**Abstract**

Pre-analytical steps contribute to an overall quality of the results of laboratory trials. The volume of blood drawn into the blood-collection tube and the anticoagulant amount introduced into the tube during its production should ensure the anticoagulant level in the recommended range; otherwise, the results can be altered. In evacuated blood-collection tubes, the internal under-pressure at the instant of the blood specimen collection affects the draw volume. During the shelf life, the internal under-pressure deteriorates. With no testing procedures in place, inappropriate anticoagulant levels can pass unnoticed. The chapter details testing procedures ensuring that the tubes are used only if, and only until, they are of the adequate quality. The reasoning behind the methodology is fully explained, and the case studies of the quality evaluations are discussed.

**Keywords:** evacuated blood-collection tubes, K3EDTA, K2EDTA, citrate, quality evaluation, draw-volume measurement, anticoagulant concentration

#### **1. Introduction**

Evacuated blood-collection tubes are evacuated containers intended for a venous blood specimen collection. They consist of a tube and a closure, which has to be tight to restrain low pressure—vacuum inside the tubes during their shelf life—but, on the other hand, it also has to be soft enough to let a sharp end of a bloodcollection device to penetrate into a tube. The collection device has a disposable needle attached to the other side for phlebotomy.

Evacuated blood-collection tubes improved patients' and medical personnel's safety and mostly replaced classical tubes, which required a syringe and a needle for a specimen collection. In a continuation, wherever a tube is mentioned, the evacuated blood-collection tube is meant.

To prevent blood coagulation, tubes contain anticoagulants, either as dry substances attached to the internal walls or as solutions. Widely known and used are sodium citrate and salts of ethylenediaminetetraacetic acid (EDTA), usually present either as dipotassium or tripotassium salts, K2EDTA or K3EDTA.

Other substances, additives might be introduced as well to ensure the adequate properties or behavior of the tubes' internal walls or closures; nevertheless, they are expected not to interfere with a determination and affect analytical results. A noncompliant constituent detected in citrate tubes was magnesium which leached

from a stopper and was consequently influencing the prothrombin time (PT) results [1]. A comprehensive study evaluating different tubes comprising also recently introduced low-magnesium version confirmed that the PT and INR differences between the tubes are correlated with the magnesium concentration differences [2].

For citrate tubes the DIN ISO 6710: 1996-12 standard recommends trisodium citrate solutions with concentrations between 100 and 136 mmol/L; however, the

Due to all these differences, the GP39-A6 standard omitted all the anticoagulant concentrations' details, leaving it entirely to a producer to bear the responsibility for securing appropriate concentration, fulfilling all the requirements, and demonstrating that they are actually met, or in other words verifying that the tubes are

The GP34-A standard provides guidance for validation and verification of tubes for venous and capillary blood specimen collection [4]. Both a manufacturer and a clinical laboratory are required to perform a comparability study on blood samples for two or more sets of tubes comprising a set which was already evaluated and approved previously. A manufacturer performs such a test after a new product was developed or where any correction actions are necessary for the production process. The laboratory needs to do it when switching from one product to another or when

A within-tube precision study requires a minimum of 20 subjects, and each sample needs to be analyzed in replicates; an appropriate number of samples, evenly distributed through the analytical measurement, are essential for trueness evaluation [4]. Several studies with sometimes dissimilar outcomes can be found in

Two blood-collection devices either with an aligned [Becton Dickinson (BD)] or

Nevertheless, contrasting outcomes were obtained for prothrombin time determinations in the glass and PVC tubes with two distinct citrate concentrations where neither material caused the significantly different results [12]. Yet another study, establishing a protocol for comparing the citrate evacuation blood-collection tubes with glass tubes employing eight measuring systems, confirmed a statistically significant but clinically not relevant difference in prothrombin time results, which were more pronounced with the tubes of the lowest 2.7 mL draw volume [13].

