Interferences in Laboratory Testing


By the end of this session the reader should be able to:

  • Describe causes of in vitro hemolysis in laboratory samples
  • Discuss the influence of hemolysis on serum sodium, potassium, calcium, acid phosphatase, folate, and aspirate aminotransferase
  • Describe the effect of circadian rhythm on serum cortisol, growth hormone, serum iron, and thyroxine
  • Describe methods by which drugs can interfere with laboratory testing (e.g., cross-reaction with antibodies, interference with enzyme reactions, enhancement or inhibition of reactions)


Additive - A substance added to a specimen that changes one or more of its physical or chemical properties.

Analyte - A substance that can be measured by an analytical technique.

Anticoagulant - A substance that can suppress, delay, or prevent coagulation of blood by preventing formation of fibrin.

Artifact - Changed state of a material resulting from artificial, rather than natural processes or conditions.

Chelation - The process of an organic molecule binding metal ions.

Circadian rhythm – Changes in concentration of analytes that occur over the course of a single day.

Clot - An aggregation of blood cells held together by fibrin, a polymerized protein.

Hemoconcentration - The process of increasing concentration of cells, proteins, and occasionally other analytes in blood through loss of water, either in vitro or in vivo.

Hemolysis - Rupture of red blood cells, releasing analytes from the cells into the surrounding serum or plasma.

Heparin - An anticoagulant that directly inhibits formation of fibrin.

Infradian rhythm – Changes in concentration of analytes that occur less frequently than once a day.

Phlebotomy - Puncturing a vein with a needle for the purpose of obtaining a sample of blood.

Plasma - The liquid part of blood in the bloodstream; obtained as a specimen by collecting blood with an anticoagulant and centrifuging the specimen.

Postprandial - After a meal; also “postcibal.”

Preanalytical variation - Factors that alter results of a laboratory test and that occur before the process of performing the test.

Proteolysis - The process of degradation of proteins, which may occur by chemical reactions or enzymatic processes.

Serum - The liquid part of blood remaining after a clot has formed.

Serum separator - A mechanical system that physically separates serum from cells (plasma separators separate plasma from cells), thereby preventing changes in concentration of serum analytes as the result of cell metabolism.

Stasis - A decrease in flow of blood to or from a part of the body.

Ultradian rhythm – Changes in concentration of analytes that occur over a period of time much less than 1 day.



Table 1
Invisible Less than 30 mg/dL hemoglobin
Barely visible 30 mg/dL hemoglobin (barely pink)
Visible 60 mg/dL hemoglobin (red) or 90 mg/dL hemoglobin (burgundy)


  • Mechanical
  • Chemical or osmotic
  • Aging
  • Temperature (both heat and cold)

Causes of Hemolysis

  • Blood drawing
  • Type of syringe or Vacutainer
  • Transfer of blood
  • Transportation of blood
  • Transportation in pneumatic automatic tube systems

NOTE: Refrigeration of whole blood inhibits the erythrocyte Na+/K+-ATPase pump, resulting in loss of potassium from the erythrocyte into the plasma and diffusion of sodium from the plasma into the erythrocyte (concentration gradient). Therefore, separate serum/plasma from cells as soon as possible.

Table 2
Concentration of Substances in Erythrocytes and Plasma***
Substance Erythrocytes Plasma Erythrocyte/Plasma Ratio
Glucose, mg/dL 74.0 90.0 0.82
Urea-N, mg/dL 14.0 16.0 0.88
Creatinine (Jaffe), mg/dL 1.8 1.1 1.63
Uric acid, mg/dL 2.5 4.6 0.55
Total cholesterol, mg/dL 139.0 194.0 0.72
*Sodium, mEq/L 16.0 140.0 0.11
*Potassium, mEq/L 100.0 4.4 22.8
Chloride, mEq/L 52.0 104.0 0.50
Bicarbonate, mEq/L 19.0 26.0 0.73
*Calcium, mEq/L 0.5 5.0 0.10
Inorganic P, mg/dL 2.5 3.2 0.78
*Acid phenylphosphatase, U/L 22.0 3.0 67
*Lactate dehydrogenase (LD), U/L 58,000 360 160
*Transaminase, AST, U/L 500 25 20
Transaminase, ALT, U/L 150 30 5
*Folate, ng/dL 200-1200 (4.6-2.7) 2.5-1.5 (0.06-0.34) 100
*Largest observed differences. **After Caraway, W.T.: Am. J. Clin. Path. 37:445, 1962.

