Micro-Raman spectroscopy study of optically trapped erythrocytes in malaria, dengue and leptospirosis infections.

Malaria, dengue and leptospirosis are three tropical infectious diseases that present with severe hematological derangement causing significant morbidity and mortality, especially during the seasonal monsoons. During the course of these infectious diseases, circulating red blood cells are imperiled to the direct ill-effects of the infectious pathogen in the body as well as to the pro-inflammatory cytokines generated as a consequence of the infection. RBCs when exposed to such inflammatory and/or pathogenic milieu are susceptible to injuries such as RBC programmed eryptosis or RBC programmed necrosis. This research aimed to explore the Raman spectra of live red cells that were extracted from patients infected with malaria, dengue, and leptospirosis. Red cells were optically trapped and micro-Raman probed using a 785 nm Diode laser. RBCs from samples of all three diseases displayed Raman signatures that were significantly altered from the normal/healthy. Distinct spectral markers that were common across all the four groups were obtained from various standardized multivariate analytical methods. Following comprehensive examination of multiple studies, we propose these spectral wavenumbers as "Raman markers of RBC injury." Findings in our study display that anemia-triggering infections can inflict variations in the healthy status of red cells, easily identifiable by selectively analyzing specific Raman markers. Additionally, this study also highlights relevant statistical tools that can be utilized to study Raman spectral data from biological samples which could help identify the very significant Raman peaks from the spectral band. This approach of RBC analysis can foster a better understanding of red cell behavior and their alterations exhibited in health and disease.

Micro-Raman Spectroscopy Analysis of Optically Trapped Erythrocytes in Jaundice.

Derangements in bilirubin metabolism and/or dysfunctions in the hepato-biliary system lead to the unhealthy buildup of bilirubin in blood, resulting in jaundice. During the course of this disorder, circulating red cells are invariably subjected to toxic effects of serum bilirubin and an array of inflammatory compounds. This study aimed to investigate the vibrational spectroscopy of live red cells in jaundice using micro-Raman spectroscopy combined with optical-trap. Red cells from blood samples of healthy volunteers and patients with jaundice were optically immobilized and micro-Raman probed using a 785 nm diode laser. Raman signatures from red cells in jaundice exhibited significant variations from the normal and the spectral-markers were obtained from multivariate analytical methods. This research gives insightful views on how different pathologies can act as "stress-milieus" for red cells in circulation, possibly impeding their normal functions and also exasperating anemia. Raman spectroscopy, an emerging bio-analytical technique, is sensitive in detecting molecular-conformations in situ, at cellular-levels and in real-time. This study could pave way in understanding fundamental red cell behavior in different diseases by analyzing Raman markers.

Computerized Morphometric Analysis of Eryptosis.

Eryptosis is the suicidal destruction-process of erythrocytes, much like apoptosis of nucleated cells, in the course of which the stressed red cell undergoes cell-shrinkage, vesiculation and externalization of membrane phosphatidylserine. Currently, there exist numerous methods to detect eryptosis, both morphometrically and biochemically. This study aimed to design a simple but sensitive, automated computerized approach to instantaneously detect eryptotic red cells and quantify their hallmark morphological characteristics. Red cells from 17 healthy volunteers were exposed to normal Ringer and hyperosmotic stress with sodium chloride, following which morphometric comparisons were conducted from their photomicrographs. The proposed method was found to significantly detect and differentiate normal and eryptotic red cells, based on variations in their structural markers. The receiver operating characteristic curve analysis for each of the markers showed a significant discriminatory accuracy with high sensitivity, specificity and area under the curve values. The software-based technique was then validated with RBCs in malaria. This model, quantifies eryptosis morphometrically in real-time, with minimal manual intervention, providing a new window to explore eryptosis triggered by different stressors and diseases and can find wide application in laboratories of hematology, blood banks and medical research.

Serial blood volume estimation in rabbits using trivalent chromium - An exploratory study.

Though often used in cardiac intensive monitoring set up, simultaneous evaluation of several variables like CVP, PCWP, SVV and hTEE for fluid volume resuscitation (especially when capillary permeability is major problem than cardiac performance) is a major challenge in many ICU setups. Therefore, repetitive determination of blood volume by trivalent chromium [51Cr (III)] as a direct single variable method may be a near ideal method during fluid volume resuscitation in cases where capillary permeability is a major problem (e.g. Burns). Hence, in the present article the repeatability and reliability of 51Cr (III) method in New Zealand white rabbits was explored. Mean blood volume values of initial measurement were 195.66 ± 47.30 ml or 89.81 ± 17.88 ml/kg body weight. Repeated mean blood volume values, measured after 1 h, was 181.98 ± 53.16 ml or 83.68 ± 22.09 ml/kg body weight. The average difference between the initial and repeated measurements was 10.93 ml (95% CI -3.33, 25.19 ml), which is not statistically significant (P = 0.128). The method using 51Cr (III) for repeat blood volume measurements after sixty minutes in rabbits is a reliable method. •Rapid•Repeatable•Reliable.

An innovative approach for serial injection in marginal vein and blood collection from auricular artery in New Zealand white Rabbit.

Serial injection into marginal vein and blood collection through auricular artery in New Zealand white Rabbit (Oryctolagus cuniculus) is an important procedure for various types of experimental studies. Limitations of the existing methods for serial injection and blood collection includes complex procedures, causes considerable discomfort to rabbits, whole blood samples obtained are highly prone to hemolysis and lastly detailed protocol is not available in the literature. Approximately 10 min before commencement of the experiment, a local anesthetic cream was applied over the right and left ear lobes. The skin at the site of sample collection and injection was prepared by shaving the area on both ears and wiping it with alcohol swab. Once prepared, a 26 GA (BD Neoflon) intravenous cannula was inserted into the marginal vein of the ear and secured with an adhesive plaster. In the other ear, a 24 GA (BD Neoflon) intravenous cannula was placed in the auricular/central artery and secured with an adhesive plaster. The novel and refined method described here has been standardized and found to be reliable. The samples obtained using this method is not susceptible to hemolysis and hence we recommend this method for serial injection and blood collection in rabbits. •Easy to perform•Not prone to hemolysis•Detailed methodology described.