Differences in some parameters were confirmed if BD plastic citrate tubes were

A research performed on a group of individuals evaluating the effect of underfilled EDTA tubes on hematological parameters by employing a particular type of

used instead of glass tubes, but they were considered unlikely to be clinically significant [14], though a comprehensiveness of a study was challenged arguing that only healthy volunteers were involved and by these means it was not yet proven that glass tubes are interchangeable with the plastic tubes [15]. But a study performed on Greiner glass citrate and plastic tubes confirmed that the tubes are substitutable as far as either untreated or patients on a traditional oral anticoagulant therapy are concerned and that this applies for the whole shelf life of the tubes [16]. Nevertheless, the plastic tubes of different brands evaluated on patients and healthy volunteers were confirmed to be statistically but not clinically significantly different [17]. For patients on oral anticoagulant therapy with vitamin K antagonists, ANOVA test confirmed statistically significant differences in prothrombin time for the tubes of four different types [18]. The study supports the claim that

validation is always necessary when there is a change in a tube type.

at an angle needle holder (Greiner Labortechnik GmbH) were evaluated either enabling a direct linear (BD) or interrupted nonlinear blood flow. A mechanical strain on blood cells was recognized as a factor potentially causing the efflux of intracellular constituents into the serum in an interrupted nonlinear flow. The magnesium, plasma hemoglobin, and prothrombin time within-subject variations were confirmed in 55 healthy individuals using a Student paired t-test. A difference in a tube material either glass or polymer was also recognized as a likely contribut-

H1-A5 standard specifies the concentrations 105, 109, and 129 mmol/L.

*Pre-Analytical Within-Laboratory Evacuated Blood-Collection Tube Quality Evaluation*

actually fit for purpose.

*DOI: http://dx.doi.org/10.5772/intechopen.80685*

changing a vendor.

the literature.

ing factor [11].

**49**

Manufacturers are obliged to specify on the label of a tube: a type of anticoagulant, a nominal draw volume, a lot number, and expiration date; within this text, we use a term expiry date as well.

Anticoagulant concentration in a blood sample after specimen collection should be within an appropriate range; otherwise, analytical results might be altered. To reach this objective, an accurate amount of anticoagulant should be introduced into a tube during production, and a draw at the moment of a specimen collection should be adequate to ensure a volume of blood entering a tube is within an acceptable range. A label on a tube provides guidance for inspection if the volume is within the suggested limits; however, this is only true if the label is precisely and accurately positioned.

The latest version of the GP39-A6 standard of the CLSI standardization body (Clinical and Laboratory Standards Institute) [3] requires of the tubes' manufacturers to ensure that until expiration date, the anticoagulant concentration remains within the 5% range of the value stated on a label. A draw volume is considered acceptable if it does not differ from the stated nominal volume for more than 10%.

The standard GP34-A recognizes the importance of appropriate blood-to-EDTA ratio for obtaining optimal examination results but avoids stating the exact limits. The EDTA can, if in a concentration which is too high hypertonically shrink red cells, affect red cell size and cause morphological changes. On the other hand, it can too extensively chelate calcium and other cations such as magnesium and zinc and affect the activity of alkaline phosphatase enzyme label used in chemiluminescent assays or reduce the efficiency of the recognition of proteins by antibodies due to the proteins' conformation changes [4].

The predecessor of CLSI, the National Committee for Clinical Laboratory Standards (NCLLS), was in the H1-A5 standard more explicit in terms of some anticoagulants' concentrations [5]. It explains that only a little bit less than a half (1.15 mmol/L) out of the total calcium concentration (2.5 mmol/L) corresponds to unbound calcium that needs to be chelated stoichiometrically with EDTA to prevent coagulation. For that reason, it suggests that EDTA concentration in blood should be between 3.7 and 5.4 mmol/L, since excessive concentration causes morphological changes in the blood.

Not consistent with this requirement was DIN ISO 6710: 1996-12 standard requiring the EDTA concentration within the 4.11–6.843 mmol/L range [6].

