Communication pubs.acs.org/jpr
Blood Plasma Reference Material: A Global Resource for Proteomic Research Johan Malm,† Pia Danmyr,‡ Rolf Nilsson,† Roger Appelqvist,‡ Á kos Végvári,‡ and György Marko-Varga‡,* †
Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, Skåne University Hospital, 20502 Malmö, Sweden ‡ Clinical Protein Science & Imaging, Biomedical Center, Department of Measurement Technology and Industrial Electrical Engineering, Lund University, BMC C13, 221 84, Lund, Sweden ABSTRACT: There is an ever-increasing awareness and interest within the clinical research field, creating a large demand for blood fraction samples as well as other clinical samples. The translational research area is another field that is demanding for blood samples, used widely in proteomics, genomics, as well as metabolomics. Blood samples are globally the most common biological samples that are used in a broad variety of applications in life science. We hereby introduce a new reference blood plasma standard (heparin) that is aimed as a global resource for the proteomics community. We have developed these reference plasma standards by defining the Control group as those with C-reactive protein levels 30 mg/L. In these references we have used both newborn children 1−2 weeks, as well as youngsters 15− 30 years, and middle aged 30−50 years, and elderly patients at the ages of 65+. In total, there were 80 patients in each group in the reference plasma pools. We provide data on the developments and characteristics of the reference blood plasma standards, as well as what is used by the team members at the respective laboratories. The standards have been evaluated by pilot sample processing in biobanking operations and are currently a resource that allows the Proteomic society to perform quantitative proteomic studies. By the use of high quality reference plasma samples, global initiatives, such as the Chromosome Human Proteome Project (C-HPP), will benefit as one scientific program when the entire human proteome is mapped and linked to human diseases. The plasma reference standards are a global resource and can be accessed upon request. KEYWORDS: C-HPP, blood plasma reference material
1. INTRODUCTION Globally, there is a major interest and need to maintain research areas that can improve on patient care and disease understanding, including “Personalized Medicine”. Targeting drug treatment bridges into alternative technologies with early indication of disease diagnosis that utilizes both imaging technologies and biomarker diagnostics. With the biomarker diagnosis developments, there is an expected shift in the demand and expectations on the future healthcare systems worldwide to chronic illnesses and aging demographics.1 These changes will create novel opportunities and challenges to the medical research community to adopt a patient-centric and technology-driven research strategy.2,3 The new entry into the medical field is the concept of quantifying proteins that are believed to have a key role in a specific disease area. Worldwide companies are being established that provide services and create a market where blood analysis is a key biofluid clinical source for diagnostics in general. We are entering into a new future, with large numbers of putative diagnostic markers to be assessed, where validations in clinical studies are needed to determine which combination of markers has the greatest diagnostic and prognostic value. Accordingly, the development of new diagnostic biomarkers has a huge potential, where both the industry as well as the academic field are investing and exploring approaches to connect technology to make innovative discoveries.4 Reference standard material as well as reference standardization methods form the cornerstones of modern clinical © XXXX American Chemical Society
protein research. Human blood as a source for clinical chemistry has its merits due to its wide applicability. The challenges, however, should not be underestimated, and those relate closely to standard operating procedure that is essential in order to guarantee the quality of the blood references.5−7 Today, sample collections from several large clinical studies have been biobanked for research applications. These biobanks at research centers and at national levels hold valuable information about diseases and pathological landmarks.8 It is of uttermost importance that a reference standard of high quality is stored in the biobanks in the same environmental conditions as the collected samples. This will allow for normalization and alignment of protein expression analysis data. There are an increasing number of clinical biobank sample collections from large-scale and population-based studies.9,10 These sample collections will be the source of important information in the years to come. One example is the BIG3 study in southern Sweden that investigates the common pathology between disease that is linked to the heart and lungs, as well as the cardiovascular system. The pathologies of those organs in the BIG3 cohort are studied within lung cancer, COPD and cardiovascular diseases (http://www.skane.se/sv/Webbplatser/Skanesuniversitetssjukhus/Forskning-och-Utveckling/Pagaendeforskning/BIG3/). Received: February 13, 2013
