Stability In Whole Blood Research Articles

The effect of a low-level yellow laser on improving the stability of stored whole blood.

To explore if low-level laser 589nm may stabilise stored whole blood and red blood cell suspension in vitro. The interventional study was conducted from November 2021 to April 2022 at the Postgraduate Medical Physics Laboratory, Mustansiriyah University, Baghdad, Iraq, and comprised blood samples from healthy adults. The samples were obtained put in tubes containing anticoagulant citrate-phosphate dextrose-adenine. Each sample was divided into eight equal aliquots and stored for 21 days at 4ºC. Of the 8 aliquots, 4 were takebn as controls, while the other 4 were irradiated by 589nm laser beam with a radiation dose of 50J/cm² for 15min. Stability test measured by the percentage of haemolysis of an overnight stored red blood cell in saline solution was done on days 0, 7, 14 and 21 for non-irradiated and radiated aliquots. Data was analysed using SPSS 24. Eight subjects (4 males and 4 females) were included, with mean age 21±3 years (range: 19-24 years). Whole blood samples showed a significant reduction in haemolysis by 40%, 20% and 42% compared to the control samples at days 0, 7 and 14, respectively (p<0.05). Red blood cell suspension showed a significant reduction in haemolysis by 9% compared to the control samples only at day 21 (p<0.05). Low-level laser 589nm beam at 50J/cm² radiation dose was more effective on the stability of stored whole blood than the red blood cell suspension.

Stability of Hemostasis Parameters in Whole Blood, Plasma, and Frozen Plasma: Literature Review and Recommendations of the SFTH (French Society of Thrombosis and Haemostasis).

Preanalytical sample management is critical for a proper assessment of hemostasis parameters, and may differ depending on prescribed tests or additional tests considered to be necessary after initial results. Although there is quite vast literature on this issue, the Working Group of the French Society of Thrombosis and Haemostasis (SFTH) deemed it necessary to make an in-depth literature review and propose recommendations for the proper handling of samples prior to hemostasis assays. This extensive assessment is accessible on-line in French at the SFTH website. Here, a more synthetic view of these recommendations is proposed, supported by easy-to-use tables. The latter respectively deal with the stability of whole blood or fresh plasma, frozen samples, and proper handling of samples forwarded on dry ice. Procedures are classified as recommended, acceptable, not conformed and lacking data. This work involved the retrieval of 125 references, first screened by a working group of 6 experts, then reviewed by 20 other experts in the field. The highly detailed conditions summarized in these tables will hopefully help hemostasis laboratories to secure the conditions recommended for sample collection and transportation. Moreover, as some conditions clearly lacked recommendations, this review can open new fields of investigation for hemostasis preanalytics.

From Bed to Bench: Pre-analytical Stability of 29 Anti-infective Agents in Plasma and Whole Blood to Improve Accuracy of Therapeutic Drug Monitoring.

Therapeutic drug monitoring requires a validated assay and appropriate conditions for sample shipment and storage based on the stability of the compound to be analyzed. This study evaluated the stability of 29 antimicrobial compounds in whole blood (WB) and plasma samples under various storage conditions. The pre-analytical stability of 22 antibiotics (amoxicillin, aztreonam, cefazolin, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftobiprole, ceftolozane, ceftriaxone, ciprofloxacin, clindamycin, cloxacillin, daptomycin, levofloxacin, linezolid, meropenem, metronidazole, moxifloxacin, piperacillin, sulfamethoxazole, and trimethoprim), 2 beta-lactamase inhibitors (avibactam, tazobactam), and 5 antituberculosis drugs (ethambutol, isoniazid, pyrazinamide, rifabutin, and rifampicin) was assessed by WB for up to 24 hours at room temperature (RT) and 72 hours at +4°C. The stability in plasma was evaluated for up to 6 hours at RT, 24 hours at +4°C, 1 month at -20°C, and 6 months at -80°C. Concerning WB stability, all investigated compounds were stable for 24 hours at RT, except meropenem and isoniazid, which were stable for 6 hours; however, for 24 hours at +4°C, all the compounds were stable. For storage durations of 48 and 72 hours at +4°C, all compounds were stable, except for ciprofloxacin, cotrimoxazole, and isoniazid. Concerning stability in plasma, all compounds were stable for 6 hours at RT, and all except isoniazid were stable for 24 hours at +4°C. All the tested compounds were stable for 7 days at -20°C, except isoniazid, for which a degradation of approximately 20% was observed. An important degradation was observed for beta-lactam antibiotics after 1 month at -20°C. All compounds were stable at -80°C for 6 months. The pre-analytical stabilities of several anti-infective compounds was described. The present results can be used to determine the appropriate conditions for shipping and storing samples dedicated to therapeutic drug monitoring of the investigated compounds.