A potential user can during time come across the tubes which were produced by not having the same set of requirements on the mind. As we already previously demonstrated, the tubes if evaluated as such not yet in contact with a blood sample are not all the same, and change in their own characteristics during their shelf life and the testing procedure which we suggested are easy to perform [7].

A concise review reflects on the behavior of EDTA as an anticoagulant in hematology and furthermore discusses its usage in proteomics, general clinical chemistry, and its applicability for measuring cytokines, protein, peptides, and cardiac markers [8]. Elsewhere, influences of a form of EDTA and its concentration on the results of hematological tests were profoundly discussed in relation to spurious counts and results regarding platelets [9], white blood cells, red blood cells, hemoglobin, red cell indices, and reticulocytes [10]; under-filled or over-filled evacuated tubes changing the anticoagulant level in a sample are exposed as an influential preanalytical source of errors.

#### *Pre-Analytical Within-Laboratory Evacuated Blood-Collection Tube Quality Evaluation DOI: http://dx.doi.org/10.5772/intechopen.80685*

For citrate tubes the DIN ISO 6710: 1996-12 standard recommends trisodium citrate solutions with concentrations between 100 and 136 mmol/L; however, the H1-A5 standard specifies the concentrations 105, 109, and 129 mmol/L.

Due to all these differences, the GP39-A6 standard omitted all the anticoagulant concentrations' details, leaving it entirely to a producer to bear the responsibility for securing appropriate concentration, fulfilling all the requirements, and demonstrating that they are actually met, or in other words verifying that the tubes are actually fit for purpose.

The GP34-A standard provides guidance for validation and verification of tubes for venous and capillary blood specimen collection [4]. Both a manufacturer and a clinical laboratory are required to perform a comparability study on blood samples for two or more sets of tubes comprising a set which was already evaluated and approved previously. A manufacturer performs such a test after a new product was developed or where any correction actions are necessary for the production process. The laboratory needs to do it when switching from one product to another or when changing a vendor.

A within-tube precision study requires a minimum of 20 subjects, and each sample needs to be analyzed in replicates; an appropriate number of samples, evenly distributed through the analytical measurement, are essential for trueness evaluation [4]. Several studies with sometimes dissimilar outcomes can be found in the literature.

Two blood-collection devices either with an aligned [Becton Dickinson (BD)] or at an angle needle holder (Greiner Labortechnik GmbH) were evaluated either enabling a direct linear (BD) or interrupted nonlinear blood flow. A mechanical strain on blood cells was recognized as a factor potentially causing the efflux of intracellular constituents into the serum in an interrupted nonlinear flow. The magnesium, plasma hemoglobin, and prothrombin time within-subject variations were confirmed in 55 healthy individuals using a Student paired t-test. A difference in a tube material either glass or polymer was also recognized as a likely contributing factor [11].

Nevertheless, contrasting outcomes were obtained for prothrombin time determinations in the glass and PVC tubes with two distinct citrate concentrations where neither material caused the significantly different results [12]. Yet another study, establishing a protocol for comparing the citrate evacuation blood-collection tubes with glass tubes employing eight measuring systems, confirmed a statistically significant but clinically not relevant difference in prothrombin time results, which were more pronounced with the tubes of the lowest 2.7 mL draw volume [13].

Differences in some parameters were confirmed if BD plastic citrate tubes were used instead of glass tubes, but they were considered unlikely to be clinically significant [14], though a comprehensiveness of a study was challenged arguing that only healthy volunteers were involved and by these means it was not yet proven that glass tubes are interchangeable with the plastic tubes [15]. But a study performed on Greiner glass citrate and plastic tubes confirmed that the tubes are substitutable as far as either untreated or patients on a traditional oral anticoagulant therapy are concerned and that this applies for the whole shelf life of the tubes [16].

Nevertheless, the plastic tubes of different brands evaluated on patients and healthy volunteers were confirmed to be statistically but not clinically significantly different [17]. For patients on oral anticoagulant therapy with vitamin K antagonists, ANOVA test confirmed statistically significant differences in prothrombin time for the tubes of four different types [18]. The study supports the claim that validation is always necessary when there is a change in a tube type.

