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Is It Really Necessary to Allow Specimens to Reach Room Temperature Before Analysis?

Introduction

A recent misunderstanding between shifts regarding work being left over for the next shift to complete resulted in this inquiry. Our "day shift" testing personnel had left several racks of chemistry samples for the "evening shift" workers to complete. As it seemed evening shift was burdened with extra workload, the explanation was that the samples were being left to reach room temperature before being tested since the courier delivered the samples a little later than usual that day. Would it have made a difference if day shift had just run the samples? Or was it necessary to hold them at room temperature for more than an hour to equilibrate, forcing the day shift to stay past their time to run them resulting in overtime; or rather, leaving them for the evening shift to pick up, causing extra work for them?

A reference lab or any other clinical laboratory that processes samples drawn and/or collected from a different site faces a unique challenge regarding specimen arrival temperature versus specimen testing temperature. Samples being transported from local and distant sites to a reference laboratory are processed for stability and shipped maintaining their integrity, for example using ice packs, for testing upon arrival, whatever time that may be. When these samples arrive, they get processed again by client service staff, meaning the samples get unpacked, checked against the manifest, and racked getting them ready for testing. As sample tubes are processed, depending on testing schedule, some samples may be tested right away, and others may be set aside to be batched and tested at a later time and/or date. That could mean refrigeration, freezing, or leaving the samples at room temperature.

The evening shift maintained to have day shift hold the samples and make them sit for more than an hour and keep them for the off-shifts to complete was unnecessary and the tubes could be loaded straight away and tested. The samples may have been shipped in a "refrigerated" state with ice packs and received cold; by the time they are processed, loaded to the MPA, or Modular-Pre Analytic section of the chemistry analyzer-COBAS, then aliquoted and run through to the COBAS, the less than a ml specimen in the testing cup has indeed reached room temperature prior to any mixing of reagent for testing. The amount pipetted is so miniscule it is sure to reach room temperature before any actual testing is done to the sample, regardless of what temperature the sample was received.

Discussion

Good laboratory practice generally means allowing samples to reach room temperature before testing (CDC, 2016.) For example, a commonly performed CAP survey for blood gas has strict directions that prior to analysis, the ampules should be brought to room temperature for at least 4 hours (College of American Pathologists, 2016.) Another readily found test kit in the average clinical laboratory is an hCG test for pregnancy. In the SA Scientific Pregnancy Ultimate (hCG) Serum/Urine kit instructions, it states the test kit itself is to be kept at room temperature and is very specific regarding the sample temperature before testing. Refrigerated serum or urine should be equilibrated to room temperature or between 15 and 30 degrees C before being tested.

A study posted on the website for the National Center for Biotechnology Information discussing the effects of temperature and time of testing of blood gas samples follows this room temperature testing line of thinking. In that report the authors admit it is common practice for blood gas samples to be stored and/or transported on ice to minimize leukocyte metabolism (Mohammadhoesini, 2015.) However, based on their study, there was a likelihood of overestimation of O2 results when samples were cooled before testing. In their study, they compared the effects of using plastic or glass syringes and how the O2 and CO2 were increased and/or decreased depending on the temperature storage or delivery to the lab. One conclusion they drew was that no matter what temperature the blood gas was kept at, iced or room temperature, the blood gas analyzer will take in the sample and warm it to its operating temperature. In the RapidPoint 500 blood gas analyzer from Siemens, the sample chamber contains a thermistor that controls the temperature of the sample during measurement (Siemens, 2011). It is certainly agreed by everyone that once blood gasses are collected, they should be sent for analysis and tested as soon as possible, with or without ice.

Conversely, another point of view is held by Dr. Alfred Plechner of the Broda O Barnes, MD Research Foundation who advises his colleagues to make sure they are using a laboratory that "processes their blood samples correctly." In his opinion, once blood is drawn, it should be spun down and immediately refrigerated. Even sitting in the centrifuge too long will allow the sample to reach room temperature, Dr. Plechner warns, and if ever brought to room temperature actually invalidates the blood serum test results (Plechner, 2014). He has seen spurious lab results from samples reaching laboratories where testing occurred at room temperatures and blames their poor processing techniques.

