Sun Diagnostics at AACC 2016-Philadelphia

Sun Diagnostics, LLC will be CLOSED Monday 8/1 through Friday 8/5 as we attend the AACC Annual Meeting in Philadelphia, PA.  We will be available via email (support@sundiagnostics.us) and will have limited access to voice mails.  PLEASE NOTE THAT NO ORDERS WILL BE SHIPPED DURING THIS WEEK as personnel will be unavailable.   If you will be in Philadelphia at the meeting, please visit us at Booth #3357.

Protein Interference with Common Laboratory Tests

Protein Interference with Common Laboratory Tests (download)

 Abstract

Objective: Protein as an interferent in underappreciated. Therefore, we evaluated interference by proteins on clinical laboratory tests. Methods: Base Pool was prepared using a serum pool. A High Pool was prepared by spiking proteins into the Base Pool (Concentrated human albumin and gamma-globulins 1:1; ASSURANCE™ Interference Test Kit, Sun Diagnostics, New Gloucester, ME). The Base Pool and High Pool were intermixed to create five levels of total protein (3.5, 6.9, 9.4, 12.4, and 15.3 g/dL). Multiple analytes were measured on the Beckman AU5800. Results: Minimal effects were seen with alkaline phosphatase (ALP), amylase (AMY), direct bilirubin (DBIL), total bilirubin (TBIL), creatine kinase (CK), gamma-glutamyltransferase (GGT), glucose (GLU), lactate dehydrogenase (LDH), lipase (LIP), phosphorus (PHOS), urea nitrogen (BUN), uric acid (UA), lipoprotein(a) (LPA), small dense low-density lipoprotein cholesterol (sdLDL-C), high sensitivity C-reactive protein (hsCRP), ferritin (FER), low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), potassium (K), chloride (CL), triglycerides (TRIG), and total cholesterol (CHOL). Positive bias with increasing protein concentration was seen with alanine aminotransferase (ALT), aspartate aminotransferase (AST), calcium (CA), creatinine (CRE), iron (FE), magnesium (MG), homocysteine (HCY), and non-esterified fatty acids (NEFA). Because proteins bind analytes such as calcium, magnesium, iron, HCY, and NEFA, and the albumin interferent is minimally processed human albumin, we believe these increases were artifactual. Negative bias was seen with carbon dioxide (CO2), apolipoprotein AI (apo AI), and apolipoprotein B (apo B). Conclusion: Human proteins (albumin and gamma-globulin) interfere with the measurement of many routine clinical chemistry assays, and manufacturers and laboratorians need to pay more attention to proteins as a potential interferents. The mechanism may be photometric or a volume depletion effect, whereby a very high protein concentration will reduce the available water so that the analyte concentration is artifactually low.

 

Introduction

Proteins as potential interferents are overlooked by laboratorians and IVD manufacturers. The assumption is that protein interference is caused by paraproteins (monoclonal immunoglobulins) [1,2], but this is an oversimplification.  Elevated proteins, including albumin and gamma-globulins may affect many assays, including bilirubin, phosphate, HDL cholesterol, GGT, CRP, and glucose [1]. The manufacturer may note on the package insert that monoclonal gammopathies may result in errors, but rarely do they perform a comprehensive study of protein interference. The Clinical Laboratory Standards Institute (CLSI) recommends testing with both albumin and gamma-globulins at a total protein concentration of 12 g/dL [3]. The interference may be due to physical or chemical alteration in the signal (e.g. increased light scattering) or it may be due to a volume displacement effect [4]. The practical effect of an increased concentration of proteins is a decrease in serum water or volume depletion: the aspirated sample is diluted with solid (protein and/or lipid) so that the analyte concentration is artifactually low. Viscosity is also a common problem with specimens containing monoclonal immunoglobulins, especially IgM, and this may affect the accuracy of sample delivery.

 

Methods

A Base Pool was prepared using leftover, de-identified patient sera. A Test Pool was prepared by adding 1 part concentrated human proteins (ASSURANCE™ Test Kit, Sun Diagnostics, LLC, New Gloucester, ME) to 1-part Base Pool. The concentrated proteins are ~50% human albumin and 50% human gamma-globulins. A Control Pool was prepared by mixing 1-part Base Pool with 1-part saline to maintain equal analyte concentrations. The Test Pool and Control Pool were intermixed to create five levels of protein interferents. Multiple analytes in each of the five levels were measured in duplicate on the Beckman AU5800 at HDL, Inc. (Richmond, VA). Bias was calculated for levels 2 to 5 versus level 1, or level 4 versus level 1. Bias < ½ the total allowable error was considered acceptable.

