Collection and handling of samples: effects on blood gases, Na, K, ionized Ca, Mg, lactate, and phosphate analyses

Introduction

There are numerous sources of preanalytical errors in laboratory testing. Among these are the following:

• Wrong test ordered

• Wrong patient collected

• Error in sample collection, including improper patient preparation

• Errors during specimen transport

• Errors during specimen processing

This chapter will address only the last three of these. As noted by Karon ( ) and Azman et al. ( ) , the top five reasons for redrawing specimens are shown in Table 10.1 .

Clearly, hemolysis and cell leakage or rupture are major causes of preanalytical errors. Contamination of a sample could include improper anticoagulant, air bubbles in the sample and contamination with IV fluids, such as saline, anticoagulants, or drugs.

Hemolysis

In vitro hemolysis is by far the most common preanalytical problem in clinical laboratory testing. It causes problems in (a) preventing hemolysis during collection, (b) detecting hemolysis in blood samples, and (c) both interpreting results and deciding whether to report the results for many tests, most notably potassium.

Preventing in vitro hemolysis . To prevent hemolysis, several guidelines may be followed:

• 1.

  Use a 23 gauge or larger needle. Smaller gauge needles can damage RBCs passing through the needle.

• 2.

  Collecting blood through an IV infusion line often causes a higher rate of hemolysis.

• 3.

  When mixing blood with anticoagulant, gently invert the tube. Never shake the tube.

• 4.

  For several reasons, blood collection tubes should be filled to their stated capacity.

• 5.

  Never forcefully expel blood from a syringe into a tube, especially an evacuated tube.

Detecting hemolysis: plasma/serum or whole blood . In former years, hemolyzed serum or plasma in evacuated tubes was usually detected by the watchful eye of skilled clinical laboratory scientists who manually handled centrifuged blood specimens. They would then grade the level of hemolysis, such as slight, moderate, or gross, then follow the laboratory's protocol for whether to report none, some, or all of the results, or request a new specimen be collected. However, visual detection of hemolysis is subject to individual interpretation and variability. Furthermore, elevated bilirubin, a common occurrence in neonates, can further complicate the visual interpretation of hemolysis ( ) .

As automation has become common in central laboratories, the samples are centrifuged, analyzed, and often reported without visual inspection of the plasma or serum. This issue has largely been solved by modern analyzers that use spectrophotometry to detect hemolysis and assign a numerical grade to its severity to alert the laboratory if the level of hemolysis significantly affects any test results. A highly debated issue is whether to report a result that is urgently requested by the physician ( ) . This is also a significant concern for any testing done at point-of-care where whole blood is analyzed and hemolysis would not likely be detected by the user. There is one commercial system that advertises a separate device that is able to detect hemolyzed blood at the point of care ( www.hemcheck.com ).

Detecting hemolysis is a significant concern for blood gas samples that are collected in syringes and must be analyzed immediately and without centrifugation. Thus, visual detection of hemolysis is not a practical option before analyzing the sample. A less than ideal option is to rapidly centrifuge the specimen after the analysis if either the results (i.e., an elevated K) or the patient's previous samples suggest hemolysis is present.

As yet, there are no blood gas analyzers that have built-in systems to detect hemolysis in uncentrifuged blood during analysis, although companies are likely working on systems that detect hemolysis during the testing process. Much of the technology is still proprietary, but the basic technology used for detection is likely based on either: (1) isolating plasma from the flow system during or before the measurement process and then using optical methods to detect levels of hemolysis; or (2) using an algorithm based on multiple analyte inputs to predict the degree of hemolysis in whole blood. While the first is similar to that used in automated clinical chemistry analyzers, the novel approach for blood gas analyzers would focus on in-line, real-time plasma isolation to determine a sample's hemolysis index. The second may have limited reliability in its reliance on whole blood alone, given the known pathological variations and nonlinear relationship between analyte changes in the presence of in vitro hemolysis. As blood gas testing continues to shift to the point of care, whatever real-time detection system of hemolysis is used, it must be both reliable and simple and have flexibility in how to alert clinicians to the impact on critical results such as potassium.

In vivo hemolysis occurs when erythrocytes are ruptured in circulation. Causes include immune reactions with cells, hemolytic anemias, and mechanical rupture from cardiac bypass, ECMO, or heart valve devices. While in vitro hemolysis that falsely affects test results is much more common and accounts for 98%–99% of hemolyzed specimens, results on samples with in vivo hemolysis will likely give a true physiologic increase of analytes in the blood. Thus, laboratory results such as potassium are appropriate and should be reported. Detecting in vivo hemolysis presents other challenges as detection often requires either a patient's history of a hemolytic process or one or more prior hemolyzed samples from the patient.

Reporting results on hemolyzed samples . Usually, the laboratory does not report any results that are significantly affected by in vitro hemolysis and requests that another specimen be collected, preferably by another person collecting the blood. The more difficult issue is when a suitable specimen cannot be recollected and the physician calls the laboratory with an urgent need for the result, which may provide some assurance in difficult clinical situations. Legal wisdom says to not report the results under any circumstances, while accommodating medical urgency says to report the result as a comment with disclaimers such as noting the result is significantly affected by hemolysis and is provided only at the request of the physician and after discussion with the laboratory director. An approach that addresses both concerns is to require one or two additional redraws, then if both specimens are still unacceptable, the laboratory provides the results if the physician requests them.

Blood gases

Blood collected for blood gas analysis is susceptible to changes, especially to p O ₂ . Anaerobic conditions during collection and handling are essential because room air has a p CO ₂ of nearly 0 and a p O ₂ of ∼150 mmHg. The factors that must be controlled are:

• Removal of all air bubbles

• Use of the proper anticoagulant

• Appropriate use of plastic syringes (glass syringes rarely used anymore)

• The temperature of storage before analysis

• The length of delay between collection and analysis of blood

• Any agitation of the specimen

The complete removal of all air bubbles is especially important before sending blood in a syringe by pneumatic transport, which will agitate the sample and markedly affect p O ₂ , with p CO ₂ much less affected by air bubbles ( , ) .

The effect of liquid heparin at <10% (vol./vol.) of the volume of blood has variable effects on blood gas and other analytes ( , ) . There appears to be little effect on pH and a relatively small effect (∼3%) on p O ₂ , although the p O ₂ also appears to be affected by the p O ₂ in the heparin diluent ( , ) . p CO ₂ , bicarbonate, and base excess are affected proportionately by dilution, about a 10% decrease. As expected, liquid heparin will dilute other constituents in blood, such as electrolytes, lactate, and glucose, which are often analyzed simultaneously in many current blood gas and electrolyte analyzers ( ) . Therefore, only dry heparin should be used as an anticoagulant.

Although plastic syringes are used for nearly all blood gas measurements, they have a potential disadvantage because they are permeable to oxygen and absorb oxygen in polyethylene ( ) . When stored in ice, because of the increased affinity of Hb for O ₂ at cold temperatures, blood can absorb oxygen within the wall of the syringe that has diffused through the plastic. This effect is most pronounced in samples with a p O ₂ of ∼100 mmHg and above; that is, when Hb is already nearly fully saturated with oxygen and is unable to buffer any added O ₂ . A p O ₂ of 100 mmHg may increase by 8 mmHg during 30 min of storage on ice. When Hb is less saturated (e.g., at a p O ₂ of 60 mmHg), it is better able to buffer the additional oxygen, causing a relatively small change in p O ₂ .