Tillgängligheten av blod kan vara livsavgörande för många personer, därför är blodgivning en viktig del av vården. För blodgivare kan en regelbunden blodgivning påverka kroppens järndepåer vilket på sikt kan leda till en järnbrist. Syftet med denna studie var att kartlägga blodgivares järndepåer i Region Jönköpings län genom att (i) utvärdera antalet och andelen nyanmälningar som ej uppfyllt godkänd S-ferritinnivå för blodgivning, (ii) jämföra nivåerna av S-ferritin vid nyregistrering och vid upprepad blodgivning, (iii) utvärdera intag av järnprofylax hos blodgivare, (iv) jämföra antalet, andelen samt orsaken till kontrollprovtagningar före och efter S-ferritin infördes som provtagningsrutin. Data insamlades och erhölls från Blodcentralen, Jönköping, Sverige, och analyserades statistiskt. Resultatet visade att fler kvinnor än män ej blev godkända som blodgivare på grund av S-ferritin <22 µg/L. Genom att sänka S-ferritinnivån kan fler kvinnor godkännas för blodgivning. Vidare visade studien att upprepad blodgivning leder till minskade järndepåer. Grupperna som erbjudits järnprofylax hade stabilare S-ferritin över tid vilket antyder att alla blodgivare skulle gynnas av järnprofylax, men vidare studier krävs. Majoriteten av blodgivare med S-ferritin ≤100 µg/L intar erbjudna järntabletter vilket visade att blodgivarna möjliggjorde fortsatt blodgivning. Slutligen visade studien att sedan S-ferritin infördes som provtagningsrutin 2017 har kontrollprovtagningar på grund av lågt B-Hb minskat, vilket visar att risken för att utveckla en järnbrist på grund av upprepad blodgivning har minskat i Region Jönköpings län. / The availability of blood could be crucial for many people; blood donation is, therefore, a vital part of healthcare. Continuous blood donation could affect the blood donors’ iron depots which could lead to an iron deficiency. The aim of the present study was to evaluate and highlight iron depots amongst blood donors in Jönköping County by (i) evaluating the number and proportion of new applicants which did not meet the approved S-ferritin levels for blood donation, (ii) compare the levels of S-ferritin in new registrations and repeated blood donations, (iii) evaluate intake of iron supplements amongst blood donors, (iv) compare the number, proportion and cause for control sampling before and after S-ferritin was introduced as a sampling routine. Data was collected from the Department of Transfusion Medicine, Jönköping, Sweden, and analysed statistically.The result showed that more women than men were not approved for blood donation because of S-ferritin <22 µg/L. By lowering S-ferritin, additional women will be approved for blood donation. Further, the study showed that continuous blood donation leads to decreased iron depots. Groups that were offered iron supplements showed more stable S-ferritin levels over time which implied that all blood donors would benefit from iron supplements, but further studies are required. The majority of the blood donors with S-ferritin ≤100 µg/L were taking their offered iron supplements which showed that the blood donors enabled future blood donations. Lastly, since S-ferritin was introduced as a sampling routine in 2017 the amount of control samplings due to low B-Hb has decreased. This shows that the risk to develop an iron deficiency due to continuous blood donation has decreased in Jönköping County.