BD Vacutainer, the leading manufacturer of specimen collection devices, maintains that using their tubes, after the venipuncture, once the serum has been removed or separated from the red blood cells (in the case of the gel barrier tube), the sample will be stable at room temperature for eight hours (Magee, 2005). They cite only specific tests that require blood to be chilled after collection in order to maintain the stability of the analyte, including acetone, ammonia, and lactic acid, among other esoteric tests. In fact, some testing requires the samples to be maintained in a refrigerated state during the collection and/or centrifugation due to the volatility and instability of the analyte. For example,

Free Phenytoin testing requires a refrigerated centrifuge for processing, ACTH requires a pre-chilled lavender tube during collection, and osmolality testing actually puts the sample in a super-frozen state before testing (Advanced Instruments, 2004). Then there are those tests that actually benefit from a clotting time spent in an incubator, such as cold agglutinins, and, in fact, some patient tubes for hematology performing CBCs must be warmed before testing, again, due to cold agglutinins. Anecdotal evidence has shown depending on a particular lab policy, the simple collection of a Prothrombin Time can range in procedure from bringing the tube to the lab on ice, to maintaining the blue top at room temperature.

Some courier schedules result in the samples from the outlying labs being delivered to the testing facility in the afternoons, after being collected from the morning draws and appointments. It could be argued that these samples have been collected and delivered within that 8 hour stability window mentioned before. Therefore, theoretically, they could be shipped at room temperature thereby negating the need to come to room temperature in the first place. It would not be necessary to transport them in a "refrigerated" condition at all since they might be tested right away once received at the reference lab.

In our day versus evening shift case above, the urine samples are given to urine bench to spin, the A1c lavender tubes are placed on the rocker to mix, and the serum is placed by the MPA for loading where all of these samples seem to be parked to wait until further notice. This processing of samples seems to stall due to staffing issues or lack of initiative to get the samples tested. Evening shift would argue that even though these specimens arriving from the outside clinics and draw sites have indeed arrived cold on ice packs, by the time they are loaded to the MPA/COBAS and sampled for testing, they have reached room temperature and it is not necessary for the racks to sit on the counter for an hour or more to "warm up." Here's why:

The urines are spun to clear them of any formed crystals or debris before testing and after coming out of the centrifuge are most certainly now at this time room temperature due to the action of the centrifuge and should not have to wait to reach room temperature. The heat generated in the rotor chamber can be quite significant (Beckman Coulter, 2013). A1c testing on lavender top tube whole blood only needs 6 to 10 inversions to completely suspend the cells. A1c testing is not the same as CBC analysis or complete blood counts where a homogenous sampling is paramount. The Roche Diagnostics reagent bulletin for the Tina-quant Hemoglobin A1c reagent states that there are many possible error flags based on the methodology of testing the ratio of the HbA1c and hemoglobin. The testing is based on the heterogeneity, or rather the vast diversity of the types of possible samples being tested, not the temperature of the sample. The temperature of the cells is irrelevant.

The serum and plasma samples received from reference sites that are received in a cold state typically take approximately 30 minutes or more to be checked in by CPA, or Central Processing Associate staff. Then, once those samples get racked and placed on the MPA, they get shuttled to the DE stopper where the caps are removed. Then the tubes move on to the aliquoter where the samples are pipetted into cups. The amount of sample being aliquoted is set in the TSM, or Total System Manager according to the needs of the testing laboratory, but typically averages about V ml. The rack of "mother tubes" are transferred on for archiving or the output buffer, wherever the MPA needs it to go and the rack of aliquots are processed along to the COBAS for analysis. This process takes approximately 10 to 15 minutes depending on how many racks are being loaded. The more samples are being pushed through, the more time it takes.

