Answering Your Questions

July 1, 2011

Can test be done with less sample?


I run a quick test for NT-proBNP with a cutoff of 450 pg/mL on patients more than 50 years of age. The test requires two drops of serum. If the patient is positive, would it be acceptable to run the test again with one drop of blood to give the physician some guideline(s) and assume that it is then positive for >900 pg/mL?


This is not a good idea. Good laboratory practice and many regulatory agencies require that all lab tests have a defined analytical measurement range (AMR) and clinical reportable range (CRR). If a lab desires to report a result beyond the AMR possible using undiluted blood, then the protocol for dilution must be validated to ensure accurate results are reported after dilution. In this case, you must validate that samples with known concentrations of NT-pro BNP can be “diluted” by applying less sample, and the results will be accurate by your method. This probably will not work, as most whole-blood immunoassays use little blood for actual testing but may require more blood to provide sample flow-through to the various stages of testing on the device. Depending on where you are located and what regulatory agencies (e.g., U.S. Food and Drug Administration) oversee laboratory testing in your area, alteration of waived or moderate complexity in this manner would turn this into a high-complexity test. This would require a different level of validation for the test, and potentially change the personnel, quality control, and proficiency-testing requirements for the test.

Brad S. Karon, MD, PhD


Hospital Clinical Laboratories

Mayo Clinic

Rochester, MN

Timing of phlebotomy for lab tests


Whenever tests are ordered for blood to be drawn at a certain time, our phlebotomists are responsible to draw as close as possible. Exact time requests are not always possible. Is there an acceptable time range for these draws (e.g., +/- 30 minutes of requested time)?


Ordering schemes and definitions vary tremendously among hospitals and facilities; I am aware of no “standard.” Timed tests are limited to those in which there is some relationship between the time a test needs to be drawn and a therapeutic decision that will be made — most commonly, drug levels or other therapeutic drug monitoring (e.g., partial-thromboplastin time [PTT] testing for heparin management). For timed tests, our policy at Mayo states that collection should occur within 15 minutes of time ordered. This is critical, especially for drugs given intravenously where levels can change quickly and decisions on next dose will be made based on previous level. Similarly, hourly glucose testing (for insulin dosing) must be done within 15 minutes of time ordered to allow physicians to interpret response to insulin dose changes.

For STAT orders, our policy states that we respond for collection within 15 minutes. The collection time in this instance is defined to allow test completion in the expected amount of time. STAT testing is often defined as being completed within one hour of the time ordered. That does not necessarily define the time from order to collection, however, as the time required to transport and analyze specimens will differ between institutions. My guess would be 10 to 20 minutes is the expected phlebotomy response time (time between order and collection) for STAT tests in many hospitals. On the other hand, for a “routine” complete blood count (CBC) ordered for a specific time, we might allow up to two hours on either side of time ordered to accommodate our lab rounding process for routine blood collections.

In our system, it is dependent upon how the test is ordered, with critical tests — such as drug levels — only orderable as “timed” tests. More esoteric tests are only orderable as routine, while many tests can be ordered routine, priority, or STAT.

The most important concept is that ordering providers must understand what will (or should) happen when they order a particular test, so the lab meets the needs of the patient population being served. Ideally, the ordering system also contains safeguards against ordering esoteric tests or tests which do not provide information needed immediately from being ordered as STAT or urgent.

Brad S. Karon, MD, PhD

Determination of MNPT and INR


Where can I find criteria to screen samples to be used to determine the mean for international normalized ratios (INRs)?


One of the methods to determine INR is to use the INR equation that is the common practice in clinical laboratories. INR is determined based on the following equation: International normalized ratio (INR) = (prothrombin time [PT]/mean normal prothrombin time [MNPT]) international sensitivity index1 — generally read as:


ISI is usually provided by manufacturers. Determining the correct MNPT is vital to reporting accurate INR results. The MNPT is defined as the geometric mean of the PT from the healthy adult population. It requires at least 20 fresh samples from healthy individuals. Geometric mean is calculated as:

X=Antilog Σ [Log(PT)/N)].

Geometric mean can also be determined automatically using an Excel spreadsheet.

Ming Xu, MD, PhD

Associate Clinical Pathologist

Department of Laboratories

Children's Hospital and Regional Medical Center

Seattle, WA


  1. Clinical and Laboratory Standards Institute. Procedures for Validation of INR and Local Calibration of PT/INR Systems; Approved Guideline. Wayne, PA: Clinical Laboratory Standards Institute; 2005. CLSI document H54-A 25(30):27.

Could elevated potassium results be due to dehydration?


Could unexpected elevated potassium results be due to patient dehydration? We have chased complaints from physicians about elevated potassium results for years. We review analyzers, quality control, calibrations, and processing but never find acceptable reasons for elevated results. Our samples tested against other labs' get comparable results. We wonder if elevations may be related to dehydration in fasting patients. When patients with elevated results are recollected non-fasting, most levels are lower. Most patients assume or are instructed erroneously that fasting means “nothing to eat or drink for X hours.” We can tell some patients are dehydrated. Are there significant contra-indications to instructing our patients to be well hydrated (with water) prior to being drawn for routine lab tests?