Converting waste into value: The sample stability of leftover EDTA whole blood. A comparative study of analyte concentrations in plasma separated from EDTA whole blood at various time intervals and serum for nine clinical chemistry analyses

Converting waste into value: The sample stability of leftover EDTA whole blood. A comparative study of analyte concentrations in plasma separated from EDTA whole blood at various time intervals and serum for nine clinical chemistry analyses

Safety and Efficacy of Mitapivat in Adult Patients with Erythrocyte Membranopathies (SATISFY)

Background: Membranopathies encompass hemolytic disorders arising from genetic defects in erythrocyte membrane proteins and include hereditary spherocytosis and stomatocytosis. Congenital dyserythropoietic anemia type II (CDA II) is associated with SEC23B gene variants and its phenotype may be similar to hereditary spherocytosis. Anemia with or without the need for chronic transfusion, jaundice, splenomegaly, iron overload are complications common in both hereditary membranopathies and CDA II. Current treatment options for membranopathies and CDA II 1, apart from splenectomy, are primarily supportive. Mitapivat, an investigational pyruvate kinase-R (PKR) activator, has demonstrated efficacy in improving anemia and reducing hemolysis in adult patients with PK deficiency, thalassemia, sickle cell disease, and a mouse model of hereditary spherocytosis. Study Design and Methods: This study (NCT05935202) is an investigator initiated, prospective, multicenter, single-arm phase 2 trial. The study will be conducted in the European Union in Denmark and The Netherlands. It is the first clinical study in the context of the European Reference Network EuroBloodNet and will be sponsored by its closely associated non-for-profit EuroBloodNet Association. A sibling study in Toronto, Canada will be registered separately, and data will be pooled in a prespecified statistical analysis plan. The study will include approximately twenty-five adult patients (aged 18 years and older) diagnosed with a membranopathy or CDA II. Diagnosis must be confirmed genetically. Average hemoglobin concentration (Hb) must be less than 13.0 g/dL for males and 11.0 g/dL for females. Patients with average Hb &amp;gt;10.0 g/dL for males and females at screening must meet at least one of the following additional criteria: Splenomegaly, fatigue attributed to hemolysis, or paraclinical hemolysis. Adequate organ function is required. Patients cannot be included if they have a diagnosis of PK deficiency or if they have received red blood cell (RBC) transfusions (≥5 units the last 12 months or any within the last 3 months). During the 8-week Dose Escalation Period, subjects will receive an initial dose of 50 mg mitapivat twice daily (BID). Based on safety and changes in Hb levels, dosing may be increased to 100 mg BID at week 4. Patients who tolerate mitapivat may be eligible to continue in two consecutive 24-week Fixed Dose Periods, with an interim analysis in between the two fixed dose periods. The primary objective of this study is to evaluate the safety of mitapivat, assessed through the occurrence of treatment-emergent adverse events. Secondary objectives include assessing the effects of mitapivat on Hb concentration (≥1.0 g/dL increase sustained at two scheduled visits), hemolysis, erythropoiesis, patient-reported outcome measures, and spleen size. Exploratory endpoints include enzyme activity and stability of PK, Hb oxygen affinity, proteomics and metabolomics. To assess how activation of PK will impact RBC function we will measure RBC deformability (osmotic gradient ektacytometry and cell membrane stability) in whole blood and in different RBC subpopulations. Acknowledgement: This project is carried out within the framework of European Reference Network on Rare Haematological Diseases (ERN-EuroBloodNet)-Project ID No 101085717. ERN-EuroBloodNet is partly co-funded by the European Union within the framework of the Fourth EU Health Programme. The study is funded by a grant from Agios Pharmaceuticals.

