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 routines for new protein biomarker discovery. Consequently, normal blood sample handling precautions apply to blood sample handling for new biomarker discovery. The blood sample handling protocol mentioned below describes room temperature, or 4C, platelet poor EDTA plasma collected within 90 min of venipuncture, handled, and screened to eliminate hemolysis. DNA can be isolated from the buffy coat that results as blood cells are sedimented to isolate the plasma. Key words: Blood, Plasma, Handling, Protein, Analysis, Proteomics

1. Introduction
1.1. Functional Protein Integrity in Blood

The possibility of blood transfusion is one indication that the functional protein properties of blood can be maintained ex vivo for a considerable amount of time. The functionality, however, degrades with time. Changes occurring in whole blood during storage result in significant deterioration of clotting factors and ultimately in a total loss of function of granulocytes and platelets (1). Even so, a red cell concentrate may be stored for up to 21days if kept at 4C, without loss of apparent function of the red cells. Platelets may be stored for up to 72h at 22C without apparent loss of hemostatic function, but not at 4C, since survival time at 4C is significantly lower than that at 22C. Furthermore, the stability of the hemostatic function, and platelet morphology, is enhanced in citrate coagulation-inhibited plasma compared to that in EDTA coagulation-inhibited plasma. Preserving active clotting factors requires processing within 6h of collection (1). The intracellular ion potassium increases in plasma over time,

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 of cells to maintain ion gradients across their cell membranes and a leakage of intracellular K+. A typical clinical requirement, therefore, is for the blood sample to be less than 4h old, and not to be hemolytic, to measure K+ ions in plasma accurately (http:/ /provtagningsanvisningar.karolinska. se/). Thus indications are that the proteins that are functional in blood remain so for at least 46 h ex vivo. This functionality would be a sensitive and broad-based bioassay for blood plasma functional protein integrity. Rapid freezing of plasma preserves the labile coagulation factors V and VIII (1). Rapid cryopreservation of plasma in less than 46 h following blood collection would, therefore, seem like a reasonable guideline for functional protein integrity.
1.2. Current Protein Biomarkers in Blood

A protein biomarker is a protein, or fragment thereof, whose altered quantity indicates a particular disease state. Presence of a protein biomarker indicates a change in expression, or state, of a protein that correlates with the risk of acute or chronic morbidity, with progression, or with susceptibility to a treatment. These proteins would be expected to vary physiologically in concentration between individuals. The reference interval is a measure of the variation that includes 95% of the variation in normal individuals, i.e., some apparently normal individuals show concentrations outside the reference interval. A separate measure, the discriminator value, or cut-off, indicates a concentration that discriminates healthy from sick individuals. Albumin is the single most abundant protein in blood plasma, constituting about half of the cell-free protein by weight. The 22 most abundant proteins constitute about 99% of the dry weight of cell-free proteins in plasma. The difference in abundance between the most abundant and the least abundant known functional proteins of current clinical importance, albumin and the cytokines, is about 1011 orders of magnitude (2). Changes in abundant plasma protein concentrations largely result from alterations in synthesis by hepatocytes in response to circulating inflammation-associated cytokines, the acute phase response (3). Other systemic changes include a tendency toward cachexia and thromboembolism in cancer (4, 5). Given that blood proteins physiologically vary in concentration and that a change of approximately 25% in plasma concentration has been suggested as a definition of an acute phase protein (3), it is clearly sometimes difficult to say if a difference in abundance corresponds to normal variation or can be used as a disease marker. This difficulty may have contributed to the significant number of initially suggested biomarkers that subsequently fail to be validated (6). It should be clear that the relatively nonspecific, systemic, alterations in the blood proteins that correspond to the acute phase response, cachexia, or increased risk of thromboembolism may well, in

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combination with more specific markers, ultimately find their way into a multiplexed disease-specific diagnostic assay. Normal tissue turnover results in the formation of tissue degradation products that are subsequently eliminated through the blood. The abundance of the proteins released by pathologic tissue turnover would be expected to depend on the type of disease process, the total mass of pathologic tissue that turns over per unit time, and the abundance of the protein in the tissue (7). The larger tissue mass that turns over per unit time, the higher the concentration of degradation products in blood, all else being equal. Proteins that are released from the pathologic tissue that are characteristic of that tissue and that are abundant enough to be detected in blood would constitute potential biomarkers. It would be preferable that the biomarkers used for diagnosis are causal of the disease or the response. Quantitation of several protein biomarkers of disease from blood samples is used in clinical practice (http://provtagningsanvisningar.karolinska.se/). The techniques employed include plasma protein electropherograms that assess any systemic inflammatory response among the most abundant plasma proteins. The increased appearance of specific characteristic proteins in blood can also serve as an indicator of specific tissue damage. Examples include aspartataminotransferase (ASAT) and alaninaminotransferase (ALAT) to assess liver damage, and creatinkinase (CK), troponinI and T (TnI and TnT), and myoglobin to assess heart tissue damage as an indication of heart infarction. These particular analytes are assessed in Li-heparin coagulation-inhibited plasma and require processing within 28 h. Other protein biomarkers for cancer, such as alpha fetoprotein, cancer-associated antigen 15-3 (CA 15-3), CA 19-9, CA 72-4, CA 125, S100 B, carcinoembryonic antigen (CEA), and prostate specific antigen (PSA), are measured in postcoagulation serum or Li-heparin plasma without specified time limit to analysis. EDTA or heparin plasma, or serum processed in 28h or less, following collection would, therefore, seem like a reasonable guideline for current biomarker protein integrity.
1.3. Establishing Optimal Conditions for Screening for New Biomarkers in Blood

