Affordable Method for Hematocrit Determination in Murine Models.

Hematocrit (Hct) is a powerful tool often used in a clinical setting for the diagnosis of blood conditions such as anemia. It is also used in the research field as a hematological parameter in both human and mouse models. Measuring Hct, however, involves the use of expensive standardized equipment (such as a CritSpin™ Microhematocrit Centrifuge). Here, we describe a novel, simple, and affordable method to determine the Hct in untreated wild-type (WT) mice and phenylhydrazine (PHZ)-induced anemic mice with reasonable accuracy, using a benchtop centrifuge commonly available in laboratories. Hct of murine samples processed with a benchtop centrifuge, when compared to the standardized method CritSpin™, showed comparable results. This approach for determining Hct of murine can prove useful to research laboratories that cannot afford specialized equipment for Hct studies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Affordable Method for Hematocrit Determination in Murine Models Basic Protocol 2: Murine Sample Validation Support Protocol: Phenylhydrazine-induced anemia in wild-type (WT) mice.

Incidental Finding of Post-Transplant Erythrocytosis After Renal Transplantation in a Patient With Chronic Kidney Disease: A Case Report.

Renal transplant aims to provide a healthy substitute for the chronically damaged kidney while also correcting the anemia of chronic disease by producing erythropoietin for effective erythropoiesis. However, in a small number of renal transplant patients, the hematocrit continues to rise even after correction of the anemia, ultimately leading to abnormally increased hemoglobin and hematocrit. This condition is termed "post-transplant erythrocytosis" (PTE). We present a case of a 50-year-old male who was diabetic, positive for hepatitis B surface antigen, and negative for polymerase chain reaction. He presented with symptoms of acute hepatitis. During the work-up, PTE was diagnosed. Our case sheds light on a common complication of renal transplant known as PTE, its possible complications in the patient, and the necessary interventions to prevent untoward outcomes. PTE, although a less common complication of renal transplant, can become serious and potentially fatal due to its sequelae of thromboembolism. The complications can range from simple thrombophlebitis and thrombosis of digital and brachial arteries to more severe events such as pulmonary embolism or stroke and cardiovascular events. Regular post-transplant follow-ups with frequent bloodwork will aid in the early diagnosis of PTE, allowing for timely intervention with appropriate treatment options such as venesection or angiotensin receptor blockers (ARBs)/angiotensin-converting enzyme (ACE) inhibitors.

Development of an LC-MS/MS method to simultaneously quantify therapeutic mAbs and estimate hematocrit values in dried blood spot samples.

Recently, monoclonal antibody (mAb) therapy has gained increasing attention in the medical field due to its high specificity. Dried blood spots (DBSs) have been used in various clinical fields due to their unique characteristics, such as easy transportation, low invasiveness, and home sampling. However, hematocrit (HCT)-associated issues may lead to inaccurate quantification; moreover, the HCT value is required for converting the drug concentration from DBS to plasma. To simultaneously measure HCT levels and quantify mAb concentrations in DBS samples, this study used volumetrically applied 15 µL DBS, and combined protein G purification and ethanol precipitation approaches as the sample preparation method. Sixty-two clinical samples were used to investigate the HCT estimation ability by using hemoglobin (Hb) peptides. Four mAbs, bevacizumab, trastuzumab, nivolumab and tocilizumab, were selected to demonstrate our method, and pembrolizumab was used as the internal standard. The optimized method could measure four mAbs and Hb peptides simultaneously within 11 min. Moreover, a correlation study revealed that the correlation coefficient for the Hb peptides and the HCT value was larger than 0.9. The HCT estimation results revealed that for over 90% of the real DBS samples the HCT could be obtained within ±20% estimation error acceptance criteria. The method was validated in terms of accuracy and precision for the four mAbs. The developed method was further applied to simultaneously quantify mAb concentrations and estimate HCT values in six patient DBS samples to demonstrate its clinical applicability. It is believed that this newly developed method could facilitate various clinical studies and provide benefits for mAb therapies in clinical fields.

The impact of initial hematocrit values after birth on peri-/intraventricular hemorrhage in extremely low birth weight neonates.

AIM: Peri-/intaventricular hemorrhage (P/IVH) is a common condition in preterm neonates and is responsible for substantial adverse neurological outcome especially in extremely low birth weight infants. As hematocrit after birth is a surrogate marker for blood volume, this study aimed to evaluate the effect of initial hematocrit values after birth on P/IVH development in extreme low birth weight (ELBW) neonates. PATIENTS AND METHODS: A prospective cohort analysis of 92 eligible ELBW neonates was performed. The relationship between initial hematocrit values in ELBW neonates after birth and subsequent development of P/IVH was examined. RESULTS: Twenty-nine of 92 infants developed P/IVH. There were significant differences in initial Hct and maximum carbon dioxide (max PCO2) in the first 3 days levels in the P/IVH group compared with no P/IVH group. Initial Hct level at birth in the P/IVH group were significantly lower than the no P/IVH group while max PCO2 in the first 3 days were found to be significantly high in the P/IVH group. There were no significant differences in other baseline demographic, perinatal, and neonatal characteristics while in univariate analysis, higher gestational age and initial Hct were associated with decreased likelihood of P/IVH. In multiple regression analysis after adjustment, only initial Hct remained significantly associated with P/IVH. There was no difference between the population by subgroups of IVH (IVH I-II and IVH III-IV) according to hematocrit and the severity of IVH. CONCLUSION: Higher initial Hct at birth is associated with decreased P/IVH in ELBW infants. We hypothesized the argument that ELBW infants who have lower initial Hct at birth have less suboptimal volume status that predisposing lower cerebral blood flow and the resultant decrease in cerebral blood flow precede the development of P/IVH.