 

Results

  • Minimal interference with total protein concentrations up to 15.5 g/dL were seen for ALP, AMY, DBIL, TBIL, CL, CHOL, CK, FER, GGT, GLU, HDL-C, HDL3-C, hsCRP, LDH, LIP, LPA, LDL-C, PHOS, K, sdLDL-C, TRIG, BUN, or UA.
  • Positive bias was seen with increasing protein concentrations for ALT, AST, CA, CRE, HCY, FE, NEFA, and MG.
  • Negative bias was seen with CO2, APOAI, and APOB.
  • Because human proteins bind calcium, homocysteine, iron, nonesterified fatty acids, and magnesium we measured these analytes in the concentrated protein interference material and subtracted this from the measured concentrations. Positive bias remained for all analytes except NEFA, which converted to a negative bias.

Table 1: Measured Protein Concentrations of Five Levels of Test Pools, g/dL

Table 1

 

 

 

Table 2: List of Analytes, Test Concentration, Allowable Error, and Bias Due to Protein

(Significant Bias due to Protein Interferent is highlighted in RED)

Table 2

 

 

 

 

 

 

 

Bias Plots: Interference Due to Protein

bias plot

 

 

 

 

 

 

 

bias plot 2

 

 

 

 

 

 

 

 

 

Conclusions:

 

  • Human proteins (albumin and gamma-globulins) interfere with a number of common laboratory tests.
  • Protein interference is not described by most manufacturers, except to note possible interference by monoclonal paraproteins.
  • Laboratorians and manufacturers need to pay greater attention to proteins as a source of interference with chemistry assays.

 

References:

  1. Dimeski G. Interference testing. Clin Biochem Rev 2008; 29 Suppl (i): S43-S48.
  2. Kroll MH, Elin RJ. Interference with clinical laboratory analyses. Clin Chem 1994; 40:1996-2005.
  3. Kazmierczak SC, Catrou PG. Analytical interference: more than just a laboratory problem. Am J Clin Pathol 2000; 113:9-11.
  4. Artiss JD, Zak B. Problems with measurements caused by high concentrations of serum solids. Crit Rev Clin Lab Sci 1987; 25: 19-41.

 

Adapted from: John H. Contois, Jason P. Nagy, and Rae-Anne Nguyen, Sun Diagnostics, New Gloucester, ME. Protein interference with common laboratory tests. Presented at the Annual Meeting of the AACC, Atlanta, GA; July 2015.

 

 

Sun Diagnostics, LLC can assist in your assay development and validation activities.

Did you know that Sun Diagnostics:

  • Welcomes custom interference material requests?  In fact, some of the interference materials, such as Atorvastatin (INT-05ATO), Ciprofloxacin (INT-05CIP), and Tamoxifen (INT-05TAM) were initially part of a customer requested special project.  Please don’t hesitate to contact us with your custom interference needs!
  • Can create custom quality control (QC) materials for your assay(s), to your specifications?  SunDx QC materials are human serum- based and more similar to patient samples.  The QC can be prepared independently of the assay calibration materials, providing an impartial assessment of assay and instrument performance. We are flexible and can accommodate small, medium and large batch sizes.  Sun Diagnostics, LLC is an FDA registered company with a strong focus on product quality and customer satisfaction.
  • Offers consulting services? The SunDx team is pleased to offer their expertise in method validation and lab operations.  We can help you develop testing procedures for validating and verifying your new assays and test methods.

 

Contact 207-926-1125 or email support@sundiagnostics.us to discuss!

Sun Diagnostics makes ASSURANCE HAMA available

Sun Diagnostics, LLC continues to simplify interference testing with the highly anticipated addition of ASSURANCE™ INT-02 HAMA Interference Test Kit.  Immunoassays are subject to interference from endogenous antibodies, especially human anti-mouse antibodies (HAMA) and rheumatoid factor (RF). These interferents are heterogeneous, which means some samples with interfering endogenous antibodies will affect a specific assay and some will not. Using multiple HAMA or RF positive samples will assess interference more thoroughly.

INT-02 contains 5 individual HAMA positive serum samples that are known to be RF negative.

INT-02 for more information.

