Anticoagulated Blood Samples Research Articles

Storage Induced Alterations in Erythrocyte Morphology and Platelet Count

Background: Examination of blood smears and various hematologic parameters is the first step in assessment of hematologic function and diagnosis of possible underlying diseases. The aim of this study was to identify artefacts in stored red blood cells and classify the occurrence of such alterations according to the age of the stored sample. This studyalso aimed to note the changes in platelet count dispatched by automated hematology coulter in such stored blood samples. Material and Methods: A prospective cross sectional study was conducted from the EDTA anticoagulated blood samples of 100 patients received in the department of Pathology after receiving ethical approval from the institutional review committee. All the samples were analyzed in 4 occasions i.e. within 2 hours, at 24 hours, at 48 hours and at 96 hours. Analysis was carried out by Sysmex 5 part analyzer for platelet counts and the Leishman stained blood films were examined to note changes in the erythroctyte morphology. Results: There was alterationsin platelet counts in 22% cases between 2-hour samples and other samples. The red blood cell morphology was not different among 2 hours samples and 24 hours sample. However, the red blood cell morphology was altered in 48 hours samples and 96 hours samples. Conclusion: Storage of EDTA anticoagulated blood even upon refrigeration can cause changes in platelet counts as well as morphology of red blood cells. Changes in red blood cells are not significant till 24 hours while the platelet counts may alter within 24 hours.

Validation of a single specimen pneumatic tube system in the clinical laboratory.

The single-specimen pneumatic tube (PTS) is a commonly used rapid specimen delivery system in modern clinical laboratories. However, its impact on sample integrity and laboratory test results remains controversial. The installation and configuration of single-specimen PTS are unique to their institution. We sought to validate our single-specimen PTS by comparing routine chemistry, immunology, and hematology results with a repeat sample integrity index for manual transport. In 2023, 30 employees were randomly selected from the company medical examination, and three tubes of procoagulant serum samples and three tubes of EDTA anticoagulant blood samples were collected from each of them. Group A uses a single specimen PTSat 8m/s, Group B uses a single specimen PTSat 15m/s, and Group C uses manual transfer. Specimens from all three groupswere simultaneously analysedfor ALT, AST, TG, TC, LDL, K, NA, CI, TSH, hs-cTnT, NSE, Cyfra21-1 and haematological analysis. The differences between the three groups of NSE and Cyfra21-1 were statistically significant (P <0.05). The differences of the rest of the items were not statistically significant. The difference in NSE was not statistically significant between groups A and B (P =0.401), B and C, and C and A (P <0.05). The difference in Cyfra21-1 was not statistically significant between groups A and B (P =0.897), B and C (P =0.052), and C and A (P =0.145). Individual sample PTS should be validated for testing prior to use to ensure the results' accuracy.

Blood stored in EDTA tubes provides accurate peanut basophil activation test results for 48 hours

BackgroundThe peanut basophil activation test (BAT) has demonstrated excellent diagnostic accuracy with heparinized blood, but its clinical utility is limited by the short stability of samples stored in this anticoagulant. ObjectiveUsing EDTA anticoagulated blood, these investigations determined if Peanut BAT sample stability can be extended to 2 days, the minimum stability requirement for diagnostic tests currently offered through American reference laboratories. MethodsPeanut non-allergic control (NAC), peanut IgE sensitized (PS), and peanut allergic (PA) children aged 6 months through 17 years were recruited from members of Kaiser Permanente Southern California. EDTA anti-coagulated blood samples were collected from participants, shipped to a centralized laboratory, and stored at 4oC for peanut BAT testing 1 and 2 days later. ResultsPeanut BAT results for 23 unblinded participants were used to establish sample rejection and interpretation criteria that were subsequently validated in a prospective double-blind study involving 112 additional children (39-NAC, 36-PS, 37-PA). Of 105 blinded blood samples tested on each study day, 88 (84%) day-1 and 90 (86%) day-2 peanut BAT results were considered interpretable, with diagnostic accuracies of 95.5% and 94.4%, respectively. Moreover, all interpretable PA results were considered positive (100% sensitivity). ConclusionUsing EDTA anti-coagulated blood samples collected remotely, 1 and 2 days before testing, study results highlight the favorable diagnostic performance characteristics of the peanut BAT and provide further evidence that the test could be readily operationalized for clinical use by interested commercial reference laboratories.