The effects of sodium glucose co-transporter (SGLT) 2 inhibitors on hematocrit levels: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Previous studies have suggested benefits of sodium glucose co-transporter 2 (SGLT2) inhibitors including improving glycemic control, lower body weight, uric acid-lowering effect and decreasing blood pressure. The aim of this study was to evaluate the effects of SGLT2 inhibitors on hematocrit (Hct) levels in patients with type 2 diabetes mellitus. METHODS: Embase, CENTRAL, PubMed and other databases were searched from the establishment of the database through to July 2020. Randomized controlled trials (RCTs) involving patients with type 2 diabetes mellitus who were treated with SGLT2 inhibitors were analyzed using the random effects model. Stata 12.0 statistical software was used to estimate the weighted mean difference (WMD) and the 95% confidence intervals (CIs). RESULTS: A total of 40 RCTs were included, comprising 21,050 patients. SGLT2 inhibitors resulted in a significant increase in Hct levels compared to patients treated with a placebo (WMD 2.67%, 95% CI, 2.53 to 2.82; P<0.001). Treatment with 2.5, 5, and 10 mg of dapagliflozin significantly increased Hct levels (WMD 1.96%, 2.27%, and 2.47%, respectively; P<0.001). Administration of 100 and 300 mg of canagliflozin also resulted in a significant increase in Hct (WMD 2.91% and 2.94%, respectively; P<0.001). Similarly, empagliflozin, at concentrations of 10 and 25 mg, caused a significant increase in Hct (WMD 3.39% and 3.44%, respectively; P<0.001). However, treatment with ipragliflozin (12.5 and 50 mg) and ertugliflozin (5 and 15 mg) only resulted in a slight increase in patient Hct levels (WMD 1.26% and 1.98%, respectively for ipragliflozin, P>0.05; WMD 2.24% and 2.64%, respectively for ertugliflozin; P>0.05). DISCUSSION: SGLT2 inhibitors, as a class of drugs, increased Hct levels in patients with type 2 diabetes, and this increase was slightly more pronounced at higher doses compared to lower doses. TRIAL REGISTRATION: The protocol of this study has been submitted to the PROSPERO platform (https://www.crd.york.ac.uk/PROSPERO/), and the registration number is CRD42020200699.

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS.

The added value of dried blood spot (DBS) samples complementing the information obtained from commonly routine doping control matrices is continuously increasing in sports drug testing. In this project, a robotic-assisted non-destructive hematocrit measurement from dried blood spots by near-infrared spectroscopy followed by a fully automated sample preparation including strong cation exchange solid-phase extraction and evaporation enabled the detection of 46 lower molecular mass (< 2 kDa) peptide and non-peptide drugs and drug candidates by means of LC-HRMS. The target analytes included, amongst others, agonists of the gonadotropin-releasing hormone receptor, the ghrelin receptor, the human growth hormone receptor, and the antidiuretic hormone receptor. Furthermore, several glycine derivatives of growth hormone-releasing peptides (GHRPs), arguably designed to undermine current anti-doping testing approaches, were implemented to the presented detection method. The initial testing assay was validated according to the World Anti-Doping Agency guidelines with estimated LODs between 0.5 and 20 ng/mL. As a proof of concept, authentic post-administration specimens containing GHRP-2 and GHRP-6 were successfully analyzed. Furthermore, DBS obtained from a sampling device operating with microneedles for blood collection from the upper arm were analyzed and the matrix was cross-validated for selected parameters. The introduction of the hematocrit measurement method can be of great value for doping analysis as it allows for quantitative DBS applications by managing the well-recognized "hematocrit effect." Graphical abstract.

Genotoxicity and subchronic toxicological study of a novel ginsenoside derivative 25-OCH3-PPD in beagle dogs.