If the sample is being front loaded directly to the COBAS, as opposed to being shuttled from the MPA, the sample probe will aspirate the appropriate amount of sample depending on the analyte, and places it in the reaction cell. Again, this will take time depending on how many samples are being pushed through the system at the same time. The more samples, the slower the process; even though the "STAT PORT" takes priority, it will sample as space allows. The amount of sample aspirated is between 1 and 35 microliters. The reaction cells where ALL testing take place: A1c samples, urine micro albumins, and chemistry panels, occur in a 37 degree C incubator bath and the E module sample cups are placed in a 37 degree incubator (CAP, 2016). This is a common step as part of the methodology for all chemistry analyzers.

Conclusion

No doubt, good lab practice means following proper specimen processing procedures to minimize pre-analytical errors before testing. From collection, to handling, processing, shipping, and storage before testing, all present any number of procedures. However, there seems to be quite a range of possibilities depending on who you ask as to what those procedures might be, not to mention any number of possible errors along the way. As traditional lab practice dictates, it is most likely a situational proposition as different locations have different circumstances to contend with. Weather conditions, long courier runs or missed flights, access to proper supplies, and staffing issues all play a part in a laboratory's procedure development.

The samples received from outside clinics and draw sites arriving for testing at a reference lab will be at the correct processing temperature, ready for testing once loaded and run on the instruments in question because the analyzers make it so. The instruments are designed to take specimens and put them in the optimal temperature, volume, or conditions necessary for testing, except in the most unique circumstances or for the most uncommon tests. It could be said if samples are drawn and tested the same day, it is not necessary to refrigerate the specimens and that would make transport and delivery more convenient and less expensive. It is probably wiser to err on the side of caution and keep patient samples refrigerated until testing, as refrigeration has proven to be a significant factor in preservation, especially if there is any possibility of delay in clinical analysis. Every laboratory must decide and develop its own policies and procedures for the best possible patient outcome.

References

(1.) Center for Disease Control Office of Surveillance, Epidemiology and Laboratory Science. (n.d.). Ready? Set? Test! [Brochure]. Author. Retrieved November 17, 2016, from https:// www.cdc.gov

(2.) College of American Pathologists. (2016). Critical Care/Aqueous Blood Gas Survey [Brochure]. Northfield: Author. AQ-C 2016 Kit Instructions

(3.) Mohammadhoesini, E., Safavi, E., & Seifi, S. (2015, March 20). Effect of Sample Storage Temperature and Time Delay on Blood Gases, Biocarbonate and PH in Human Arterial Blood Samples. Retrieved November 18, 2016, from https://www. ncbi.nlm.nih.gov/

(4.) Operators Guide. (2011). Tarrytown, NY: Siemens. RAPID-Point 500 System Blood Gas

(5.) Magee, L. S., MBA, MT(ASCP). (2005). Preanalytical Variables in the Chemistry Laboratory. Lab Notes, 15(1).

(6.) Plechner, A., MD. (2014, February 10). Is Your Blood Sample Being Handled and Processed Correctly? [Web log post]. Retrieved November 18, 2016, from http://drplechner.com/

(7.) The Advanced Osmometer Model 2020. (2004). Norwood, MA: Advanced Instruments. User's Guide

(8.) Imai, K., Watari, S., Sakazume, T., & Mitsuyama, S. (2008). Clinical Chemistry and Immunoassay Testing Supporting the Individual Healthy Life. [Scholarly project]. In Hitachi Review. Retrieved November 17, 2016, from www.hitachi.com

(9.) Allegra X-30 Series Centrifuges [Pamphlet]. (2013). Brea, CA: Beckman Coulter. Instructions for Use

(10.) Tina-quant Hemoglobin A1c Gen-2 [Reagent Bulletin]. (2012). Roche Diagnostics, Indianapolis.

(11.) Cobas 6000 analyzer series, http://www.mylabonline.com/ products/cobas6000/c6000.php

Roberta Fern Pizarro, MPA, MT(AMT), PBT(ASCP)C, HCM, Medical Technologist at Dept. of Veteran Affairs at the Puget Sound Health Care System in Seattle, WA
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Title Annotation:Article 441 .5 Clock Hour
Author:Pizarro, Roberta Fern
Publication:Journal of Continuing Education Topics & Issues
Date:Apr 1, 2017
Words:2316
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