If your lab staff have been chasing after physician complaints about unexpected potassium results, they are not alone. Most clinical labs face similar challenges with potassium reporting; few find obvious causes or meaningful lasting solutions.

Your questions arise from a suspicion that fasting and dehydration are factors that influence plasma potassium; these seem unlikely causes. Review a detailed list of common causes of potassium problems — an excellent table of issues to consider is available from BD that may help design processes to detect and avoid these problems1 (e.g., put a sign in the phlebotomy room to discourage fist clenching. Draw plasma instead of serum for renal patients — with relatively high potassium and exposed to anticoagulants. Avoid temperature changes in blood that will affect potassium moving into or out of red cells).

Potassium levels decrease transiently following feeding. Following a carbohydrate meal, insulin is released and stimulates the uptake of glucose into many tissues with phosphate, potassium, and magnesium. In extreme situations, this can manifest with symptoms of refeeding syndrome with hypokalemia.2 It is possible that ambulatory and recently fed patients could return for repeat phlebotomy and have lower potassium levels than during an initial blood draw when they were fasting. This would not explain why fasting potassium levels were unexpectedly elevated in these patients during the first phlebotomy.

The physiology of water and sodium are closely linked. While patient rehydration promotes hemodilution, the regulation of plasma potassium is tightly regulated and seldom varies in the absence of changes to acid-base balance, sodium, glucose, and insulin or potassium administration. In contrast, BD's table1 does include a comment that patient dehydration can be associated with inherently higher potassium levels. This may be attributed to the difficulty in performing phlebotomy on dehydrated patients with difficult-to-find veins that collapse during the procedure.

Patients are often told “no food or drink” because the instructions are clear and leave no room for interpretation. After sleeping overnight, this is the way most people awake; and this basal state is often sampled to create reference intervals used in interpretation of test results. “No food or drink” is a reasonable request, so long as the patient is not distressed or dehydrated. Dehydration can lead to hemoconcentration (elevating hematocrit, cell counts, lipid, and protein levels up by up to +20%). Drinking water to ensure adequate hydration while fasting is not contraindicated for the vast majority of clinical lab tests.

Andrew W. Lyon, PhD, F(CACB), D(ABCC)

Clinical biochemist,

Calgary Laboratory Services;

and associate professor,

Department of Pathology

and Laboratory Medicine,

University of Calgary

Calgary, Alberta, Canada


  1. BD. Troubleshooting Erroneous Potassiums in a Clinical Laboratory Setting. Accessed June 4, 2010.
  2. Mehanna HM, Moledina J, Travis J. Refeeding syndrome: what it is, and how to prevent and treat it. BMJ . 2008;336:1495-1498.

Do plastic and ice affect pO2?


We are in the process of correlating blood gases on the i-Stat, and the respiratory technicians are taking the samples to a local facility for comparison. We have found that the results coming back from the samples that have been sent out are showing elevated pO2 results. In our investigation, we noted that the samples obtained by the respiratory techs were being collected in plastic syringes and then put on ice. According to the articles we have located about collection requirements for arterial blood gas (ABG), plastic syringes should not be put on ice or in ice baths but should be left a room temp and tested as soon as possible and not over 30 minutes. If the time extends beyond 30 minutes, the sample should be collected in a glass syringe and put in an ice bath. What is the standard for blood-gas collection if transporting takes 30 minutes or longer? Is ice or ice baths for transport of ABG an acceptable practice and/or is this more likely what is causing our discrepancies of pO2results between our i-Stat and the reference facility?


CLSI guidelines C46-A (Blood Gas and pH Analysis and Related Measurements) and H11-A4 (Procedures for the Collection of Arterial Blood Specimens) are references available for arterial blood-gas collection and analysis.

Plastic syringes are recommended for blood-gas collection because gas transport across plastic is much less than occurs across glass syringes. For this reason, I would not recommend using glass syringes for crosschecks. Our own internal stability studies have shown that pO2 values are relatively stable up to 60 minutes, either at room temperature or on ice. Recommendations against leaving samples on ice for longer than 30 minutes arise out of concerns that electrolyte (specifically potassium) concentrations may rise after this amount of time on ice. In fact, guidelines suggest that analysis of blood gas is appropriate after 60 minutes on ice, but that electrolyte measurement should not be performed in this case.

Several variables could be involved in the findings of increased pO2 values after samples are iced and transported for comparison on a reference analyzer. If using samples from patients or volunteers breathing room air then you would expect that any bubbles present in the samples would cause pO2 to increase as a function of time and amount of disturbance of the sample. This would be analogous to studies that have found sending samples via pneumatic-tube system results in increased pO2 for samples with small (and sometimes undetectable) bubbles. Another possibility is simply analytic bias between the two instrument platforms. One simple experiment can be performed to understand the discrepancy is to use some samples from patients who are mechanically ventilated with very high FI-O2. These samples should display a decrease in pO2 as a function of time and transport rather than an increase. It is also advisable to compare results from proficiency-testing material (after proficiency testing has been completed) or commercial standards or controls.

Brad S. Karon, MD, PhD

MLO's “Tips from the clinical experts” column provides practical, up-to-date solutions to readers' technical and clinical issues from experts in various fields.

Readers may send questions to tips < at >