The effect of common anticoagulants in detection and quantification of malaria parasitemia in human red blood cells by ATR-FTIR spectroscopy

Abstract

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) has the potential to become a new diagnostic tool for malaria and other diseases. For point-of-care testing, the use of ATR-FTIR in malaria diagnosis enables the analysis of blood in the aqueous state, which represents an enormous advantage by minimising the sample preparation by removing the need for cell fixation. Here we report the quantification of malaria parasitemia in human RBCs in their normal physiological aqueous state. A potential confounding variable for spectroscopic measurements performed on blood are the various anticoagulants that are required to prevent clotting. Accordingly, we tested the effects of 3 common anticoagulants; Sodium Citrate (SC), Potassium Ethylenediaminetetraacetic Acid (EDTA) and lithium heparin on plasma and whole blood in the aqueous and dry phase. Principal Component Analysis (PCA) revealed the model was heavily influenced by the anticoagulants in the case of dry samples, however, in aqueous whole blood samples, the effect was less pronounced as the water in the sample presumably diluted the amount of anticoagulant in contact with the ATR crystal. The possible influence of the anticoagulant effect on the ability to quantify parasitemia levels was tested using Partial Least Squares Regression Analysis (PLS-R). There was no influence of anticoagulants on quantification in the 0-1% range, however attempts to quantify at lower levels (0-0.1%) was best achieved with heparin compared to the other two anticoagulants. The results demonstrate ability to diagnose malaria using ATR-FTIR spectroscopy using wet RBC samples as well as underscoring the desirability to perform wet measurements as these minimise the possible confounding influence of anticoagulants used in blood collection.

Investigating pre‐analytical requirements for serum and plasma based infrared spectro‐diagnostic

“…An ATR‐FTIR study has reported a significant contribution of sodium citrate, EDTA and lithium heparin anticoagulants to dried plasma and whole blood spectra compared to wet samples. A better detection at low range of malaria parasitemia in red blood cells was achieved for samples prepared in lithium heparin tubes . Our previous study has shown that serum and plasma samples were affected mainly in the protein region when fresh samples were compared to the first freeze‐thaw cycle .…”

Infrared spectroscopy coupled to cloud-based data management as a tool to diagnose malaria: a pilot study in a malaria-endemic country

“…For example, the spectral region 1354–1186 cm −1 contains bands assigned to proteins. Protein levels in blood cells decrease in samples due to the consumption of haemoglobin by malaria parasites, and this is detected by spectroscopy [20]. The lower wavenumber region (1185–1006 cm −1 ) includes bands from nucleic acids shown in the laboratory studies [15, 20] to be strongly loaded in models used to discriminate malaria infected samples.…”

“…Other potential drawbacks have been identified. For example, it has been shown that anticoagulants used in blood collection tubes can affect ATR-FTIR spectroscopy-based malaria diagnosis [20], depending on the volume of blood that is drawn into the collection tube and the type of anticoagulant employed. Only one type of anticoagulant (EDTA) was employed in this study, which did not yield spectral bands that could confound malaria diagnosis.…”

“…Ideally, a POC test should employ finger-prick blood volumes that can be measured directly at POC, rather than venous samples processed in a central laboratory as reported here. It has also been demonstrated [20] that malaria parasites can be detected with parasitaemia levels < 0.1% directly from unfixed aqueous blood samples. This approach, requiring less than 200 mL of blood, could easily be used in POC clinical settings, and would be particularly suited to diagnosing malaria in infants.…”

Biofluid diagnostics by FTIR spectroscopy: A platform technology for cancer detection

“…For blood plasma collection, a number of anticoagulants -including heparin and EDTA -are used in clinical practise in order to prevent blood clotting. It has been shown that the choice of anticoagulant is noticeable in the resultant infrared spectrum, which may conceal underlying biological information [55,56]. Whilst the use of serum may overcome this collection issue, other factors (e.g.…”