists prospectively collected and analyzed clinical samples using locally available IVD commercial hematology analyzers. Eight hundred and fifty-five total samples were collected at 6 sites using 9 distinct analyzer types. Samples were assessed for reticulocyte percent (RET%), immature reticulocyte fraction (IRF), and reticulocyte hemoglobin content (RHC). Method comparison and regression statistics were calculated. These analyses were used to determine whether statistical recalibration offered a potential avenue for increasing comparability between these methods. RESULTS: While methods producing reticulocyte percent were the most comparable in this study, the state of harmonization for the IRF and RHC was reduced with pearson correlation coefficients ranging from 0.955 to 0.77 and 0.927 and 0.680, respectively. Nevertheless, use of parameters from the Passing Bablok regression substantially improved the comparability of the results. In addition, precision data was derived which also demonstrated substantial differences between analyzer systems. CONCLUSION: While reticulocyte counting is correlated between the automated methods evaluated in this study, the current state of harmonization of other reticulocyte parameters is not as strong. A major challenge in moving this field forward is the need for commutable materials to facilitate comparisons between analyzers not co-located. A potential alternate approach to improve the current state would be instrument re-calibration. However, this is challenging both technically and due to national regulatory frameworks.
Assuntos
Hematologia , Reticulócitos , Humanos , Contagem de Reticulócitos/métodos , Padrões de Referência , HemoglobinasRESUMO
Two kinds of reticulocytes, and atypical erythroid cells, were found in the blood of apparently healthy ducks with Wright-Giemsa (W-G) stain. The reticulin of network-type examples (nRtc) is of a large complex (3-D) form. The punctate reticulocyte (pRtc) contains small pin-point cytoplasmic granulations; both cells are distinct from a polychromatic RBC (pRBC). Atypical erythroid cells with oval or irregular shapes reminiscent of primary or yolk-sac RBCs (ysRBC) accompanied the Rtc. Rare binuclear cells (bi) were either polychromatic (pRBC and pRtc) or full hemoglobin (Hb) types (RBC). Some bi with equal-sized daughter nuclei were presumed mitotic products. Conversely, unequal daughter nuclei were amitotic products, a nuclear division without chromosomes or a spindle. Erythrocytes formed tight aggregations with thrombocytes or other cells called "toroids"; further indicating a reactive hemogram. Erythroplastids (ep) anuclear erythroid cells, found along with other atypia, were either pRBC or full Hb types. The total white blood counts (TWBC) of the study set ranged from 5 K/µL (embryo E [d24]) to >100 K/µL (older ducks) with heterophil ratios (H/L) ranging from 0.5 to >4.0. Atypical erythroid cells, like atypical leukocytes, indicate a transition from homeostasis to a reactive state. Recognition of nRtc, pRtc, and toroids as reactive forms, expands the utility of hematologic data in assessing stress levels, indicating pathology, and exploring welfare questions. The present observations support earlier work showing some reticulocytes are detected by W-G alone and do not require vital dyes. Cells and behaviors described here demonstrate the benefit that a description of overall cytology adds to H/L ratios in evaluating a hemogram.
Assuntos
Patos , Reticulócitos , Animais , Galinhas , Eritrócitos , LeucócitosRESUMO
BACKGROUND: The Plasmodium vivax merozoite restrictively invades immature erythrocytes, suggesting that its ligand(s) might interact with corresponding receptor(s) that are selectively abundant on reticulocytes to complete the invasion. Finding the ligandâreceptor interaction involved in P. vivax invasion is critical to vivax malaria management; nevertheless, it remains to be unraveled. METHODS: A library of reticulocyte receptors and P. vivax ligands were expressed by a HEK293E mammalian cell expression system and were then used to screen the interaction using enzyme-linked immunosorbent assay (ELISA). A flow cytometry-based erythrocyte binding assay and bio-layer interferometry experiment were further utilized to cellularly and quantitatively identify the ligandâreceptor interaction, respectively. RESULTS: Plasmodium vivax merozoite-specific thrombospondin-related anonymous protein (PvMTRAP) was found to interact with human CD36 using systematic screening. This interaction was specific at a molecular level from in vitro analysis and comparable to that of P. vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) (KD: 37.0 ± 1.4 nM and 7.7 ± 0.5 nM, respectively). Flow cytometry indicated that PvMTRAP preferentially binds to reticulocytes, on which CD36 is selectively present. CONCLUSIONS: Human CD36 is selectively abundant on reticulocytes and is able to interact specifically with PvMTRAP, suggesting that it may function as a ligand and receptor during the invasion of reticulocytes by P. vivax.