Types of Interferences of Hemolysis with Laboratory Tests

Interference Type 1

Interference with chemical procedure

a) participation of hemoglobin in the reaction through augmentation or inhibition
b) spectrophotometric interference

All are totally dependent upon the particular analytical methodology utilized for measurement. Manufacturers continually improve methodology and minimize interferences. New methods working in the near infrared are presently being developed for use with blood substitutes. These methods have no or minimal interference from hemolysis.

Table 3
Analyte Examples Degree of Hemolysis Allowable (Depends on analytical method) Mechanism
Ammonia Slight hemolysis Interferes with color development and increases value
Amylase Slight hemolysis Increases value
Bilirubin Slight hemolysis Interferes with diazotization
Carotene No hemolysis Increases value
Ceruloplasmin No hemolysis or turbidity Decreases value
Digoxin Slight to moderate hemolysis alright but preferably none
Folic acid Slight to moderate hemolysis acceptable but preferably none
Protein electrophoresis No hemolysis May augment beta1 between alpha2 and beta
Insulin No hemolysis Causes destruction of hormone for RIA
Testosterone Slight to moderate Interference with liquid scintillation counting
Triglycerides No hemolysis

Interference Type 2

Leakage of constituents of red cells into plasma or serum (Analytes higher in erythrocytes than in serum)

Table 4
Analyte Examples Degree of Hemolysis Allowable (Depends on analytical method) Mechanism
Alkaline phosphatase Slight hemolysis Greater concentration in RBC’s than in serum
Creatine phosphokinase (CK) Slight hemolysis Greater concentration in cells than in serum
Folate Slight to moderate hemolysis acceptable but preferably none
LDH Absolutely no hemolysis Much greater concentration in RBC’s than in serum
Potassium (as above) (as above)
Protein Slight hemolysis No hemolysis, lipemia or icteric serum with refractometer method
AST Very slight hemolysis Greater concentration in RBC’s than in serum
ALT Slight hemolysis (as above)

Interference Type 3

Dilution of plasma or serum constituents (Analytes lower in erythrocytes then in serum)

Table 5
Analyte Examples Degree of Hemolysis Allowable (Depends on analytical method) Mechanism
Chloride Slight hemolysis Dilutional effect
Lithium Slight to moderate hemolysis Gross hemolysis has a dilutional effect
Magnesium No hemolysis Dilutional effect
Phosphorus Slight to moderate hemolysis Concentration in serum a little greater than in RBC’s
Sodium Slight hemolysis Dilutional effect

Interference Type 4

Interference by blood contamination of other body fluids

a) Amniotic fluid scan for bilirubin
b) L:S ratio in amniotic fluid
c) Spinal fluid protein

Table 6
Circadian Rhythms
Cortisol (secondary to ACTH secretion) AM levels > PM levels
Growth hormone Elevated during sleep
Serum iron AM levels > PM levels (30%)
Thyroxine PM levels > AM levels (15%)


Virtually all drugs may affect the results of clinical laboratory tests by:
a) In vivo effects, and/or
b) Interference with the analytical procedure (in vitro effects)

Because of the large number of prescribed drugs in use and the multiplicity of effects by some of these agents, the problem of drug interference with laboratory tests is extremely complex. A vast amount of information on this topic has accumulated, particularly within the last decade, and the aim of this session is to raise the level of awareness of this problem.

