Executive Summary The Complete Blood Count (CBC) is considered one of the most commonly performed screening tests in medical diagnostics. The CBC is performed using a hematological analyzer, which counts the numbers and types of different cells within the blood. However, due to the high concentration of cells in the blood samples to be counted, the dilution process is considered an essential factor for obtaining accurate counting results. Below is an investigation for an invention of a unique method and device for blood sample dilution in hematology analyzers. As mentioned, before starting an analysis a hematology cell counter device must dilute a precise defined volume of a whole blood sample with a diluent reagent (in this study 20ml of a blood sample is diluted with 4,5ml of diluent reagent). This dilution process must be accurate and repeatable with a high precision to produce the target dilution ratio (here 1:200). Exclusive to almost all hematology analyzers today, shear values (SV) are used to obtain highly precise volumes. These SV components are however very costly and add a higher complexity to the systems. This thesis was therefore aimed as an experimental evaluation for a novel passive dilution tool called shearing block (SB), which could possibly then replace the SV and be used in the coming Haematology Analyzer Devices manufactured by Boule Medical AB. The SB has the advantages of being low cost, having a simple mechanism, being much more flexible for integration with any microfluidic system and also eliminates the need for complex control systems or equipment, thereby lowering the need for calibration and maintenance. If a SB could replace the SV with an equally precise accuracy on the 20ml blood volume it would be highly beneficial. The set hypothesis was that the dilution process via the SB, will only be affected by blood viscosity. Through changing the blood’s viscosity, via changing the hematocrit concentration (HCT) and blood sample temperature, this study aimed to conclude if this gave a significant effect on the blood sample dilution via the SB. This was achieved through two performed experiments both including the same control group (CG) along with a test group (TG). All tests were performed using the same blood sample, the same reference measuring device (Medonic M32) and experimental setup. The experimental setup included the control group, CG, (N=30) consisting of 20µl blood samples aspirated through an electronic pipette in room temperature condition being mixed with 4,5ml of diluent reagent that had been automatically dispensed by the Medonic M32 instrument. The 20µl blood together with the 4,5ml diluent resulted in the targeted 1:200 dilution ratio. The test group, TG, (where N=30 for each respective group) was diluted through the SB through a fully automated process for the targeted 1:200 dilution. All diluted blood samples were then analyzed on the Medonic M32 for all hematology parameters and the hemoglobin (HGB) parameter was used as an indicator to quantify the blood volume in the TG runs as compared to the CG reference runs. Two test groups (TG) were analyzed: the first investigating the effect of the HCT and the second of the sample temperature on the blood volume (before dilution) in the SB. The HCT concentration levels were tested at 15%, 27%, 33% and 58% with samples and test run and kept at a constant 25°C. The second test group investigated the effect of 15°C, 25°C, 35°C and 39°C on a blood sample with a constant HCT of 33%. The main aim of this experimental study was to validate the SB simulation. However, the experiments results, showed that the diluted blood volume via SB was strongly influenced by changes in hematocrit concentration and that the unwanted additional blood volume, was increasing directly proportionally with hematocrit concentration. On the other hand, there seemed to be no apparent change in the blood volume on the SB diluted samples for the various temperature differences between 15-35°C. To conclude, the experiments results were not consistent with predictions of SB simulation model and there are two reasons that could explain that. Firstly, in the simulation the blood was approximated with a homogeneous fluid with a given viscosity. The second reason is the mismatch between the dilution process via the SB and the simulation (the simulation started with an idealized initial status, whereas