B-097 Storage Stability of HbA1c in Whole Blood Tubes at Room Temperature on the Abbott Alinity c Clinical Chemistry System

Abstract Background Hospital laboratories frequently receive requests to order additional tests on an existing specimen. For this reason, laboratories routinely save specimens after initial testing has been completed for a defined period of time. The current literature lacks specific information regarding HbA1c stability in whole blood and the effects of red blood cell settling. In addition, literature references are not inclusive of various conditions which can cause gaps in data collection such as samples in queue for prolonged periods. To obtain information on prolonged storage of whole blood tubes, HbA1c was assessed for storage at room temperature (RT) (20–25°C) for up to 4 hours on the Alinity c system. In addition, potential carryover from a whole blood to a serum sample was assessed. Methods A minimum of 40 whole blood sample pools (approx. 8 mLs) were prepared from remnant samples. Sample pools were mixed, aliquoted, and stored at RT on the Alinity c system. The samples were tested as a single replicate continuously for up to 4 hours without mixing (minimum of 20 cycles) and compared to the baseline sample values. Carryover from a whole blood to a glucose serum sample was assessed after 4 hours continuous testing of whole blood to mimic a high volume testing laboratory. The stability of each HbA1c sample and carryover into a glucose sample were evaluated by comparing the mean absolute or percent difference from the respective sample baseline measurement. The allowed deviation from baseline was 5%. Results After 4 hours at RT, the whole blood cells had settled to approximately 50% from the top of the tube. The red blood cell settling had no significant impact on the results. Within sample precision for the 20 cycles of repetitive testing was &amp;lt;/ = 1%. The 48 individual samples showed insignificant difference from the baseline value, with a mean change of 1.0% (Range 0–3.2%). Sample carryover from the whole blood samples to a serum glucose sample was not significant (2.71 Baseline vs 2.64 first replicate). Conclusion This study demonstrated that the storage stability of HbA1c is stable for up to 4 hours at RT on the Alinity c system.

Development and validation of a hybrid immunoaffinity LC–MS/MS assay for quantitation of total antibody (TAb) from an antibody drug conjugate (ADC) PYX-201 in human plasma

A hybrid immunoaffinity LC-MS/MS assay was developed and validated for the quantitation of total antibody (TAb) from an antibody drug conjugate (ADC) PYX-201 in human plasma. PYX–201 was proteolyzed using trypsin, and a characteristic peptide fragment PYX-201 P1 with ten amino acids IPPTFGQGTK from the complementarity-determining regions (CDRs) was used as a surrogate for the quantitation of the TAb from PYX-201. Stable isotope labelled (SIL) peptide I(13C6, 15N)PPTFG(13C9, 15N)QGTK was used as the internal standard (IS). We performed chromatographic analysis using a Waters Acquity BEH Phenyl column (2.1 mm × 50 mm, 1.7 µm). Quantification of PYX-201 TAb was carried out on a Sciex triple quadrupole mass spectrometer API 6500 using multiple reaction monitoring (MRM) mode with positive electrospray ionization. To validate PYX–201 TAb, a concentration range of 0.0500 µg/mL to 20.0 µg/mL was used, yielding a correlation coefficient (r) of ≥ 0.9947. For intra-assay measurements, the percent relative error (%RE) ranged from –23.2% to 1.0%, with a coefficient of variation (%CV) of ≤ 14.2%. In terms of inter–assay measurements, the %RE was between −10.5% and −5.7%, with a %CV of ≤ 12.7%. The average recovery of the analyte was determined to be 81.4%, while the average recovery of the internal standard (IS) was 97.2%. Furthermore, PYX–201 TAb demonstrated stability in human plasma and human whole blood under various tested conditions. This assay has been successfully applied to human sample analysis to support a clinical study.

