Determination of trace elements in thrombocytes by ICP-MS

Amanda Hultén

November 19th 2019

Degree Project C in Chemistry, 1KB010

Bachelor Program in Chemistry

Department of Chemistry BMC – Uppsala University

Supervisor: Jean Pettersson, Uppsala University

Abstract Analyses were carried out on a trace elements reference whole blood specimen from horse and on a thrombocyte mixture solution from multiple human donors which had been donated at Akademiska sjukhuset, Uppsala University Hospital. The study was carried out to potentially use the element profile of the trace element analytes Fe, Cu, Zn, Se and Sr in thrombocytes of healthy patients to be able to detect analyte deficiencies in patients with deficiency related diseases using ICP-MS. The goal of the report was to identify analyte isotopes suitable for determining the trace element analyte concentrations in human thrombocyte mixture solution along with a suitable sample preparation method validated with samples prepared from the reference whole blood specimen. A sample preparation method with microwave acid digestion was validated in this report. The validation of the sample preparation method indicated promising results for future research, the trace element analytes were all above LOD and LOQ in the reference whole blood samples. Recovery tests indicated that the calibration standards were well matched to the sample matrix, and that the sample did not affect the sensitivity of the analytes in the sample solution. Due to higher concentrations of Fe and Zn than the calibrated range there were uncertainties determining their concentrations. Measured values of Cu lied above the reference values which was likely due to interferences of PO2 and SO2 not corrected for by the helium KED. For the thrombocyte mixture solution no reliable LOD or LOQ were able to be calculated from blanks due to no series of thrombocyte blanks being measured. Problems concerning presentation of concentration values, cell counting and integrity of thrombocyte cells are discussed in the report.

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Table of content Abstract 2

Table of content 3

1 Abbreviations 4

2 Introduction 5

3 Theory 6 3.1 Thrombocytes 6 3.2 ICP-MS instrument 6 3.3 Sample preparation ICP-MS 6

Whole blood 6 Thrombocytes 7

3.3 Internal standard 7 3.4 Calibration 7

4 Materials and Method 8 4.1 Chemicals 8 4.2 Standard preparation ICP-MS 8 4.3 Sample preparation ICP-MS 8

Microwave acid digestion 8 Whole blood 9 Thrombocytes 9

4.4 Measurements with ICP-MS 10

5 Results and discussion 11 5.1 ICP-MS measurements 11

Whole blood 11 Thrombocytes 18

6 Conclusion 22

7 Future work 22

8 Acknowledgement 23

9 References 24

10 Appendix 25 9.1 ICP-MS settings 25 9.2 Sample preparation 28

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1 Abbreviations AS - Autosampler CI - Confidence interval Helium KED - Helium kinetic energy discrimination HG-AAS - Hydride Generation Atomic Absorption Spectrometry ICP-MS - Inductively Coupled Plasma Mass Spectrometry LOD - Limit Of Detection LOQ - Limit Of Quantification MPS - Microwave sample Preparation System m/z - mass to charge MQ - Milli-Q purified PIXE - Proton Induced X-ray Emission ppb - parts per billion ppm - parts per million ppt - parts per trillion QID - Quadrupole Ion deflector sd - standard deviation (v/v) - volume/volume

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2 Introduction In this study Inductively Coupled Plasma Mass Spectrometry, henceforth referred to as ICP-MS, was used to analyze concentrations of selected isotopes of the elements Fe, Cu, Zn, Se and Sr in a human thrombocyte mixture solution, i.e. a blood platelet sample. At first, sample preparation method was tested on a trace elements reference whole blood specimen from horse in order to find a suitable sample preparation method, validated by measurement data analysis. Previous trace elements biochemistry studies (e.g. [1][2][3]) have been performed on e.g. whole blood, erythrocytes, neutrophil granulocytes and thrombocytes using ICP-MS, HG-AAS(Hydride Generation Atomic Absorption Spectrometry) and PIXE(Proton Induced X-ray Emission). Among other functional biological compounds the cell metabolism of thrombocytes involves trace elements such as metal ions that e.g. interacts as signal substances in numerous biological interactions [4][1]. Analyzes of trace elements in thrombocytes is of interest as thrombocytes have a shorter life span (10-14 days) than erythrocytes, i.e. red blood cells, which have a mean lifespan of 120 days[1]. This shorter lifespan would make it possible to analyse more recent events in the human metabolism. This short life span of thrombocytes combined with the ability to measure on a ppt/ppb (parts per trillion/parts per billion) level[5] with the ICP-MS instrumentation would make it possible for early on diagnosis of trace element deficiency related diseases. This would be executed by analyzing the trace element analyte profile in healthy patients to compare against real patient samples with the potential outcome of successful early on diagnosis. After method validation, a thrombocyte mixture solution from multiple donors, donated at Akademiska sjukhuset, Uppsala University Hospital, was used for further measurements. This study was carried out to optimistically find a small piece of the puzzle in the research of Alzheimers, Parkinson's disease, prostate cancer as these diseases have been found to potentially be associated with deficiencies or concentration alterations of the elements of interest[6][7].

