The measurement of free hemoglobin (free Hb) in blood is crucial for assessing the risk of organ damage in patients with hemolytic diseases. However, the colorimetric method, commonly used in clinical practice, does not distinguish between free Hb and the hemoglobin-haptoglobin complex (Hb-Hp) in the blood, instead reflecting the total Hb level. Although size-exclusion high-performance liquid chromatography (SEC-HPLC) can specifically measure free Hb, its clinical use is limited by long assay times. Here, we developed a novel assay method for the rapid quantification of free Hb in serum, distinguishing it from Hb-Hp, using a latex agglutination immunoturbidimetric assay (LATIA). This method could be used to measure free Hb in sera in the range of 1-100 μg/mL in approximately 15 min using an automatic biochemistry analyzer. Using Hb-spiked serum samples from healthy adults, there was a high correlation with Hb levels determined using the newly developed method and SEC-HPLC, indicating a high specificity for free Hb. This novel assay can be used to monitor levels of free Hb in patients with various hemolytic diseases and to design therapeutic strategies based on measured values. However, further studies are required to assess its clinical performance.

Removing free hemoglobin generated during extracorporeal circulation remains a challenge. Currently, there is no adsorbent with specificity and good biosafety for removing hemoglobin. In this study, a new chitosan/sodium alginate/carbon nitride (CS/SA/C3N4) hydrogel adsorbent was prepared by blending SA with C3N4 to drop into CS/CaCl2 solution. The physicochemical properties of CS/SA/C3N4 hydrogel were evaluated using some techniques, including scanning electron microscope, Zeta potential measurement, and thermogravimetric analysis. Hemoglobin adsorption in vitro, stability, hemocompatibility, cell compatibility, inflammatory reaction and blood extracorporeal circulation in vivo were also evaluated. The findings revealed that the CS/SA/C3N4-0.4 % hydrogel exhibited an impressive adsorption capacity of 142.35 mg/g for hemoglobin. The kinetic data of hemoglobin adsorption were well-described by pseudo second-order model, while the isothermal model data conformed to the Langmuir model. The hardness and modulus of CS/SA/C3N4-0.4 % was 11.7 KPa and 94.66 KPa respectively, which indicated robust resistance to breakage. CS/SA/C3N4 demonstrated excellent hemocompatibility, biocompatibility and anti-inflammatory properties. In addition, the results of in vivo rabbit extracorporeal blood circulation experiment demonstrated that CS/SA/C3N4 could adsorb free hemoglobin from blood while maintaining high biosafety standard. Consequently, CS/SA/C3N4 hydrogel emerges as a promising candidate for use as a hemoglobin adsorbent in extracorporeal blood circulation system.

Gemtuzumab ozogamicin (GO) is a CD33 monoclonal antibody-drug conjugate currently in use to treat myeloid malignancies. A unique adverse effect of this medication is destruction of CD33 positive macrophages resulting in reduced clearance of free hemoglobin leading to grossly red plasma. This build-up of free hemoglobin can potentially lead to end organ damage and prevent performance of clinically necessary laboratory evaluation. We present a case of a pediatric patient who developed this adverse effect and was successfully treated with therapeutic plasma exchange (TPE). We also present results from a systematic review of the medical literature and share data from a query of the United States Food and Drug Administration (FDA) Adverse Event Reporting system for GO-related hemoglobin scavenging impairment. Among reported cases, patients undergoing TPE and those receiving steroids had improved outcomes. Practitioners should be aware of this rare drug side-effect and the potential utility of TPE for these patients.

BACKGROUND: Methods based on paper-based analytical devices (PAD) and smartphone photographic colorimetric detection have become representative instrument-independent point-of-care testing (POCT) platforms due to their low cost and simplicity. However, the detection of target components from whole blood sample still presents challenges in terms of field preparation of small amounts of blood sample and detection sensitivity. This paper presents a rapid online processing method for whole blood samples on PAD based on plasma separation membrane (PSM), and combined with electrokinetic stacking and selective chromatic reaction. Real-time smartphone-based colorimetric detection of free hemoglobin (FHb) and human serum albumin (HSA) was successfully demonstrated.
RESULTS: With the proposed method, both detections for low and high concentration analytes could be implemented. The limits of detection of 16.6 mg L-1 for FHb and 0.67 g L-1 for HSA were obtained, respectively, with RSD below 8 %. The reliability of the method was verified by the recovery test and desktop spectrophotometric method. The detection results for real blood samples were in agreement with that by clinical methods.
UNASSIGNED: The PAD method is inexpensive, simple and fast, and detection of a whole blood sample of 5 μL can be finished in 5 min. This work shows that POCT of biomarkers from whole blood with PAD is possible without using any desktop facilities.

Background: The authors report the relevance of using a point of care test (Helge®) for free hemoglobin determination and concordance of the values the with Cobas® 8000 and spectrophotometer methods. Results: The within-run of the point of care test was <3%. Good correlations among the three methods were observed and an acceptable concordance for hemolysis index values from 50 mg/dl. An excellent agreement between the Cobas 8000 and the spectrophotometer was found. Conclusion: Automated methods represent methods of choice for free hemoglobin determination. An advantage of the Helge system is that it can be applied to samples experiencing a delay in evaluation due to the long distance between the collection site and the central laboratory. Another advantage is its use at the bedside, in the monitoring of extracorporeal membrane oxygenation patients.

