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Best Practices in Tube Handling

Handling all the threats to blood-sample integrity is an enormously complex undertaking. Human blood is complicated. We’ve been studying it for centuries and we still don’t know everything there is to know about it. And what we do know is exceedingly difficult to apply every time we handle a sample.

That’s because the preanalytical phase, or pre-examination phase, is predominantly a manual process that requires human programming. At the point of collection, machines don’t draw blood, mix blood, or label samples. In most places their handling is manual. That’s where the “practices” of Best Practices comes in. We’re talking about human behavior here, and how people practice the art of drawing and handling samples.

Happy tube, happy test

A test result is accurate only if the tube is properly filled and handled. A happy tube means a happy test. Tubes leave their manufacturer happy, but what happens when they get to your place? Do they lose their good nature? Does the way you handle them make them cranky? Stubborn? Nervous? Let’s take a look at what we might be doing that changes their disposition.

One cannot discuss tube handling completely without first addressing how to handle tubes before they’re filled. Tube manufacturers sweat this part because once the tubes are in your hands, how they perform is completely and entirely out of their control. While they’re made with high tolerances for a wide variety of environments, they still have to be handled properly before they’re filled.

Make sure you’re rotating your stock, first in/first out. An expired tube on your shelf or in your draw stations or phlebotomy trays is not only hard to explain to inspectors, but not likely to fill to the proper level.

According to the CLSI standard on tubes and additives (GP39-A6), at the end of a tube’s shelf life, the draw volume shall be no more than 10% below the stated draw volume. So you’re assured an in-date tube will fill at least 90 percent. With an expired tube, there’s no guarantee.

One of the most commonly overlooked pre-collection tube handling aspects is their storage temperature. Has your facility’s air conditioning units ever failed? When they do, and they will, are you certain someone is tasked with checking the impact the high temperature might have had on the tubes in stock? If not, when tubes start underfilling three months later, will it be traced to the a/c failure or blamed on the tube manufacturer or phlebotomy team? Connect the dots early and you’ll solve big problems before they even happen.

Tubes in storage should always be kept away from sunlight and direct heat, as well. They can wreak the same havoc as a malfunctioning a/c unit. Direct sunlight will be easy to detect, but are your stored tubes in close contact with heating ducts? If so, relocate them or have your facility-management team redirect the air flow so that tubes are not directly impacted. Don't forget about tubes used by a courier or mobile draw team. Are they subjected to high temperatures prior to use? If so, put a procedure in place to keep your transient inventory of tubes at the manufacture’s stated temperature range.

If your altitude is more than 5000 feet (1500 meters) above sea level, make sure the tubes you stock are high-altitude versions. Altitude affects the tubes’ vacuum in no small way, so don’t underestimate the importance of this factor.

All these factors will reduce the vacuum in the tubes. So if you’re finding a sudden increase in the number of underfilled tubes, ask yourself these questions: • Is our inventory being properly rotated or are we using expired tubes? • Are the tubes exposed to higher temperatures, or were they in the last few weeks or months? • For those of you above 5000 feet: are we using high-altitude tubes, or did we accidentally receive regular tubes?

Filling factors

On to tube-handling tips during the draw. Are you careful about how the blood enters the tube? You should be. Some patient's red blood cells are more delicate than others, Think of your red blood cells as fragile crystal orbs that don’t tolerate a lot of force, Instead of letting the blood directly impact the bottom full-force, angle the tube so the incoming stream hits the side at an angle. It's as easy as raising the tube with a finger just enough to let the blood impact the side instead of the bottom.(See right.)

No discussion on tube handling is complete without stressing the importance of the order in which tubes must be filled. The order for venipunctures, which has not changed since 2003, is as follows: 1) blood cultures, 2) citrate, 3) serum, 4) heparin, 5) EDTA, 6) glycolytic inhibitors. The order is the same for syringes as it is for tube holders. For capillary draws, the order is 1) EDTA, 2) other additive tubes, 3) serum tubes. This order has not changed since at least 1999. [Answers to questions on the order of draw may be found by entering "order of draw," including the quotation marks, in the search window at]

One way to tell is the order of draw was not followed is if the laboratory's chemistry department starts questioning the reliability of their potassium results. When the EDTA tube is filled before a serum or plasma tube to be tested for potassium, EDTA, which is rich in potassium, can carry over and contaminate the next tube. The same contamination can happen---to a much greater degree---if the contents of an EDTA tube (lavender stopper) is poured into a tube to be tested for potassium.