Adapting the SMART tube technology for flow cytometry in feline full blood samples.

Flow cytometry of blood samples is a very valuable clinical and research tool to monitor the immune response in human patients. Furthermore, it has been successfully applied in cats, such as for infections with feline immune deficiency virus (FIV). However, if cells are not isolated and frozen, analysis of anticoagulated blood samples requires mostly prompt processing following blood collection, making later analysis of stored full blood samples obtained in clinical studies often impossible. The SMART Tube system (SMART TUBE Inc., California, United States; SMT) allows fixation and long-term preservation of whole blood samples at -80°C. However, this system has so far only been applied to human biological samples. In the present study, a new flow cytometry SMART Tube protocol adapted for feline whole blood samples was successfully established allowing quantification of T-helper cells, cytotoxic T-cells, B-cells, monocytes, and neutrophils up to 2 years post sampling. Results obtained from frozen stabilized and fresh blood samples were compared for validation purposes and correlated to differential blood counts from a conventional hematology analyzer. Clinical applicability of the new technique was verified by using samples from a treatment study for feline infectious peritonitis (FIP). Using the new SMT protocol on retained samples, it could be demonstrated that long-term storage of these SMT tubes is also possible. In summary, the newly adapted SMT protocol proved suitable for performing flow cytometry analysis on stored feline whole blood samples, thus opening up new avenues for veterinary research on a variety of aspects of clinical interest.

Evaluation of early African swine fever virus detection using CP204L gene encoding the p30 protein using quantitative polymerase chain reaction.

The African swine fever virus (ASFV), spanning 170-193 kb, contains over 200 proteins, including p72 and p30, which play crucial roles in the virus's entry and expression. This study investigated the capability of detecting ASFV early through the analysis of genes B646L and CP204L, encoding p72 and p30 antigen proteins, by employing ASFV, diagnosis, immunohistochemistry (IHC), quantitative polymerase chain reaction (qPCR), and IHC techniques. Samples were taken from both experimentally and field-infected pigs to evaluate the effectiveness of qPCR and IHC in detecting ASFV. Twenty-two infected pigs were necropsied at 3-, 5-, 7-, and 9-day post-infection to obtain the first set of samples, collecting anticoagulated blood and tissues each time. The thymus, spleen, and lymph nodes were processed by fixing in 10% formalin, paraffin-blocking, and undergoing IHC staining. Forty anticoagulated blood samples were collected from clinically infected sows at a pig farm for the second batch of samples. Based on the lowest Ct values, three blood samples were diluted fivefold for qPCR DNA testing, and their tissues were used for both qPCR and IHC analyses. At 1-day post-infection, p30-qPCR identified more ASFV-positive pigs and measured lower Ct values compared to p72-qPCR. At later time points, both methods showed similar levels of detection. ASFV was detected earlier and with lower Ct values in lymphoid tissues using p30-qPCR compared to p72-qPCR, particularly in the spleen and lymph nodes. In a field outbreak study, p30-qPCR demonstrated superior sensitivity and lower Ct values when detecting ASFV in blood samples compared to p72-qPCR. The early detection of the CP204L gene encoding p30 and its corresponding antigenic protein in ASFV diagnosis compared to the gene encoding p72 suggests that CP204L and p30 are promising candidates for the development of more effective antigen and antibody testing methods.