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3 Theory 3.1 Thrombocytes In previous studies [2] it has been found that the selenium concentration in thrombocytes have a significant effect on the concentrations of iron and zinc in thrombocytes; with a higher selenium concentration the zinc concentration is increased simultaneously but the iron decreases. It is assumed that selenium affects the transportation of iron and zinc into, and out from, the thrombocytes[8]. Selenium is thought to affect iron transport via the endoplasmic reticulum[8] and according to Johansson (1994)[9] “it is suggested that when the concentration of selenium is too low in the thrombocytes iron may produce sticky thrombocyte reactive to cell walls” which potentially could have an effect of the thrombocyte’s function. 3.2 ICP-MS instrument The ICP-MS instrument is composed of several parts, at the sample inlet there is a nebulizer which creates an aerosol and a spray chamber which excludes large droplets [10]. In the torch the fine spray is heated in a pre heating zone, argon gas being used as coolant gas, then the sample is vaporized, atomized and ionized to create a plasma of the sample, the ions are then passed through to a Quadrupole Ion deflector (QID)[11]. QID works by deflecting ions in an electric field, uncharged atoms/molecules will not continue past the deflector as a charge is needed for them to be able to bend through the curve [11]. Following the QID is the collision cell, in this case helium KED. Helium KED is a collision cell with kinetic energy discrimination, helium gas collides with the spray of the injected sample. This is used to filter out atoms and/or molecules that interfere with the analyte signal due to the interfering atoms and/or molecules having the same m/z (mass to charge) ratio as the analyte. Without helium KED atoms/molecules with the same m/z ratio reach the detector grid at the same time and the analyte can therefore not be detected separately. The theory behind the usage of helium KED is that the atom/molecule with the larger surface will be hit more times, by the helium gas particles, than the smaller atom/molecule. This causes the larger atom/molecule to lose more momentum and will not be able to pass an energy potential barrier located at the end of the collision cell, therefore optimally only the analyte travels through the quadrupole mass separator and get singled out and counted in the grid detector and gives a pulse intensity signal. This gives a more accurate reading of analyte signal as the interfering atoms/molecules to the different analyte isotopes are minimized. [11][12] Different isotopes are present in different abundances of an element, isotopes can also be affected by potential interferences[5]. Isotopes to be measured were therefore selected optimally by as high of an abundance as possible that had little to no potential interference. 3.3 Sample preparation ICP-MS

Whole blood A freeze dried reference whole blood specimen from horse was used in the whole blood sample preparation in the analysis to validate the method against the given reference values. When

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validated, the method can be applied for the human thrombocyte mixture solution. The reference whole blood specimen from horse was used for validation as it is more commercially available since larger quantities of blood can be drawn from horses rather than humans.

Thrombocytes In the sample preparation of thrombocytes from a frozen solid, sodium citrate was used for its anticoagulant properties to wash the blood platelets in between the centrifugation steps. This was because the thrombocytes were still mixed with blood plasma which contain fibrinogen that in contact with the enzyme thrombin form insoluble fibres of fibrin that bind to the thrombocyte surface and thus clotting the blood platelets together, the washing method prevents this from happening[13]. [8] In the washing of thrombocytes, centrifugation is used to separate the thrombocytes from blood plasma, possible remaining erythrocytes i.e. red blood cells, neutrophil granulocytes i.e. white blood cells and other compounds that might still reside in the thrombocyte mixture solution. 3.3 Internal standard A thallium internal standard was used in all calibration standards, samples and blanks with a fixed concentration. An internal standard is used to compensate for physical interferences that disturb the transportation of the plasma, such as the spray, droplet size and transportation of the analyte through the ICP-MS. This corrects for the physical interferences since the internal standard is transported in the same way as the analytes through the system. The internal standard also enables us to calculate the analyte to internal standard ratio of the measured intensity signals. This ratio can then be used to calculate the actual analyte concentration in the samples for more accurate concentration calculations. The internal standard can because of this compensate for minor deviations in sample volume, as the ratio corrects for this. [11] In the sample and blank preparation of thrombocytes, a rhodium internal standard was used in the washing solution. The intensity signal of the Rh internal standard showed how much of the washing solution that still remained in the thrombocyte pellet after the washing steps. This signal was then corrected for with the Rh signals of the blanks giving accurately comparable signals of samples and blanks. This is because the sample blanks were prepared with an aliquot of sodium citrate solution estimated optically from the volume of the thrombocyte pellet samples. Thallium and rhodium were selected as internal standards because of their similarity to the analytes and because according to the reference values of the reference whole blood specimen they should not be present in the prepared samples (<0.01 µg/L). 3.4 Calibration An interval of different concentration standards were used to make an evenly calibrated range for the measurements based on the present analyte concentrations in the reference whole blood specimen. In theory a calibration curve should extend beyond the highest concentration measured, so that no concentration need to be extrapolated.