the SB channel was prefilled by blood). This means that the simulation excluded any effect that may occur as a convection effect during blood flow inside the channel and whilst encountering diluent reagent. These two reasons explain why the results of the simulation was not consistent with that of the experiments, regarding the unwanted blood volume. Therefore, a new simulation is required. Recommendations for future actions: undoubtedly there are several optimizations that may increase the accuracy of the proposed SB design such as; removing the diluent's reservoir for eliminating the effect of bubbles, changing the geometrical angles or use a smaller diameter for the inlets and outlets of the microchannels to reduce the convection and diffusion effect, (which in turn would reduce the unwanted blood volume). Therefore, determining the best SB's microchannel structure to perform the dilution process with minimum unwanted blood volume remains a near future next step follow-up project. / Abstract Föreliggande uppfinning avser en unik metod och anordning för blodprovspädning i hematologiska analysatorer. Innan en analys startas, måste en hematologicellräknare utspäda exakt definierad volym helblodsprov med ett utspädningsreagens. (i detta fall 20ml av ett blodprov med 4,5 ml utspädningsreagens). Denna spädnings process måste vara repeterbar med en extremt hög prestanda och spädningsratio (i detta fallet 1:200). I de flesta fall används så kallade vridventiler (”shear valves”) för att göra mycket exakta blod volymsbeskäringar. Dessa komponenter är i hematologisystemen dock oftast extremt kostsamma och utger en mycket högre komplexitet på systemet på många sätt. Denna avhandling var därmed en utredningsstudie för att undersöka en ny passiv avskavnings mekanism och metod så kallad ”shear block”, (SB) som skulle kunna ersätta nuvarande vridventiler i Boules nuvarande hematologisystem. Denna metod har många fördelar så som lägre kostnad, simplifierad mekanism, flexibilitet inför integration med microfluidiska system samt att färre komplexa kontrollsystem och utrustning vilket minskar kalibrering och underhållsbehov. Hypotesen inför denna studie är därmed att Shear Block spädningsmetoden endast påverkas av blodets viskositet. Genom förändringar av blodets viskositet, via hematokrit- och temperaturändringar, försökt påvisa om det skapar en signifikant effekt på spädnings processen i SB. Detta testades genom två experiment som vardera innehöll två testgrupper: en kontrollgrupp (KG) och en testgrupp (TG). Alla tester genomfördes med samma blodprov, mätdonsinstrument (Medonic M32) och test uppsättning. I testuppsättningen var KG (N=30) 20ul uppmätta blodprover som med hjälp av en elektronisk pipette aspirerats under rumstemperatur innan det sedan blandats med 4,5ml spädningsreagens som automatiskt dispenseras från Medonic M32 instrumentet. Denna 20ul blod med 4,5ml reagens skapar den 1:200 spädningsratio. TG (var N=30 för varje respektive testgrupp) var spätt genom SB med en fullt automatiserad process för den 1:200 spädningen. Alla spädda prover var sedan analyserade på Medonic M32 för alla hematologiparametrar och HGB värdet användes som en indikator för att kvantifiera spädningsprovernas blodvolym i TG körningarna med KG värden som referens. TG bestod av två grupper: Den första undersökte påverkan av HCT och den andra temperaturen på blodets volym, innan spädningen, i SB. HCT nivåer på 15, 27, 33 samt 58 % testades först under en konstant 25°C. Andra testgruppen undersökte sedan effekten av 15, 25, 35 och 39°C på ett prov med HCT=33%. Poängen med dessa TG var att validera SB funktionen. Utfallet visade dock att blodprover spädda genom SB var högst påverkade av HCT koncentrationen och därmed den oönskade extrablodvolymen var direkt proportionerlig med den ökade HCT koncentrationen. Däremot var det ingen volympåverkan på prover spädda via SB med temperaturskillnader mellan 15 till 35°C. Sammanfattningsvis var tyvärr inte experimentets resultat konsekventa med våra förutsägelser för SB simulatorn då SB inte innefattade och tog hänsyn till hela spädningsprocessen.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:kth-223711 |
| Date | January 2017 |
| Creators | Alsaeede, Mustafa |
| Publisher | KTH, Medicinteknik och hälsosystem |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | TRITA-STH ; 2017:99 |
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