Diagnosis and treatment of anemia in creole horses with two iron-rich diets

The aim of this study was to diagnose and treat anaemia in Creole horses from the province of Piura, Peru. The study was conducted from June to December 2022. Anticoagulant blood samples were taken to determine haemoglobin, haematocrit, and erythrocyte values to identify individuals considered anemic. A total of 8 horses (4 female and 4 male) from 3 to 8 years old were selected. The animals were negative for anaplasmosis and piroplasmosis and were dewormed orally with 1% ivermectin. The animals were distributed into two groups and were fed two diets (D1 and D2), rich in iron, for 60 days. D1 contained 3.0 Mcal/kg, 18% protein, 4% fat, 8.5% fibre, 8% ash and 345 mg/kg iron and D2 contained 3.3 Mcal/kg, 18% protein, 4% fat, 15% fibre, 12% ash and 533.9 mg/kg of iron. At 30 and 60 days, the presence of anaemia was evaluated by haematological examination and body weight. The horses on Diet 1 increased (p&lt;0.05) the haemoglobin concentration by 0.7 and 0.9 g/dL at 30 and 60 days, respectively, and gained 15.3 kg, while those on Diet 2 increased (p&lt;0.05) the haemoglobin concentration in 0.8 and 1.5 g/dL, erythrocytes in 0.33 and 0.90x106/µL, and haematocrit in 2.1 and 5.1% at 30 and 60 days, respectively, and they gained 27.8 kg. It is concluded that Diet 2 made it possible to repair the problem of anaemia in the Creole horses of Piura.

Optimizing Platelet-Rich Plasma: Spin Time and Sample Source.

The buff-colored layer separating the plasma from red blood cells (RBCs) in centrifuged blood was named the "buffy coat" in the late 19th century. The division of platelets (PLTs) and leukocytes (WBCs) between the buffy coat, plasma, and RBC layers in centrifuged blood has not been described before. In this study, we centrifuged 8.5 mL anticoagulated blood samples at 1000× g for 1, 2, 3, 5, 10, and 20 min. We then divided each sample into ten layers and analyzed each layer for cellular composition and mean platelet volume (MPV). Our results show that even after 20 min of centrifugation, about 15% of platelets remain in the plasma layers and 65% in the RBC layers. We found that the platelet count achieved from aspiration of 1 mL volume was optimal, with aspiration beginning 1/2 mL below the buffy coat and extending 1/2 mL above the buffy coat rather than beginning at the buffy coat itself and aspirating only plasma. Using this method of aspiration, we found that the total platelet count means reached a maximum in the 1 mL around the buffy coat after only 5 min of centrifugation.

Molecular epidemiological investigation of piroplasms carried by pet cats and dogs in an animal hospital in Guiyang, China.

Piroplasmosis is a zoonotic disease mainly caused by the Babesia and Theileria parasites. Piroplasmosis is often a subclinical infection in dogs and cats that is difficult to detect and is often suspected when clinical signs such as anemia are present. It has been reported to be prevalent in China. However, molecular evidence of the disease has not been reported in pet dogs and cats in Guiyang. In this study, we collected 307 anticoagulated blood samples from an animal hospital in the Wudang District of Guiyang during the period March 2021 to November 2021 and extracted DNA from the samples. The 18S rDNA gene was amplified using PCR, and the positive amplification product was sequenced. The sequences were then analyzed for homology and phylogeny. Of the 307 samples collected, 164 were feline and 143 were canine, with a total of 23 amplifying a target band of approximately 400 bp. The percentage of positives of piroplasms infection in pet cats was 4.27% (7/164), with the pathogens being T. uilenbergi (3) and T. luwenshuni (4). One Colpodella sp. and two undetermined species were also detected in the cat samples. The percentage of positives of piroplasms infection in pet dogs was 7.69% (11/143), with the pathogen being T. uilenbergi (11). One Colpodella sp. was also detected in the dog samples. The results confirmed that T. uilenbergi and T. luwenshuni are prevalent in pet cats and dogs in this area. In addition, the study found a rare zoonotic pathogen, Colpodella sp., in cats and dogs. Therefore, this study is expected to serve as a valuable reference for decision-making regarding animal health management and public health work.

Comparison between Hematology and Serum Biochemistry of Qinling and Sichuan Giant Panda (Ailuropoda melanoleuca qinlingensis and sichuanensis).