Reliable capillary sampling of carcinoembryonic antigen at home: the CASA feasibility study.

Follow-up after colorectal cancer requires frequent surveillance of the tumour marker carcinoembryonic antigen (CEA). Home-based blood sampling could be beneficial in terms of patients' well-being and societal cost-effectiveness. Blood sampling by venepuncture is unsuitable for home-based sampling. The aim of this feasibility study is to evaluate the long-term whole-blood stability of CEA. In this prospective feasibility study capillary blood withdrawal was collected in a Hem-Col® microtube containing a patented stabilization buffer using an internal lithium standard to correct for dilution. Long-term whole-blood stability was considered adequate if the relative bias in CEA concentration between delayed analysis of capillary samples and directly processed venepuncture is within the total error margin of CEA. Twenty two colorectal cancer patients were included to determine the stability of CEA in capillary sampling compared with directly processed (i.e. within 2h) venepuncture sampling. The median time between venous sampling and CEA analysis and capillary sampling and CEA analysis was 2h (interquartile range 1-4h) and 76 h (interquartile range 74-95 h), respectively. A Bland-Altman difference plot excluding outliers showed an overall relative bias of -1.23%. The two capillary samples in our outlier analysis also showed the highest lithium concentrations. Home-based capillary sampling with the use of the Hem-Col® buffer is a feasible method for CEA determination when analysed within 4 days after blood withdrawal, allowing monitoring for colorectal cancer patients from home. High lithium concentrations due to insufficient filling of the Hem-Col® tube suggest less reliable CEA measurements.

Guiding fibrinogen replacement by rotational thromboelastometry in liver transplantation: bridging the evidence gaps

Rotational thromboelastometry (ROTEM) and thromboelastography (TEG) are viscoelastic assays of blood coagulation that can be performed as point-of-care (POC) tests at the bedside or during surgical procedures [1]. Both methods monitor clot formation, firmness, and stability in whole blood under low shear stress; however, they utilize different technologies to detect clot properties. ROTEM and TEG are commonly used in transfusion algorithms to guide the administration of hemostatic products in trauma, postpartum hemorrhage, liver transplantation, and cardiac surgery [1,2].

Quantitative impact of pre-analytical process on plasma uracil when testing for dihydropyrimidine dehydrogenase deficiency.

Determining dihydropyrimidine dehydrogenase (DPD) activity by measuring patient's uracil (U) plasma concentration is mandatory before fluoropyrimidine (FP) administration in France. In this study, we aimed to refine the pre-analytical recommendations for determining U and dihydrouracil (UH2 ) concentrations, as they are essential in reliable DPD-deficiency testing. U and UH2 concentrations were collected from 14 hospital laboratories. Stability in whole blood and plasma after centrifugation, the type of anticoagulant and long-term plasma storage were evaluated. The variation induced by time and temperature was calculated and compared to an acceptability range of ±20%. Inter-occasion variability (IOV) of U and UH2 was assessed in 573 patients double sampled for DPD-deficiency testing. Storage of blood samples before centrifugation at room temperature (RT) should not exceed 1h, whereas cold (+4°C) storage maintains the stability of uracil after 5hours. For patients correctly double sampled, IOV of U reached 22.4% for U (SD = 17.9%, range = 0-99%). Notably, 17% of them were assigned with a different phenotype (normal or DPD-deficient) based on the analysis of their two samples. For those having at least one non-compliant sample, this percentage increased up to 33.8%. The moment of blood collection did not affect the DPD phenotyping result. Caution should be taken when interpreting U concentrations if the time before centrifugation exceeds 1hour at RT, since it rises significantly afterwards. Not respecting the pre-analytical conditions for DPD phenotyping increases the risk of DPD status misclassification.

Serum GFAP- reference interval and preanalytical properties in Danish adults.

