CC2 LAB MIDTERMS FLASHCARDS

COVERAGE: LDH, AMYLASE, LIPASE

Tissue Source/s of LDH

Tissue Source/s of LDH

Heart, liver, muscle, kidney

Clinical Significance of LDH

↑ myocardial infarction, liver disease, renal disease, certain forms of anemia, malignant disease, progressive muscle dystrophy

Clinical significance of amylase

Diagnosis of acute pancreatitis

Clinical significance of lipase

Evaluate conditions associated with pancreas

LDH equation

Amylase equation

Lipase equation

Reagent stability for LDH

Reconstituted: 14 days (ref temp) 8 hours (room temp)

Reagent Composition LDH

• L-lactate (75mM)

• NAD (5.5 mM)

• Buffer (80 mM)

• pH 9.0

• Non-reactive stabilizers and fillers

Specimen Stability LDH

Serum: 2-3 days at room temp

Interfering Substances

oxalate

oxamate

EDTA

LDH: Reagent Volume

1ml

LDH: Pre-incubation (time and temp)

37°C for 3 minutes

LDH: Sample Volume

25uL

Wavelength LDH

340nm

Multiplier of LDH

6592

LDH: Time interval (reading)

2 minutes A1: read after 1 minute

LDH: expected values

Males

50-166 IU/L (30°C)

80-285 IU/L (37°C)

Females

60-132 IU/L (30°C)

103-227 IU/L (37°C)

formula for LDH

△Abs/min × 6592

Limitations (LDH)

Samples with values above 800 IU/L should be diluted 1:1 with saline, re-assayed and the results multiplied by two (2).

Temperature Correction

1. If the assay is performed at 37°C but is to be reported at 30°C; multiply the results by 0.6.

2. If the assay is performed at 30°C but is to be reported at 37°C; multiply the results by 1.7

LDH: procedure linearity

800 IU/L

millimolar absorptivity of NADH

6.22

Formula to acquire multiplier in LDH

First quantitative method for Amylase assay

Iodemetric method

Who introduced iodometric method?

Wohlegemuth

Disadvantages of old methods in amylase assays

long incubation times

possible endogenous glucose interference

Who introduced the current method/assay used to quantify amylase?

Wallenfels

Substrate(s) in the method that Wallenfels introduced.

p-Nitrophenylglycosides or p-Nitrophenyl-D-Maltoheptaoside (PNPG7)

Lipase hydrolyzes _____?

glycerol esters of long-chain fatty acids

Advantage in the current method of amylase assay

short lag time

greater stability

Final products of the reaction in amylase equation

p-Nitrophenol

Glucose

What is measured in the LDH assay?

rate of formation of NADH

What color is produced when PNPG1 is hydrolyzed by glucosidase to glucose and p-Nitrophenol?

yellow

What are the three enzymes that are involved in the amylase assay?

amylase

glucoamylase

glucoasidase

Wavelength (Amylase)

405nm

Reagent stability (amylase)

Reconstituted:

10 days (room temp)

30 days (ref temp)

Unreconstituted:

until expiry (ref temp)

Reagent Composition (Amylase)

• p-Nitrophenyl D-Malthoheptaoside 0.9 mM

• Glucosidase (yeast) (25, IU/L)

• Glucoamylase (Rhizopus Sp.) 10,000 IU/L

• Sodium chloride (50mM)

• Calcium chloride (5mM)

• Buffer (50mM)

• pH 6.9

specimen for amylase assay

serum and urine

Specimen Stability (amylase)

Serum & Urine:

1 week (room temp)

several months (ref temp)

Plasma: heparin tube

Urine: 24 hr period

Interfering Substances

Macroamylasemia ↑ pancreatic amylase in serum

Lipemia and hemolysis ↑ amylase

Insulin and some bacteria ↑ amylase

Reagent Volume (amylase)

1ml

Pre-incubation (amylase)

37°C for 3 minutes

amylase: specimen volume

25uL

Multiplier (amylase)

4824

Millimolar absorptivity of p-Nitrophenol

8.5

Time interval (reading)

Every 30 seconds for 2 minutes A1: read after 15 seconds

Note: 5 readings in total

Expected Values Amylase

Serum: up to 96 IU/L Urine: 18 to 330 IU/L

formula for amylase assay

△Abs/min × 4824

amylase procedure limitations

Samples that exceeded the linearity limit (1,500 IU/L) should be diluted with an equal volume of saline and rerun. Multiply the result by two

To convert to nkat/L, the IU/L value should be multiplied to?

16.67

Formula for corrected ΔAbs. /5min

Corrected ΔAbs. /5min = ΔAbs test- ΔAbs blank

why should hemolysis be avoided in LDH assay?

Erythrocytes release large amount of LDH (falsely elevated result)

he modified the method introduced by Voget

Shibabi

in the method proposed by Voget, what is measured in the assay?

rate of change in turbidity over a specific unit of time

Serum lipase hydrolyzes ____?

olive oil emulsion

TRUE OR FALSE. The increase in turbidity at 400nm is proportional to lipase activity in the specimen.

FALSE. (must be decrease in turbidity)

Reagent composition (lipase)

• Substrate: olive oil in alcohol

• Buffer: tris buffer (69mM), sodium deoxycholate (10mM), pH 9.0

Reagent stability (lipase)

Reconstituted: 7 days (refrigerated)

Unreconstituted: until expiry (room temp)

In preparing the reagent for lipase assay, what volume of distilled water must be added?

25 ml

Specimen stability (lipase)

Serum:

1 week (room temp)

3 weeks (ref temp)

several months (frozen)

Interfering substance (lipase)

Hemolyzed specimen

Reagent volume (lipase)

3 ml

Pre-incubation

37°C for 5 minutes

Sample volume (lipase)

100uL

Wavelength (lipase)

400nm

Multiplier (lipase)

1953

concentration of olive oil (micromoles/liter) in the working solution

315

Time interval (reading)

5 minutes after initial absorbance reading A1: read after adding sample and mixing (initial)

Expected values (lipase)

Adults: 10-150 U/L (age more than 60 years old 18-180 IU/L)

Procedure limitation (lipase)

Samples with values above 280 IU/L should be diluted 1:1 with distilled water, re-assayed and final results multiplied by two.

To convert IU/L to Cherry-Crandall units, divide IU/L by

70

One International Unit (IU) is defined as

the amount of enzyme that catalyzes the transformation of one micromole of substrate per minute under decreed conditions.