Giant pandas are the flagship species in world conservation, and include two subspecies, Ailuropoda melanoleuca qinlingensis (A. m. qinlingensis) and Ailuropoda melanoleuca sichuanensis (A. m. sichuanensis). Hematology and serum biochemistry studies are crucial to protecting giant pandas. Even though research on hematology and serum biochemistry are well-established in A. m. sichuanensis, research in A. m. qinlingensis is scarce. The study aimed to (1) establish a baseline for hemogram and reference intervals (RIs) for hematological and serum biochemical parameters in A. m. qinlingensis, (2) assess the possible variations in these parameters of A. m. qinlingensis based on age, gender, and storage condition of blood samples, and (3) compare the parameters to those of A. m. sichuanensis. Blood samples (n = 42) were collected from healthy A. m. qinlingensis (n = 21) housed in Shaanxi (Louguantai) Rare Wildlife Rescue and Breeding Research Center, and hematological (n = 25) and serum biochemical parameters (n = 18) were analyzed in March and December of 2019. The results showed no significant abnormality in the blood smears of all individuals in this study, except for a few serrated red blood cells, platelet aggregations, and occasionally giant platelets. Between sub-adult and adult A. m. qinlingensis, there were significant differences in five hematological and one serum biochemical parameter (p < 0.05), whereas six serum biochemical parameters were present when α = 0.1 (p < 0.1). Gender influenced % NEU, % LYM, % EOS, LYM, EOS, GGT, and CHOL of A. m. qinlingensis. The majority of the hematological and serum biochemical parameters of A. m. qinlingensis were different from those of A. m. sichuanensis regarding age and gender. The anticoagulant whole blood samples of A. m. qinlingensis stored at 2-8 °C for 24 h and the serum samples stored at -18 °C for 48 h had little influence on the values of hematological and serum biochemical parameters. In conclusion, this study provided a baseline of hemogram and established RIs for hematological and serum biochemical parameters of A. m. qinlingensis. RIs of A. m. sichuanensis reported before were not completely fit for A. m. qinlingensis, and age, gender, or the storage condition of blood samples influenced some of the parameters of A. m. qinlingensis. To the authors' knowledge, this is the first report of a hemogram baseline and RIs for hematological and serum biochemical parameters of A. m. qinlingensis.

Clinical application of a fully automated blood collection robot and its assessment of blood collection quality of anticoagulant specimens.

To investigate the application of intelligent puncture blood collection robots in anticoagulated blood specimens, the satisfaction of subjects with the two blood collection methods, and the feasibility of intelligent blood collection devices to replace manual blood collection methods in clinical work. A total of 154 volunteers from Zhongshan Hospital Fudan University were recruited to compare the test results of anticoagulant blood samples between blood collection robot and manual blood collection, a questionnaire was used to inquire about the volunteers' feelings about the two blood collection methods; the blood collection data of 6,255 patients willing to use the robot for blood collection were collected to analyze the success rate of blood collection. The blood collection robot is superior to manual specimen collection in terms of volume and pain of specimen collection, and the puncture success rate is 94.3%. The anticoagulated blood specimens collected by the robot had 11 indexes statistically different from the results of manual blood collection, but the differences did not affect the clinical diagnosis and prognosis. The intelligent robotic blood collection is less painful and has better acceptance by patients, which can be used for clinical anticoagulated blood specimen collection.

First identification and characterization of ovine gammaherpesvirus type 2 in horses and artiodactyla from an outbreak of malignant catarrhal fever in Mexico.

Ovine gammaherpesvirus 2 (OvHV-2), a member of the genus Macavirus, causes sheep-associated malignant catarrhal fever (SA-MCF), a fatal lymphoproliferative disease affecting a wide variety of ungulates in addition to horses. This study described an outbreak of SA-MCF in Mexico and the identification of the OvHV-2 virus in primary rabbit testis cultures through the generation of intranuclear inclusion bodies, syncytia, immunofluorescence (IF), immunocytochemistry (ICC), immunohistochemistry (IHC), endpoint polymerase chain reaction (PCR), and partial sequencing of the ORF75 gene. The animals involved in this outbreak showed mucogingival ulcers in the vestibule of the mouth and tongue, hypersalivation, corneal opacity, reduced food consumption, and weight loss of variable severity. These clinical signs and the histopathological findings suggested the diagnosis of SA-MCF. Buffy coat fractions from the anticoagulated blood samples of ill animals were collected and analyzed by PCR. Positive buffy coats were used to inoculate the primary cell cultures of rabbit testis to identify the virus. Small clusters of refractile cytomegalic cells, characteristic of viral cytopathic effects, were observed between 48 and 72 h post-infection. Furthermore, intranuclear acidophilic inclusion bodies (IBs) were identified in the inoculated primary culture cells, and the cytoplasm showed immunoreactivity with hyperimmune rabbit serum against OvHV-2. Moreover, in the liver histological sections from sick deer, immunoreactive juxtanuclear IBs were identified with the same rabbit hyperimmune serum. The obtained sequences were aligned with the OvHV-2 sequences reported in GenBank and revealed a nucleotide identity higher than 98%. Based on the evidence provided in this study, we conclude that the outbreak of SA-MCF in the municipality of Tequisquiapan in the state of Queretaro, Mexico, was caused by OvHV-2. This is the second study reporting that horses are susceptible to OvHV-2 infection and can develop SA-MCF. We identified for the first time in Mexico, the presence of OvHV-2 in buffy coats from horses and Artiodactyla.