Mixing by the numbers

Probably one of the most frustrating problem any laboratory has is when samples aren’t mixed properly. Not only can clots wreak havoc on sensitive laboratory instruments, it delays test results by at least an hour waiting for a new sample. Mixing isn’t hard. In fact, it’s so simple that a lot of people think it isn’t important. Don't be one of the many who underestimate this important step. Mixing must be slow and deliberate. No sideways jiggles and no furious shaking. Tubes with additives must be inverted all the way down, paused for the air bubble to rise to the top, and all the way back up again. That’s one inversion. Follow your tube manufacturer's recommendation, but serum, EDTA, and heparin tubes should be inverted 5-10 times, and citrate tubes 3-5 times.

If you're an educator or trainer, drive the point home using visual images. Take a filled anticoagulated tube and show trainees how the air bubble rises as the tube is inverted. Explain it’s the air bubble that erodes the additive off the wall and into the blood, and if that bubble isn’t allowed to rise all the way to the top, it can’t do the job. That’s why inversions have to be slow and deliberate, with a pause every time it’s turned over.

Fill 'er up

The last tube handling issue to address during the draw is filling the tube sufficiently. Do you and those who work with you or for you know what the fill lines on the label represent? Are they the minimum, maximum or the ideal volume? If the mark on the tubes you use represents the minimum and someone thinks it’s the maximum, then they’re going to submit short samples thinking there’s a fudge factor they get to work with. Just remember, manufacturers put a carefully calibrated quantity of anticoagulant into their tubes. There’s a certain sweet spot with the concentration of every additive. Stay within that sweet spot and the tube performs as it should. Tinker with the concentration by overfilling or underfilling and the blood no longer represents the patient’s status.

Make sure you and your staff or students know how to interpret these lines. Greiner Bio-One uses a black arrow on it's label. The point of the arrow indicates 100 percent of the stated volume. The top and bottom of the arrow represent a ten-percent variance. If the level of the filled blood tube is above or below the upper and lower extremes of the arrow, the tube should not be submitted for testing. The BD Vacutainer® Plus sodium citrate tube has an etched line on the plastic itself that indicates the minimum fill line. Tubes with a blood level that falls below this line should not be submitted. For Sarstedt tubes, the line on the label indicates the optimal fill.

Submitting underfilled citrate tubes risks adjustments to the patient's blood thinner that may put him/her at risk of stroke or other complications from circulating clots that can be catastrophic, even deadly.

It's not just coags that are affected by underfilling. The following analytes are affected when underfilling a heparin tube: ALT, AST, amylase, LDH, lipase, potassium, troponin, CK and GGT. It’s not just speculative.(1-3) On the other side of the equation, is your lab testing underfilled tubes for analytes known to be affected? If so, those who test underfilled samples are not being managed properly.

How much can you underfill an EDTA tube and not affect results? Underfill an EDTA and you get falsely lower hematocrits and MCVs because EDTA in excess concentrations shrink the red blood cells. But at what level of underfill does it become significant? Studies on this are scant, which is why we must adhere to the tube manufacturer's recommendations. To avoid underfilling, stock a wide variety of tubes in your trays and draw stations that include low draw-volume tubes. That way when there’s a difficult draw, the phlebotomist can employ tubes that have lower vacuum and submit lower volume tubes filled to their sweet spot, the only place that can possibly render an accurate lab result.

It's no surprise whey we refer to phlebotomy as the most underestimated procedure in health care. There’s so much to it, and most of the mistakes we make when drawing or handling a sample that changes the test result can’t be fixed by the lab. Most of the time they can’t even be detected. Make this your mantra for the month: Best practices proactively protects patients, or BP4 for short.

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