Clinical Chem 2 - Lab Practical 2 Flashcards
Glucose Assay?
> Normal Range, Principle, Specimen, Interference, Clinical Significance, SI units, and Calculations.
Normal Range: 70-105 mg/dl
Principle:
Glucose is oxidized by glucose oxidase to gluconate and hydrogen peroxide.
Phenol + 4-AAP + hydrogen peroxide, in the presence of peroxidase,
produces a quinoneimine dye that is measured at 500 nm
Specimen Storage/Collection:
Non-hemolyzed serum or heparinized plasma is recommended
Serum must be separated from the clot promptly since the rate of glucose
decrease is approximately 7% per hour in whole blood
Glucose in serum is stable for twenty-four hours when stored refrigerated (2-
8°C)
Interference:
Grossly lipemic samples may cause falsely elevated glucose values.
Bilirubin to the level of 20 mg/dl and Hemoglobin to a level of 500 mg/dl have
both been found to exhibit negligible interference (<3%) in this assay
Clinical Significance:
The determination of glucose in serum is most commonly performed for the
diagnosis and treatment of diabetes mellitus
mg/dl
Abs. (Patient)/ Abs. (Standard) x Concentration of Standard (mg/dl)
= Glucose (mg/dl)
Triglyceride Assay
> Normal Range, Principle, Specimen, Interference, Clinical Significance, SI units, and Calculations.
Normal Range: 44-148 mg/dl
Principle:
Serum triglycerides are hydrolyzed to glycerol and free fatty acids by lipase.
In the presence of ATP and glycerol kinase (GK), the glycerol is converted to
glycerol-1-phosphate.
The glycerol-1-phosphate is then oxidized by glycerol phosphate oxidase (GPO) to yield hydrogen peroxide.
The condensation of hydrogen peroxide with 4-chlorophenol and 4-aminophenazone (4-AA) in the presence of peroxidase (POD) produces a red colored quinonimine dye which absorbs at, or near 500 nm
Specimen Storage/Collection:
Fresh, clear, unhemolyzed serum is the specimen of choice.
The serum should be collected following a 12 hour fast, and separated from the clot as soon as possible. Avoid anticoagulants containing fluoride or oxalate
Serum of plasma may be stored for one week at 2-8°C or for three months at –20°C
Frozen samples should be thawed at room temperature and mixed
completely before analysis. Thawed samples should not be refrozen
Interference:
The method is not influenced by hemoglobin values up to 100mg/dl or by
bilirubin levels up to 12 mg/dl (<5%)
Detergents can interfere with the action of lipase
Clinical Significance:
Triglycerides determinations are of interest in the diagnosis and treatment of
atherosclerosis, poorly controlled diabetes mellitus, nephrosis, liver disease,
or other diseases involving lipid metabolism
mg/dl
Abs Unk/ Abs Std x Conc. Std = Triglycerides (mg/dl)
Cholesterol Assay
> Normal Range, Principle, Specimen, Interference, Clinical Significance, SI units, and Calculations.
Cholesterol: < 200 mg/dl
Principle:
Cholesterol esters are broken down into cholesterol and fatty acids by cholesterol esterase.
Cholesterol and O2 in the presence of cholesterol oxidase forms hydrogen peroxide and cholesterol-3-one
H2O2 + 4-AAP + phenol in the presence of peroxidase produces water and quinonimine (a red product) that is measured spectrophotometrically.
Specimen Collection/Storage:
Use non-hemolyzed serum.
Cholesterol is stable for 7 days at room temperature (18-25*C) & for 6 months when frozen and protected against evaporation.
Interferences:
A number of drugs and substances that affect cholesterol concentrations.
Clinical Significance:
The determination of cholesterol is mostly used for determinations of total cholesterol, which is used to determine risk for heart disease, heart attacks, and other cardiovascular related conditions.
mg/dl
[Abs. (patient)/Abs. (standard)] x concentration of standard = cholesterol
Direct Bilirubin Assay
> Normal Range, Principle, Specimen, Interference, Clinical Significance, SI units, and Calculations.
Direct Bilirubin: 0 - 0.5 mg/dl
Principle:
Sulfanilic acid and sodium nitrite react and produce diazotized Sulfanilic acid (diazo).
Direct bilirubin couples with diazo ro produce azobilirubin. The intensity of the color produced is directly proportional to the direct bilirubin present in the sample.
Sample Collection/Storage:
Fresh, non-hemolyzed serum.
If kept at room temperature & in the dark, analyze within 2 hours.
Samples can store for up to 12 hours when refrigerated and protected from light.