Neuron-specific enolase (NSE) is one of the biomarkers used as an indicator of brain disorder, but since it is also found in blood cell components, there is a concern that a spurious increase in NSE may occur after cardiovascular surgery, where cardiopulmonary bypass (CPB) causes hemolysis. In the present study, we investigated the relationship between the degree of hemolysis and NSE after cardiovascular surgery and the usefulness of immediate postoperative NSE values in the diagnosis of brain disorder. A retrospective study of 198 patients who underwent surgery with CPB in the period from May 2019 to May 2021 was conducted. Postoperative NSE levels and Free hemoglobin (F-Hb) levels were compared in both groups. In addition, to verify the relationship between hemolysis and NSE, we examined the correlation between F-Hb levels and NSE levels. We also examined whether different surgical procedures could produce an association between hemolysis and NSE. Among 198 patients, 20 had postoperative stroke (Group S) and 178 had no postoperative stroke (Group U). There was no significant difference in postoperative NSE levels and F-Hb levels between Group S and Group U (p = 0.264, p = 0.064 respectively). F-Hb and NSE were weakly correlated (r = 0.29. p < 0.01). In conclusion, NSE level immediately after cardiac surgery with CPB is modified by hemolysis rather than brain injury, therefore it would be unreliable as a biomarker of brain disorder.

Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.

BACKGROUND: Ex situliver machine perfusion at subnormothermic/normothermic temperature isincreasingly applied in the field of transplantation to store and evaluateorgans on the machine prior transplantation. Currently, various perfusionconcepts are in clinical and preclinical applications. Over the last 6 years ina multidisciplinary team, a novel blood based perfusion technology wasdeveloped to keep a liver alive and metabolically active outside of the bodyfor at least one week.
METHODS: Within thismanuscript, we present and compare three scenarios (Group 1, 2 and 3) we werefacing during our research and development (R&D) process, mainly linked tothe measurement of free hemoglobin and lactate in the blood based perfusate. Apartfrom their proven value in liver viability assessment (ex situ), these twoparameters are also helpful in R&D of a long-term liver perfusion machine and moreover supportive in the biomedical engineering process.
RESULTS: Group 1 (\"good\" liver on the perfusion machine) represents the best liver clearance capacity for lactate and free hemoglobin wehave observed. In contrast to Group 2 (\"poor\" liver on the perfusion machine), that has shown the worst clearance capacity for free hemoglobin. Astonishingly,also for Group 2, lactate is cleared till the first day of perfusion andafterwards, rising lactate values are detected due to the poor quality of theliver. These two perfusate parametersclearly highlight the impact of the organ quality/viability on the perfusion process. Whereas Group 3 is a perfusion utilizing a blood loop only (without a liver).
CONCLUSIONS: Knowing the feasible ranges (upper- and lower bound) and the courseover time of free hemoglobin and lactate is helpful to evaluate the quality ofthe organ perfusion itself and the maturity of the developed perfusion device. Freehemoglobin in the perfusate is linked to the rate of hemolysis that indicates how optimizing (gentle blood handling, minimizing hemolysis) the perfusion machine actually is. Generally, a reduced lactate clearancecapacity can be an indication for technical problems linked to the blood supplyof the liver and therefore helps to monitor the perfusion experiments.Moreover, the possibility is given to compare, evaluate and optimize developed liverperfusion systems based on the given ranges for these two parameters. Otherresearch groups can compare/quantify their perfusate (blood) parameters withthe ones in this manuscript. The presented data, findings and recommendations willfinally support other researchers in developing their own perfusion machine ormodifying commercially availableperfusion devices according to their needs.

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Blood transfusions are mainly given to intensive care patients; therefore, additional complications that could arise from storage lesions in preserved blood should be avoided. It has been shown that human stored red blood cells are subject to changes that are considered to be a number of interdependent processes involving metabolic disarrangement and oxidative stress. The aim of our study was to determine alterations in selected hematological and biochemical parameters and to assess whether and when oxidative stress is a significant phenomenon in stored dog CPDA-1 whole blood. Ten ½ unit bags of whole blood donated from dogs and preserved with CPDA-1 (anticoagulant containing citrate, phosphate, dextrose and adenine) were stored for 5 weeks. Each week, a 9 ml sample was drawn aseptically to measure hematological parameters, selected metabolites, free hemoglobin content, osmotic fragility, antioxidant enzyme activity, total antioxidant capacity, malondialdehyde concentration and protein carbonyl content.The results revealed an MCV decrease in the first week of storage and then a gradual increase; osmotic fragility decreased at that time and remained low throughout the study period. Leukodepletion became significant in the fourth week of storage. The free hemoglobin concentration continuously increased, with the greatest changes observed in the last two weeks of storage. The total antioxidant capacity changed in a reverse manner. Superoxide dismutase and glutathione peroxidase activities decreased from week 0 to week 3, and catalase activity tended to decrease over time. The highest malondialdehyde concentrations in blood supernatant were measured in the first week of storage, and the carbonyl concentration increased after 35 days.Hematological changes and oxidative stress are already present in the first week of storage, resulting in depletion of the antioxidant system and subsequent accumulation of oxidation products as well as erythrocyte hemolysis, which are most pronounced at the end of the storage period.