A research performed on a group of individuals evaluating the effect of underfilled EDTA tubes on hematological parameters by employing a particular type of

from a stopper and was consequently influencing the prothrombin time (PT) results [1]. A comprehensive study evaluating different tubes comprising also recently introduced low-magnesium version confirmed that the PT and INR differences between the tubes are correlated with the magnesium concentration differences [2]. Manufacturers are obliged to specify on the label of a tube: a type of anticoagulant, a nominal draw volume, a lot number, and expiration date; within this text, we

Anticoagulant concentration in a blood sample after specimen collection should be within an appropriate range; otherwise, analytical results might be altered. To reach this objective, an accurate amount of anticoagulant should be introduced into a tube during production, and a draw at the moment of a specimen collection should be adequate to ensure a volume of blood entering a tube is within an

acceptable range. A label on a tube provides guidance for inspection if the volume is within the suggested limits; however, this is only true if the label is precisely and

The latest version of the GP39-A6 standard of the CLSI standardization body (Clinical and Laboratory Standards Institute) [3] requires of the tubes' manufacturers to ensure that until expiration date, the anticoagulant concentration remains within the 5% range of the value stated on a label. A draw volume is considered acceptable if it does not differ from the stated nominal volume for more than 10%. The standard GP34-A recognizes the importance of appropriate blood-to-EDTA ratio for obtaining optimal examination results but avoids stating the exact limits. The EDTA can, if in a concentration which is too high hypertonically shrink red cells, affect red cell size and cause morphological changes. On the other hand, it can too extensively chelate calcium and other cations such as magnesium and zinc and affect the activity of alkaline phosphatase enzyme label used in chemiluminescent assays or reduce the efficiency of the recognition of proteins by antibodies due to

The predecessor of CLSI, the National Committee for Clinical Laboratory Standards (NCLLS), was in the H1-A5 standard more explicit in terms of some anticoagulants' concentrations [5]. It explains that only a little bit less than a half

(1.15 mmol/L) out of the total calcium concentration (2.5 mmol/L) corresponds to unbound calcium that needs to be chelated stoichiometrically with EDTA to prevent coagulation. For that reason, it suggests that EDTA concentration in blood should be between 3.7 and 5.4 mmol/L, since excessive concentration causes morphological

Not consistent with this requirement was DIN ISO 6710: 1996-12 standard requiring the EDTA concentration within the 4.11–6.843 mmol/L range [6]. A potential user can during time come across the tubes which were

produced by not having the same set of requirements on the mind. As we already previously demonstrated, the tubes if evaluated as such not yet in contact with a blood sample are not all the same, and change in their own characteristics during their shelf life and the testing procedure which we suggested are easy to

A concise review reflects on the behavior of EDTA as an anticoagulant in hematology and furthermore discusses its usage in proteomics, general clinical chemistry, and its applicability for measuring cytokines, protein, peptides, and cardiac markers [8]. Elsewhere, influences of a form of EDTA and its concentration on the results of hematological tests were profoundly discussed in relation to spurious counts and results regarding platelets [9], white blood cells, red blood cells, hemoglobin, red cell indices, and reticulocytes [10]; under-filled or over-filled evacuated tubes changing the anticoagulant level in a sample are exposed as an influential pre-

use a term expiry date as well.

*Biochemical Testing - Clinical Correlation and Diagnosis*

accurately positioned.

changes in the blood.

perform [7].

**48**

analytical source of errors.

the proteins' conformation changes [4].

analyzer [19] leads to contrasting outcomes not necessarily aligned with other studies [20] and general principles and recommendations.

Validations and verifications as required by the standard GP34-A are complex to perform, time demanding, and require resourceful personnel [4].

The standard exposes a blood collection as a pre-analytical (preexamination) source causing varying degrees of errors. It brings to light a lack of a mechanism that would enable systematic evaluations of the influences of pre-analytical (preexamination) variables on laboratory test (examination) results [4].