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4 Materials and Method 4.1 Chemicals For the calibration standards, samples and sample blanks the following chemicals were used; sub. boiled nitric acid 68%, VWR Chemicals, France and Hydrogen peroxide 30% for traces analysis, VWR Chemicals, France. The Fe single element standard was of the brand SpectrosoL®, BDH Chemicals, Ltd Poole England, with the concentration 1000 µg/mL. All the other single element standards were of the brand Spectrascan, Teknolab Sweden with the individual concentrations: Se 1003 +/- 2 µg/mL 1.4% HNO3 (v/v) (volume/volume), Sr 1004 +/- 5 µg/mL 0.1% HNO3 (v/v), Cu 1003 +/- 5 µg/mL 3% HNO3 (v/v), Zn 1006 +/- 3 µg/mL 2% HNO3 (v/v), Tl 1001 +/- 5 µg/mL 0.7% HNO3 (v/v), Rh 1002 +/- 18 µg/mL 15% HCl (v/v) The reference whole blood specimen used was Seronorm™ Trace elements Whole blood L-1, lot MR4206, exp 200908 ref 201505, SERO, Norway and the thrombocyte mixture solution was Thrombocytes, leukocyte- and pathogen reduced, D0167V00, PAS-E added, drawn 20171108 at Akademiska sjukhuset, Uppsala University Hospital. For the washing solution Tri-Sodium citrate dihydrate, Merck, Germany was used. All dilutions of standards, samples and sample blanks were done with Milli-Q Purified water (MQ), Merck Millipore, USA. 4.2 Standard preparation ICP-MS A standard solution of 1.0 ppm (parts per million) multi-element standard with 1.0% (v/v) HNO3 was prepared from 1000 ppm Fe, Se, Sr, Cu, Zn single element standards. Calibration standards with 10.0% (v/v) HNO3 were prepared from the standard solution with final analyte concentrations of 0.0; 0.2; 0.5; 1.0; 5.0 ppb and a 5.0 ppb Tl-internal standard. 4.3 Sample preparation ICP-MS

Microwave acid digestion The microwave acid digestion was performed in a microwave sample preparation system, Titan MPS, equipped with teflon bombs to digest the samples and sample blanks. For the digestion 10.0% (v/v) HNO3 and 10.0% (v/v) H2O2 was used, which is a common acid digestion method on blood samples[1]. Samples and sample blanks were prepared directly in the bombs where the sample aliquots were weighed in with differential weighing. Tl-single element standard was added to give a final concentration of 5.0 ppb in the samples and sample blanks. Normal volume in the samples and sample blanks preparation was 6 mL due to it being the smallest possible sample volume of the bombs and that only 1 mL is sufficient for one ICP-MS run. For the microwave digestion runs; temperature, pressure, ramp time, hold time and power settings found in Table 1 were used.

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Table 1: Programme settings of microwave digestion runs.

Step T(°C) p (bar) Ramp (min) Hold (min) Power (%)

1 165 30 5 10 80

2 190 30 1 10 90

3 50 30 1 10 0

Whole blood The freeze dried reference whole blood specimen was rehydrated with 5.0 mL MQ according to the manufacturer's instructions and the same specimen vial was used throughout all whole blood sample preparations. Samples were prepared in the teflon bombs containing 10.0% (v/v) HNO3, 10.0% (v/v) H2O2, 5.0 ppb Tl-internal standard, and an aliquot of the rehydrated reference whole blood specimen. For the normal 6 mL sized samples 100 µL aliquots of the reference whole blood specimen were used. For a batch of 12 mL samples 200 µL aliquots of the reference whole blood specimen were used, these samples were then spiked and standard addition was performed, see Appendix Table A.7. For a linearity test 0.0, 25.0, 75.0 and 125.0 µL reference whole blood specimen was used in the normal 6 mL sample volume. Corresponding sample blanks were prepared for all samples without an aliquot of the reference whole blood specimen.

Thrombocytes At the time of each sample preparation a sufficient aliquot, for sample preparation, of the frozen thrombocyte mixture solution were broken off and thawed at room temperature. A suitable washing method[14] was used with a solution of 3.8% sodium citrate that was prepared with a rhodium internal standard of 333.0 ppb which gave a 5.0 ppb concentration when diluted in the 6 mL blank samples. The thrombocyte mixture solution was washed three times with the sodium citrate solution and centrifuged in between each washing at 3000G [14][15][8] at room temperature for 5 minutes at the time. In each of the washing steps, a 1:1 volume of thrombocyte mixture solution:sodium citrate solution were used. After the first washing step enough supernatant was removed for the remaining volume to be composed of 1 mL supernatant and the acquired thrombocyte pellet. After the final washing step all supernatant was removed, leaving the thrombocyte pellet. The thrombocyte samples were then prepared in the teflon bombs with the same acid digestion, as for the reference whole blood specimen, with the Tl-internal standard and the aliquot consisting of the washed and centrifuged thrombocyte pellet. 4 grams of the thrombocyte mixture solution was washed for each sample, which corresponds to a small final pellet volume. Corresponding sample blanks were prepared without the thrombocyte mixture solution however an aliquot of sodium citrate solution was added. The aliquot was estimated to be about 90 µL based on the volume of the thrombocyte pellet.

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4.4 Measurements with ICP-MS Measurements were carried out on an ICP-MS, NexIONTM 300D equipped with an AS-93plus autosampler. Scan mode was set to peak hopping mode (collects data from pre-set masses), isotopes measured were Fe-57, Cu-65, Zn-68, Se-82(corrected for Kr) and Sr-88 which were selected with the help of the SyngistixTM software based on least possible potential interferences to highest possible abundance. Helium KED was used for all selected isotopes to minimize potential interference by other atoms and/or molecules of the same m/z ratio as the trace elements of interest. A washing solution of 10% (v/v) HNO3 was used in between each of the calibration standards, samples and sample blanks to match the acid content of calibration standards, samples and sample blanks. Tl-205 was the isotope measured for the Tl-internal standard of the samples and sample blanks. Rh-103 was the isotope measured for the additional internal standard of the sodium citrate solution used to wash the thrombocytes and used in the thrombocyte sample blanks. Potential isotopes to be measured included Fe-56, Cu-63, Zn-66, Se-76, Sr-86 but these were excluded because of interferences that could not be eliminated using helium KED. Fe-56 have too much interference of ArO (mass 56) which derives from the argon gas as it is passed through the plasma. Se-76 was excluded because of the interference of argon gas, Ar2, which is present in high concentrations. Cu-63, Zn-66 ended up being excluded because of higher potential matrix effects. Sr-86 ended up being excluded because of its lower abundance. Because of these isotopes having interferences they could not be compared to the measured isotopes and therefore the isotope ratios in the samples could not be examined, the ratios of the isotopes are therefore assumed to be the same in the calibration standards and whole blood and thrombocyte samples. For further ICP-MS instrument setting see Appendix Table A.1-A.5.