Complete blood count (CBC) analysis, often provided by an automated hematology analyzer, is a fundamental diagnostic test for evaluating a patient’s overall health status as a tool in diagnosing various medical conditions. CBC provides insights into the composition of the blood cells with parameters such as Mean Corpuscular Volume (MCV) and Hemoglobin (HGB). MCV represents the average size and volume of red blood cells, while HGB indicates the oxygen-carrying capacity of the blood. Different technologies are used in hematology analyzers, such as the impedance method and spectrophotometry, to achieve precise measurements of MCV and HGB. However, exploring other methodologies is of interest to potentially reduce instrument complexity and cost. Flow cytometry, based on light scatter, provides detailed information on the characteristics of individual cells and is commonly used in CBC analysis to differentiate white blood cells and reticulocytes. However, while the potential of this method for investigating MCV and HGB levels is well established, it is of significant interest to determine if the measurement techniques can be streamlined from three to one by solely using flow cytometry in this prototype analyzer. In this thesis, the possible use of measuring MCV and HGB using a flow cytometry system based on optical scatter data from a prototype hematology analyzer has been examined. The need to sphere the red blood cells before the measurements have also been investigated to evaluate reagent needs. The results have been evaluated based on the correlation factor, accuracy and precision of the proposed optical method. It is shown that the optical method in this thesis, can be used to measure MCV and HGB. However, the necessity of sphering the cells remains. Furthermore, a comparison is made between the optical method and the Sysmex XN-1000 to evaluate the accuracy of the obtained values. Finally, possible improvements and future work are suggested. / Blodstatus, utförd av automatiserade hematologiinstrument, är ett grundlägggande diagnostiskt test för att utvärdera en patients allmänna hälsotillstånd som ett verktyg för att diagnostisera olika medicinska tillsånd. Blodstatus ger inblick i blodets sammansättning med parameterar som Medelvolym av röda blodkroppar (MCV) och Hemoglobin (HGB). MCV representerar den genomsnittliga storleken och volymen av röda blodkroppar, medan HGB indikerar blodets syrebärande förmåga. Olika metoder används i hematologiinstrument, såsom impedansmetoden och spektrofotometri för att mäta MCV och HGB värden. Undersökning med hjälp av andra metoder för mätning av dessa parameter är dock utav intresse för att potentiellt minska instrument komplexitet och kostnader. Flödescytometri, baserad på ljusspridning, ger detaljerad information om egenskaperna hos enskilda celler och används ofta för att differentiera vita blodkroppar och reticulocyter. Även om potentialen att undersöka MCV- och HGB-nivåer med denna metod redan är väletablerad, är det av betydande intresse att undersöka om man kan minska mätmetoderna från tre till en, genom att enbart använda flödescytometri i detta prototypinstrument. I detta examensarbete har möjligheten att mäta MCV och HGB med hjälp av flödescytometri baserat på optisk spridningsdata från en prototyp hematologiinstrument undersökts. Behovet av att ”sfära” de röda blodkropparna innan mätningarna har också undersökts för att utvärdera reagensbehov. Resultaten har utvärderats utifrån den förslagna optiska metodens korrelationsfaktor, noggrannhet och precision. Resultatet visar att den optiska metoden som presenteras i denna uppsats, kan användas för att mäta MCV och HGB. Dock kvarstår behovet av att ”sfära” cellerna. Dessutom görs en jämförelse mellan den optiska metoden och Sysmex XN-1000 för att utvärdera noggrannheten hos de erhållna värdena. Till sist ges förslag på möjliga förbättringar och vidareutveckling av den optiska metoden för framtiden.

Master of Science / Department of Kinesiology / Thomas J. Barstow / Near infrared spectroscopy is currently routinely used to assess tissue (muscle) oxygenation at rest and during exercise. While most investigators assume that hemoglobin ([Hb]) is the major contributor to the responses seen during exercise, the relative contribution of myoglobin ([Mb]) to the NIRS signals remains controversial. PURPOSE: a) To calculate the range of light absorbing potential (LAP) of hemoglobin and myoglobin in mammalian skeletal muscle at rest based on analysis of published chemical and morphometric data in humans and other mammals (Part 1), and b) use the information in a) to interpret changes in total [Hb+Mb] from NIRS during exercise (Part 2). METHODS: Part 1: Information was retrieved from five published studies with regard to capillary density (#caps/mm2) and [Mb] in skeletal muscle of human, horse and rat. Preference was given to studies in which both measurements were provided for the same muscles. [Hb] in skeletal muscle was estimated as a function of capillary density, [Hb] in systemic blood, and the ratio of capillary-to-systemic hematocrit at rest and during exercise. Part 2: Changes in total [Hb] + [Mb] (as t[Hb+Mb]) from published NIRS data obtained from human subjects performing cycling or knee extension exercise were interpreted in the context of the results of Part 1. RESULTS: Part 1: Individual group mean values for skeletal muscle [Mb] in the literature ranged from 0.25-0.67 mM in human samples, with a similar range for muscles of the rat hindlimb; horse limb muscles tended to be higher (up to 1.0 mM). Capillary densities ranged from ~200 to 600 caps/mm2 in human and rat muscles, and up to 800 caps/mm2 in horse muscle. Assuming a resting capillary hematocrit of 22% and 4 fold greater LAP for each mole [Hb] vs [Mb], the resulting estimation of capillary [Hb] ranged from ~0.03 to 0.09 mM in human and rat muscles, and up to ~0.13 mM in horse muscles. The results suggest that [Mb] could contribute ~50-70% of the total LAP at rest in human skeletal muscle. Part 2: With exercise, total heme by NIRS can increase ≥ 30% in individual human subjects. Assuming this increase reflects only increased [Hb], this fits well with the observed increase in capillary hematocrit with exercise. CONCLUSIONS: 1) In skeletal muscle at rest, [Mb] is likely to be at least as significant a light absorbing heme as is [Hb] in most mammalian muscles, including the human leg. 2) Observed increases in t[Hb+Mb] with NIRS during exercise can be explained by an increase in capillary hematocrit, even in the presence of significant [Mb].