BACKGROUND: Ginsenosides have been widely used clinically for many years and were regarded as very safe. However, a few researches on the toxicities of these kinds of agents showed that some ginsenosides may have side-effect on the rats or dogs. So it is extremely necessary to further clarify the potential toxicity of ginsenosides. This study was carried out to investigate long-term toxicity and genotoxicity of 25-methoxydammarane-3, 12, 20-triol (25-OCH3-PPD), a new derivative of ginsenoside, in beagle dogs. METHODS: Twenty-four beagle dogs were divided randomly into four treatment groups and repeatedly orally administered with 25-OCH3-PPD capsule at 60, 120, and 240 mg/kg/day for 91 consecutive days. Ames, micronucleus, and chromosomal aberration tests were established to analyze the possible genotoxicity of 25-OCH3-PPD. RESULTS: There was no 25-OCH3-PPD-induced systemic toxicity in beagle dogs at any doses. The level of 25-OCH3-PPD at which no adverse effects were observed was found to be 240 mg/kg/day. The result of Ames test showed that there was no significant increase in the number of revertant colonies of 25-OCH3-PPD administrated groups compared to the vehicle control group. There were also no significant differences between 25-OCH3-PPD administrated groups at all dose levels and negative group in the micronucleus test and chromosomal aberration assay. CONCLUSION: The highest dose level of 25-OCH3-PPD at which no adverse effects were observed was found to be 240 mg/kg per day, and it is not a genotoxic agent either in somatic cells or germs cells. 25-OCH3-PPD is an extremely safe candidate compound for antitumor treatment.

Drug monitoring by volumetric absorptive microsampling: method development considerations to mitigate hematocrit effects.

AIM: GSKA is a compound that was in development in clinical trials. A bioanalysis method to quantify GSKA using volumetric absorptive microsampling (VAMS) was developed and hematocrit (HCT) related assay bias was investigated. METHODOLOGY: After accurate sampling of 10 µl blood, VAMS tips were air dried approximately 18 h and desorbed by an aqueous solution containing internal standard. The recovered blood underwent liquid-liquid extraction in ethyl acetate to minimize matrix suppression. Assay accuracy, precision, linearity, carryover, selectivity, recovery, matrix effects, HCT effects and long-term quality control stability were evaluated. CONCLUSION: HCT-related assay bias was minimized in 30-60% blood HCT range, and all validation parameters met acceptance criteria. The method is suitable for quantitative analysis of GSKA in human blood.

Hematocrit and plasma albumin levels difference may be a potential biomarker to discriminate preeclampsia and eclampsia in patients with hypertensive disorders of pregnancy.

BACKGROUND: We evaluated whether alterations of hemoglobin (HB), hematocrit (HCT), serum albumin level (ALB), and the difference of HCT and ALB can be used in the diagnosis of preeclampsia and eclampsia in patients with hypertensive disorders of pregnancy (HDP). METHODS: A total of 509 individuals were recruited and divided into 4 groups: Group 1, 170 healthy non-pregnant women; Group 2, 125 normal pregnant women; Group 3, 105 pregnant women diagnosed with gestational and chronic hypertension; Group 4, 109 pregnant women diagnosed as having preeclampsia and eclampsia. Data of HB, HCT, ALB, globulin (GLB) were collected at the time of a prenatal examination during the third trimester. RESULTS: Alterations in the HCT and the ALB levels in these groups were significantly different. Group 4 had a higher mean HCT-ALB value (P<0.01), but lower ALB and GLB values compared with the other three groups. We used Groups 2 and 3 as the respective reference to draw the receiver operating characteristic (ROC) curves of HCT-ALB in Group 4, and found that the threshold values of maximum index corresponding were 12.95 and 12.65 (sensitivity>57.0%, specificity>98.9%), respectively. CONCLUSIONS: The value of HCT-ALB>12.65 might be used as a potential biomarker for the auxiliary diagnosis of preeclampsia and eclampsia in HDP.

Effects of aerobic exercise on hematologic indices of women with rheumatoid arthritis: A randomized clinical trial.

BACKGROUND: To investigate the effects of moderate aerobic exercise on the hemoglobin, hematocrit, and red blood cell (RBC) mass of women with rheumatoid arthritis (RA). MATERIALS AND METHODS: This randomized clinical trial was conducted at the Specialized Clinic of Physical Medicine and Rehabilitation, Al-Zahra Hospital of Isfahan, during a 4-month period in 2014. We included patients with RA who did not have any malignancy and hematologic disorder. Two groups - one group receiving aerobic therapy along with medical therapy (N = 16) and the other group receiving medical therapy alone (N = 17) both for a period of 8 weeks. The levels of RBC mass, Hb, and HCT were measured before and after the intervention. The changes in these parameters were compared between the two study groups. RESULTS: There was no significant difference between the two study groups regarding the baseline characteristics. The aerobic exercise resulted in increased RBC mass (P < 0.001), Hb (P < 0.001), and HCT (P < 0.001). However, those who received medical therapy alone did not experience any significant changes in these parameters. We found that the RBC mass (P = 0.581), Hb (P = 0.882), and HCT (P = 0.471) were comparable between the two study groups after 8 weeks of intervention. CONCLUSION: Although the aerobic exercise results in increased Hb, HCT, and RBC mass in patients with RA, the increase was not significant when compared to that in controls. Thus, the increase in the HB, HCT, and RBC could not be attributable to aerobic exercise.