Blood film preparation and staining procedures

In this work, a liquid crystal mesophase as a potential bone-repairing booster has been induced from a polyacrylic acid (PAAc) gel loaded with synthetic hydroxyapatite nanoparticles (nHA) under physiological conditions of pH and temperature that resemble the early stages of bone formation. Dynamic processes at the microscale in biological systems require a material capable of behaving following a specific order and fluency. Besides, they must fulfill conditions such as biocompatibility and good mechanical properties. Our study aimed to obtain new data about the safety, hemostasis, biocompatibility (activated partial thromboplastin time (APTT) test, prothrombin time (PT) test, lactate dehydrogenase (LDH) test, red blood cell morphology observation, hemolysis percentage) and physicochemical/mechanical behavior (Attenuated Total Reflection (ATR), Infrared spectroscopy and rheological assays) of nanoparticles, and materials built with them. Both systems have shown biocompatibility with red blood cells and adequate flow properties, demonstrating promising qualities for applicability. The formation of lyotropic liquid crystals at physiological conditions was evaluated with crossed polaroids microscope with a temperature control plate. The formation of these structures under bone remodeling conditions shows the potential application of PAAc-nHA materials. In literature, the study of materials applied as bone fillers is mainly focused on mechanical and osteoblasts compatibility assays. However, the hematological effect, consequences on the coagulation cascade, the integrity of cell membranes, and hemolysis percentage have not been addressed thoroughly. The strength of our work is focused on considering these aspects when tridimensional arrangements essential for bone formation are formed.

Hydrogen cyanamide (Dormex) is a plant growth regulator that is classified as a highly toxic poison. There are no definite investigations to help in its diagnosis and follow-up. This study aimed to investigate the role of hypoxia-inducible factor-1α (HIF-1α) in the diagnosis, prediction, and follow-up of Dormex-intoxicated patients. Sixty subjects were equally divided into two groups: group A, the control group, and group B, the Dormex group. Clinical and laboratory evaluations, including arterial blood gases (ABG), prothrombin concentration (PC), the international normalized ratio (INR), a complete blood count (CBC), and HIF-1α, were done on admission. CBC and HIF-1α were repeated for group B 24 and 48 h after admission to track abnormalities. Group B also had brain computed tomography (CT). Patients with abnormal CT scans were referred for brain magnetic resonance imaging (MRI). Significant differences in levels of HB, WBCs, and platelets were also detected in group B up to 48 h after admission, as white blood cells (WBCs) rose with time and hemoglobin (HB) and platelets diminished. The results described a highly significant difference in HIF-1α between the groups, and it depended on the clinical condition; therefore, it can be used in the prediction and follow-up of patients up to 24 h after admission.

Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi and Babesia caballi in equids, including horses, donkeys, zebras, and mules. It is globally endemic with significant economic impact on the equine industry. Infected animals may serve as carriers, and they may be a source of infection for ticks, thereby posing a great challenge for disease management. Sri Lanka is a tropical country, where infections by various tick-borne parasites are common among livestock animals. However, infections by T. equi and B. caballi remain unstudied in Sri Lanka. Therefore, in the present study, we conducted an epidemiological survey to investigate the presence of T. equi and B. caballi in apparently healthy free-roaming donkeys. Blood samples were randomly taken from 111 donkeys in Mannar (n = 100) and Kilinochchi (n = 11) districts in Sri Lanka. Thin blood smears were prepared from the blood samples and subjected to microscopic examination. Additionally, blood DNA samples were prepared and screened for T. equi and B. caballi infections using species-specific PCR assays. Our results showed that 64 (57.7%) and 95 (85.6%) of the donkeys were positive for T. equi by microscopy and PCR, respectively. However, all samples were negative for B. caballi. Phylogenetic analysis of the T. equi 18S rRNA sequences detected two distinct genotypes, namely C and D. To our knowledge, this is the first report of T. equi in Sri Lanka and of genotype C in donkeys. The present study highlights the importance of monitoring the shrinking donkey population in Sri Lanka owing to EP caused by T. equi.

Application of modern and innovative spectroscopic and microscopic approaches to biomedical analysis opens new horizons and sheds new light on many unexplored scientific territories. In this review, we critically summarize up-to-date Raman-based methodologies for red blood cells (RBCs) analysis used in biology and medicine, and compare them with both classical, as well as other spectroscopic and microscopic approaches. The main emphasis is placed on the advantages, disadvantages and capabilities of each technique for detection of RBC deteriorations and RBC-related diseases. Although currently used classical techniques of medical analysts serve as a gold standard for clinicians in diagnosis of erythropathies, they provide insufficient insight into RBC alterations at the molecular level. In addition, there is a demand for non-destructive and label-free analytical techniques for rapid detection and diagnosis of erythropathies. Their recognition often requires multimodal methodology comprising application of methods including sophisticated spectroscopy-based techniques, where Raman-based approaches play an important role.

The external quality assessment (EQA) scheme is particularly important for laboratory performance evaluation. Peripheral blood smears are necessary to identify morphological features, and the procedure for preparing such smears must be robust to meet the ISO 15189 standard. Although blood smear preparation is a routine activity in medical laboratories, an appropriate procedure for preparing a series of blood smears with high homogeneity and durable stability for EQA purposes has not yet been published elsewhere. For this reason, a robust procedure was developed and validated in this study.

Various factors affecting blood smear preparation, such as the amount of time collected blood samples are stored before fixation, suitable reagents, and specification parameters for each step, including fixation, staining, and timing of the staining steps, were studied. Each experiment was evaluated based on homogeneity and stability characteristics.

Whole blood mixed with EDTA anticoagulant was used to make the blood smears. Samples were fixed with pure anhydrous alcohol and stained in Coplin jars using the Wright-Giemsa method.

The homogeneity and stability of two lots of smears suitable for EQA purposes was confirmed based on intact morphology of the smears for more than 8 months at room temperature.