Blood Plasma Handling for Protein Analysis

7/30/2019 Blood Plasma Handling for Protein Analysis

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Chapter 20

Blood Plasma Handling for Protein Analysis

Christer Ericsson and Monica Nistr

Abstract

Blood handling routines have been worked out that result in consistent protein analytic results in

clinical practice. It would seem reasonable to build on this experience when devising handling routinesor new protein biomarker discovery. Consequently, normal blood sample handling precautions apply toblood sample handling or new biomarker discovery. The blood sample handling protocol mentionedbelow describes room temperature, or 4C, platelet poor EDTA plasma collected within 90 min o

venipuncture, handled, and screened to eliminate hemolysis. DNA can be isolated rom the buy coatthat results as blood cells are sedimented to isolate the plasma.

Key words: Blood, Plasma, Handling, Protein, Analysis, Proteomics

The possibility o blood transusion is one indication that theunctional protein properties o blood can be maintained ex vivoor a considerable amount o time. The unctionality, however,degrades with time. Changes occurring in whole blood duringstorage result in signicant deterioration o clotting actors andultimately in a total loss o unction o granulocytes and platelets(1). Even so, a red cell concentrate may be stored or up to 21 days

i kept at 4C, without loss o apparent unction o the red cells.Platelets may be stored or up to 72 h at 22C without apparentloss o hemostatic unction, but not at 4C, since survival time at4C is signicantly lower than that at 22C. Furthermore, thestability o the hemostatic unction, and platelet morphology, isenhanced in citrate coagulation-inhibited plasma compared tothat in EDTA coagulation-inhibited plasma. Preserving activeclotting actors requires processing within 6 h o collection (1).The intracellular ion potassium increases in plasma over time,

1. Introduction

1.1. Functional Protein

Integrity in Blood

Joakim Dillner (ed.), Methods in Biobanking,Methods in Molecular Biology, vol. 675,DOI 10.1007/978-1-59745-423-0_20, Springer Science+Business Media, LLC 2011


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indicating an inability o cells to maintain ion gradients acrosstheir cell membranes and a leakage o intracellular K+. A typi-cal clinical requirement, thereore, is or the blood sample to beless than 4 h old, and not to be hemolytic, to measure K+ ions inplasma accurately (http://provtagningsanvisningar.karolinska.se/). Thus indications are that the proteins that are unctional inblood remain so or at least 46 h ex vivo. This unctionality

would be a sensitive and broad-based bioassay or blood plasmaunctional protein integrity. Rapid reezing o plasma preservesthe labile coagulation actors V and VIII (1). Rapid cryopreserva-tion o plasma in less than 46 h ollowing blood collection

would, thereore, seem like a reasonable guideline orfunctionalprotein integrity.

A protein biomarker is a protein, or ragment thereo, whosealtered quantity indicates a particular disease state. Presence o aprotein biomarker indicates a change in expression, or state, o aprotein that correlates with the risk o acute or chronic morbidity,

with progression, or with susceptibility to a treatment. These pro-teins would be expected to vary physiologically in concentrationbetween individuals. The reerence interval is a measure o the

variation that includes 95% o the variation in normal individuals,i.e., some apparently normal individuals show concentrationsoutside the reerence interval. A separate measure, the discrimina-tor value, or cut-o, indicates a concentration that discriminateshealthy rom sick individuals.

Albumin is the single most abundant protein in blood plasma,constituting about hal o the cell-ree protein by weight. The 22most abundant proteins constitute about 99% o the dry weighto cell-ree proteins in plasma. The dierence in abundancebetween the most abundant and the least abundant known unc-tional proteins o current clinical importance, albumin and thecytokines, is about 1011 orders o magnitude (2). Changes inabundant plasma protein concentrations largely result rom alter-ations in synthesis by hepatocytes in response to circulating

infammation-associated cytokines, the acute phase response (3).Other systemic changes include a tendency toward cachexia andthromboembolism in cancer (4, 5). Given that blood proteinsphysiologically vary in concentration and that a change o approx-imately 25% in plasma concentration has been suggested as a de-nition o an acute phase protein (3), it is clearly sometimes dicultto say i a dierence in abundance corresponds to normal varia-tion or can be used as a disease marker. This diculty may havecontributed to the signicant number o initially suggested bio-markers that subsequently ail to be validated (6). It should be

clear that the relatively nonspecic, systemic, alterations in theblood proteins that correspond to the acute phase response,cachexia, or increased risk o thromboembolism may well, in