Assuntos
Malária Vivax , Plasmodium vivax , Animais , Humanos , Reticulócitos , Ligantes , Merozoítos , Trombospondinas , MamíferosRESUMO
Safrole oxide (SAFO), a metabolite of naturally occurring hepatocarcinogen safrole, is implicated in causing DNA adduct formation. Our previous study first detected the most abundant SAFO-induced DNA adduct, N7-(3-benzo[1,3] dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SAFO-G), in mouse urine using a well-developed isotope-dilution high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (ID-HPLC-ESI-MS/MS) method. This study further elucidated the genotoxic mode of action of SAFO in mice treated with SAFO 30, 60, 90, or 120 mg/kg for 28 days. The ID-HPLC-ESI-MS/MS method detected N7γ-SAFO-G with excellent sensitivity and specificity in mouse liver and urine of SAFO-treated mice. Our data provide the first direct evidence of SAFO-DNA adduct formation in rodent tissues. N7γ-SAFO-G levels in liver were significantly increased by SAFO 120 mg/kg compared with SAFO 30 mg/kg, suggesting rapid spontaneous or enzymatic depurination of N7γ-SAFO-G in tissue DNA. Urinary N7γ-SAFO-G exhibited a sublinear dose response. Moreover, the micronucleated peripheral reticulocyte frequencies increased dose-dependently and significantly correlated with N7γ-SAFO-G levels in liver (r = 0.8647; p < 0.0001) and urine (r = 0.846; p < 0.0001). Our study suggests that safrole-mediated genotoxicity may be caused partly by its metabolic activation to SAFO and that urinary N7γ-SAFO-G may serve as a chemically-specific cancer risk biomarker for safrole exposure.
Assuntos
Adutos de DNA , Safrol , Camundongos , Animais , Safrol/toxicidade , Espectrometria de Massas em Tandem , Espectrometria de Massas por Ionização por Electrospray/métodos , Guanina , Reticulócitos/química , Reticulócitos/metabolismo , Fígado/metabolismo , Cromatografia Líquida de Alta PressãoRESUMO
OBJECTIVES: To compare serum ferritin and RET-He values among extremely low gestational age neonates ELGANs with other markers of iron-deficient erythropoiesis. STUDY DESIGN: This is a secondary analysis of the NICHD Darbepoetin Trial. Study data from placebo recipients who had a serum ferritin, a RET-He, and a mean corpuscular volume (MCV) measurement within a 24-hour period were analyzed for correlation. RESULTS: Mixed linear regression models showed no association between ferritin and RET-He at both early (ß = 0.0016, p = 0.40) and late (ß = -0.0001, p = 0.96) time points. Positive associations were observed between RET-He and MCV at baseline, early, and late time points (p < 0.01, =0.01, <0.001, respectively), while ferritin was not associated with MCV at any time point. CONCLUSIONS: Our study shows that RET-He is better correlated with MCV as a marker of iron-limited erythropoiesis than ferritin. The results suggest that ferritin is limited as a marker of iron sufficiency in premature infants. STUDY IDENTIFICATION: FDA IND Number 100138; ClinicalTrials.gov number NCT03169881; NRN ID number NICHD-NRN-0058 (Darbe).