The emergence of high-throughput, multiplexed protein analysis is expected to provide a platform for discovering new protein biomarkers in a time-efficient and comprehensive fashion. This potential, however, largely remains to be realized. The reasons may include that not enough time has passed for the early biomarker candidates to be validated on a large scale, the limited sensitivity of the current high-throughput technologies (8, 9), physiologic variability, and the variability in handling and analysis of blood samples. There seems to be a good reason to use the existing experience from clinical chemical practice and blood transfusion in

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devising optimal conditions for screening for new biomarkers in blood. That experience would indicate that a preanalytic lag of 2h at room temperature would be acceptable. It should, however, be clear that multiplex analysis potentially is more demanding than analysis of single or a few analytes, since the possibility exists that individual critical analytes degrade with a unique time course. In addition, the sample collection should be as economical on personnel and financial resources as possible in order to make the collection of large sample volumes possible. This would specifically mean not to require more stringent collection requirements than is supported by evidence. This review discusses the current state-of-the-art protocol for standardized blood sample handling in maintaining consistent analytic utility of blood plasma for protein analysis. It would be preferable if blood samples could be collected continuously in a healthcare system according to reasoned and standardized protocols, in order to obtain large numbers of representative and comparable samples with known patient outcomes. As discussed above, there exist protocols for clinical assays for protein biomarkers that specify the use of either (postcoagulation) serum, EDTA plasma, citrate plasma, or heparin plasma. Citrate and EDTA inhibit coagulation by chelating divalent cations, which subsequently inhibit enzymes involved in blood clotting. Heparin functions through the activation of antithrombin III. One of the current challenges is to determine which one blood derivative would be optimal in most cases. The top choice may not need to be optimal for all conceivable analytes, as long as the artifacts introduced can be documented. The Plasma proteome Project (PPP) of the Human Proteome Organization (HUPO) has determined that for protein analysis, EDTA coagulationinhibited plasma is preferable to citrate- or heparin-inhibited plasma and to serum (10, 11). Platelet depletion was found to be beneficial in reducing contamination with platelet-derived proteins (12). Serum production causes reduction in proteins involved with clot formation and an increase in the number of detectable peptides by about 40% (12) and has been shown to be difficult to standardize. Nevertheless, it should be recalled that several of the current cancer biomarkers are measured in serum. Therefore, serum should probably not be recommended for biomarker screening, but may, after validation studies, be well suited for targeted assays of individual analytes. The original assessment of a preference for EDTA plasma left open the maximum allowable time before separation of plasma and blood cells for optimal results, and also which temperature is optimal for most studies. It would seem preferable that a standardized blood plasma handling paradigm be based on systematic studies of the influence of time, temperature, and coagulation status and any other relevant variable on defined, identified, analytes,

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and that any changes observed fitted into a reasonable hypothesis of cause and effect. Specifically, higher temperatures may conceptually favor degradation, while low temperature may cause coldinduced activation of platelets in certain buffers and potentially subsequent release of platelet-derived proteins. Several recent studies have addressed the question of changes in the protein composition of serum and plasma during processing, some looking at time before separation of plasma or serum (1315) and some looking at effects of incubation after separation (13, 16). They have used chromatographic fractionation of the more abundant blood proteins and recorded any differences between various handling parameters by mass spectrometry. The methods used generate mass-to-charge spectra of peptides and proteins, where the identity of the corresponding protein is initially unknown. Clearly, the findings in those cases need to be validated with other technologies and sample sets to identify individual analytes and fit any changes into reasonable hypotheses, especially since it is noted that it can be difficult to obtain stable, reproducible SELDI-TOF MS results (17). Serum and plasma samples show differences in their protein spectra, supporting the HUPO PPP conclusion that a choice between blood derivatives is necessary (13, 14, 16). The effect of visibly detectable hemolysis was readily detectable in the protein pattern, but overnight fasting, or not, had no apparent effect on the observed proteome (16). In one study, many of the changes in the pre-centrifugation serum samples were readily apparent within 30min of venipuncture, whereas virtually all significant changes in the plasma samples did not occur until 4 h after venipuncture (14). Many of the observed serum peaks arose directly from platelets or during coagulation, as determined by comparisons. In contrast, another study concluded that serum quality is compromised only if it is left to clot at room temperature for more than 3h, or more than 24 h at 4C (15). The reason for the discrepancy is not clear, but may be methodological. In a third study, it is concluded that keeping blood at room temperature for 1, 6, or 24h changes the proteome profile considerably (13). Postcentrifugation serum samples showed profound timedependent changes in proteome profiles compared with EDTA or heparin plasma samples. Most changes within MS spectra occurred after a storage time of 4h at room temperature (16). In contrast, another study found that only minimal changes of the serum proteome were noted within 6 h of incubation, while the changes became observable after 8h of incubation. For serum and plasma samples stored up to 24 h at 4C, the proteomes did not present with significant changes (13). While it would seem like these data need to be followed up using identified proteins and specific reagents for each potential marker,