A Comparative Evaluation of Performance of Sysmex XN 3000 and Horiba Yumizen H2500 Automated Complete Blood Count Analysers

Modern automated laboratory haematology analysers use various methods to measure different haematological parameters. These parameters are useful in the diagnostic and clinical interpretation of patient symptoms. So, it is very important to compare the performance of different analysers measuring the same parameter. Hence, a comparison of complete blood counts analysed by Sysmex XN 3000 and Horiba Yumizen H2500 was performed. Total 296 EDTA anti-coagulated blood samples were processed in both the analysers in duplicate within 4 h of collection. The white blood cell count, red blood cell count, erythrocyte indices, differential leukocyte count, platelet count and platelet indices and reticulocyte count were compared. A good level of correlation and agreement between different parameters were obtained. A strong correlation was observed (r > 0.9) between Sysmex XN 3000 and Yumizen H2500 for WBC (0.997), RBC (0.997), Haemoglobin (0.999), haematocrit (0.974), MCV (0.902), MCH (0.99),, platelet count by impedance (0.989), mean platelet volume (0.954), plateletcrit (0.971), platelet distribution width (PDW) (0.916), neutrophils (0.997), lymphocytes (0.989), monocytes (0.943), and eosinophils (0.991) counts. A moderate correlation was observed for RDW-CV (0.75). The basophils count showed poor correlation (r < 0.5) possibly because of sample selection with mostly low basophils count. An acceptable bias was observed for most of the parameters like WBC, RBC, Haemoglobin, Haematocrit, platelet counts, neutrophils, lymphocytes, eosinophils and monocytes. The studied instruments ensured satisfactory interchangeability except for few parameters, thus facilitate substitution of one analyser by another without affecting the clinical decision making.

Analysis of Novel Immunological Biomarkers Related to Rheumatoid Arthritis Disease Severity.

Rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs) are the most frequently used rheumatoid arthritis (RA) diagnostic markers, but they are unable to anticipate the patient's evolution or response to treatment. The aim of this study was to identify possible severity biomarkers to predict an upcoming flare-up or remission period. To address this objective, sera and anticoagulated blood samples were collected from healthy controls (HCs; n = 39) and from early RA (n = 10), flare-up (n = 5), and remission (n = 16) patients. We analyzed leukocyte phenotype markers, regulatory T cells, cell proliferation, and cytokine profiles. Flare-up patients showed increased percentages of cluster of differentiation (CD)3+CD4- lymphocytes (p < 0.01) and granulocytes (p < 0.05) but a decreased natural killer (NK)/T lymphocyte ratio (p < 0.05). Analysis of leukocyte markers by principal component analysis (PCA) and receiver operating characteristic (ROC) curves showed that CD45RO+ (p < 0.0001) and CD45RA+ (p < 0.0001) B lymphocyte expression can discriminate between HCs and early RA patients, while CD3+CD4- lymphocyte percentage (p < 0.0424) and CD45RA+ (p < 0.0424), CD62L+ (p < 0.0284), and CD11a+ (p < 0.0185) B lymphocyte expression can differentiate between flare-up and RA remission subjects. Thus, the combined study of these leukocyte surface markers could have potential as disease severity biomarkers for RA, whose fluctuations could be related to the development of the characteristic pro-inflammatory environment.

