Hot Topic

Common Test-Ordering Errors
Part 5: Overordered Tests

Plasma Thiamin
Serum Folate
Zinc Protoporphyrin (ZPP)


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Published: August 2010

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Dr. Dale reviews test-ordering errors commonly seen at Mayo Medical Laboratories. This is the fifth presentation in the series and addresses overordered tests including thiamin, folate, and zinc protoporphyrin.

Presenter: Jane C. Dale, MD

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Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this 8-part series is Dr. Jane Dale, a consultant in the Department of Laboratory Medicine and Pathology at Mayo Clinic. Dr. Dale reviews test-ordering errors commonly seen at Mayo Medical Laboratories. This is the fifth presentation in the series and addresses over-ordered tests including thiamin, folate, and zinc protoporphyrin.

Testing at Mayo Medical Laboratories

Here at Mayo Medical Laboratories we receive over 800,000 test orders each month. While by our estimation most of the orders we receive are appropriate orders, we do experience some recurring test-ordering problems. In this presentation, I will describe the most common problems that we see. I will also describe our efforts to decrease these problems and recommend ways that you too can help reduce these errors. Together, by decreasing inappropriate test orders, we can improve patient care and reduce costs.

Common Mayo Medical Laboratories Test-Ordering Errors

In this 8-part series, I address 2 types of errors—misordered tests and overordered tests. In parts 1 through 3, I addressed the most commonly misordered tests, that is, tests ordered for the wrong purpose.

Overordered Tests

This is a list of the most frequently overordered tests that we receive at Mayo Medical Laboratories—tests that we believe are ordered too frequently, including a few that we do not recommend doing at all. In part 4, I addressed the first 2 bullet items. In this presentation, part 5, I will discuss the thiamin, folate, and zinc protoporphyrin.

Thiamin (Vitamin B1)

Plasma thiamin is a test that we believe should not be ordered. Thiamin (also known as vitamin B1) is an essential vitamin that is required for a variety of the body’s biochemical pathways, including those involved with metabolism of carbohydrates and fats, brain function, and peripheral nerve myelination.

Thiamin is obtained from the diet—body stores are limited—and deficiencies can develop quickly. Thiamin deficiency is most prevalent in chronic alcoholics due to poor nutrition, although it can occur in individuals with chronic gastrointestinal problems or those who are elderly, on cancer treatment, or receiving diuretic therapy. Severe thiamin deficiency causes congestive heart failure, peripheral neuropathy, Wernicke encephalopathy (which is a medical emergency that can progress to coma and death), and Korsakoff syndrome (which can follow Wernicke encephalopathy, resulting in memory loss that is often irreversible and dementia). Importantly, rapid treatment of Wernicke encephalopathy with thiamin can prevent Korsakoff syndrome.

Thiamin diphosphate (also called thiamin pyrophosphate) is the active form of thiamin and is the best measure of thiamin status. Thiamin diphosphate in circulating blood is primarily present in erythrocytes, with <10% in the plasma phase of blood. Thus, testing of serum or plasma doSes not provide a useful result.

HPLC analysis of thiamin diphosphate in whole blood or in erythrocytes is a sensitive, specific, and precise method for determining the nutritional status of thiamin and is a reliable indicator of total body thiamin stores. "Thiamin (Vitamin B1), Whole Blood" is available at Mayo Medical Laboratories. This assay specifically quantitates the active form of vitamin B1 as an indicator of vitamin B1status.

For a good while, we encouraged our customers to order the whole blood thiamin test but when we received orders for plasma thiamin, we forwarded the specimens on to another laboratory. Recently, because of the demonstrated lack of utility of serum and plasma B1 levels, we took this a step further and discontinued forwarding those tests.

Folate—Serum and RBC

Serum folate is another overordered vitamin test. Folate is a B vitamin and, along with vitamin B12, is required for normal red blood cell maturation. Deficiency of either of these nutrients causes macrocytic anemia.