Glial fibrillary acidic protein (GFAP) is a promising biomarker that could potentially contribute to diagnosis and prognosis in neurological diseases. The biomarker is approaching clinical use but the reference interval for serum GFAP remains to be established, and knowledge about the effect of preanalytical factors is also limited. Serum samples from 371 apparently healthy reference subjects, 21-90 years of age, were measured by a single-molecule array (Simoa) assay. Continuous reference intervals were modelled using non-parametric quantile regression and compared with traditional age-partitioned non-parametric reference intervals established according to the Clinical and Laboratory Standards Institute (CLSI) guideline C28-A3. The following preanalytical conditions were also examined: stability in whole blood at room temperature (RT), stability in serum at RT and-20°C, repeated freeze-thaw cycles, and haemolysis. The continuous reference interval showed good overall agreement with the traditional age-partitioned reference intervals of 25-136ng/L, 34-242ng/L, and 5-438ng/L for the age groups 20-39, 40-64, and 65-90 years, respectively. Both types of reference intervals showed increasing levels and variability of serum GFAP with age. In the preanalytical tests, the mean changes from baseline were 2.3% (95% CI:-2.4%, 6.9%) in whole blood after 9h at RT, 3.1% (95% CI:-4.5%, 10.7%) in serum after 7days at RT, 10.4% (95% CI:-6.0%, 26.8%) in serum after 133days at-20°C, and 10.4% (95% CI: 9.5%, 11.4%) after three freeze-thaw cycles. The study establishes age-dependent reference ranges for serum GFAP in adults and demonstrates overall good stability of the biomarker.

Fabrication of a Polyvalent Aptamer Network on an Electrode Surface for Capture and Analysis of Circulating Tumor Cells.

Capture and analysis of circulating tumor cells (CTCs) from complex matrixes is pivotal for the prediction of cancer metastasis and personalized treatment of cancer. Herein, we propose a strategy for CTC capture by design and fabrication of a polyvalent aptamer network on an electrode surface, which can be further used for the sensitive analysis of CTCs. In our design, the polyvalent aptamer network, which is constructed via a rolling circle amplification reaction, can significantly enhance the cell-binding abilities. Meanwhile, tetrahedral DNA structures previously assembled on the electrode surface will promote the spatial orientation and reduce the steric hindrance effect of the cell capture, thus improving the cell capture efficiency. Importantly, a detectable electrochemical signal can be obtained without additional signal probes by means of target-induced allostery of the DNA hairpin structures. Further studies reveal that the electrochemical response is proportional to the logarithm of the CTC abundance ranging from 102 to 5 × 104 cell mL-1 with a low limit of detection of 23 cell mL-1. Moreover, the proposed capture strategy exhibits excellent stability and anti-interference in human whole blood, indicating its promising potential in clinical diagnosis.

Stability of Anti-thyroid Stimulating Hormone Receptor Antibody in Whole Blood and Serum: Caution Required for Reflective and Batch Testing

Anti-thyroid stimulating hormone receptor antibody (TRAb) stability is stated as 7h at 20-25°C in the Roche Elecsys assay kit insert. The purpose of this study was to determine TRAb stability in whole blood and serum to assess the suitability of samples for reflective and weekly batch testing (with a single freeze-thaw cycle). In the first study, blood from n = 5 volunteers was used to assess: (1) stability in whole blood at room temperature up to 24h, and (2) stability in serum at 4-8°C up to 72h. In the second study, n = 21 patient samples were analysed in serum stored at 4-8°C for two and fivedays post-preliminary analysis. There was a statistically significant decrease in TRAb concentration caused by the single freeze-thaw cycle of -8.9% ± 5.2% (p = 0.03). TRAb concentration decreased in whole blood between 0 and 24h by -16.5% ±9.2%, and declined in serum over time by -11.6% ±6.6% (at 12h). In the patient samples, serum TRAb concentration decreased over time by -4.6% ± 2.5% at day two and -6.5% ± 4.0% at day five. TRAb concentration decreases over time in both whole blood at room temperature and serum samples stored at 4-8°C. A single freeze-thaw cycle also has a statistically significant effect on TRAb concentration.

