An Overview

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Science

In 2002 the World Health Organization recommended that plasma was more applicable than serum in most clinical tests since plasma, with less interference, was better at reflecting the pathological situation of a patient.^1-5 There are existing trends that plasma will replace serum in clinical biochemical tests, although there are reports of discrepancy between the testing results between serum and plasma.^6-9

Gennaro and colleagues¹⁰ reported that the difference of the matrix effect between serum and plasma was too minimal to be neglected using the high performance liquid chromatography (HPLC) method for the measurement of anti- tuberculosis drugs. Other researchers^11-13 investigated analytes in plasma and serum immediately separated from cells and concluded that serum and lithium-heparin plasma samples could be used interchangeably. These reports indicated the matrix effect was too minimal to be neglected. Plasma has some advantages to serum as a clinical specimen. First is the prevention of coagulation-induced interferences. In some serum samples with poor coagulability, clot formation can be still ongoing when centrifugation is finished. Inadequate clot formation with residual fibrin strands can clog the sampling pipette. The use of plasma circumvented these clotting-related problems.¹⁴ Second is turnaround time (TAT) savings. It takes 20–30 minutes for the blood sample to completely clot prior to being spun-down when serum is used to be tested.

Anti-coagulated blood samples are ready to be run once they are drawn from patients.¹⁵ Owning to this time-saving virtue, plasma samples are preferable in urgent tests. Third is the prevention of coagulation-induced interferences. The coagulation process changes the concentrations of numerous constituents of the extra-cellular fluid beyond their maximum allowable

limit,¹⁶ while plasma can better reflect the pathological situation of a patient than serum. Fourth, the serum yield from a given volume of whole blood is always less than plasma, however, certain analyses cannot be performed on plasma.¹⁷

Heparin is the recommended anticoagulant for many de- terminations using whole blood or plasma specimens because of its minimal chelating properties, minimal effects on water shifts, and relatively low cation concentration. Heparin induces the in- hibition of thrombin and Factor X to prevent clotting or activa- tion of thrombin, which in turn prevents the formation of fibrin from fibrinogen. Lithium heparin is recommended because it is the least likely to interfere with the results when performing tests for other ions, like sodium.¹⁸

Banfi and colleagues⁷conducted an inquiry among Italian laboratories and reported that gel separator tubes were more extensively used than plain tubes for obtaining serum. The gel separator was made up of an organic hydrophobic substance and Cab-O-sil, which was inert to blood. It showed no in- fluence on immunological and clinical chemical technology contacted with serum. Gel separator is an inert, thixotropic polymer gel with a specific gravity of 1.04,¹⁹ which moves to the interface based on a density gradient and forms an imper- meable barrier between the serum and the clot. Serum separator tubes provide a closed system allowing for the collection, transport, processing, and sampling of specimens. However, we have not found any systematic report on the clinical application of lithium-heparin plasma with gel separator.

Bebecca and colleagues²⁰ reviewed much of the literature regarding a sample type that may affect the measurement of hormone concentration, while most literature reported that analyte concentrations showed no difference between serum and heparin

The Feasibility of Using Lithium-Heparin Plasma From a Gel Separator Tube as a Substitute for

Serum in Clinical Biochemical Tests

Yuan-hua Wei, MD, Chun-bing Zhang, MD, PhD, Xue-wen Yang, MD, PhD, Ming-de Ji, MD

(Department of Laboratory Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing, People’s Republic of China)

DOI: 10.1309/LMIXVAI70KS0UWQI

Abstract

Background: Although plasma is preferable to serum as a clinical specimen, there has been little data published to the widespread use of plasma with gel separator. The aim of this study was to investigate the feasibility of lithium-heparin plasma with gel separator as the substitute of serum in clinical biochemical tests.

Methods: Three specimen types were labeled as serum with gel separator (S), lithium

heparin-plasma (P), and lithium heparin-plasma with gel separator (G). Primarily 120 specimens were centrifuged and analyzed within 2 hours (T₀), 24 hours (T₂₄), and 48 hours (T₄₈).

Differences in analyte concentrations between tubes at T₀ and following storage times (T₂₄ to T₄₈) were evaluated for statistical significance.

Wright’s staining of serum and plasma smears was used to investigate the formed elements in the upper liquid.