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5 Results and discussion 5.1 ICP-MS measurements As mentioned, this study was carried out to investigate a small piece of the puzzle concerning detecting trace element analyte deficiencies in patient samples for potential early on diagnosis of trace element deficiency related diseases. Hence this, the goal of this project was to identify analyte isotopes suitable for determining trace element analyte concentrations in human thrombocyte mixture solution along with a suitable validated sample preparation method.

Whole blood The in-solution concentrations of the trace element analytes in the whole blood samples were measured on four 12 mL samples, with 200 µL reference whole blood specimen, to be compared to the limit of detection (LOD) and the limit of quantification (LOQ) of the method. The mean +/- CI (confidence interval) for the in-solution concentration can be found in Table 2. Table 2. Measured in-solution concentration of the trace element analytes in whole blood samples (n=4).

Element Measured in-solution concentrations mean ± CI (n=4)

Fe57(µg/L) 5000 ± 128

Cu65(µg/L) 10.1 ± 0.2

Zn68(µg/L) 67.4 ± 1.3

Se82(µg/L) 0.9 ± 0.1

Sr88(µg/L) 0.3 ± 0.0

The LOD of the assembled method was calculated from the standard deviation (sd) of a series of eight 6 mL whole blood sample blanks by the equation: LOD = . the sd was derived fromds × 3 the calculated trace element analyte concentration of the sample blank solutions. The LOQ of the method was also calculated from the sd of the same blank series using LOQ = . Thed 0s × 1 calculated LOD and LOQ for the analyte elements can be found in Table 3.

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Table 3. Calculated LOD and LOQ from a series of whole blood sample blanks (n=8).

Element

LOD sd )( × 3

LOQ sd 0)( × 1

Fe57(µg/L) 2.0 6.5

Cu65(µg/L) 0.2 0.5

Zn68(µg/L) 1.0 3.4

Se82(µg/L) 0.1 0.5

Sr88(µg/L) 0.1 0.2

From the LOD and LOQ validation and the measured in-solution concentration of the trace element analytes one can see that all the measured in-solution values of the analytes were above the LOD and LOQ so the measured values are reliable. Measured values of trace element analytes in the reference whole blood specimen, calculated from the in-solution concentrations, to be compared with reference values, were calculated from four 12 mL samples with 200 µL reference whole blood specimen. The mean +/- CI for the calculated measured values and reference values can be found in Table 4 and Figure 1-5. Table 4. Calculated measured values and reference values in the reference whole blood specimen. Mean ± CI. (n=4), *outside reference values

Element Measured values mean ± CI (n=4)

Reference values mean ± CI

Fe57(mg/L) 294 ± 7* 432 ± 28

Cu65(µg/L) 619 ± 14* 564 ± 33

Zn68(µg/L) 3961 ± 108* 5500 ± 300

Se82(µg/L) 57 ± 5* 79.8 ± 5,4

Sr88(µg/L) 30 ± 1 27.8 ± 1,7

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Figure 1. Calculated measured values and reference values of Fe57 in the reference whole blood specimen. mean ± CI. (n=4).

Figure 2. Calculated measured values and reference values of Cu65 in the reference whole blood specimen. mean ± CI. (n=4).

Figure 3. Calculated measured values and reference values of Zn68 in the reference whole blood specimen. mean ± CI. (n=4).

Figure 4. Calculated measured values and reference values of Se82 in the reference whole blood specimen. mean ± CI. (n=4).

Figure 5. Calculated measured values and reference values of Sr88 in the reference whole blood specimen. mean ± CI. (n=4).