Stability of hematologic analytes in New Zealand white rabbits using an impedance‐based analyzer

The validity of hematologic data is crucial for correctly diagnosing certain conditions and providing accurate research results. However, delays in blood testing are inevitable. This study aimed to determine whether prolonged storage at different temperatures affects CBC measurand stability in rabbit whole blood using the impedance-based Scil Vet ABC hematology analyzer. Hematologic tests were performed on whole blood samples collected from 20 New Zealand white rabbits at <1hour post-blood collection as the baseline and at 6, 24, 48, and 72hours post-blood collection, with aliquots stored at 21 and 4°C. CBC measurand stability was determined by comparing the obtained values at each time point to the baseline value. Statistically significant variations were compared with current quality specifications to determine if a meaningful bias existed. Hematocrit, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration remained stable for 72hours in blood stored at 4°C. No meaningful bias was observed for mean corpuscular volume after 48 and 72hours of storage at 4°C. Only the white blood cell count was stable for 72hours at 21°C. The platelet count, hemoglobin concentration, and mean platelet volume were not stable at 6hours post-blood collection at either temperature. The results of this study showed that the hematocrit can be tested for 72hours in blood stored at 4°C, without a significant change from the baseline value. However, if all hematologic measurands are of interest, the immediate testing of blood samples, namely <1hour post-blood collection, remains highly recommended.

Development, validation and application of a UHPLC-MS/MS method for quantification of the adiponectin-derived active peptide ADP355 in rat plasma.

A UHPLC-MS/MS method for the quantification of ADP355, an adiponectin-derived active peptide, was developed and validated. The extraction method employed simple protein precipitation using methanol and chromatographic separation was achieved on anAccucore™ RP-MS C18 column (100 × 2.1mm, 2.6μm, 80 Å), using 0.1% formic acid in both water and acetonitrile with gradient elution at the flow rate of 400μl/min within 4.0min. Detections were performed under positive ion mode with multiple reaction monitoring ion transitions m/z 1109.2 → 309.8 and 871.4 → 310.1 for ADP355 and Jt003 respectively at unit resolution. The linearity range of the calibration curve was 2-1,000 ng/ml with a lower limit detection of 0.5ng/ml. The selectivity, linearity, precision, accuracy, recovery, matrix effect and stability were validated, and all items met the requirement of US Food and Drug Administration guidance. This method was successfully applied to an intravenous pharmacokinetic study of ADP355 in rats and the in-vitro stability in rat serum, plasma and whole blood was also assessed.

Analyte stability in whole blood using experimental and datamining approaches

The analytical stability of laboratory tests relies mostly on internal and external quality control procedures. Summarized patient data has in several studies been shown to be a good supplement for monitoring analytical stability. In our present investigation, we evaluate a datamining method for retrospective evaluation and assessment of analyte stability in whole blood. Results from the laboratory information system were used as the basis for the datamining approach. Blood tests were requested by the general practitioners and drawing of the blood sample was either at the general practitioner's or at the hospital outpatient clinics. We were able to split data into groups based on sample collection place and time to analysis. The datamining approach was compared to experiments where samples were incubated at a single temperature as well as an experiment where the temperatures were changed during incubation. To demonstrate the method, we selected three laboratory tests considered representative: potassium, phosphate, and lactate dehydrogenase. The datamining approach showed results similar to the reference experiment. Furthermore, our results show that the analytes phosphate and potassium were not stable after short storage at a lower temperature.

An LC-MS/MS assay for analysis of equilibrium angiotensin II in human serum.

The current first-line screening test for primary hyperaldosteronism is the plasma aldosterone:renin ratio; however, renin assays have several disadvantages and the ARR is affected by medications and physiological factors. Angiotensin II is a key biologically active hormone in the renin-angiotensin-aldosterone system. It has been suggested that measurement of equilibrium levels of this peptide, involving an in vitro incubation of serum prior to analysis, may provide a better marker of renin-angiotensin-aldosterone system activity than renin. An eqAng II LC-MS/MS assay was developed, optimized and validated. Serum samples were incubated at 37°C for 45 min prior to stabilization with cold EDTA solution, solid phase extraction and LC-MS/MS analysis. Stability in whole blood and the effect of cryoactivation were assessed. For comparison to the current screening test, 150 anonymized patients' samples were analysed for eqAng II, renin activity and aldosterone (all by LC-MS/MS). The assay had good precision, minimal bias and acceptable recovery. EqAng II did not change significantly when whole blood samples were stored for up to 72 h, and cryoactivation was only observed for pregnant patients. EqAng II was significantly correlated with renin, and the aldosterone:eqAng II ratio had a strong positive correlation with the aldosterone:renin ratio. An LC-MS/MS assay for eqAng II has been developed which shows promise as an alternative screening test for primary hyperaldosteronism. Compared to renin assays, it is quicker, simpler and less likely to be affected by anti-hypertensive medications. Further clinical validation in hypertensive patients would be required prior to implementation.