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they generally support the conclusion that serum preparation is difficult to standardize and that there are smaller changes in the plasma proteomes, than in the serum proteome, in the first hours. It will be interesting to test the hypothesis originally derived from the experience from transfusion and existing biomarker analysis that a preanalytic lag of about 2h at room temperature would be acceptable, with respect to protein integrity, cellular integrity, and platelet activation.
1.4. Future Developments in Blood Handling and Validation

Unless new physical or chemical principles are brought to bear on blood plasma handling, we cannot at present reasonably expect any major improvements in existing blood handling paradigms. On the contrary, with the current state of information, we cannot even say that they unequivocally are needed. What we can expect is an examination and a validation of the current framework, and an optimization based on those findings. It currently remains to be determined what the optimal time and temperature would be for blood processing, and to develop validation markers for blood handling. One such study is underway (Ismail etal., manuscript in preparation). Subsequently, we can expect studies of the stability of individual analytes under the conditions of that optimal handling protocol, to serve as a reference for the study of alterations in disease.

2. Materials
1. Personal barrier protection, gloves, face shield, protective clothing 2. EDTA blood sample tubes 3. Crushed wet icemaker 4. Centrifuge capable of achieving 2500RCF at 4C or room temperature 5. One milliliter handheld pipette with disposable sterile tips 6. Sarstedt Filtropur S 0.2-mm filter (No./REF 83.1826.001) 7. Cryotube 1.8ml (NUNC 375418) 8. Freezer-safe barcoded labels 9. Liquid nitrogen 10. 80C freezer 11. Sample database 12. Container for safely disposing of potential biohazardous materials

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3. Method
Based on (12). 1. The blood sample should be taken as a venous sample, without clenching or pumping of the fist (see Note 1). 2. Blood samples are collected into four 10-ml K2EDTA plastic tubes, and inverted carefully ten times to distribute the anticoagulant (see Note 2). 3. The tubes should either be precooled on ice or remain at room temperature at all times. 4. Note the time of collection. 5. Allow the blood samples taken at room temperature to cool to room temperature for 20min (see Note 3). 6. Note the time of cooling, if applicable. 7. The plasma is separated from the cells by centrifugation for 10min at 2000 RCF at either 4C or at room temperature (see Notes 1, 4, 5). 8. From each tube, 2 ml of the supernatant is removed and subjected to filtration through a low protein-binding 0.2-mm filter to remove any remaining platelets or other cells. 9. The buffy coat containing white cells, at the interface between the plasma supernatant and the erythrocyte pellet, can be saved separately as a source of DNA (see Notes 6, 7, 8). 10. The plasma samples are aliquoted into 1.8ml cryovials and frozen by immersion in liquid nitrogen, without delay (see Notes 8, 9). 11. All aliquoting and freezing should be complete within 90min. 12. Note the actual time of freezing (even if target times are exceeded), so actual processing time can be calculated. Note the temperature: 4C or room temperature. 13. Enter data into database in accordance with legal and contractual requirements. 14. The plasma and buffy coat should be stored at 80C. 15. Dispose of biological material waste in accordance with local requirements.

4. Notes
1. Apply NIH universal precautions to avoid contamination from or to the sample.

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2. Sample collection at room temperature is less demanding than that at 4C, and so should be used consistently if a reliable cold collection is not practically possible. For the sake of consistency, it is important to use one protocol or the other, and not to mix protocols. 3. Slow cooling avoids water condensation that may cause osmotic hemolysis. 4. Centrifugation is only partially effective in sedimenting platelets. Platelets are a major source of released proteins. Decant the plasma at the top of the tube to minimize contamination with platelets. 5. For an even more complete removal of platelets, filtration is added as a second step. The filtration should be performed with filters having sufficiently small pores to remove platelets and made from a low protein-binding material. 6. Carefully observe the buffy coat layer atop the erythrocytes as you aspirate it, in order to optimize the yield and purity. Buffy coat can be collected in the same kind of cryotubes as plasma. 7. We recommend snap freezing the buffy coat in liquid nitrogen. 8. Snap-freeze in liquid nitrogen while holding the tubes upright with forceps to keep the cap free of frozen liquid. 9. Apply local precautions when handling liquid nitrogen.
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