Results: At T₀, 7 analytes in G showed the most valid results reflecting the in vivo state over P and S, and the other 24 analytes in G, P, and S were indistinguishable. The number of formed elements in P was larger than S and G.

Conclusions: The lithium-heparin plasma with gel separator has advantages over serum with gel separator and plasma in clinical biochemical tests.

Submitted 9.23.09 | Revision Received 11.19.09 | Accepted 11.23.09

Downloaded from https://academic.oup.com/labmed/article/41/4/215/2504888 by guest on 01 June 2024

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plasma. If plasma is finally found to be more suitable for clinical lab testing, gel separator should be continuously employed in the anticoagulated vacuum tubes. But nothing published system- atically reported the advantage of lithium heparin-plasma with gel separator (G) over plasma only with lithium heparin (P), and serum (G).²¹ In this study, we compared values of G with those of P and S in 31 tests, and focused on 7 biochemical tests to assess whether G is optimal. If so, it may pave the way for the future application of plasma samples in vacuum tubes with gel separator in clinical laboratory processes.

Materials and Methods

This study was approved by the ethics committee of the Jiangsu Province Hospital of Traditional Chinese Medicine.

Each donor was informed of the purpose of the investigation.

In 1 day, 120 blood specimens were collected from 40 donors in the following types of plastic tubes:

S: 5 mL Serum Gel Separator BD Vacutainer SST II Ad- vance Tube (REF 367954);

P: 6 mL Plasma Heparinized BD Vacutainer (REF 367885), without gel separator;

G: 3 mL Plasma BD Vacutainer PST Gel and Lithium Heparin (REF 367960).

Triplicate blood samples (S, P, and G) from a single ve- nipuncture were obtained sequentially from the antecubital vein. The sequence was randomized to negate the effect of draw order. All patients were >18 years of age.

We selected 31 biochemical tests which were certificated by the accreditation of ISO15189:2007 (Medical Laborato- ries-Particular Requirements for Quality and Competence).

Therefore, these tests could be traced to the source and results should be reliable. The list of analytes was Aspartate aminotransferase (AST), total protein (TP), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glucose (GLU), potas- sium (K⁺), inorganic phosphorus (P), alanine aminotransferase (ALT), albumin (ALB), L-γ-Glutamyll transferase (GGT), total bilirubin (TB), direct bilirubin (DB), total bile acids (TBA), cholinesterase (CHE), adenosine deaminase (ADA), urea, creatinine, sodium (Na⁺), chloride (Cl^-), calcium (Ca²⁺), carbon dioxide (CO2), uric acid (UA), magnesium (Mg), a- amylase (AMY), lipase (LIP), creatine kinase (CK), cholesterol (CHO), triglycerides (TG), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), lipoprotein (a) (Lp[a]). The 31 analytes and daily controls were analyzed on the Olympus biochemis- try analyzer AU2700 (Olympus, Tokyo, Japan).

Serum was allowed to clot for 30 minutes at RT and centrifuged at 3000 × g for 10 minutes. Plasma was centrifuged immediately at 3000 × g for 10 minutes.

All specimens were transported to the laboratory for analysis within a maximum of 2 hours (T₀). The AST, TP, ALP, LDH, GLU, K⁺, and phosphorus were further repeated at the 24th hour (T₂₄) and the 48th hour (T₄₈). The other 24 analytes were only analyzed at T₀for less related to coagulation processes and limited volume of serum and plasma. Except

The determination of sample size was calculated by the formula of sample size determination of 2-way ANOVA tests:

=34, so 40 patients were calculated as the suitable statistical numbers.

Where u_α=1.64, u_β=1.28 were determined by the type I and II error, σ_d equals the standard deviation of paired samples, and δ equals the difference of paired samples. σ_d/δ=2 was believed to be the average ratio in the study.

At T₀, all results of 31 analytes were performed using a 2-way ANOVA and LSD-t test to establish which pair of conditions differed significantly using software package SPSS 14.0 (Chicago, IL). The statistical significance was considered to be as important as the clinical significance since triplicate blood samples (S, P, and G) were drawn at the same time from a single patient.