Samples can store for 3 months frozen (-20*C)
Interferences:
Presence of interfering drugs.
Improper storage/exposure to direct sunlight can cause a 50% decrease in bilirubin within 1 hour.
Serum hemoglobin levels of up to 1 g/dl do not interfere with results.
Clinical Significance:
The determination of direct bilirubin is used to diagnose hyperbilirubinemia
It is also useful when used with total or indirect bilirubin in determining various stages of hepatic non functionality (prehepatic, hepatic, and posthepatic).
mg/dl
[ (Abs. of Unk - Abs. of Unk blank) / (Abs. of Cal - Abs. of Cal blank) ] x concentration of Cal = direct bilirubin
Lactate Dehydrogenase Assay
> Normal Range, Principle, Specimen, Interference, Clinical Significance, SI units, and Calculations.
Male: 50-166 U/L (30°C), 80-285 U/L (37°C)
Female: 60-132 U/L (30°C), 103-227 U/L (37°C)
Principle:
Lactate dehydrogenase catalyzes the oxidation of lactate to pyruvate with simultaneous reduction of NAD to NADH.
The rate of NAD reduction can be measured as an increase in absorbance at 340 nm.
This rate is directly proportional to LD activity in serum
Specimen Collection/Storage
Non-hemolyzed serum is recommended. Red cells contain large concentrations of LD
The serum should be removed from the clot promptly
Samples should be assayed soon after collection. LD in serum is reported stable for two to three days at room temperature
Do not freeze or expose the serum to high temperatures (37°C) as this may inactivate thermolabile LD isoenzymes
Interferences:
Certain drugs and substances affect LD activity
Bilirubin to the level of 20 mg/dl has been found to exhibit negligible interference (≤ 5%) in this assay
Hemolysis has been shown to significantly interfere with the assay at levels as low as 100 mg/dl.
Clinical Significance:
Increased levels of LD are associated with myocardial infarction.
Levels reach a maximum approximately 48 hours after the onset of pain and persist about ten days.
The degree of elevation is of value in assessing the extent of damage and in developing a prognosis.
LD elevations are also observed in liver disease, pernicious anemia, in some cases of renal disease, and in some cases of skeletal muscle trauma
U/L
Measure absorbance at 30 seconds (A1) and 1 minute (A2)
The change in absorbance (A2 -A1) multiplied by the factor 3376 will yield results in U/L.
GGT Assay
> Normal Range, Principle, Specimen, Interference, Clinical Significance, SI units, and Calculations.
Male: 8-37 U/L at 30°C, 9-54 U/L at 37°C
Female: 6-24 U/L at 30°C, 8-35 U/L at 37°C
Principle:
GGT in the sample catalyzes the transfer of the glutamyl group from GLUPA-
C to glycylglycine according to the above reaction.
The amount of 5-amino-2-nitrobenzoate formed is proportional to GGT activity and may be measured kinetically at 405 nm
Specimen Collection/Storage:
Use serum only. GGT activity is inhibited by most anticoagulants.
Serum GGT is reported stable in serum for up to seven days when stored at
2-25°C, up to one month when stored at 4°C, and up to one year at (-20°C)
and protected from evaporation
All specimens and controls should be handled in accordance with good laboratory practices using appropriate precautions as described in the CDC/NIH
Interferences:
Most anticoagulants used in blood collection tubes inhibit GGT activity
Anti-epileptic drugs (phenytoin and barbiturates) may falsely elevate GGT
levels
Bilirubin to the level of 20 mg/dl has been found to exhibit negligible
interference (< 5%) in this assay
Hemoglobin from 100-500 mg/dl has been found to show minimal depression (approximately 5-7%) of recovered GGT activities
Clinical Significance:
GGT measurements are used in the diagnosis and treatment of liver diseases such as alcoholic cirrhosis, and primary and secondary tumors.
Elevated GGT levels appear earlier and are more pronounced than those of other liver enzymes, in cases of obstructive jaundice and metastatic neoplasms
U/L
Get absorbance every 60 seconds for 2 minutes
Calculate the average absorbance difference/minute (∆Abs/Min.)
The ∆Abs./Min. multiplied by the factor 1158 will yield results in U/L
Alanine Aminotransferase Assay
> Normal Range, Principle, Specimen, Interference, Clinical Significance, SI units, and Calculations.
4 to 24 IU/L (30°C). 4 to 36 IU/L (37°C)
Principle:
ALT catalyzes the transfer of the amino group from L-alanine to α-
ketoglutarate resulting in the formation of pyruvate and L-glutamate.
