Blakes Practical Two Flashcards
Liquid GGT clinical significance on pointe scientific
-used in the diagnosis and treatment in liver disease
- such as Alcoholic cirrhosis, primary and secondary tumors
- Elevated GGT levels appear more pronounced in cases of obstructive jaundice and metastatic neoplasms.
GGT reaction
L-y-Glutamyl-3-carboxy-4-nitroanilide + Glycylglycine ————–
GGT reaction by word
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 405nm.
To prepare the working reagent then of GGT
Reagents are supplied as ready to use liquids. To prepare working reagent, mix 5 parts of R1 reagent with 1 part R2 reagent.
Reagent storage and stability of GGT
Store reagents at 2-8°C. The reagents are stable until the expiration date if stored as directed. The working reagent is stable for 21 days at 2-8°C. NOTE: The R2 reagent is temperature sensitive and can be affected by prolonged exposure to room temperature. Return reagent to 2-8°C as soon as possible after use.
reagent precautions GGT
- This reagent is for in vitro diagnostic use only.
- Do not use the reagent if the initial absorbance of the working reagent
is greater than 0.800 when measured at 405 nm against water or if the reagent fails to meet the stated parameters of performance - Do not pipette by mouth. Avoid ingestion and contact with skin as toxicity has not been established.
- Reagents in this kit contain sodium azide as a preservative. Sodium azide may form explosive compounds in metal drainlines. When disposing of reagents through plumbing fixtures, flush with copious amounts of water. For further information, refer to “Decontamination of Laboratory Sink Drains to Remove Azide Salts,” in the Manual Guide-Safety Management No. CSC-22 issued by the Centers for Disease Control, Atlanta, Georgia.
GGT specimen collection and storage
- Use serum only. GGT activity is inhibited by most anticoagulants.
- It is recommended that specimen collection be carried out in accordance with NCCLS document M29-T2. No method can offer complete assurance that human blood samples will not transmit infection. Therefore, all blood
samples should be considered potentially infectious. - 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.7 - All specimens and controls should be handled in accordance with good
laboratory practices using appropriate precautions as described in the CDC/NIH Manual, “Biosafety in Microbiological and Biomedical Laboratories
GGT interferences
- Most anticoagulants used in blood collection tubes inhibit GGT activity.8
- Anti-epileptic drugs (phenytoin and barbituates) may falsely elevate GGT
levels.9,10 - 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.
NOTE: GGT level was 45 U/L for the bilirubin study and 48 U/L for the hemoglobin study.
GGT expected values
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
GGT linearity
Linearity: 0-800 U/L.
LDH 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.
LDH reaction and in words
L-Lactate + NAD+ with enzyme LDH————> Pyruvate + NADH + H+
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 340nm. This rate is directly
proportional to LD activity in serum.
To prepare working reagent LDH
Reagents are supplied as ready to use liquids. To prepare a working
reagent, mix 5 parts Buffer (R1) with 1 part Co-enzyme (R2)
Reagent storage and stability LDH
Reagent Storage and Stability
Reagents are stable until stated expiration if stored as directed. If a single
working reagent is required, prepare only the amount required to complete
current day’s testing. Discard remaining reagent after completion of testing.
Protect from light. Avoid microbial contamination
LDH precautions
Precautions
1. This reagent is for in vitro diagnostic use only.
2. The reagents contain sodium azide (0.1%) as a preservative. Do not
ingest. Avoid skin and eye contact. Sodium azide may react with lead
and copper plumbing fixtures giving rise to explosive metal azides.
Flush with large volumes of water when disposing of the reagent.
3. All specimens and controls should be handled in accordance with good
laboratory practices using appropriate precautions as described in the
CDC/NIH
LDH specimen and storage
- Non-hemolyzed serum is recommended. Red cells contain large
concentrations of LD. 5 - 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.9 - Do not freeze or expose the serum to high temperatures (37°C) as this may
inactivate thermolabile LD isoenzymes. 10 - Specimen collection should be carried out in accordance with NCCLS M29-
T2.11 No method can offer complete assurance that human blood samples
will not transmit infection. Therefore, all samples should be considered
potentially infectious
Interferences of LDH
- Certain drugs and substances affect LD activity. See Young, et al. 12
- 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
LDH linearity
Assay: 0-1000 U/L.
LDH expected values
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)
GGT reference range and stability
range= 5-40U/L
Specimen stability: Best to maintain stability at RT, but can be stored for up to 14 days at refrigerated or frozen temperatures
GGT primary role
Primary role is the detection and differential diagnosis of hepatobiliary disease.
GGT examples of conditions where the highest elevations are
Examples of conditions where the highest elevations of GGT occur would be extra hepatic obstructions I.e. gallstones or a tumor blocking the bile duct (5 to 30X the upper reference range)
Moderate GGT elevations
Moderate elevations can occur in injury caused by chronic alcoholism or drug ingestion
GGT is useful in differentiating liver disease from what
Useful in differentiating liver disease from bone disease when it is elevated in the absence of an elevated ALP
Amylase clinical significance
The determination of amylase activity in serum is most commonly performed
for the diagnosis and treatment of diseases of the pancreas.
Amylase reaction
10 CNPG3 with enzyme a-amylase ——————–> 9 CNP + CNPG2 + 9G3 + G
-Amylase hydrolyzes the 2-chloro-p-nitrophenyl-
a-D-maltotrioside
(CNPG3) to release 2-chloro-nitrophenol and form 2-chloro-p-nitrophenyl-a-
D-maltoside (CNPG2), maltotriose (G3) and glucose (G). The rate of
increase in absorbance is measured at 405 nm and is proportional to the a-
amylase activity in the sample
A-amylase reagent storage
Do not use if:
1. The absorbance of the working reagent is greater than 0.600 when
measured at 405 nm against water in a cuvette with a 1 cm path length.
2. The reagent fails to meet stated parameters of performance.
3. The reagent is turbid or displays other evidence of bacterial contamination.
amylase interferences
- A number of drugs and substances affect the determination of amylase.15,16
Young et al have published a comprehensive list of such substances.17 - Macroamylase 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.18 - Bilirubin (30mg/dl) and hemoglobin (500mg/dl) have each been found to
have a negligible effect on this procedure. - Lipemic samples up to 1000 mg/dl have been reported to have no effect on
serum amylase determinations.1
Specimen Collection and Handling amylase
- Unhemolyzed serum is the specimen of choice. Specimens should be
collected as per NCCLS document H4-A3.13 - 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.
amylase expected values and clinical significance
Serum: 25-125 U/L
Linearity: 0-2,000 U/L
Amylase physiology
-Catalyzes the hydrolysis of alpha-glycosidic bonds in order to release various sugars from starches (releases sugars from starches)
-Produced in the acinar cells of the pancreas and salivary glands
amylase clinical significance
-Highest elevations occur in pancreatitis and inflammation of the salivary glands
-To diagnose acute pancreatitis, you should measure both amylase and lipase, Amylase peaks at 12 hours after onset and returns to normal after 3-4 days
Lab methods
-Laboratory Methods
-Couples enzyme reactions resulting in the measurement of NAD+ at 340nm
-Testing is available to measure the activities of p-amy and s-amy
Amylase reference range
27-131 IU/L
High levels of amylase
Acute pancreatitis (4-6X the upper limit of normal), pancreatic duct obstruction, pancreatic abscess, cholecystitis, mumps, intestinal obstruction, mesenteric thrombosis
Low levels of amylase
Cystic Fibrosis, Hepatitis, Cirrhosis