Values of 7 focused analytes were analyzed to evaluate within-tube stability. Results for each tube at T₀ were used as a reference to evaluate within-tube stability over T₄₈. The ratio of variation (RV) was equal to (T₄₈ - T₀)/T₀. The higher the RV, the more instable the tube type. The ratio of variation was deemed to be clinically significant if it was higher than the total error (TE) range. The TE was calculated from the following equation, which was generated by a combina- tion of imprecision and bias. TE=1.65× (imprecision, I) + (allowable bias, B) (a<0.05)

Results

Results of Three Tube Types From T₀ to T₄₈ The statistical data of 7 focused analytes in the 3 tube types from T₀ to T₄₈ are summarized in Table 1. Other statistical data of 24 analytes at T₀ are not shown for non-statistical differences among the 3 tube types (Table 1).

Most of the analytes displayed a non-statistical significance for differences between tube types at T₀. However, AST, ALP, LDH, K⁺, and phosphorus, which had much higher concentrations in blood cells than plasma, were higher in S than G at T₀. Total protein, which was composed of fibrinogens and other proteins, was higher in G than S. Glucose, which appeared in blood cells metabolisms, was lower in P than G.

The RVs reflected the level of time-dependent changes of analytes. The RVs showed the sample in the S tubes produced similar results from T₀ to T₄₈, which indicated the analytes in the serum were stable for 48 hours at 4°C. However, the analytes in plasma were less stable in the G tubes and poorly stable in P tubes. Specifically, AST, TP, ALP, LDH, GLU, and K⁺ in the serum collected by the S tubes showed no clinical significance over 48 hours, while only AST, TP, ALP, and LDH were stable in the plasma from the G tubes within 48 hours. Except GLU, the other 6 analytes continuously increased within 48 hours.

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RBCs, platelets, or WBCs. The results showed that the serum sample contained the fewest micro-particles in the superna- tant and gel-separated plasma (G) and was better than plasma (P) in bearing tiny mass residues.

Discussion

Figures 1-3 showed there were some RBCs, WBCs, or platelets in the upper liquid, and the formed elements in S or G were fewer than in P. These suspended blood cells increased the

determining imprecision and explained why G was more stable than P as the RV showed. Many analyte concentrations in the intracellular fluid are much higher than those in extracellular fluid, such as a higher concentration of AST, ALT, LDH, K⁺, and phosphate in RBCs and platelets as well as higher concentrations of ALP in WBCs. When P was centrifuged at a high speed, there were still a few blood cells in the plasma, which resulted in a higher RV. While the barrier gel (G) formed a stable barrier from the liquid and cells, it reduced the number of blood cells providing more accurate results.

Table 1_Data Summary of 7 Analytes for T₀ Comparison and Time-Course Trending

Means at 3 Times (h)

Type of Statistical RV (%) Clinical

Analyte Analyte N 0 24 48 Significance at T₀ (T₄₈- T₀)/T₀ TE (%) Significance of RV

0 24 48

AST (U/L) S 40 27.7 27.5 28.2 S-P(n) 1.8 15.2 n

P 40 27.4 28.6 29.6 P-G(s) 8.0 n

G 40 26.5 27.3 28.0 S-G(s) 5.7 n

TP (g/L) S 40 68.0 67.8 69.1 S-P(s) 1.6 3.4 n

P 40 70.4 72.1 74.6 P-G(n) 6.0 s

G 40 70.5 70.7 72.8 S-G(s) 3.3 n

ALP (U/L) S 40 79.6 79.3 80.0 S-P(s) 0.5 11.7 n

P 40 78.0 75.4 75.6 P-G(n) –3.1 n

G 40 78.1 76.5 75.8 S-G(s) –2.9 n

LDH (U/L) S 40 183.1 181.4 191.7 S-P(s) 4.7 11.4 n

P 40 172.8 181.5 204.5 P-G(n) 18.3 s

G 40 171.1 171.5 189.5 S-G(s) 10.8 n

GLU (mmol/L) S 40 4.84 4.76 4.82 S-P(s) –0.4 6.9 n

P 40 4.74 3.93 3.19 P-G(n) –32.7 s

G 40 4.79 4.22 3.80 S-G(n) –20.7 s

K⁺(mmol/L) S 40 4.45 4.28 4.57 S-P(s) 2.7 5.8 n

P 40 4.08 4.99 6.48 P-G(n) 58.8 s

G 40 4.07 4.13 4.40 S-G(s) 8.1 s

Phosphorus (mmol/L) S 40 1.24 1.31 1.40 S-P(s) 12.9 10.2 s

P 40 1.19 1.38 1.70 P-G(n) 42.9 s

G 40 1.20 1.25 1.37 S-G(s) 14.2 s

S, serum; P, lithium heparin-plasma; G, lithium heparin-plasma with gel separator; N, number of specimens; S-P, paired-samples T tests between S and P; P-G, paired-samples T tests between P and G; S-G, paired-samples T tests between S and G; (s), significantly different, P<0.05; (n), no significant; RV, ratio of variation.

