Corriente de fuga de sustitución - S

Agilent MassHunter data acquisition, quantitative analysis (version B.05.00/B5.0.291.0) and qualitative analysis (version B.04.00) software were used for LC-MS/MS data analysis. Agilent LC-MS/MS 1200/6400 general operation manual was available in the LC-MS/MS laboratory. Collected data was stored and backed-up in the main computer of the LC-MS/MS laboratory and an external memory drive; both of which are password protected.

Microsoft Excel software (scatter chart, trend-line equation) was used for calibration curve generation and Hb-drv concentration calculation. “Method Validator” software (Philippe Marquis 1999, version; 1.19) was used for accuracy testing and method correlation studies (Linear regression, Passing-Bablok and Difference Plot). Assay linearity was assessed with LinChecker software (Philippe Marquis 2001, version; 1.1.2.0).

Rosita Zakariaee Abkoo 62

Chapter 3

Determination of Haemoglobin

Derivatives in Aged Dried Blood Spots to

Estimate Haematocrit

Rosita Zakariaee Abkoo 63

3

Determination of Haemoglobin Derivatives in Aged Dried

Blood Spot to Estimate Haematocrit

The levels of the haematocrit and haemoglobin at extreme high or low ends may influence the accuracy of the measurements quantification of the DBS sample. To alleviate the potential errors associated with the dried matrix of DBS sample which could span the total testing process, we aimed to predict the haematocrit of the punched dried blood spot through primary spectrophotometric estimation of its haemoglobin content. This correction could be incorporated into DBS standard operation procedures for measurement of different types of biomarkers.

This chapter has been peer reviewed and published with contribution of other co-authors as an analytical full paper (316). My contribution to this study and subsequent publication was included; developing the concept for the study in conjunction with my senior supervisor (RG), performing the experiments and method development, analysis and interpretation of data, writing the first draft of the publication and reviewing and incorporating all suggestions from the co-authors.

3.1 Introduction

“The micro-sampling technique associated with dried blood spot (DBS) samples is considered to be less invasive, requiring only a small sample volume (usually capillary) applied to filter paper, that is easy to transport and store with a lower risk of infection transmission (119, 121, 317). These advantages have prompted the interest in utilising DBS for clinical assessment outside of newborn blood spot screening (NBS). As such, DBS applications now encompass analytes related to clinical diagnosis, epidemiological studies, therapeutic drug monitoring, pharmacokinetics and toxicokinetic studies (10). However, the accuracy of DBS quantitation requires additional considerations over the conventional “liquid” blood sample. This is due to the physical characteristics of the blotted whole blood sample affecting the quality of the measurement. In particular, potential irregular errors associated with haematocrit (Hct) span the DBS total testing process:

Rosita Zakariaee Abkoo 64

Pre-analytically, the levels of a patient’s haemoglobin (Hb), and therefore Hct, at extreme high or low ends may influence the viscosity and fluidity of the spreading sample (241). Hence, there would be sample volume dissimilarity for a fixed size punch from the DBS originating from whole blood samples with different levels of Hct (Figure 1) (10, 240, 241, 254).

Figure 3.1. Calibrators set for estimation of Hb-drv of DBS samples. Haemoglobin concentration effects on the measured vitamin D of DBS

sample and whole blood samples with different levels of Hb do not diffuse similarly on the Guthrie card.

Analytically, Hct can influence the extraction of a measurand from the filter paper. This has been demonstrated in the quantitative bio-analysis of drugs where Hct has been identified as a causal factor of assay bias due to variations in the efficiency of the extraction process (305).

Post-analytically, the reference intervals and decision points for biomarkers and clinical assessments are predominantly described for samples with a liquid matrix (serum/plasma). The accurate conversion of dry matrix results (i.e. from the DBS) requires a consistent relationship with the liquid form (serum/plasma). Hence it is useful to know the Hct value (306).

DBS laboratories are often keen to know the Hct in order to account for, and ideally correct for, associated sample variation across the total testing procedure. The additional collection of a liquid sample (venous or capillary) for Hct measurement at the time of the DBS collection is recommended (318). However, there are many archived DBS samples that have limited biological information (including Hct), where prediction of the spotted blood Hct level is warranted. As such, the indirect (e.g. potassium measurement) or direct determination of Hct from the DBS cards is proposed (319-323).

To alleviate the DBS-Hct problem, scientists have employed a variety of correction techniques. The strategies used can be broadly divided into the following groups: 1) measuring the Hb and/or Hct

Rosita Zakariaee Abkoo 65

independently at the time of collection from a whole blood liquid sample; 2) determining the Hb and/or Hct from the DBS sample; or 3) not determining Hb and/or Hct at all. Where a Hct correction option is sought, practical solutions to minimise bias have been devised. These include: analysis of the entire blood spot; determination of the spotted blood volume by weighing the whole spot; using dried plasma collection; pre-cutting the filter paper prior to application of the blood; assessment or prediction of Hct from the spotted whole blood; and normalising the DBS measurement using the average value of Hct for males/females (10, 167, 318, 324). Whilst each of these correction approaches has merit, none have been widely adopted due to inherent limitations within the individual approach.

Over time, Hb converts to various Hb derivatives (Hb-drv) (Figure 3.2). Estimation of Hb-drv from the actual DBS punch could potentially be used as a surrogate for Hct to account for physiologically low or high Hct levels. We propose this might be possible spectrophotometrically using the eluted (solubilised) punched DBS (hence taking into account both diffusion and extraction differences). In this study, we aimed to predict the Hct of the punched archived DBS through primary spectrophotometric estimation of its Hb-drv content. Successful confirmation of this hypothesis would enable this technique to be incorporated into DBS standard operation procedures, allowing the laboratory to account for Hct in association with their DBS analyte of interest.

Figure 3.2. Haemoglobin is an oxygen transport protein in the form of oxyhaemoglobin (Hb-O2). As soon as fresh whole blood is air exposed

outside the body, Hb is completely oxygen saturated and converts into Hb-O2. Over time, Hb-O2 is auto-oxidised to met-haemoglobin (met-

Hb). Met-Hb eventually denatures into hemi-chrome (HC) (known as the most stable Hb derivative) . HC is a common name for group of low spin forms of the ferri-Hb (met-Hb) constituting ferri-alpha / beta subunits. The distinct feature of the met-Hb is its six liganded state of the haem iron that formed through the separate alterations of the globin conformation so that atoms endogenous to protein became bound as a sixth ligand of the haem iron. The abundant majority of HC have a proximal (F8) and a distal (E7) His as fifth and sixth ligands respectively (325, 326). Following our observations using direct spectrometry method after week 8 HC is measured.

Rosita Zakariaee Abkoo 66