1.2. Current Protein

Biomarkers in Blood
http://provtagningsanvisningar.karolinska.se/http://provtagningsanvisningar.karolinska.se/http://provtagningsanvisningar.karolinska.se/http://provtagningsanvisningar.karolinska.se/


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335Blood Plasma Handling for Protein Analysis

combination with more specic markers, ultimately nd their wayinto a multiplexed disease-specic diagnostic assay.

Normal tissue turnover results in the ormation o tissue deg-radation products that are subsequently eliminated through theblood. The abundance o the proteins released by pathologic tis-sue turnover would be expected to depend on the type o diseaseprocess, the total mass o pathologic tissue that turns over perunit time, and the abundance o the protein in the tissue (7).The larger tissue mass that turns over per unit time, the higherthe concentration o degradation products in blood, all else beingequal. Proteins that are released rom the pathologic tissue thatare characteristic o that tissue and that are abundant enough tobe detected in blood would constitute potential biomarkers. It

would be preerable that the biomarkers used or diagnosis arecausal o the disease or the response.

Quantitation o several protein biomarkers o disease romblood samples is used in clinical practice (http://provtagningsan-

visningar.karolinska.se/). The techniques employed include plasmaprotein electropherograms that assess any systemic infammatoryresponse among the most abundant plasma proteins. The increasedappearance o specic characteristic proteins in blood can alsoserve as an indicator o specic tissue damage. Examples includeaspartataminotranserase (ASAT) and alaninaminotranserase(ALAT) to assess liver damage, and creatinkinase (CK), troponin Iand T (TnI and TnT), and myoglobin to assess heart tissue damageas an indication o heart inarction. These particular analytes areassessed in Li-heparin coagulation-inhibited plasma and requireprocessing within 28 h. Other protein biomarkers or cancer,such as alpha etoprotein, cancer-associated antigen 15-3 (CA15-3), CA 19-9, CA 72-4, CA 125, S100 B, carcinoembry-onic antigen (CEA), and prostate specic antigen (PSA), aremeasured in postcoagulation serum or Li-heparin plasma withoutspecied time limit to analysis. EDTA or heparin plasma, or serumprocessed in 28 h or less, ollowing collection would, thereore,seem like a reasonable guideline or current biomarker protein

integrity.

The emergence o high-throughput, multiplexed protein analysisis expected to provide a platorm or discovering new protein bio-markers in a time-ecient and comprehensive ashion. Thispotential, however, largely remains to be realized. The reasonsmay include that not enough time has passed or the early bio-marker candidates to be validated on a large scale, the limitedsensitivity o the current high-throughput technologies (8, 9),physiologic variability, and the variability in handling and analysis

o blood samples.There seems to be a good reason to use the existing experi-

ence rom clinical chemical practice and blood transusion in

1.3. Establishing

Optimal Conditions

for Screening for New

Biomarkers in Blood
http://provtagningsanvisningar.karolinska.se/http://provtagningsanvisningar.karolinska.se/http://provtagningsanvisningar.karolinska.se/http://provtagningsanvisningar.karolinska.se/


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devising optimal conditions or screening or new biomarkers inblood. That experience would indicate that a preanalytic lag o2 h at room temperature would be acceptable. It should, how-ever, be clear that multiplex analysis potentially is more demand-ing than analysis o single or a ew analytes, since the possibilityexists that individual critical analytes degrade with a unique timecourse. In addition, the sample collection should be as economi-cal on personnel and nancial resources as possible in order tomake the collection o large sample volumes possible. This wouldspecically mean not to require more stringent collection require-ments than is supported by evidence. This review discusses thecurrent state-o-the-art protocol or standardized blood samplehandling in maintaining consistent analytic utility o blood plasmaor protein analysis.