Wright’s staining of samples from the same patient in each of 3 tubes (S, P, and G) at T₀ under microscope at x1000 magnification.

Figure 1_Wright’s staining of S (x1000). There is 1 micro-particle residue.

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The coagulation process changed the concentrations of numerous constituents of the extra-cellular fluid beyond their maximum allowable limit.¹⁶ The changes were induced by the following 2 mechanisms: (1) increased concentrations caused by platelet, RBC, and WBC components in serum as compared with plasma (eg, AST, LDH, K⁺,^13,22 phosphorus,^23,24 ALP, etc); and (2) decreased concentration of constituents in serum as a result of cellular metabolism and the coagulation process (eg,

Mg, AMY, LIP, CK, CHO, TG, HDL-C, LDL-C, and Lp(a), were indistinguishable at T₀ between serum and plasma. The differences shown in Table 1 were greatly due to the changes of constituents caused by the coagulation and metabolism process.

Therefore, we concluded that tests of AST, ALP, LDH, K⁺,¹³ and phosphorus in plasma at T₀ better reflected the in vivo state of constituents than serum.

The higher TP concentration in plasma must be attrib- Figure 2_Wright’s staining of P (x1000). There are 6 micro-particle residues.

Figure 3_Wright's staining of G (x1000). There are 2 micro-particle residues.

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induced by coagulation consumed glucose, which resulted in a negative bias.^12,23,24 For accurate glucose values, blood specimens should be analyzed as soon as possible. Our study showed the GLU values in P were lower than in S and G, so P may have a negative bias. Therefore, we concluded that tests of GLU in G or S at T₀ better reflected the in vivo state of constituents than in P.

The RV was equal to the level of time-dependent changes of analytes from T₀ to T_48.Obviously, analytes in P were the least stable. Analytes in S were more stable than G; however, it might be due to the higher baseline values of S than G at T₀, which resulted in less time-dependent changes. The gel separator moved upwards into the serum and plasma interface during centrifugation, where it formed a stable barrier from liquid and cells to keep analytes stable. Lowbeer and colleagues²⁵ reported that NTproBNP in G was stable for 3 days at room temperature, which was better than in S and P.

Above all, we concluded that AST, ALT, LDH, K⁺, and phosphate in S showed positive biases compared with the in vivo state of a constituent, while TP and GLU in S showed negative biases. We did not consider analyzing the difference of the 3 samples on the condition of delayed centrifugation to simplify and control the experiment better. While some authors²³ studied the difference of samples for delayed centrifugation very well, many of them concluded that most of the tests were indistinguishable from serum or plasma after 2 hours. Only with K⁺, AST, GLU, etc, can plasma and serum not be used interchangeably. As a clinical reporter, it is important to reflect the in vivo state of constituents at T₀.²³ In this study, all of the 31 analytes studied in G could better reflect the in vivo state of constituents at T₀ over S and P, and G was more suitable than P for sample storage.

No hospitals in China use lithium-heparin plasma with gel separator as a routine practice. This study sheds new light on the application of the heparin-anticoagulated vacuum tubes with gel separator in clinical biochemical tests. Although serum was still the preferred assayed material for historical and traditional reasons, plasma in lithium-heparin tubes with gel separator can be a substitution for a serum sample for most common biochemical tests, even though other analytes such as drugs, hormones, immunosuppressive, and cardiac markers were not analyzed in this study. A series of reference ranges for plasma analysis including AST, ALT, TP, ALP, LDH, GLU, K⁺, phosphorus, etc, should be established for clinical use. ^LM

Acknowledgements: We gratefully thank Bing Bai in Pharmacology at Emory University for writing assistance.

Keywords: plasma, serum, specimen types, gel separator, analytes

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