Dried blood spots is an alternative method to collect blood samples from research subjects. However, little is known about how hemoglobin and hematocrit affect bead-based multiplex assay performance. The purpose of this study was to determine how bead-based multiplex assays perform when analyzing dried blood spot samples. A series of four experiments outline the study each with a specific purpose. A total of 167 subject samples were collected and 92 different biomarkers were measured. Median fluorescence intensity results show a positive correlation between filtered and non-filtered samples. Utilizing a smaller quantity of sample results in a positive correlation to a larger sample. Removal of hemoglobin from the dried blood spot sample does not increase detection or concentration of biomarkers. Of the 92 different biomarkers measured 56 were detectable in 100-75% of the attempted samples. We conclude that blood biomarkers can be detected using bead-based multiplex assays. In addition, it is possible to utilize a smaller quantity of sample while avoiding the use of the entire sample, and maintaining a correlation to the total sample. While our method of hemoglobin was efficient it also removed the biomarkers we wished to analyze. Thus, an alternative method is necessary to determine if removing hemoglobin increases concentration of biomarkers. More research is necessary to determine if the biomarkers measured in this study can be measured over time or within an experimental model.

Prior work in our lab has indicated that reduced Hydroxocobalamin (B12r) can be added to human blood and is able to convert carbon monoxide (CO) into carbon dioxide. This has great potential as a direct antidote to mitigate the toxic effects of CO poisoning which is a public health risk. In the first part of our work, we use highly specific wavelengths of light and Raman spectroscopy to study changes in Carboxyhemoglobin (COHb) between blood treated with oxygen and blood treated with oxygen and B12r in a flowing circuit of blood. Using Raman spectroscopy, we found that the addition of B12r hastens the conversion of the COHb Raman signals to Oxyhemoglobin (HbO2) Raman signals. In addition, the B12r absorbance of light energy within the Raman spectrum is an exploitable relationship that can be used to measure B12r presence in the blood. In part two of our study we focused on the neurobehavioral testing of rats injured by CO exposure, however, we were not able to find statistical differences in the behavioral tests between exposed and unexposed rats.

The purpose of this study was to identify the effects of a hemoglobin-based oxygen carrier, HBOC-201, on the cardiovascular system. Systemic cardiovascular parameters of mean arterial pressure (MAP), pulse pressure, heart rate, and oxygen saturation, as well as vascular resistance, were examined. A murine model of the cardiovascular system and microvasculature was employed. Sprague-Dawley rats (male; 230-530g; N = 13) were anaesthetised and surgically prepared for intravital microscopy of the spinotrapezius muscle. Increasing doses of HBOC-201 (2 mg/kg, 22 mg/kg, 230 mg/kg, and 780 mg/kg) and an iso-oncotic volume control were administered to assess for a dose-response relationship. MAP displayed a significant increase from baseline for both treatment groups, with no significant difference between the two. Arteriolar diameter displayed no changes from baseline, or between treatment groups or across doses. Based on these results, the noted changes in MAP were due to hypervolemia, and not a property of HBOC-201, itself.

Based upon experimental evidence from the 1970’s we proposed that a reduced form of hydroxocobalamin should be capable of producing carbon dioxide (CO2) from carbon monoxide (CO) in blood, and that this conversion should be detectable. Using resonance raman spectroscopy we demonstrated that a mixture of hydroxocobalamin and ascorbic acid could create the reduced form of hydroxocobalamin. We used a closed-loop circulation system with a hollow-fiber membrane oxygenator to produce carboxyhemoglobin. Using sensitive gas monitoring equipment to the gas-out port of the oxygenator we analyzed the CO and CO2 concentrations coming from the oxygenator. The mixture of hydroxocobalamin and ascorbic acid caused a 5-fold increase in the CO2 concentration of the gas-out flow, in comparison to baseline and negative controls. These findings offer initial support for the potential use of a mixture of hydroxocobalamin and ascorbic acid as an injectable antidote for carbon monoxide poisoning.

In the US today, blood transfusion is safer than ever. Nevertheless, the century-old quest for a suitable blood substitute persists. The elimination of unwanted side effects, especially transfusion-transmitted diseases, the problems and high cost factor involved in collecting and storing human blood, the pending worldwide shortages, and the need for compatibility testing are the driving forces contributing towards the development of blood substitutes. The leading research is focusing on hemoglobin-based oxygen carriers (HBOCs), which are limited in clinical application due to the pressor effect they induce. In this study, the mechanisms through which HBOCs affect mean arterial pressure (MAP), arteriolar diameter, and nitric oxide levels in the microcirculation were investigated, using Oxyglobin (HBOC-301), a third generation glutaraldehyde-polymerized bovine hemoglobin. The spinotrapezius muscle of female Sprague-Dawley rats was exteriorized for microcirculatory observations. HBOC in doses of 0.1, 1.0, 10.0, and 100.0 μM i.v., LNAME (30 mg/kg, i.v.), and papaverine (100 μM, topically) were given to the rat. Heparinized saline (0.1 ml and 0.5 ml, i.v.) served as control. MAP was monitored continuously through a cannula in the right carotid artery. Images of the feed, arcade and transverse arterioles were captured using a Zeiss Axioplan microscope, equipped with a digital camera, and imaging software. All doses of HBOC produced an overall vasoconstriction of the arterioles leading to an elevated MAP. Following L-NAME pretreatment, HBOC administration alone and with papaverine produced no significant elevation in MAP, indicating that the increase in resistance required basal amounts of nitric oxide (NO). This study concludes that the constriction of the arterioles correlated with the level of hypertension, and that these effects occur in a dose-dependent manner as a consequence of NO scavenging.