It would be preerable i blood samples could be collectedcontinuously in a healthcare system according to reasoned andstandardized protocols, in order to obtain large numbers o rep-resentative and comparable samples with known patient out-comes. As discussed above, there exist protocols or clinical assaysor protein biomarkers that speciy the use o either (postcoagula-tion) serum, EDTA plasma, citrate plasma, or heparin plasma.Citrate and EDTA inhibit coagulation by chelating divalentcations, which subsequently inhibit enzymes involved in bloodclotting. Heparin unctions through the activation o antithrombinIII. One o the current challenges is to determine which onebloodderivative would be optimal in most cases. The top choice maynot need to be optimal or all conceivable analytes, as long as theartiacts introduced can be documented. The Plasma proteomeProject (PPP) o the Human Proteome Organization (HUPO)has determined that or protein analysis, EDTA coagulation-inhibited plasma is preerable to citrate- or heparin-inhibitedplasma and to serum (10, 11). Platelet depletion was ound to bebenecial in reducing contamination with platelet-derived pro-teins (12). Serum production causes reduction in proteinsinvolved with clot ormation and an increase in the number o

detectable peptides by about 40% (12) and has been shown to bedicult to standardize. Nevertheless, it should be recalled thatseveral o the current cancer biomarkers are measured in serum.Thereore, serum should probably not be recommended or bio-marker screening, but may, ater validation studies, be well suitedor targeted assays o individual analytes.

The original assessment o a preerence or EDTA plasma letopen the maximum allowable time beore separation o plasmaand blood cells or optimal results, and also which temperature isoptimal or most studies. It would seem preerable that a standard-

ized blood plasma handling paradigm be based on systematic stud-ies o the infuence o time, temperature, and coagulation statusand any other relevant variable on dened, identied, analytes,


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and that any changes observed tted into a reasonable hypothesiso cause and eect. Specically, higher temperatures may concep-tually avor degradation, while low temperature may cause cold-induced activation o platelets in certain buers and potentiallysubsequent release o platelet-derived proteins.

Several recent studies have addressed the question o changesin the protein composition o serum and plasma during process-ing, some looking at time beore separation o plasma or serum(1315) and some looking at eects o incubation ater separa-tion (13, 16). They have used chromatographic ractionation othe more abundant blood proteins and recorded any dierencesbetween various handling parameters by mass spectrometry. Themethods used generate mass-to-charge spectra o peptides andproteins, where the identity o the corresponding protein is ini-tially unknown. Clearly, the ndings in those cases need to be

validated with other technologies and sample sets to identiy indi-vidual analytes and t any changes into reasonable hypotheses,especially since it is noted that it can be dicult to obtain stable,reproducible SELDI-TOF MS results (17).

Serum and plasma samples show dierences in their proteinspectra, supporting the HUPO PPP conclusion that a choicebetween blood derivatives is necessary (13, 14, 16). The eect o

visibly detectable hemolysis was readily detectable in the proteinpattern, but overnight asting, or not, had no apparent eect onthe observed proteome (16).

In one study, many o the changes in the pre-centriugationserum samples were readily apparent within 30 min o venipunc-ture, whereas virtually all signicant changes in the plasma sam-ples did not occur until 4 h ater venipuncture (14). Many o theobserved serum peaks arose directly rom platelets or duringcoagulation, as determined by comparisons. In contrast, anotherstudy concluded that serum quality is compromised only i it islet to clot at room temperature or more than 3 h, or more than24 h at 4C (15). The reason or the discrepancy is not clear, butmay be methodological. In a third study, it is concluded that

keeping blood at room temperature or 1, 6, or 24 h changes theproteome prole considerably (13).