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The 95% confidence interval of the measured and reference value of Sr overlapped while in Fe they were divided by a gap of 103 ppm, for Cu they were divided by 8 ppb, for Zn 1131 ppb and Se 12.4 ppb. The measured calculated values and reference values of the trace element analytes were however within the same tenfold range of each other. The reference whole blood specimen was expired by 10 years but this should not affect the trace element profile. The measured values of Fe, Zn, and Se were below the reference values but Cu was ever-so-slightly above the reference values. Since all analytes were above the LOQ and that some of the analytes gave measured values under the reference values (i.e. Fe, Zn, Se) this could maybe indicate interferences of these analytes resulting in lower concentrations. A more possible explanation for the lower measured values of Fe and Zn is that these analytes in the in-solution concentrations were far outside of the calibrated range of 0.0-5.0 ppb where their concentrations were Zn: 67.4 ± 1.3 ppb and Fe: 5000 ± 128 ppb. These concentrations were estimated from repeated measurements of the samples under the conditions that the concentrations were assumed to be within the linear area of the calibration. At least for Fe this large deviation from the calibrated range derive great uncertainty when determining concentrations, a wider calibrated range that cover the expected in-solution concentrations should therefore be considered in future measurements. In hindsight the calibrated range should have been larger, or the sample dilution greater, considering the reference values of Fe, Cu, Zn. The higher measured values of Cu could potentially derive from the helium KED not being able to make all PO2 and SO2 loose enough momentum in the collision cell i.e. some PO2 and SO2 might pass the energy potential barrier and reach the detector. Standard addition was performed on two 12 mL samples, with a 200 µL aliquot of reference whole blood specimen, each sample was divided into three smaller 3 mL samples where one was not spiked and the other two were spiked with different known concentrations, see Appendix Table A.7. These spiked concentrations were supposed to be x2 and x4 concentration but a calculation error was made and the spiked concentrations ended up being higher by a factor of four. The samples were not spiked with Fe due to the already above-calibration curve-concentrations present in the samples. The reference values and calculated measured values of the standard addition can be found in Table 5 and Figure 6-9.

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Table 5. Reference values and calculated measured values of standard addition in reference whole blood specimen. mean ± CI. *outside reference values

Element Reference values mean ± CI

Measured values standard addition mean ± CI [16(p.129)]

Fe57(mg/L) 432 ± 28 -

Cu65(µg/L) 564 ± 33 517 ± 0,007*

Zn68(µg/L) 5,500 ± 300 5,354 ± 0,004

Se82(µg/L) 79,8 ± 5,4 103 ± 0,000*

Sr88(µg/L) 27,8 ± 1,7 27 ± 0,001

Figure 6. Reference values and calculated measured values of Cu65 from standard addition in reference whole blood specimen. mean ± CI.

Figure 7. Reference values and calculated measured values of Zn68 from standard addition in reference whole blood specimen. mean ± CI.

Figure 8. Reference values and calculated measured values of Se82 from standard addition in reference whole blood specimen. mean ± CI.

Figure 9. Reference values and calculated measured values of Sr88 from standard addition in reference whole blood specimen. mean ± CI.

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The measured and calculated concentrations of the standard addition were not deviating too much from the reference values, indicating some improvement in this method compared to the first calibration method. However, the calculated values contained some uncertainty due to the accidentally four times higher added concentrations than intended which ended up beyond the calibrated trace element analyte range. Recovery of the digestion method for elements Cu, Zn, Se, Sr were calculated from standard additions as spikings. Samples used were three 12 mL, with a 200 µL aliquot of reference whole blood specimen, each sample was divided into three smaller 3 mL samples each which were spiked with known concentrations, see Appendix table A.7. The samples were not spiked with Fe due to the already above-calibration curve-concentrations present in the samples. The calculated recoveries can be found in Table 6. Table 6. Recovery of digestion method for elements Cu, Zn, Se, Sr. Samples used were three 12 mL, with 200 µL reference whole blood specimen, divided into three 3 mL samples each which were spiked with known concentrations.

Element Measured values Recovery ± sd (%)

Cu65 99 ± 7

Zn68 100 ± 10

Se82 97 ± 27

Sr88 96 ± 9

The recovery describes how well the calibration standards were matrix-matched to the sample matrix, this also shows that the sample did not affect the sensitivity of the analytes in the sample solution. The high standard deviation for selenium derived from the in-solution concentrations being low, close to the LOQ of selenium. None of the recoveries indicated a significant deviation from the null hypothesis with a student t-test H0=100%, P < 0.05. Note that this did not mean that significant deviations did not exist, only that they were not demonstrated i.e. the null hypothesis could not be rejected [16]. For a linearity test 0.0, 25.0, 75.0 and 125.0 µL reference whole blood specimen was used on eight normal (6 mL) samples, see Appendix Table A.6.. The samples were measured and the analyte signal to the internal standard signal ratios were plotted against blood fraction weight. These plotted diagrames can be seen in Figure 10-14.

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Figure 10. In-solution concentration ratios of Fe/Tl in samples with reference whole blood specimen fractions of 0.0, 25.0, 75.0, 125.0 µL.

Figure 11. In-solution concentration ratios of Cu/Tl in samples with reference whole blood specimen fractions of 0.0, 25.0, 75.0, 125.0 µL.

Figure 12. In-solution concentration ratios of Zn/Tl in samples with reference whole blood specimen fractions of 0.0, 25.0, 75.0, 125.0 µL.

Figure 13. In-solution concentration ratios of Se/Tl in samples with reference whole blood specimen fractions of 0.0, 25.0, 75.0, 125.0 µL

Figure 14. In-solution concentration ratios of Sr/Tl in samples with reference whole blood specimen fractions of 0.0, 25.0, 75.0, 125.0 µL

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Figure 10-14 shows that enough reagents were added to facilitate complete sample digestion in the range 25-125 µl.

Thrombocytes The in-solution concentration of the trace element analytes in thrombocyte samples were measured on four 6 mL samples with 4 grams of the thrombocyte mixture solution washed down to an approximately 90 µL sized pellet. The in-solution concentrations to be compared to the LOD and LOQ of the method. The mean +/- CI for the in-solution concentration can be found in Table 7. Table 7. Measured in-solution concentration of the trace element analytes in thrombocyte samples (n=4).