The characteristics of the tubes entering the pre-analytical phase are such variables, and this is where this chapter tends to contribute.

Differences between tubes of different brands examined 5 years apart in time are going to be enlightened, and the testing procedures which are fast, cheap, and easy to implement into laboratory practice are explained in full details. Robustness of personal profiles of athletes and validation studies performed on blood samples can profit from knowing the attributes of the tubes that were actually used or evaluated.

**Figure 2.**

**Figure 3.**

**Figure 4.**

**51**

*Anticoagulant concentrations and draw volumes of the B brand tubes obtained 5 years apart in time (orange/*

*Pre-Analytical Within-Laboratory Evacuated Blood-Collection Tube Quality Evaluation*

*Anticoagulant concentrations and draw volumes of the C brand tubes obtained 5 years apart in time (orange/*

*Anticoagulant concentrations and draw volumes of the D brand tubes obtained 5 years apart in time (orange/*

*blue); the numbers in the labels indicate the time until the expiration.*

*DOI: http://dx.doi.org/10.5772/intechopen.80685*

*blue); the numbers in the labels indicate the time until the expiration.*

*blue); the numbers in the labels indicate the time until the expiration.*

### **2. Quality of evacuated blood-collection tubes for hematological tests reevaluated in 5 years' time**

In this section, we compare the results of a quality evaluation of the bloodcollection tubes, on which we previously reported [7], with the results obtained in 5 years' time. The same producers were considered, namely, Becton Dickinson, Greiner Bio-One, and Laboratorijska tehnika Burnik d. o. o.

**Figures 1**–**4** are dedicated to the tubes of four different brands. The abscise axis stands for a draw volume. Ordinate on the right indicates an anticoagulant concentration expected for a blood sample after a specimen collection. We are going to explain the meaning of the left ordinate axis, which relates to a testing procedure, later. A frame in green indicates the limits set by the H1-A5 standard [5]. The horizontal lines confine the range of the acceptable anticoagulant concentration; the left vertical line represents a limit under which a draw volume is expected not to fall to prevent the anticoagulant concentration to rising too high.

We kept the assigned marks A, B, and C from the previous study for the K3EDTA tubes, and D for the K2EDTA tubes, not disclosing the producers' identity. We additionally included the tubes F, not previously evaluated. The more recent

#### **Figure 1.**

*Anticoagulant concentrations and draw volumes of the A brand tubes obtained 5 years apart in time (orange/ blue) and of the tubes F not previously included (the numbers in the labels indicate time until the expiration).*

*Pre-Analytical Within-Laboratory Evacuated Blood-Collection Tube Quality Evaluation DOI: http://dx.doi.org/10.5772/intechopen.80685*

#### **Figure 2.**

analyzer [19] leads to contrasting outcomes not necessarily aligned with other

Validations and verifications as required by the standard GP34-A are complex to

The standard exposes a blood collection as a pre-analytical (preexamination) source causing varying degrees of errors. It brings to light a lack of a mechanism that would enable systematic evaluations of the influences of pre-analytical (preexamination) variables on laboratory test (examination) results [4].

The characteristics of the tubes entering the pre-analytical phase are such vari-

Differences between tubes of different brands examined 5 years apart in time are going to be enlightened, and the testing procedures which are fast, cheap, and easy to implement into laboratory practice are explained in full details. Robustness of personal profiles of athletes and validation studies performed on blood samples can profit from knowing the attributes of the tubes that were actually used or

**2. Quality of evacuated blood-collection tubes for hematological tests**

In this section, we compare the results of a quality evaluation of the bloodcollection tubes, on which we previously reported [7], with the results obtained in 5 years' time. The same producers were considered, namely, Becton Dickinson,

We kept the assigned marks A, B, and C from the previous study for the K3EDTA tubes, and D for the K2EDTA tubes, not disclosing the producers' identity. We additionally included the tubes F, not previously evaluated. The more recent