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dx.doi.org/10.1021/pr400131r | J. Proteome Res. XXXX, XXX, XXX−XXX
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Table 1. Summary of the Biomarker Data Generated in Technical Duplicates α1-antitrypsin (g/L) ALAT (μkat/L) Albumin (g/L) ALP (alk phosphatase) (μkat/L) ApoA1 (g/L) ApoB (g/L) ASAT (μkat/L) Bilirubin (μmol/L) Calcium (mmol/L) CK (creatinine kinase) (μkat/L) CKMB (μg/L) Cl (mmol/L) Creatinine (μmol/L) CRP (C-reactive protein)(mg/L) Cystatin C (mg/L) Estradiol (pmol/L) Fe (μmol/L) Ferritin (μg/) Folate (nmol/L) Free PSA (ratio free/total PSA) FSH (IE/L) FreeT3 (pmol/L) FreeT4 (pmol/L) Bile acid (μmol/L) Glucose (mmol/L) GT (glutamyltransferase) (μkat/L) Haptoglobin (g/L) HCG (IE/L) Homocysteine (μmol/L) HDL (mmol/L) IgA (g/L) IgG (g/L) IgM (g/L) K (mmol/L) Bilirubin conj. (μmol/L) Cobalamin (pmol/L) Cholesterol (mmol/L) Cortisol (nmol/L) LD (μkat/L) LDL (mmol/L) LH (IE/L) Mg (mmol/L) Myoglobin (μg/L) Na (mmol/L) Orosomucoid (g/L) Pancrease amylase (μkat/L) Procalcitonin (μg/L) Phosphate (mmol/L) Prolactin (mIE/L) Pro-BNP (ng/L) Progesterone (nmol/L) PSA (μg/L) PTH (pmol/L) SHBG (nmol/L) T3 (nmol/L) T4 (nmol/L) TCO2 (mmol/L) Testosterone (nmol/L) Triglyceride (mmol/L) TIBC (μmol/L) Troponin (ng/L)
control 1
control 2
disease 1
disease 2
1.22 0.15 41.5 1.09 1.58 0.8 0.66 5.2 2.36 2.24 2.61 102.1 84 1.34 1.05 147.2 12.9 152.4 37.56 0.163 23.51 5.52 18.08 7.8 6.66 1 1.24 0.575 13.5 1.33 1.93 10 1.2 4.19 0 579.5 4.56 594 4.25 2.55 13.29 0.83 263.4 141.1 0.73 0.51 0.06 1.15 506.8 456.7 3.42 0.573 4.74 55.25 1.92 99.92 18 4.1 1.49 65.26 13.35
1.24 0.14 40.8 1.1 1.58 0.8 0.66 5.8 2.32 2.29 2.66 100.4 82 1.39 1.07 150.9 12.7 149.7 38.29 0.161 23.25 5.43 18.37 8 6.58 0.96 1.21 0.633 13.7 1.34 1.95 9.89 1.22 4.14 0 582.2 4.56 609.1 4.29 2.54 13.41 0.82 257.6 139.6 0.73 0.5 0.058 1.14 508.5 453.7 3.47 0.553 4.71 54.32 1.96 97.03 18.2 4.37 1.5 65.57 12.71
1.96 0.28 32.5 1.89 1.15 0.78 0.74 9.2 2.33 2.67 2.8 98.3 123 115.33 1.84 1370 5 518 45.4 2.13 18.48 4.09 16.64 8 7.26 1.37 2.73 653.6 14.5 1.14 2.57 9.72 0.99 4.14 3.19 973.3 4.03 844 4.87 2.22 11.25 0.82 145.8 137.3 1.71 0.44 2.24 1.09 571.4 3549 10.59 6.77 5.95 58.79 1.49 90.14 18.8 2.8 1.41 50.76 43.76
1.95 0.3 32.9 1.9 1.16 0.79 0.75 9.4 2.33 2.67 2.79 97.3 124 116.98 1.8 1396 4.9 520.8 45.4 2.15 18.04 4.25 16.18 8 7.24 1.39 2.72 655.7 14.5 1.15 2.56 9.71 0.99 4.1 3.4 988.3 4.01 850.4 4.89 2.23 11.39 0.8 144.7 135.7 1.7 0.43 2.19 1.09 565.9 3503 10.68 6.95 5.78 59.95 1.48 90.54 19 2.76 1.43 50.63 42.12
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dx.doi.org/10.1021/pr400131r | J. Proteome Res. XXXX, XXX, XXX−XXX
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Table 1. continued Total Protein (g/L) TSH (mIE/L) Urate (μmol/L) Urea (mmol/L)
control 1
control 2
67 2.55 305 5.9
67.8 2.56 302 5.9
disease 1 67.7 1.77 345 8.4
disease 2 67.8 1.71 347 8.6
plasma fraction was then transferred and sampled repeatedly to give a 100 mL plasma pool of respectively control and disease groups. Seven thousand blood fractions were generated in total to give 10 μL aliquots, dispensed into the 384-rack tubes. The collection of blood samples was approved by the ethical board at Lund University (approval number: LU 532−03).
This clinical initiative is screening the three major smokinginduced diseases in asymptomatic smokers in the general public (http://www.youtube.com/watch?v=m3ZGNIi0jwI). Another large scale clinical study initiative by the UK Biobank is an epidemiological study intended to create a resource for research in all medical disciplines, enabling new and ground breaking research on the connections between heredity, environment and lifestyle (http://www.ukbiobank.ac.uk/). The reference plasma samples developed in this project initiative will give great value to global initiatives, such as the CHPP, that will benefit as one scientific program when the entire human proteome is mapped and linked to human diseases.11−17 In this study, we focused on the development of a reference blood plasma standard that has been characterized and is available as a resource for Proteomics research teams around the world.