Element Measured in-solution concentrations mean ± CI (n=4)

Fe57(µg/L) 46.1 ± 6.3

Cu65(µg/L) 6.6 ± 0.5

Zn68(µg/L) 54.5 ± 1.5

Se82(µg/L) 3.4 ± 0.9

Sr88(µg/L) 1.3 ± 0.1

Here the in-solution concentration of the thrombocyte samples Fe and Zn did not deviate as much from the calibrated range (factor of ~10) as in the in-solution concentration of the whole blood samples did (factor ~1000 and ~15 respectively). Since no big series of blanks were prepared the LOD of the assembled thrombocyte method was calculated from the standard deviation of a series of four 6 mL thrombocyte sample blanks in the same way as above. The sd was derived from the calculated trace element analyte concentration of the thrombocyte sample blanks. LOQ of the thrombocyte method was also calculated from the sd of the same blank series as above. The calculated LOD and LOQ for the analyte elements can be found in Table 8.

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Table 8. Calculated LOD and LOQ from a series of thrombocyte sample blanks (n=4).

Element

LOD sd )( × 3

LOQ sd 0)( × 1

Fe57(µg/L) 0.1 0.2

Cu65(µg/L) 1.0 3.4

Zn68(µg/L) 0.7 2.5

Se82(µg/L) 0.4 1.4

Sr88(µg/L) 0.8 2.6

From the LOD and LOQ validation of the thrombocyte method and the measured in-solution concentration of the trace element analytes in thrombocyte samples one can see that all measured in-solution values of the analytes, except for strontium, were all above the LOQ, strontium was above the LOD though. But since the LOD and LOQ were calculated from only four thrombocyte sample blanks we can not say if the measured in-solution concentration values truly are reliable for determination of iron, copper, zinc and selenium. If instead the measured in-solution concentrations of the trace element analytes are compared to the LOD and LOQ validation of the whole blood method, (the difference between the two types of blanks was the 90 µL sodium citrate aliquot with the rhodium internal standard) we see that the measured in-solution values of the trace element analytes were all above the LOD and LOQ. By this comparison can we draw a conclusion about the in-solution concentration trace element analytes being reliable or not though, since the whole blood sample blanks had a different matrix compared to the thrombocyte samples. The LOD and LOQ calculated from the four thrombocyte sample blanks were the best of the estimated LOD and LOQ in this sense, though a series of four blanks will give only a rather imprecise estimate of the LOD and LOQ. Measured values of trace element analytes in the thrombocyte mixture solution were calculated from four 6 mL samples with 4 grams of the thrombocyte mixture solution washed down to an approximately 90 µL sized pellet. The mean +/- CI for the calculated measured values can be found in Table 7 and Figure 15.

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Table 7. Calculated measured values of trace element analytes in the thrombocyte mixture solution (n=4).

Element Measured values mean ± CI

Fe57(µg/L) 254 ± 1.0

Cu65(ng/L) 1792 ± 9

Zn68(µg/L) 35 ± 0.2

Se82(ng/L) 715 ± 4

Sr88(ng/L) 2094 ± 11

Figure 15. Calculated measured values of trace element analytes in the thrombocyte mixture solution (n=4). Since the trace element analyte concentrations of the thrombocytes was expressed in concentration per gram of the donated thrombocyte mixture solution, it was not possible to correctly compare these data with other data from previous reports. If one would have measured the thrombocyte count per gram thrombocyte mixture solution, one could have expressed the concentration per thrombocyte cell. One could also have used an average mean weight of thrombocytes [17] to recalculate the concentrations to concentration per gram which would allow for comparison with some studies(eg. for selenium in [3][18]). Unfortunately these recalculations

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would pose problems as the thrombocyte pellet did not solely consist of pure platelets, such recalculations would therefore give false trace element analyte concentrations for the thrombocytes. Also the reason as to why the number of thrombocytes were not analyzed in this report was firstly because the thrombocyte mixture solution used for this project had previously been frozen, thawed and frozen again, which possibly may have altered the physics of thrombocyte mixture solution and therefore the element profile if they were broken or leaking. As platelets are delicate as well they could easily break when handled which potentially could alter their analyte composition [3][8]. The washing of the thrombocytes could potentially also have had this side effect. Optimally the thrombocytes would have been counted after the washing steps but this was not possible due to the inability to declot thrombocyte pellet without injuring the platelets. So as the thrombocyte cells were not counted by Akademiska Sjukhuset nor in this study no conclusion can be drawn concerning damage to the thrombocytes nor that it was a consequence of thawing or washing i.e. a possible analyte alternation can not be determined. Based on the rhodium signal in the thrombocyte samples and thrombocyte sample blanks the small 90 µL thrombocyte pellets consisted of about 60 µL sodium citrate solution which means that the remaining 30 µL consisted of thrombocytes. Realistically the remaining 30 µL also contained different residues derived from blood components and residues of added solutions to the thrombocyte mixture solution such as conservatives. Also the thrombocyte pellet aliquot was estimated to be 90 µL from looking at it, but this could instead have been estimated by differential weighing which would have been more accurate. As measurement times were 75 milliseconds per element, instead 25 milliseconds could have been used for trace element analytes of high concentrations i.e. Fe, Zn and 250 milliseconds could have been used for the trace element analytes of low trace element analyte concentrations i.e. Cu, Se, Sr, in order to receive a better accuracy of the pulse intensities. A longer reading for lower concentration trace elements gives a more accurate estimate of the average signal.