Postcentriugation serum samples showed proound time-dependent changes in proteome proles compared with EDTA orheparin plasma samples. Most changes within MS spectra occurredater a storage time o 4 h at room temperature (16). In contrast,another study ound that only minimal changes o the serum pro-teome were noted within 6 h o incubation, while the changesbecame observable ater 8 h o incubation. For serum and plasmasamples stored up to 24 h at 4C, the proteomes did not present

with signicant changes (13).While it would seem like these data need to be ollowed up using

identied proteins and specic reagents or each potential marker,


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they generally support the conclusion that serum preparation isdicult to standardize and that there are smaller changes in theplasma proteomes, than in the serum proteome, in the rst hours.It will be interesting to test the hypothesis originally derived romthe experience rom transusion and existing biomarker analysisthat a preanalytic lag o about 2 h at room temperature would beacceptable, with respect to protein integrity, cellular integrity, andplatelet activation.

Unless new physical or chemical principles are brought to bear onblood plasma handling, we cannot at present reasonably expectany major improvements in existing blood handling paradigms.On the contrary, with the current state o inormation, we cannoteven say that they unequivocally are needed. What we can expectis an examination and a validation o the current ramework, andan optimization based on those ndings. It currently remains tobe determined what the optimal time and temperature would beor blood processing, and to develop validation markers or bloodhandling. One such study is underway (Ismail et al., manuscriptin preparation). Subsequently, we can expect studies o the stabil-ity o individual analytes under the conditions o that optimalhandling protocol, to serve as a reerence or the study o altera-tions in disease.

1. Personal barrier protection, gloves, ace shield, protectiveclothing

2. EDTA blood sample tubes

3. Crushed wet icemaker

4. Centriuge capable o achieving 2500 RCF at 4C or roomtemperature

5. One milliliter handheld pipette with disposable sterile tips

6. Sarstedt Filtropur S 0.2-mm lter (No./REF 83.1826.001)

7. Cryotube 1.8 ml (NUNC 375418)

8. Freezer-sae barcoded labels

9. Liquid nitrogen

10. 80C reezer

11. Sample database

12. Container or saely disposing o potential biohazardousmaterials

1.4. Future

Developments

in Blood Handling

and Validation

2. Materials


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Based on (12).

1. The blood sample should be taken as a venous sample, with-out clenching or pumping o the st (see Note 1).

2. Blood samples are collected into our 10-ml K2EDTA plastic

tubes, and inverted careully ten times to distribute the anti-coagulant (see Note 2).

3. The tubes should either be precooled on ice or remain atroom temperature at all times.

4. Note the time o collection.

5. Allow the blood samples taken at room temperature to cool

to room temperature or 20 min (see Note 3).

6. Note the time o cooling, i applicable.

7. The plasma is separated rom the cells by centriugation or10 min at 2000 RCF at either 4C or at room temperature(see Notes 1, 4, 5).

8. From each tube, 2 ml o the supernatant is removed and sub-jected to ltration through a low protein-binding 0.2-mm l-ter to remove any remaining platelets or other cells.

9. The buy coat containing white cells, at the interace

between the plasma supernatant and the erythrocyte pellet, canbe saved separately as a source o DNA (see Notes 6, 7, 8).

10. The plasma samples are aliquoted into 1.8 ml cryovials androzen by immersion in liquid nitrogen, without delay (seeNotes 8, 9).

11. All aliquoting and reezing should be complete within 90 min.

12. Note the actual time o reezing (even i target times areexceeded), so actual processing time can be calculated. Notethe temperature: 4C or room temperature.

13. Enter data into database in accordance with legal and con-tractual requirements.

14. The plasma and buy coat should be stored at 80C.

15. Dispose o biological material waste in accordance with localrequirements.

1. Apply NIH universal precautions to avoid contaminationrom or to the sample.

3. Method

4. Notes


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2. Sample collection at room temperature is less demandingthan that at 4C, and so should be used consistently i a reli-able cold collection is not practically possible. For the sake oconsistency, it is important to use one protocol or the other,and not to mix protocols.

3. Slow cooling avoids water condensation that may causeosmotic hemolysis.

4. Centriugation is only partially eective in sedimenting plate-lets. Platelets are a major source o released proteins. Decantthe plasma at the top o the tube to minimize contamination

with platelets.