High-Throughput CD36 Phenotyping on Human Platelets Based on Sandwich ELISA and Mutant Gene Analysis

Background: CD36 deficiency is closely associated with fetal/neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness, and other hemorrhage disorders, particularly in Asian and African populations. There is a clinical need for rapid and high-throughput methods of platelet CD36 (pCD36) phenotyping to improve the availability of CD36 typing of donors and assist clinical blood transfusions for patients with anti-CD36 antibodies. Such methods can also support the establishment of databases of pCD36-negative phenotypes. Study Design and Methods: A sandwich enzyme-linked immunosorbent assay (ELISA) for CD36 phenotyping of human platelets was developed using anti-CD36 monoclonal antibodies. The reliability of the assay was evaluated by calculating the intra-assay and inter-assay coefficients of variation (CV). A total of 1,691 anticoagulant whole blood samples from healthy blood donors were randomly selected. PCD36 expression was measured using a sandwich ELISA. PCD36 deficiency was confirmed by flow cytometry (FC). Mutations underlying pCD36 deficiency were identified using polymerase chain reaction sequence-based typing (PCR-SBT). Results: The sandwich ELISA for pCD36 phenotyping had high reliability (intra-assay CV, 2.1–4.8%; inter-assay CV, 2.3–5.2%). The sandwich ELISA was used to screen for CD36 expression on platelets isolated from 1,691 healthy blood donors. Of these, 36 samples were pCD36-negative. FC demonstrated absence of CD36 expression on monocytes in three of the 36 cases. In the present study population, the frequency of CD36 deficiency was 2.13% (36/1,691), of which 0.18% (3/1,691) was type I deficiency and 1.95% (33/1,691) was type II deficiency. In addition, we used PCR-SBT to characterize the gene mutations in exons 3–14 of the CD36 gene in 27 cases of CD36 deficiency and discovered 10 types of mutations in 13 pCD36-negative samples. Conclusion: The present study describes the development and characterization of a highly reliable sandwich ELISA for high-throughput screening for pCD36 expression. This novel method is feasible for clinical applications and provides a useful tool for the establishment of databases of pCD36-negative phenotype donors.

(092) Leptin Increases T-cells in Testis to Modulate Leydig Stem Cell Function

Abstract Introduction Testis microenvironment which is comprised of cells like Sertoli and peritubular cells releases paracrine factors which could potentially modulate testicular functions. In our previous studies, we identified Leptin as a key paracrine factor in the testicular microenvironment, released by Sertoli and peritubular cells and goes to bind its receptor in Leydig cells. Once this association happens it further induces the expression of the desert hedgehog signaling pathway, which is known to play an important role in the Leydig Stem cell’s differentiation to Adult Lydig Cells, which are capable of producing testosterone. However, one of the limitations in our study was that the model we used were from human cells that were extracted from testes’ biopsies which lacked an intact immune microenvironment. In the present study we explored the influence of Leptin on an intact testicular immune microenvironment. Objective To explore the influence of Leptin on an intact testicular immune microenvironment. Methods We used 6-weeks old C57BL6 mice which were exposed to two different concentrations of Leptin, 10 and 100 mg/ml for seven days. After seven days of treatment, these animals were humanly sacrificed and we collected their blood and testes. Blood was subjected to the CBC differential profile performed using EDTA anti-coagulated blood samples with a Hemavet Mascot Multispecies Hematology System Counter 1500R (CDC Technologies, Inc., Oxford, CT). Samples were counted no longer than five minutes after blood was drawn. In addition to that, the testes were used and subjected to comprehensive immunophenotyping that allowed us to evaluate in detail the impact of Leptin treatment at different doses on different immune cell types which are found within the testes. Results Results showed that Leptin at low dose (10mg/ml) showed increased levels of serum Testosterone (13.20 + 0.37 ng/dl) but were not significant, LH and FSH (LH 0.016 + 0.02 ng/ml, FSH in 51.71 + 12.93 ng/ml) showed significant (p&amp;lt;0.05) increase. On the contrary, animals which received Leptin at 100mg/ml showed no significant changes in hormone levels. CBC profiling suggested that amongst all the factors that were compared, Lymphocytes and Platelets were significantly influenced specifically upon low doses of Leptin. Interestingly, comprehensive immunophenotyping highlighted that the animals that received Leptin at low doses had a significant increase in the total number of CD4+ (helper T cells) and CD8+ (Cytotoxic T lymphocytes) cells. This pattern was not observed in animals which received high doses of Leptin. Conclusions The observation made in this study highlights unexplored aspects of influence of paracrine factors released by Sertoli and peritubular myoid cells on modulating immune microenvironment within the testis to potentially influence testosterone production. In future studies we will be exploring how the changes in the different immune cell types are correlated directly with the increase in T cells and how this correlation can influence the Leydig Stem Cell differentiation into Leydig Adult Cells. Disclosure No