A proteólise limitada de certas proteínas leva à liberação de peptídeos opióides endógenos. Vários relatos apontam que peptídeos derivados da hemoglobina como hemorfinas e hemopressinas têm efeito antinociceptivo, pela atividade de modulação de receptores acoplados a proteínas G. No presente estudo, um ensaio de captura do substrato (ECS) foi combinado com a marcação isotópica e LC-MS/MS para identificar e caracterizar um novo fragmento da hemoglobina que se liga à EP24.15. O peptídeo AGH, identificado neste trabalho, inibe respostas de hipernocicepção periféricas através de receptores opióides do tipo <font face=\"Symbol\">m . A persistência do peptídeo AGH no tecido nervoso perfundido sugere relevância fisiológica. Embora o AGH seja derivado de hemoglobina e tenha atividade opióide, falta-lhe a sequência chave das hemorfinas (YPWT), indicando que ele pode pertencer a uma nova classe de peptídeos derivados da hemoglobina. Adicionalmente, o AGH modula as interações entre as proteínas 14-3-3<font face=\"Symbol\">e e EP24.15 in vitro, podendo estar relacionado com a secreção não convencional da EP24.15. / Limited proteolysis of certain proteins leads to the release of endogenous opioid peptides. Several reports have shown that hemoglobin-derived peptides such as hemorphins and hemopressins have an antinociceptive effect by modulating GPCR activity. In the present study, a substrate capture assay (SCA) was combined with isotopic labeling and LC-MS/MS to identify and characterize a new bioactive hemoglobin fragment that binds to EP24.15. AGH, a new peptide identified in this work, inhibits peripheral hyperalgesic responses through <font face=\"Symbol\">m opioid receptors (MOR). The persistence of AGH peptide in perfused nervous tissue suggests its physiological relevance. Although AGH is derived from hemoglobin and it is a peptide with opioid activity, it lacks the key sequence of hemorphins (YPWT), indicating that it is part of a new class of peptides derived from hemoglobin. Additionally, the AGH modulates interactions between 14-3-3<font face=\"Symbol\">e and EP24.15 proteins in vitro and may be related to the unconventional EP24.15 secretion.

Neste trabalho, analisamos a interação de pequenas moléculas, marcadores hidrofóbicos, com hemoglobina, em diferentes estados: monocristal, pó e solução aquosa. Os métodos de análise empregados, são baseados nas teorias de relaxação em líquidos e técnicas não lineares de ressonância ST-RPE (transferência de saturação), fornecendo informações sobre mudanças locais nas vizinhanças desses marcadores. Um dos marcadores hidrofóbicos, o TEMPO, mostrou uma anisotropia considerável nos espectros de RPE do monocristal de hemoglobina que está relacionado com o empacotamento molecular da proteína do cristal. A associação desses métodos de análise conduziu a importantes informações sobre mudanças na camada de hidratação em várias proteínas: hemoglobina, mioglobina e lisozima, monitoradas pelo marcador covalente derivado da maleimida, e induzidas pela temperatura, sob diferentes condições de hidratação. Desta forma o uso da espectroscopia de RPE especialmente com simulação espectral e DT-RPE mostro ser um método potente e ainda não explorado no estado de hidratação de proteínas / In this work, the interactions of small molecules, hydrophobic spin labels, with hemoglobin, under different states was analysed: single crystal, powder and aqueous solution. The methods of analysis employed, are based in relaxation theory in liquids and non-linear techniques, saturation transfer (ST-EPR), giving informations on the local changes in neighbourhood of the labels. One of the hydrophobic labels, TEMPO, showed a considerable anisotropy in the hemoglobin single crystal spectra, a result related to the molecular packing in the protein single crystal. The association of the techniques of analysis all together lead to important informations an temperature induced changes in the hydration layer in several proteins: hemoglobin, myoglobin, NEM*, a maleimide derivated, in different conditions of hydration. In this way the use of EPR spectroscopy and particularly with spectral simulations and ST-EPR, proved to be a powerful and not yet very much explored method to study the problem of protein hydration