5. For an even more complete removal o platelets, ltration isadded as a second step. The ltration should be perormed

with lters having suciently small pores to remove platelets

and made rom a low protein-binding material.

6. Careully observe the buy coat layer atop the erythrocytes asyou aspirate it, in order to optimize the yield and purity. Buycoat can be collected in the same kind o cryotubes asplasma.

7. We recommend snap reezing the buy coat in liquidnitrogen.

8. Snap-reeze in liquid nitrogen while holding the tubes uprightwith orceps to keep the cap ree o rozen liquid.

9. Apply local precautions when handling liquid nitrogen.

References

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Proteomics, 1(11): 84567.3. Gabay, C. and I. Kushner, (1999) Acute-phase

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6. Finlay, J.A., E.W. Klee, C. McDonald, J.R.Attewell, D. Hebrink, R. Dyer, B. Love, G.Vasmatzis, T.M. Li, J.M. Beechem, and G.G.Klee, (2006) A Systematic method or selectiono promising serum protein biomarkers to

improve prostate cancer (PCa1) detection.Clinical Chemistry,52(11): 2159-2162.

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8. Ericsson, C., Franzn, B. and Nistr, M.,(2006) Frozen Tissue Biobanks; TissueHandling, cryopreservation, extraction anduse or proteomic analysis. Acta Oncologica,45: 643.

9. Hortin, G.L., (2006) The maldi-to massspectrometric view o the plasma proteomeand peptidome. Clin Chem, 52(7): 1223-37.

10. Omenn, G.S., D.J. States, M. Adamski, T.W.Blackwell, R. Menon, H. Hermjakob, et al.,(2005) Overview o the HUPO PlasmaProteome Project: results rom the pilot phase

with 35 collaborating laboratories and multipleanalytical groups, generating a core dataset o3020 proteins and a publicly- available data-base. Proteomics,5(13): 322645.


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11. Rai, A.J., C.A. Geland, B.C. Haywood, D.J.Warunek, J. Yi, M.D. Schuchard, et al., (2005)HUPO Plasma Proteome Project specimencollection and handling: towards the stan-dardization o parameters or plasma pro-teome samples. Proteomics, 5(13): 326277.

12. Tammen, H., I. Schulte, R. Hess, C. Menzel,M. Kellmann, T. Mohring, et al., (2005)Peptidomic analysis o human blood speci-mens: comparison between plasma specimensand serum by dierential peptide display.Proteomics, 5(13): 341422.

13. Hsieh, S.Y., R.K. Chen, Y.H. Pan, and H.L.Lee, (2006) Systematical evaluation o theeects o sample collection procedures onlow-molecular-weight serum/plasma pro-teome proling. Proteomics, 6(10): 318998.

14. Banks, R.E., A.J. Stanley, D.A. Cairns, J.H.Barret, P.C. Clarke, D. Thompson, and P.J.Selby, (2005) Infuences o Blood Sample

Processing on Low-Molecular-Weight ProteomeIdentied by Surace-Enhanced LaserDesorption/Ionization Mass Spectrometry.Clinical Chemistry, 51(9): 163749.

15. West-Nielsen, M., E.V. Hogdall, E. Marchiori,C.K. Hogdall, C. Schou, and N.H. Heegaard,(2005) Sample handling or mass spectromet-ric proteomic investigations o human sera.

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16. Findeisen, P., D. Sismanidis, M. Riedl, V.Costina, and M. Neumaier, (2005) Preanalyticalimpact o sample handling on proteome prol-ing experiments with matrix-assisted laser des-orption/ionization time-o-fight massspectrometry. Clin Chem,51(12): 2409-11.

17. Karsan, A., B.J. Eigl, S. Flibotte, K. Gelmon,P. Switzer, P. Hassell, et al., (2005) Analyticaland preanalytical biases in serum proteomicpattern analysis or breast cancer diagnosis.Clin Chem, 51(8): 15258.

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Blood Plasma Handling for Protein Analysis