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6 Conclusion In the whole blood method, all measured in-solution concentration values of the trace element analytes were above the LOD and LOQ and therefore reliable. There was no proven significant deviation in the recoveries but note that the null hypothesis was not rejected. All the calculated concentrations, in the reference whole blood specimen, of the spikings of the whole blood samples were also above the LOD and LOQ but because of the unintentionally higher spike concentration (a factor of four), a large difference between the concentration of added spike and the calibration curve concentration interval was present. The recovery test indicated that the calibration standards were well matched to the sample matrix, and that the sample did not affect the sensitivity of the analytes in the sample solution. In the whole blood samples, Fe’s (deviating with a factor of 1000), and maybe Zn’s(deviating with a factor of 15) in-solution concentration’s large deviations from the calibrated range derived great uncertainty when determining concentrations. In the thrombocyte samples the in-solution concentration of Fe and Zn did not deviate as much from the calibrated range (factor of ~10). Since the concentrations were not presented per thrombocyte cell in this report, the concentrations were instead presented per gram thrombocyte mixture solution. This does not give good comparable data for future references as we do not know how many thrombocytes there were per gram thrombocyte mixture solution. Neither do we know for a fact if the trace element profile had been altered by the previously repeated thawing. In spite of all these reasons as to why further measurements are required to validate the performance of the thrombocyte method, there were indications that this sample preparation and ICP-MS method could be a suitable method for analyzing normal Fe, Cu, Zn, Se, Sr concentrations on this type of sample. That is if further tests prove that the measured in-solution concentrations of the trace element analytes are indeed reliable according to LOD and LOQ for the thrombocyte method.

7 Future work Further complementary standard addition measurements would be needed to achieve more correct standard addition values and a wider calibrated range that cover the expected in-solution concentrations, or a higher dilution factor for Fe and Zn, should be considered in future measurements. A new fresh thrombocyte mixture solution should also be used in order to analyze the trombocyte concentration of the trace element analytes per thrombocyte cell. Concerning the lack of proper LOD and LOQ validation of the thrombocyte method further tests are needed in order to estimate the thrombocyte method more precisely. Further measurements would later also be needed to find out if this method is also compatible to detect these analytes in the small blood fractions available in patient samples, both in healthy patients and in patients with trace element deficiency diseases.

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8 Acknowledgement First I would like to take this opportunity to thank my benevolent, genuine, supervisor Jean Pettersson for his devoted guidance, extensive expertise, and tireless patience throughout the course of this project. Secondly I would like to thank Erland Johansson for his astonishing engagement in this project, for evoking my curiosity in this problem at issue and for providing me and Emma with the thrombocyte mixture solution needed. His dedication in this area of research is truly inspiring. I would also like to thank Jean ́s PhD student Marcus Korvela for his passion for teaching and help with the ICP-MS instrument. Lastly I would like to thank all the amazing people on the Analytical Chemistry Department of BMC for this wonderful time. I will truly miss all the interesting conversations, the fantastic ambiance and the incredible fika breaks.

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9 References [1] Johansson, E. Westermarck, T. Ek, P. Atroshi, F. Metabolism changes as indicated by the Erythrocytes of patients with Alzheimer’s disease, Pharmacology and Nutritional Intervention in the Treatment of Disease, Faik Atroshi. (May 28th 2014) DOI: 10.5772/57511. [2] Johansson, E. Lindh, U. Availability of selenium compounds in man. Gustaf Werner Institute, Department of physical biology, Box 531, S-75121 Uppsala, Sweden.Proceedings of STDA’s third international symposium. (15-17th october 1984) Stockholm. [3] Hansson, L. Pettersson, J. Eriksson, L. Olin A. Atomic absorption spectrometric determination of selenium in human blood components. Clinical Chemistry (Apr 1989), Vol. 35 No. 4, 537-540 [4] Boije, Henrik. Syllabus for Exploring the Brain II, the nervous system. Verbal information 20190123, Uppsala university. [8] Johansson, Erland. Verbal information 20191008, Uppsala university. [9] Johansson, E. The Biochemistry of selenium in humans. KI, Clinical Research Center, Huddinge Hospital, S-14186 Huddinge, Sweden. Proceedings of STDA’s Fifth international symposium. (8-10th May 1994) Brussels. [5] PerkinElmer®, The Nexion® Series of ICP-Mass Spectrometers, 008906A_08, (2011). [6] Mattson, M. P. Calcium and Neuronal Injury in Alzheimer’s Disease. Ann. N. Y. Acad. Sci. 747, 50–76 (2010). [7] Huang, X. et al. Zinc and Health: Current Status and Future Directions Alzheimer’s Disease,  - Amyloid Protein and Zinc. Am. Soc. Nutr. Sci. 130, 1488–1492 (2000). [10] PerkinElmer®, Single Cell ICP-MS Analysis: Quantification of Metal Content at the Cellular Level, 013586_01 PKI, (2017) [11] Harris, D. C. 2010. Quantitative Chemical Analysis. Freeman 2010. [12] Collision/Reaction Cells in ICP-MS Cell Design Considerations for Optimum Performance in Helium Mode with KED. Agilent Technologies, Inc., 2010 June 08, (2010) 5990-5955EN [13] Nationalencyklopedin, fibrinogen. Available at: http://www.ne.se.ezproxy.its.uu.se/uppslagsverk/encyklopedi/lång/fibrinogen (Date: 2019-10-08) [14] Dhurat, R., & Sukesh, M. (2014). Principles and Methods of Preparation of Platelet-Rich Plasma: A Review and Author's Perspective. Journal of cutaneous and aesthetic surgery, vol. 7,4 (2014), 189–197. doi:10.4103/0974-2077.150734 [15] Abcam®, Isolation of human platelets from whole blood, pdf. Available at www.abcam.com (Date: 2019-10-7) [16] Miller, J. N. & Miller, J. C. 2010. Statistics and Chemometrics for Analytical Chemistry. Prentice Hall, Pearson (2010). [17] Kiem J, Iyengar GV, Borberg H, et al. Sampling and sample preparation of platelets for trace element analysis and determination of certain selected bulk and trace elements in normal human platelets by means of neutron activation analysis. Nucl Act Tech Life Sci, Proc hit Symp. Vienna, Austria: hit. Atomic Energy Agency, (1979):143-64. [18] Kasperek K, Iyengar GV, Kiem J, Borberg H, Feinendegen LE. Elemental composition of platelets. Part ifi. Determination of Ag, Au, Cd, Co, Cr, Cs, Mo, Rb, Sb, and Se in normal human platelets by neutron activation analysis. Clin Chem 1979;25:711-5.