Role of direct antiglobulin test in assessing the severity of extravascular hemolysis in autoimmune hemolytic anemia – analysis from a cross-sectional study

Background: Autoimmune hemolytic anemia (AIHA) is an immune hemolytic disease characterized by hemolysis and anemia which results from increased red cell destruction due to auto antibodies directed against self-antigens on red cells. Clinical characteristics and potential associated hemolysis in AIHA can greatly depend on the serological characteristics. Aims and Objectives: This study was done to find out the association of Grade of polyspecific Direct Antiglobulin test (DAT), complement involvement and immunoglobulin G (IgG) subclass with severity of hemolysis in an AIHA patients. Materials and Methods: This was a cross-sectional study conducted for a period of 18 months from March 1, 2012, to August 31, 2013. Fifty consecutive AIHA patients diagnosed by a positive DAT in EDTA anticoagulated blood samples using poly specific anti human globulin reagent were followed up and results of various immunohematological tests such as polyspecific and monospecific DAT, IgG subtyping, and thermal amplitude were performed. Hematological and biochemical parameters such as hemoglobin, percentage of reticulocyte, total serum bilirubin, and serum lactate dehydrogenase also were recorded. Data were analyzed using SPSS v17. Results: Majority of patients were belonging to 31–40 year age group and there was a remarkable female predilection. Secondary AIHA was more common (56%) than primary. SLE was identified as the most common underlying disorder in secondary AIHA. Majority (46%) were Grade 4 DAT positive. Predominant autoantibody identified was IgG (52%). Of the total IgG positive cases, IgG1 was the predominant subclass. C3d alone was detected in 18% of cases and C3d and IgG together in 30%. About 48% of our patients were of warm type, 32% mixed type, and 20% cold type. About 80% of the study population were treated with steroids alone. A small percentage of cases (16%) needed immunosuppressant in addition to steroids and still a smaller percentage (4%) needed splenectomy. Higher grades of DAT positivity were associated with more severe degree of anemia and hyperbilirubinemia. The study also demonstrated a positive correlation between DAT grade and severity of hemolysis. Conclusion: AIHA patients in this study were predominantly of Warm AIHA with more of IgG1 involvement. A significant association is found between higher grades of DAT results and severity of anemia and jaundice in this study group. A positive correlation exists between grades of DCT and hemolysis as well.

Genotyping for Scianna blood group system polymorphism in Pakistan.

Objective: To establish the occurrence of Scianna blood group alleles via genotyping in Pakistani blood donors. Study Design: Cross-sectional Analytical study. Setting: Department of Hematology, Army Medical College (AMC) in association with the Armed Forces Institute of Transfusion (AFIT) Rawalpindi. Period: January 2019 and January 2020. Material &amp; Methods: In this study 300 blood donors were incorporated after taking informed consent; DNA was extracted from their EDTA anticoagulated blood samples and tested for SC*01 and SC*02 alleles by Sequence-Specific Primer Polymerase Chain Reaction (PCR-SSP), which was ensued by gel electrophoresis. The allele frequencies were established and Hardy Weinberg equilibrium was determined. Chi-square test was applied to compare the observed and expected genotype frequencies as well as for comparison with published allele frequencies of other populations. We used SPSS version 22 for the analysis of study data. Results: All donors were homozygous for SC*01, demonstrating allele frequency for SC*01 nearly 1.00. Genotype frequencies were congruent with the Hardy Weinberg equilibrium. The difference between the allele frequencies of SC*01 and SC*02 alleles of population of Pakistan was not statistically significant when compared to those of other populations. Conclusion: This maiden research illustrates Scianna blood group distribution in Pakistani community by PCR-SSP. This method is cheap and proficient and can be applied in developing nations. The findings of this study can aid in the production of local red cell panels, detection of uncommon blood types, and setting up of a domestic rare blood donor platform.