Development and Validation of a Bioanalytical LC-MS/MS Method for Simultaneous Determination of Sirolimus in Porcine Whole Blood and Lung Tissue and Pharmacokinetic Application with Coronary Stents.

Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and ascomycin (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 0.5–50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.

AT1R-Specific Ligand Angiotensin II as a Novel SPECT Agent for Hepatocellular Carcinoma Diagnosis.

Hepatocellular carcinoma (HCC) is characterized by a high mortality and early diagnosis and treatment are critically needed. Ang II type 1 receptor (AT1R) has recently emerged as a potential molecular target for cancer diagnosis and intervention. Here, we labeled angiotensin II (Ang II), an AT1R ligand that is overexpressed in various solid cancers, with the near-infrared fluorescent dye, MPA, and radionuclide technetium-99m, and evaluated its capacity for HCC detection. These analyses were done in vitro using HepG2 (AT1R-positive) and BxPC3 (AT1R-negative) cell lines, and in vivo using a subcutaneous and orthotopic xenograft mouse model by fluorescence and SPECT imaging. Both Ang II-PEG4-MPA- and [99mTc]Tc-HYNIC-PEG4-Ang II-bound AT1R exhibited a high affinity in vitro and [99mTc]Tc-HYNIC-PEG4-Ang II displayed an acceptable level of in vitro stability in rat plasma and whole blood. In vivo imaging revealed excellent specific tumor-targeting in HepG2 mouse xenografts models. In vitro and in vivo competition experiments revealed specific Ang II-PEG4-MPA and [99mTc]Tc-HYNIC-PEG4-Ang II uptake by HepG2 cells and tumors. Altogether, AT1R-positive tumors were successfully detected via fluorescence and SPECT imaging using Ang II-PEG4-MPA and [99mTc]Tc-HYNIC-PEG4-Ang II, respectively. Given their superior targeting capacity, Ang II-PEG4-MPA and [99mTc]Tc-HYNIC-PEG4-Ang II are promising tools for HCC detection and warrant clinical translation.

Stability of Whole Blood Lactate Specimens at Room Temperature Versus Slushed Ice Conditions.

There are limited data on lactate stability in whole blood. The purpose of this study was to determine whole blood lactate stability at room temperature and in slushed ice conditions. An equal number of arterial and venous samples were obtained from 202 subjects hospitalized for various pathophysiological conditions. Whole blood lactate concentration was measured over 5 different times spanning 80-90 min in a blood gas lab at a major hospital center. Samples were stored at room temperature (22-24°C) or in slushed ice conditions (0.1-0.2°C) before analysis. The mean increase in lactate concentration was 0.001 mmol/L/min in samples on slushed ice over 90 min. However, at room temperature conditions, the mean increase in lactate concentration was 0.008 mmol/L/min regardless of whether the sample was arterial or venous. An increase in whole blood lactate concentration of ≥ 0.4 mmol/L occured after 45 min at room temperature, with 5% of all whole blood specimens demonstrating a meaningful change at ≤ 20 min. The ≥ 0.4 mmol/L change in whole blood lactate is considered significant based on the College of American Pathologists instrument peer-group standards. Considering that a change in whole blood lactate concentration of ≥ 0.4 mmol/L is unacceptable instrument peer-group variation as defined by the College of American Pathologists, ice is no longer needed to stabilize whole blood lactate specimens when the draw time to analyze time is < 45 min. Samples remain stable even at 90 min when left on ice.