While laboratory testing for patients with macrocytic anemia typically begins with measurement of serum folate and vitamin B12 concentrations, folate levels are relatively poor predictors of total body folate stores. Serum folate is neither sensitive nor specific:

Serum folate levels typically fall within a few days after dietary folate intake is reduced and may be low even in the presence of adequate tissue stores. Alcohol intake and medications may cause similar changes. Conversely, serum folate concentration may rapidly rise to a normal level after just 1 balanced meal, and may be normal despite the presence of low tissue stores.

Red blood cell folate concentration is less subject to short-term dietary changes and is thus considered more reliable, but that test also has sensitivity and specificity problems.

Diagnostic Algorithm for Macrocytic Anemia

This algorithm, which was published by one of Mayo's hematologists, shows an approach for diagnosing the cause of macrocytic anemia that avoids the folate assay limitations. This diagnostic approach substitutes methylmalonic acid and/or homocysteine testing for folate.

Simplified Versions of Vitamin-Dependent Pathways

This next slide shows simplified versions of 2 vitamin-dependent pathways. The first involves both folate and B12; and the second involves B12 only. I show these, not only because disturbances in these pathways are central to the pathologic changes that occur in folate and B12 deficiencies, but also to demonstrate how homocysteine and methylmalonic acid levels can help determine the cause of megaloblastic anemia.

In B12 deficiency, both homocysteine and methylmalonic acid concentrations are increased; whereas in folate deficiency, only homocysteine is increased and MMA is normal.

Lead Toxicity

The next issue I'd like to talk about is lead toxicity, as it relates to the overordered test zinc protoporphyrin or ZPP. While the ban on lead paint in the late 1970s greatly decreased the incidence of lead toxicity in our country, it did not eliminate it because lead-based paint is still present in some homes and lead toxicity remains a threat to children in the United States.

Lead can have adverse effects throughout the body, but it is especially harmful to the developing brain and nervous system of young children. It is estimated that a child's IQ will fall 1 to 3 points for every 10 mcg/dL increase in blood lead. However, even lead levels lower than 10 mcg/dL (which is the cut point that has been recommended for intervention) can be harmful.

A number of studies have demonstrated an association between learning difficulties, behavioral problems, and IQ and blood lead levels between 5 and 9 mcg/dL. Furthermore, according to the CDC "There is no safe level of lead in the blood. Any confirmed level of lead in the blood is a reliable indicator that the child has been exposed to lead." Consequently, it is important to have sensitive and specific tests available for all at risk children.

Heme Synthesis

Before blood lead assays were widely available, red blood cell zinc protoporphyrin was used as an indicator of lead toxicity. That test works because lead inhibits several enzymes in the heme synthesis pathway, including the last step, which is catalyzed by the enzyme ferrochetolase. In the presence of lead-induced inhibition, protoporphyrin intermediates accumulates within the RBC; thus, RBC zinc protoporphyrin is a marker for lead toxicity.

Laboratory Assessment of Lead Toxicity

However, the test is not specific for lead toxicity. For example, in iron deficiency, because iron is not available for heme synthesis, zinc protoporphyrin also accumulates within red blood cells. Zinc protoporphyrin also is not a very sensitive indicator of lead exposure—it cannot reliably detect exposure to lead at the level necessary to protect children. According to the CDC, and I quote: "Since erythrocyte protoporphyrin is not sensitive enough to identify more than a small percentage of children with blood lead levels between 10 and 25 microgram per deciliter and misses many children with blood lead levels greater than or equal to 25 microgram per deciliter (McElvaine et al, 1991), measurement of blood lead levels should replace the erythrocyte protoporphyrin test as the primary screening method."

For all these reasons, blood lead is the test of choice for screening children for lead toxicity. In our laboratory, the blood lead test is performed using inductively coupled plasma-mass spectrometry. The assay is sensitive and specific, providing the accurate results that are necessary to identify those children for whom interventions are required.

Because of poor sensitivity and specificity, ZPP is no longer recommended for lead screening in children.


This concludes Part 5 of this series on some of the tests that, in our experience as a reference laboratory, are commonly ordered inappropriately.