2.3. Biomarker Analysis
The following tests were run using standard methods from Roche: α1-antitrypsin, ALAT (alanine aminotransferase), albumin, ALP (alkaline phosphatase), apoA (apolipoprotein A1), apoB (apolipoprotein B), ASAT (aspartate aminotransferase), bilirubin, calcium, CK (creatinine kinase), CKMB (phosphocreatine kinase isoform MB), Cl, creatinine, C-reactive protein, cystatin C, estradiol, Fe, ferritin, folate, free PSA, FSH, freeT3, freeT4, bile acid, glucose, GT (glutamyltransferase), haptoglobin, HCG, homocysteine, HDL, IgA, IgG, IgM, potassium, conjugated bilirubin, cobalamin, cholesterol, cortisol, LD (lactate dehydrogenase), LDL, LH, magnesium, myoglobin, Na, orosomucoid (α1-acid-glycoprotein), pancreas amylase, procalcitonin, phosphate, prolactin, NT-proBNP (Nterminal pro brain natriuretic peptide), progesterone, PSA, PTH, SHBG, T3, T4, TCO2, testosterone, triglyceride, TIBC, troponin, total protein, TSH, urate, urea (see Table 1). All tubes were analyzed on a Cobas 8000 modular analyzer from Roche (Basel, Switzerland).
2. EXPERIMENTAL SECTION 2.1. Materials and Instruments
A Hamilton Microlab STAR liquid handling robotic workstation (Hamilton, Reno, NV) was used for automated aliquoting of blood samples. Conductive CO-RE tips (1000 μL), high volume with filters (cat. Nr: 235905), wide bore high Mod-Tip 3.20 mm ID (300 μL) without filters (cat. Nr: 235444) and Slim tips (300 μL) with filters (cat. Nr: 235647) were purchased from Hamilton (Bonaduz Ag, Switzerland). The Matrix storage tubes and seal 0.1 mL 384 2D tubes (cat. Nr: 3815), 20 μm peelable heat sealing foil (cat. Nr: AB-3729), Matrix storage tubes and seals, 0.5 mL 96 2D tubes (cat. Nr: 3734) and 96 caps Sepra seal (cat. Nr: 4463) were from Thermo Fisher Scientific (MA, USA). The variable temperature and time heat sealer instrument (ALPS 3000 50 V, Thermo Scientific, MA) was used for sealing 384-tubes with the foil. For registration of aliquots a VisionMate scanner was used (Thermo Scientific, MA). Nautilus LIMS* (Thermo Scientific, MA) was used throughout the entire study documenting the sample origins and specific 2D barcodes on tubes as unique identifiers.
3. RESULTS AND DISCUSSION Blood is the most commonly used patient sample type where clinical chemistry assays are at place, and out of the various blood fractions, plasma with heparin as the anticoagulant is used most of the time when patients are diagnosed for various diseases in the hospital. The heparin plasma type is used throughout the world for medical diagnosis and healthcare controls in routine practice. This is the major reason for us to choose the heparin plasma as our primary target for the first generation of a plasma reference standard (Figure 1). From that
2.2. Blood Samples
Patient blood samples were obtained from the Department of Clinical Chemistry, Skåne University Hospital, Malmö, Sweden. The reference plasma standard Control group is defined as those with C-reactive protein levels 30 mg/L. In these plasma references, we have used both newborn children 1−2 weeks, as well as youngsters 15−30 years, middle aged 30−50 years, and elderly patients at the ages of 65+. In total there are 80 patients in each group in the reference plasma pools. Blood was collected and sampled in primary tubes with heparin as anticoagulant. Five milliliters of blood was sampled in primary tubes and centrifuged for 10 min at 2000 rpm, which is the standard procedure at the hospital. These sample tubes were stored at +4 °C during and used for the present study 1−16 h after collection. According to the C-reactive protein value, the
Figure 1. Blood plasma collected from a multiethnic donor group of 0−65+ year old men and women. C
dx.doi.org/10.1021/pr400131r | J. Proteome Res. XXXX, XXX, XXX−XXX
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Communication
Figure 2. Workflow of the reference blood samples from the collection at the hospitals until the delivery to research groups.
material, healthy is not really applicable to these patient samples that were available within the selection process. Finally, we agreed with the clinical chemistry colleagues that C-reactive protein, a known and widely applied inflammatory marker, would be a good choice as the molecular group indicator. We analyzed a large set of heparin plasma samples within the two groups to validate the suitability to use C-reactive protein as the biomarker in order to build the two reference plasma biofluid sample groups. We chose concentration levels 30 mg/L. The Creactive protein levels were determined before freezing the samples at −80 °C.
the needle went into the patient arm, until pooling took place ranging between 2 and 8 h, during which period the samples were kept at +4 °C. This is a shorter time than in large-scale studies, like UK Biobank (http://www.ukbiobank.ac.uk/) and LifeGene, (https://www.lifegene.se/), where storage at +4 °C for 24−36 h is used. The globalization and best common practices in clinical chemistry have made it possible to run multicontinent studies with a positive outcome in learning, which relates to optimal patient treatments. The current study builds on the principle that samples should be stored in tubes at ultralow temperatures (−80 °C) and at small sample volumes (
