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Detektion av fetala erytrocyter med flödescytometri och fetalt hemoglobin med spektrofotometri
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
2018 (Swedish)Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
Abstract [sv]

Fetomaternell blödning (”Fetomaternal hemorrhage”, FMH) innebär förekomst av fetalt blod i maternellt blod. FMH hos RhD-negativa kvinnor med RhD-positiva foster kan ge upphov till immunisering. Massiv FMH kan leda till allvarliga konsekvenser för fostret. Flödescytometriska metoder används oftast för att kvantifiera FMH. Denna studie syftade till att bestämma detektionsgränsen för analys av fetala erytrocyter med Fetal Cell CountTM kit vilket bygger på en kombination av antikroppar mot fetalt hemoglobin och antikroppar mot karbanhydras. Vuxenblod spetsat med olika mängder navelsträngsblod preparerades med Fetal Cell CountTM kit och analyserades med 200 000 händelser på flödescytometern Navios. Resultatet av sex serier av spetsade prover visade att CV-värdena vid koncentrationerna 0,014% och 0,045% var 17,69% respektive 8,41%. Resultatet av upprepade mätningar av ett 0,045% spetsat prov visade att CV-värdet var 8,59% och medelvärdet var 0,045%. Resultaten indikerar att precisionen och riktigheten var bra vid koncentrationen 0,045%, vilket indikerar metodens detektionsgräns vid 0,045%. FMH kan även påvisas genom att mäta fetalt hemoglobin (HbF) i ett hemolysat. Analys av HbF på spetsade prover med blodgasinstrumentet ABL 835 utfördes i denna studie för att ta reda på metodens detektionsgräns. Resultatet visade att ju lägre koncentrationen av HbF, desto sämre precision. Vid de lägsta koncentrationerna som kunde detekteras var CV-värdena höga, upp till 34%. En mycket grovt uppskattad detektionsgräns var 5-10%. Slutsatserna var att detektionsgränsen för analys av fetala erytrocyter med Fetal Cell CountTM kit var 0,045% vid analys av 200 000 händelser; detektionsgränsen för analys av HbF med blodgasinstrumentet är 5-10%. 

Abstract [en]

Fetomaternal hemorrhage (FMH) refers to the entry of fetal blood into the maternal circulation. FMH can lead to give rise to immunization when an RhD-negative mother has an RhD-positive fetus. Massive FMH can lead to devastating consequences for the fetus. Flow cytometric methods are usually used to quantify FMH. The objective of this study was to obtain the limit of detection for the analysis of fetal erythrocytes using Fetal Cell CountTM kit which is based on a combination of antibodies against fetal hemoglobin and antibodies against carbonic anhydrase. Adult blood samples spiked with various amount of cord blood were treated with Fetal Cell CountTM kit and analyzed with Navio flow cytometer with counting of 200 000 events. The results of six series of spiked samples showed that coefficient of variation (CV) obtained at concentration (conc.) 0014% and 0.045% were 17.69% and 8.41%, respectively. The results of repeated measurement of a 0.045% spiked sample showed that the CV value was 8.59% and the mean value was 0.045%. These results indicate that the accuracy and precision are good at the concentration of 0.045%. FMH can be qualified by measuring the fetal hemoglobin (HbF) in a red cell lysate. Analysis of HbF in spiked samples using the blood gas instrument ABL835 was carried out in order to figure out the limit of detection. The results showed that the lower the conc. HbF was, the worse the precision was; at the lowest concentrations which could be detected, the CV values were high, up to 34%. The limit of detection is likely between 5% and 10% HbF av total hemoglobin. The conclusions were that the limit of detection for analysis of fetal cell using Fetal Cell CountTM kit was 0.045% fetal erythrocytes for 200,000 events; the limit of detection for HbF analysis using the blood gas instrument was 5-10% HbF.

Place, publisher, year, edition, pages
2018. , p. 20
Keywords [sv]
Fetala erytrocyter, fetomaternell blödning, fetalt hemoglobin, F-celler, flödescytometri, blodgasinstrument
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-76392OAI: oai:DiVA.org:lnu-76392DiVA, id: diva2:1223593
External cooperation
Blekingesjukhuset i Karlskrona
Educational program
Biomedical Laboratory Science Programme, 180 credits
Supervisors
Examiners
Available from: 2018-06-26 Created: 2018-06-25 Last updated: 2018-06-26Bibliographically approved

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