CLINICAL LABORATORY ANALYSIS
Approximately 8% of total human body weight is represented by blood, with an average volume in
females and males of 5 and 5.5 L, respectively [1].
Blood consists of a cellular fraction, which includes erythrocytes, leukocytes, and thrombocytes, and a liq- uid fraction, which transports these elements through- out the body. Blood vessels interconnect all the organ systems in the body and play a vital role in communi- cation and transportation between tissue compart- ments. Blood serves numerous functions, including delivery of nutrients to tissues; gas exchange; transport of waste products such as metabolic by-products for disposal; communication through hormones, proteins, and other mediators to target tissues; and cellular protection against invading organisms and foreign material. Given these myriad roles, blood is an ideal specimen for measuring biomarkers associated with various physiological conditions, whether it is direct measurement of cellular material and surface markers or measurement of soluble factors associated with cer- tain physiological conditions.
Plasma consists of approximately 93% water, with the remaining 7% composed of electrolytes, small organic molecules, and proteins. Various constituents of plasma are summarized inTable 3.1. These analytes are in transit between cells in the body and are present in varying concentrations depending on the
physiological state of the various organs. Therefore, accurate analysis of the plasma is crucial for obtaining information regarding diagnosis and treatment of diseases. In clinical laboratory analysis, plasma can be obtained from whole blood through the use of anticoa- gulants followed by centrifugation. Consequently, plasma specimens for the clinical laboratory contain anticoagulants such as heparin, citrate, EDTA, oxalate, and fluoride. The relative roles of these anticoagulants in affecting analyte measurements are discussed later in this chapter. In contrast to anticoagulated plasma specimens, serum is the clear liquid that separates from blood when it is allowed to clot. Further separa- tion of the clear serum from the clotted blood can be achieved through centrifugation. Given that fibrinogen is converted to fibrin in clot formation during the coag- ulation cascade, serum contains no fibrinogen and no anticoagulants.
In the clinical laboratory, suitable blood specimens include whole blood, plasma, and serum. Key differ- ences in these sample matrices influence their suitabil- ity for certain laboratory tests (Table 3.2).
Whole Blood
In addition to the obvious advantage of whole blood for the analysis of cellular elements, these specimens are also preferred for analytes that are concentrated within the cellular compartment. Erythrocytes can be considered to be a readily accessible tissue with mini- mal invasive procedures and may more accurately reflect tissue distribution of certain analytes. Examples of such analytes include vitamins, trace elements, and certain drugs (Table 3.3). Erythrocytes are the most abundant cell type in the blood. In adults, 1μL of blood contains approximately 5 million erythrocytes compared to 400011,000 leukocytes and 150,000450,000 platelets [4]. The volumetric fraction of erythrocytes is clinically referred to as the hemato- crit, expressed as a percentage of packed erythrocytes in a blood sample after centrifugation. The normal range for adult males is 4151%, and that for adult females is 3645% [4]. Clearly, alterations in hemato- crit will directly alter the available plasma water con- centration, which in turn affects the measurement of water-soluble factors in whole blood.
A major use for whole blood specimens is for point- of-care analysis. Although point-of-care meters can be located in the clinical laboratory, the primary advan- tage of this technology is near-patient testing, which offers rapid and convenient analysis and the use of small sample volumes while the clinician is examining the patient. The most common point-of-care specimens are taken by skin puncture. These blood samples are TABLE 3.1 Principal Components of Plasma
Component Quantity Units
Sodium 144 mmol/L
Potassium 4 mmol/L
Bicarbonate 25 mmol/L
Chloride 105 mmol/L
Hydrogen ions 40 mmol/L
Calcium 2.5 mmol/L
Magnesium 0.8 mmol/L
Inorganic phosphate 1.1 mmol/L
Glucose 4.5 mmol/L
Cholesterol 2.0 g/L
Fatty acids 3.0 g/L
Total protein 7085 g/L
Albumin 45 g/L
α-Globulins 7 g/L
β-Globulins 8.5 g/L
γ-Globulins 10.6 g/L
Fibrinogen 3 g/L
Prothrombin 1 g/L
Transferrin 2 g/L
21
DIFFERENCES BETWEEN WHOLE BLOOD, PLASMA, AND SERUM SPECIMENS FOR CLINICAL LABORATORY ANALYSIS
composed of a mixture of blood from the arterioles, venules, and capillaries and may be diluted with inter- stitial fluid and intracellular fluid. Furthermore, the extent of dilution with interstitial and intracellular fluid is also affected by the hematocrit. Given these physiological differences, analytes measured in whole blood do not exactly match results obtained from anal- ysis of plasma or serum samples. Indeed, there is less water inside erythrocytes compared to the plasma;
therefore, levels of hydrophilic analytes such as glu- cose, electrolytes, and water-soluble drugs will be lower in the capillary whole blood[5].
As mentioned previously, it is apparent that changes in both hematocrit level and plasma water content contribute to the discrepancy in analyte mea- surements between whole blood and plasma meth- ods. However, in the case of point-of-care glucose meters, it was proposed and later adopted by the International Federation of Clinical Chemistry that a general conversion factor of 1.11 be applied to obtain plasma-equivalent glucose molarity [6,7]. Although this was an attempt to produce more harmonized results regarding glucose measurement and reduce clinical misinterpretations, the application of a gen- eral conversion factor does not take into account the wide variations in both hematocrit and plasma water levels exhibited by some patient subpopulations.
