Table of Contents
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OBJECTIVES
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)
KEY TERMS
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.
INTERFERENCE OF HEMOLYSIS WITH LABORATORY TESTS
Hemolysis
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) |
Mechanisms
- 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
Examples:
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%) |
DRUG INTERFERENCES
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) | |
Albumin | |
Decrease (false) | Aspirin |
Increase | Heparin, increases dye binding to other serum proteins |
Bisalbuminemia | Penicillin binding to albumin causes two albumin peaks on serum electrophoresis |
Genetic | |
Alkaline phosphatase | |
Decrease | Anticoagulants (oxalate, fluoride, citrate bind Mg++) |
Increase | Estrogens, gentamicin (hepatotoxicity) |
Human albumin injections as plasma expanders (made from human placenta) | |
Amylase | |
Decrease | Anticoagulation with oxalate or citrate |
Increase | Opiates cause spasm of sphincter of Oddi |
Oral contraceptives (effect on liver) | |
Bilirubin | |
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 | |
Calcium | |
Decrease | Diuretics enhance excretion |
Phenytoin (Dilantin) may cause osteomalacia | |
Increase | Antacids (large amounts), e.g., TUMS |
Vitamin D | |
Cholesterol | |
Decrease | Androgens decrease synthesis |
Neomycin forms salts with bile acids in gut | |
Increase | Bilirubin (methodological) |
Corticosteroids (methodological) | |
Creatinine | |
Decrease | Marijuana (?) |
After meals | |
Increase | Acetoacetic acid, acetone and ascorbic acid are chromogenic in picrate reaction; glucose same as above |
Digoxin | |
Increase | Quinidine releases digoxin from heart muscle and also decreases renal excretion of digoxin, causing substantial increases in serum digoxin |
Glucose | |
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 |
Phenobarbital | |
Increase | Valproic acid causes increase in serum hime lide by inhibiting its renal excretion |
Potassium | |
Decrease | Diuretics, cathartics, aspirin, steroids |
Increase | Blood transfusions |
Hemolysis (non-drug) | |
Refrigeration of whole blood (inhibition of Na/K-ATPase pump) | |
(non-drug) | |
Marijuana (?) | |
Spironolactone inhibits Na/K-exchange in renal tubules | |
Sodium | |
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