Sun Diagnostics Posters Presented at AACC 2015

8/5/15

Sun Diagnostics presented two AACC Annual meeting in Atlanta, GA.

Protein Interference with Common Laboratory Tests was presented during the “Factors Affecting Test Results” session.  The research highlights the fact that proteins as potential interferents are often overlooked; many laboratory tests are affected by high protein concentration.

Validation of a New Lipoprotein(a) Particle Number ELISA was presented during the “Lipids/Lipoproteins” session.  The SunDx Lp(a)-P ELISA is accurate, precise, and linear over a wider range than most Lp(a) assays, and is strongly correlated to the reference method.  Importantly, the assay shows no bias due to apo(a) isoform size.

Testing Interference from Common Drugs – Guest Blog, Pradip Datta, PhD.

Assay interference caused by common drugs is a challenging and often overlooked problem. Dr. Pradip Datta has written a brief blog discussing drugs as potential interferents in clinical assays.  We think you will find his insights interesting and thought provoking. With the thousands of drugs in the marketplace, how can manufacturers and laboratorians recognize and test the drugs that may affect their assays?

Sun Diagnostics is now offering a test kit to help assess potential drug interferents. The kit includes drugs that were recommended by an expert panel as “…likely to present frequent, clinically significant interference.” There are many newer over-the-counter and prescription drugs that may also be potential interferents, but the drugs recommended by the expert panel, packaged in a convenient kit, represent a great first step in addressing this unmet need for better drug interference testing.

Testing Interference from Common Drugs

Pradip Datta, PhD

Interference from commonly used drugs in laboratory tests is a major problem for both manufacturers and end-users of in-vitro diagnostic assays. The drugs can be common over the counter drugs (analgesics, antihistamines or anti-inflammatory agents), or prescribed medications (antibiotics, anticancer, neurologic); some of the latter are prescribed for lifetime usage (for example, anti-asthmatic, cholesterol-lowering, immunosuppressant, etc.). Such interference affects not only immunoassays (by cross-reactivity), but also chemistry assays (by enzyme interference or chromogenic side reactions). Serious errors in reported results may happen without knowledge of such interference. Manufacturers of commercial diagnostic assays may report some such interference in their assay Inserts.  But with some 20,000 drugs approved by FDA and an average of 340 new drugs entering the formulary every year, it is a formidable task to study the drug interference of a new assay. As with any interference, the effect becomes more prominent and critical at low analyte levels; and with increasing emphasis on the clinical utility of very low endogenous concentrations of some analytes like cardiac troponin or prostate specific antigen (PSA), this problem will get worse. An additional difficulty in evaluating drug interference is the possibility of interference from drug metabolites as well.

Sonntag and Scholer1 observed a variety of interference (positive or negative) from 18 common drugs in some 70 assays. There is a larger compendium of >40,000 drug effects on laboratory tests2. In recognition of the problem of interference, the Clinical and Laboratory Standards Institute (CLSI) developed a protocol to study interference in any assay before adopting it in the laboratory3. The protocol measures both positive and negative assay interferences.

In addition to therapeutic drugs, laboratorians must evaluate effects of abused drugs as well in a new assay. The commonest drug of abuse (DAU) is ethanol. With increasing recognition of chronic pain as a debilitating disease, abuse or misuse of pain medications is a growing problem.

Every clinical laboratory should be aware of the potential of drug interference, especially from the drugs prevalent in the population they serve, and be careful in reporting their results. It is easier to spot an aberrant result outside the reference range of the assay, but more difficult when the interference has the opposite effect: causing a clinically important result to fall inside the reference range. Knowledge of patient’s drug history and drug interference will help the laboratorian in such cases.

REFERENCES:

  1. Sonntag O, Scholer A. Drug interference in clinical chemistry: recommendations of drugs and their concentrations to be used in drug interference studies. Ann Clin Biochem 2001; 38:376-385.
  2. Young DS. Effects of drugs on clinical laboratory tests, 5th ed. Washington: AACC Press, 2000.
  3. Clinical and Laboratory Standards Institute (CLSI). CLSI document EP7-A2 (ISBN 1-56238-584-4). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005.

2015 Product Pricing Notification

Due to a significant increase in the cost of raw materials required to manufacture the triglyceride-rich lipoprotein component of the ASSURANCE Interference Test Kit for routine interferents, the price of these two items will increase as of January 1, 2015.  This is our first ever price increase.  We are pleased to let you know that our other products will maintain their current pricing.  Please download this letter for your records.