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10 Appendix 9.1 ICP-MS settings ICP-MS computer programme: SyngistixTM for ICP-MS - Instrument control session. Table A.1. Timing settings for ICP-MS measurement runs

Scan Mode Peak Hopping

MCA Channels 1

Dwell time per AMU 75 ms

Integration Time 4500 ms

Corrections Se corrected for Kr

Profile Helium KED

Ammonia flow 0

Helium flow 4.5

RP a 0

RP q 0.25

Table A.2. Processing settings for ICP-MS measurement runs.

Detector Dual

Process Spectral Peak Average

QID On

Isotope Ratio Mode Off

Blank Subtraction After internal standard

Measurement Unit cps

Process Signal Profile Average

Baseline Readings 0

Smoothing Applied, factor 5

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Table A.3. Equation setting for ICP-MS measurement runs.

Analyte Mass (amu)

Abundance Corrections Potential interferences

Fe-57 56.9354 2.119000 CaO

Cu-65 64.9278 30.830000 PO2, SO2, TiO, Ba++

Zn-68 67.9249 18.750000 VO, ClO2, ArS, TiO, So2, Ba++, Ba++, Ce++

Se-82 81.9167 8.730000 -1.007833*Kr-83 Kr, Ar2H, BrH, Er++, Ho++, Dy++

Sr-88 87.9056 82.580000 Yb++, Lu++

Tl-205 204.9750 70.257600 -

Rh-103 102.9050 100.000000 SrO Table A.4. Sampling time and speed settings for the ICP-MS measurement runs.

Step Time (sec) Speed (+/- rpm)

Sample flush 45 -24

Read delay 15 -20

Wash 45 -24

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Table A.5. Profile Helium KED settings for the ICP-MS measurement runs.

Helium KED Active DAC values

Description Step value

Setting time (sec)

Minimum value

Maximum value

0.96 Nebulizer gas flow [NEB]

0.02 10 0 1.5

1.2 Auxiliary gas flow

0.025 10 0.6 2

18 Plasma gas flow

0.5 10 10 20

1600 ICP RF power 50 15 400 1600

-2000 Analog stage voltage

-100 2 -3000 0

1050 Pulse stage voltage

50 2 0 2500

15 Discriminator threshold

5 0 0 1000

-10.75 Deflector voltage

0.25 0 -100 20

-12 Quadrupole rod offset [QRO]

0.5 1 -26 26

-8 Cell entrance voltage

1 1 -60 20

-25 Cell exit voltage

1 1 -60 20

-15 Cell rod offset [CRO]

1 1 -40 10

475 Axial field voltage

25 1 -500 500

0 RPa 0.005 0 0 0.24

0.25 RPq 0.05 0 0.05 0.9

4.5 Gas flow 0.05 10 0 7.288

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9.2 Sample preparation Table A.6. Weights of differentially weighted whole blood aliquots for the 6 mL samples used to see concentration linearity at small sample aliquots.

Sample Sample aliquot (µL) Weighed in sample (g)

1 0 0

2 0 0

3 25 0.023

4 25 0.023

5 75 0.075

6 75 0.077

7 125 0.123

8 125 0.125

Table A.7. Added known concentrations of trace element analytes in standard addition and spiking tests in three 3 mL aliquot samples per three 12 mL samples with 200 µL whole blood.

Sample Add conc Cu (ppb)

Add conc Zn (ppb)

Add conc Se (ppb)

Add conc Sr (ppb)

Sample 1 x0 0.0 0.0 0.0 0.0

Sample 1 x2 14.0 89.3 3.6 2.1

Sample 1 x4 128.7 803.3 11.0 6.3

Sample 2 x0 0.0 0.0 0.0 0.0

Sample 2 x2 14.0 89.3 3.6 2.1

Sample 2 x4 128.7 803.3 11.0 6.3

Sample 3 x0 0.0 0.0 0.0 0.0

Sample 3 x2 14.0 89.3 3.6 2.1

Sample 3 x2 14.0 89.3 3.6 2.1

Sample 3 x2 14.0 89.3 3.6 2.1

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