DNA extraction from clotted blood in genotyping quality.

DNA extraction from frozen blood clots is challenging. Here, the authors applied QIAGEN Clotspin Baskets and the Gentra Puregene Blood Kit for DNA extraction to cellular fraction of 5.5ml whole blood without anticoagulating additives. The amount and quality of extracted DNA were assessed via spectrophotometer and gel electrophoresis. Results from array-based genotyping were analyzed. All steps were compared with DNA isolated from anticoagulated blood samples from a separate study. The quality and concentration of DNA extracted from clotted blood were comparable to those of DNA extracted from anticoagulated blood. DNA yield was on average 27μg per ml clotted blood, with an average purity of 1.87 (A260/A280). Genotyping quality was similar for both DNA sources (call rate: 99.56% from clotted vs 99.49% from anticoagulated blood).

Simultaneous determination of cordycepin and its metabolite 3'-deoxyinosine in rat whole blood by ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry and its application to accurate pharmacokinetic studies.

Cordycepin from Cordyceps possesses excellent pharmacological properties, including anti-inflammation and anti-tumor effects, therefore representing a potential alternative medicine. However, doubts about the pharmacokinetic results of cordycepin had been raised in the previous study due to its rapid deamination. The organic solvent methanol was immediately added to terminate the degradation of cordycepin in anticoagulated blood samples and enable the accurate evaluation of pharmacokinetics in vivo. A sensitive and selective ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry method was developed and validated to simultaneously determine cordycepin and its deamination metabolite 3'-deoxyinosine using 2-chloroadenosine as an internal standard in rat whole blood. The calibration curves of cordycepin and 3'-deoxyinosine showed excellent linearity within the concentration range of 1.05-10 000.00 ng/ml with acceptable accuracy, precision, selectivity, recovery, matrix effect, and stability. This method was successfully applied to the pharmacokinetic study of cordycepin and its metabolite in rat blood. The effect of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride on the pharmacokinetics of cordycepin was investigated. In summary, the reliable pharmacokinetic parameters of cordycepin and its deamination metabolite 3'-deoxyinosine in rat blood were successfully elucidated. Erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride considerably prolonged the half-life of cordycepin in vivo.

Detection of Androgen Receptor Gene Polymorphism in Sudanese Patients of Prostate Cancer in Khartoum State

Background: Prostate cancer (PCa) is the most commonly diagnosed solid tumor among men. Genetic susceptibility had been proposed among the risk factors for the development of this cancer.&#x0D; Aim: To investigate the effect of androgen receptor gene polymorphism in the susceptibility of prostate cancer among Sudanese patients.&#x0D; Methods: This study was conducted in Khartoum State during the period from December 2021 to May 2022. The study population that was selected consisted of one hundred patients, who had prostate cancer, who attended for routine follow-up assessment following their chemotherapy treatment. A total of 5 ml EDTA anti-coagulated venous blood samples were obtained from all participants. Prostate specific antigen (PSA) was measured by competitive chemiluminescence immunoassay. DNA extraction was performed for all samples by chemical method and genotyping was performed by PCR-RFLP method using Eco147I enzyme.&#x0D; Results: The Androgen receptor genotype showed that wild (G/G) type was more frequent (89%) than heterozygous (G/A) type (11%), and allele G was more frequent (94.5%) than allele A (5.5%). The mean serum PSA level among prostate cancer patients was 18.71+31.68 ng/ml. Comparison of the mean serum PSA levels between different AR genotypes revealed no significant association between the genotype and the hormonal level (p. values &gt; 0.05).&#x0D; Conclusion: Androgen receptor gene polymorphism was detected in this study population as heterozygous type. Moreover; there was no significant association between AR genotype and serum PSA level.