Pre-Analytical Within-Laboratory Evacuated Blood ...

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 blood-collection device to penetrate into a tube. The collection device has a disposable needle attached to the other side for phlebotomy.

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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].

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 : -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 pre-analytical source of errors.

For citrate tubes the DIN ISO : -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.

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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 under-filled EDTA tubes on hematological parameters by employing a particular type of 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.

When we speak of &#;order of draw&#; what exactly are we talking about?

In phlebotomy, this term has special meaning as it is the order in which certain tubes can be used. There are seven tubes which make up the order of draw. 

They are listed by chemical additives that have been introduced to the tube by the manufacturer to perform a specific function when mixed with the blood.

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