Lactate dehydrogenase catalyzes the reduction of pyruvate and the simultaneous oxidation of NADH to NAD. The resulting rate of decrease in absorbance is directly proportional to ALT activity
Specimen Collection/Storage:
Hemolyzed samples cannot be used as red cells contain ALT
ALT in serum is stable for three days at room temperature (15-30°C), seven
days refrigerated (2-8°C), and thirty days frozen (-20°C).
Interferences:
Bilirubin to at least 30 mg/dl, and hemoglobin to at least 400 mg/dl, have
been found to have a negligible effect on this procedure.
Clinical Significance:
Elevated levels of ALT are often only observed in liver diseases such as cirrhosis, hepatitis, or metastatic carcinoma.
However, there can be elevated levels of ALT with infectious mono, muscular dystrophy, and dermatomyositis
IU/L
Get absorbance every 60 seconds for 2 minutes
Calculate the average absorbance difference/minute (∆Abs/Min.)
The ∆Abs./Min. multiplied by the factor 1768 will yield results in IU/L
Aspartate Aminotransferase Assay
> Normal Range, Principle, Specimen, Interference, Clinical Significance, SI units, and Calculations.
8-22 IU/L at 30°C, 5-34 IU/L at 37°C
Principle:
AST catalyzes the transfer of the amino group from L-aspartate to ɑ-Ketoglutarate to yield oxalacetate and L-glutamate.
The oxaloacetate undergoes reduction with simultaneous oxidation of NADH
to NAD in the malate dehydrogenase (MDH) catalyzed indicator reaction.
The resulting rate of decrease in absorbance at 340 nm is directly
proportional to the AST activity.
Lactate dehydrogenase (LDH) is added to prevent interference from endogenous pyruvate which is normally present in
serum.
Specimen Collection/Storage:
Non-hemolyzed serum is recommended. Red cells contain AST which can
give falsely elevated results.
AST in serum is reported stable for ten days when refrigerated (2-8°C), two weeks when frozen (-20°C), and four days when stored at room temperature
(15-30°C)
Interferences:
Patients with severe vitamin B6 deficiency could have a decreased recovery
of AST, presumably due to a lack of pyridoxal phosphate
Bilirubin to at least 18 mg/dl, and hemoglobin to at least 300 mg/dl, have
been found to have a negligible effect on this procedure
Clinical Significance:
Diseases involving liver, heart, skeletal muscle and kidneys can lead to elevated levels of AST in serum.
Following myocardial infarction, AST levels are elevated and reach a peak after 48 to 60 hours.
Hepatobiliary diseases such as cirrhosis, metastatic carcinoma and viral hepatitis can show increased levels of AST.
Other disorders which can lead to an elevated level of AST are muscular dystrophy, dermatomyositis, acute pancreatitis and infectious mononucleosis
IU/L
Get absorbance every 60 seconds for 2 minutes
Calculate the average absorbance difference/minute (∆Abs/Min.)
The ∆Abs./Min. multiplied by the factor 1768 will yield results in IU/L
Amylase Assay
> Normal Range, Principle, Specimen, Interference, Clinical Significance, SI units, and Calculations.
25-125 U/L
Principle:
α-Amylase hydrolyzes the 2-chloro-p-nitrophenyl-α-D-maltotrioside (CNPG3) to release 2-chloro-nitrophenol and form 2-chloro-p-nitrophenyl-α-D-maltoside (CNPG2), maltotriose (G3) and glucose (G).
The rate of increase in absorbance is measured at 405 nm and is proportional to the α-amylase activity in the sample
Specimen Collection/Storage:
Non Hemolyzed serum is the specimen of choice.
Anticoagulants, such as Citrate and EDTA, bind calcium that is needed for
amylase activity. Plasma with these anticoagulants should not be used.
Amylase in serum is reported stable for one week at room temperature (18-
25 °C) and for two months when stored refrigerated at 2-8 °C.
Interferences:
Macro-amylase in the specimen can cause a measured hyperamylasemia,
that could lead to a false diagnosis of acute pancreatitis. However, no
clinical symptoms are usually associated with macroamylasemia
Bilirubin (30 mg/dl) and Hgb (500 mg/dl) have a negligible effect
Lipemic samples up to 1000 mg/dl have been reported to have no effect
Clinical Significance:
Determination of amylase activity in serum is most commonly performed for the diagnosis and treatment of diseases of the pancreas
U/L
Get absorbance every 60 seconds for 2 minutes
Calculate the average absorbance difference/minute (∆Abs/Min.)
The ∆Abs./Min. multiplied by the factor 3178 will yield results in IU/L