The main effects of drug interferences are:

a) In vivo effects (pharmacological effects)
1. Intended or therapeutic effects
2. Side effects

While the clinician usually is aware of the main therapeutic effects of drugs, other changes in certain biochemical parameters are often neglected and unexpected, since they may be unrelated to the main action of a drug, and these “side effects” are often ignored. Well known examples are, for instance, the enzyme induction in the liver by barbiturates and phenytoin (Dilantin) or the increases in thyroxine binding globulin (TBG), cortisol binding globulin (CBG), transferring, and fibrinogen following intake of oral contraceptives. Lesser known examples are, for instance, elevations of amylase with oral contraception or decreases in thyroxine (T4) values due to penicillin.

b) In vitro interferences with analytical procedure (methodological effects)
Results may be increased or decreased.
The most frequent modes of action are:
1. Alterations of chemical reactions (enhancement or inhibition)
2. Cause of turbidity in the reaction system
3. Interference with enzyme reactions
4. Cross-reaction with antibodies
5. Radioactive interference, due to in vivo use of radioactive compounds

Table 7
Common drug-induced modifications of clinical chemistry test values (All are dependent upon the particular analytical methodology utilized for measurement)
Decrease (false) Aspirin
Increase Heparin, increases dye binding to other serum proteins
Bisalbuminemia Penicillin binding to albumin causes two albumin peaks on serum electrophoresis
Alkaline phosphatase
Decrease Anticoagulants (oxalate, fluoride, citrate bind Mg++)
Increase Estrogens, gentamicin (hepatotoxicity)
Human albumin injections as plasma expanders (made from human placenta)
Decrease Anticoagulation with oxalate or citrate
Increase Opiates cause spasm of sphincter of Oddi
Oral contraceptives (effect on liver)
Decrease Daylight, 30% per hour!! (non-drug)
Barbiturates induce glucuronyl transferase in newborns
Increase Any drug with liver toxicity or causing cholestasis
Methyldopa reacts with diazo reagent
Decrease Diuretics enhance excretion
Phenytoin (Dilantin) may cause osteomalacia
Increase Antacids (large amounts), e.g., TUMS
Vitamin D
Decrease Androgens decrease synthesis
Neomycin forms salts with bile acids in gut
Increase Bilirubin (methodological)
Corticosteroids (methodological)
Decrease Marijuana (?)
After meals
Increase Acetoacetic acid, acetone and ascorbic acid are chromogenic in picrate reaction; glucose same as above
Increase Quinidine releases digoxin from heart muscle and also decreases renal excretion of digoxin, causing substantial increases in serum digoxin
Decrease Ascorbic acid interferes with glucose oxidase procedures
Increase Ascorbic acid interferes with procedures which utilize reduction (e.g., reduction of cupric ion, ferricyanide, etc.)
Occult Blood
False negative Ascorbic acid inhibits tetramethylbenzidine reaction
False positive Large amounts of meat or undigested meat fibers; aspirin in over 70% of patients when given >3 g/day due to bleeding into GI tract
Increase Valproic acid causes increase in serum hime lide by inhibiting its renal excretion
Decrease Diuretics, cathartics, aspirin, steroids
Increase Blood transfusions
Hemolysis (non-drug)
Refrigeration of whole blood (inhibition of Na/K-ATPase pump)
Marijuana (?)
Spironolactone inhibits Na/K-exchange in renal tubules
Decrease Refrigeration of whole blood; inhibition of Na/K-ATPase pump (non-drug)
Total T4
Decrease Phenytoin (Dilantin) and salicylates compete with binding sites on TBG
Increase Oral contraceptives increase TBG

Listing of Effects of Drugs on Clinical Laboratory Tests

While a number of reference works exist, the most complete is that by Young, et al. (see next page). The listing consists of two main files of reported interferences.

These are arranged in
a) alphabetic listing by drug name
b) alphabetic listing by laboratory test

Each file contains close to 20,000 entries of reported effects of drugs and also those of posture, sleep, insomnia, diurnal variation, sex differences, age differences, blindness, ovulation, menstruation, menopause, etc.

Consultation of these listings whenever drug or other effects are suspected is highly recommended. All listed items of interference are referenced and a total of 5,476 literature sources are cited. The following sample pages are shown for the drug by drug and the test by test listings (with permission).

The pertinent abbreviations as used in these listings are:
B- blood
C- cerebrospinal fluid
dec- decreased
inc- increased
M- methodological effect
0- other fluid
P- plasma
S- serum
T- test conditions
U- urine
V- in vivo effect
Z- No effect

Unless otherwise stated, the content of this page is licensed under Creative Commons Attribution-ShareAlike 3.0 License