*Anticoagulant concentrations and draw volumes of the A brand tubes obtained 5 years apart in time (orange/ blue) and of the tubes F not previously included (the numbers in the labels indicate time until the expiration).*

**Figures 1**–**4** are dedicated to the tubes of four different brands. The abscise axis stands for a draw volume. Ordinate on the right indicates an anticoagulant concentration expected for a blood sample after a specimen collection. We are going to explain the meaning of the left ordinate axis, which relates to a testing procedure, later. A frame in green indicates the limits set by the H1-A5 standard [5]. The horizontal lines confine the range of the acceptable anticoagulant concentration; the left vertical line represents a limit under which a draw volume is expected not to fall

studies [20] and general principles and recommendations.

*Biochemical Testing - Clinical Correlation and Diagnosis*

ables, and this is where this chapter tends to contribute.

Greiner Bio-One, and Laboratorijska tehnika Burnik d. o. o.

to prevent the anticoagulant concentration to rising too high.

**reevaluated in 5 years' time**

evaluated.

**Figure 1.**

**50**

perform, time demanding, and require resourceful personnel [4].

*Anticoagulant concentrations and draw volumes of the B brand tubes obtained 5 years apart in time (orange/ blue); the numbers in the labels indicate the time until the expiration.*

#### **Figure 3.**

*Anticoagulant concentrations and draw volumes of the C brand tubes obtained 5 years apart in time (orange/ blue); the numbers in the labels indicate the time until the expiration.*

#### **Figure 4.**

*Anticoagulant concentrations and draw volumes of the D brand tubes obtained 5 years apart in time (orange/ blue); the numbers in the labels indicate the time until the expiration.*

results are marked with an asterisk; a number indicates the number of days until the expiration.

It also needs to be mentioned that if the draw-volume inspection relays on the label mark a judgment can be false. During our first study, we found out that only one brand of the tubes had a label positioned precisely; in all others the indicators

*Pre-Analytical Within-Laboratory Evacuated Blood-Collection Tube Quality Evaluation*

Easy-to-perform testing procedures as we used here which do not require blood samples can as a precautious measure ensure that the tubes are used only if they are of the adequate quality and their quality does not fluctuate too much during the time. It can alert a laboratory when it would be advisable to perform a much more complex and time-demanding verification study on blood samples. Archived data on the tubes' characteristics and quality during a longer period of time can provide a piece of evidence for other studies and rule the tubes out as a potential cause for

In this section, we explain the changes in a behavior of the tubes during their

The tubes of different brands differ in their drawing capability and, in a way, how it reduces during the time, as **Figure 5** demonstrates. No container is entirely tight and leaks to some extent. The conditions to which the tubes were exposed or under which they were stored contribute. The tubes of the same lot would behave

Even though tubes are of a high quality, are purchased at the same time, and

are of the same lot, they are not all the same if used during their shelf life.

The tubes' drawing capability depends on a difference between the external (*p*st) and internal (*p*int\_20°C) pressure. The lower the internal pressure, and the higher the difference to the external pressure, the higher a drawing capability. A tube's internal

**2.3 Tubes' drawing capability reduces during the time**

*Drawing capability of the tubes reduces continuously during their shelf life.*

differently in different circumstances.

volume (*V*tube) also contributes to higher capacity to draw a liquid.

on the tube were misleading.

*DOI: http://dx.doi.org/10.5772/intechopen.80685*

variations.

shelf life.

**Figure 5.**

**53**

Ellipses in **Figures 1**–**4** provide an insight into a spread of results obtained for the tubes within a series of measurements. Ellipses in blue correspond to the more recent results; those in orange originate from a previous study. A length of a horizontal axis of an ellipse equals a standard deviation of the draw-volume measurements; a vertical axis indicates a standard deviation of the anticoagulant concentration as expected for blood samples. A crossing of the two axes of an ellipse is defined by the mean values of the two parameters. The smaller the ellipse, the higher the quality of the produced tubes in terms of their precision or, in other words, a repeatability of a product.