Indeed, the proportion of total errors exceeding 10 and 15% in glucose measurements has been found to increase with patient acuity[8]. For this reason, inter- pretation of analyte measurements in whole blood should be sensitive to the hemodynamic status of the patient.
Plasma Versus Serum Specimens
Although it is clear that there are certain advantages to whole blood specimens in point-of-care and hemato- logical testing, plasma and serum are the preferred blood specimens for measuring soluble factors in the clinical laboratory. In addition to their previously mentioned discrepancies in composition, plasma and serum exhibit variations in the concentration of certain analytes.
Certainly, the coagulation cascade contributes to consumption of some substances (e.g., fibrinogen, plate- lets, and glucose) and to the release of others (e.g., potas- sium, lactate, lactate dehydrogenase, phosphate, and ammonia). For example, the presence of fibrinogen in plasma contributes significantly to the higher levels (65%) of total protein compared to serum [9].
Conversely, the release of elements or cell lysis associ- ated with the coagulation cascade is responsible for the increase in potassium (66%), inorganic phosphate (611%), ammonia (638%), and lactate (622%) in serum compared to plasma [9]. Furthermore, anticoagulants, preservatives, and other additives that aid or inhibit coagulation may interfere with the assay, as discussed later. Also, the presence of fibrinogen may interfere with chromatic detection or binding in immunoassays or the appearance of a peak that may simulate a false monoclonal protein in the gamma region during protein electrophoresis[9,10].
Serum Versus Plasma for Clinical Laboratory Tests
There are many advantages to using plasma over serum for clinical laboratory analysis. However, for TABLE 3.2 Components of Clinical Whole Blood, Plasma, and Serum Matrices
Whole Blood Plasma Serum
Cellular elements Erythrocytes Leukocytes Thrombocytes
Proteins Proteins Proteins (excluding fibrinogen)
Electrolytes Electrolytes Electrolytes
Nutrients Nutrients Nutrients
Waste (metabolites) Waste (metabolites) Waste (metabolites)
Hormones Hormones Hormones
Gases Gases Gases
Maycontain anticoagulants Containsanticoagulants Containsnoanticoagulants
Patient on therapeutics Specimen additive
some analytes, serum is preferred over plasma. These issues are addressed in this section.
Larger Sample Volume
If blood is allowed to clot and is then centrifuged, approximately 3050% of the original specimen volume is collected as serum. Conversely, plasma constitutes approximately 55% of the volume of uncoagulated blood after centrifugation. Therefore, the higher yield associated with plasma samples is generally preferred, especially when sample volume may be critical as in the case of the pediatric population, smaller patients, or in special cases in which blood volume needs to be conserved.
Less Pre-Analytical Delay
The process of clotting requires at least 30 min under normal conditions without coagulation
accelerators. Furthermore, coagulation may still occur postcentrifugation in serum samples. Therefore, another advantage of plasma is that analyte determina- tions can be achieved in whole blood prior to plasma separation provided that a suitable anticoagulant has been used. For example, an anticoagulated whole blood specimen may be used for point-of-care mea- surements followed by plasma separation, which would avoid the delay associated with obtaining an additional specimen for laboratory analysis.
Reduction of in Vitro Hemolysis
In addition to the time delay associated with blood clotting, there is an increased risk of lysis and conse- quent false increases in many intracellular analytes such as potassium, iron, and hemoglobin released from erythrocytes in serum specimens. Therefore, it is TABLE 3.3 Examples of Analytes Measured in Blood Cell Lysates[2,3]
Hematology Vitamins Trace Elements Drugs Toxic Elements
Hemoglobin Direct measurement Chromium Cyclosporine Cyanide
Red cell indices Vitamin E Selenium Sirolimus (rapamycin) Lead
Porphyrias Vitamin B1(thiamine) Zinc Tacrolimus (FK506, Prograf) Mercury
Cytoplasmic porphyrin metabolic enzyme activity
Vitamin B2(riboflavin)
Also FMN, FAD Vitamin B6(pyridoxine,
pyridoxamine, pyridoxal) PLP
Biotin
Folic acid (folate) Pantothenic acid Functional activity
Vitamin B1(thiamine) Transketolase Vitamin B2(riboflavin)
FAD-dependent glutathione reductase Vitamin B6(pyridoxine,
pyridoxamine, pyridoxal) AST, ALT activity
Vitamin B12(cyanocobalamin) Deoxyuridine suppression test Niacin
NAD/NADP ratio
ALT, alanine aminotransferase; AST, aspartate aminotransferase; FAD, flavin adenine dinucleotide; FMN, riboflavin-50-phosphate; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; PLP, pyridoxal-50-phosphate.
23
DIFFERENCES BETWEEN WHOLE BLOOD, PLASMA, AND SERUM SPECIMENS FOR CLINICAL LABORATORY ANALYSIS
advised to separate the serum as quickly as possible.
Conversely, plasma separation can be achieved at higher centrifugal speeds without risking the initiation of hemolysis and thrombocytolysis[9].
Advantages of Serum Over Plasma
Anticoagulants and additives in plasma specimens can directly interfere with the analytical characteristics of the assay, protein binding with the analyte of inter- est, and sample stability. Furthermore, liquid anticoa- gulants may lead to improper dilution of the sample.
For example, blood drawn in tubes with sodium citrate is diluted by 10%, but this may increase depending on whether the draw is complete. Moreover, incomplete mixing with anticoagulants can lead to the risk of clot formation. Also, the choice of anticoagulant will depend on their respective influences on the various assays offered by the clinical laboratory, and tubes with anticoagulants and additives are often more expensive.