Glucose Flashcards by Kyra Catedral

SPECIMEN CONSIDERATIONS

PCWPUS

-Whole blood
-Plasma
-CSF
-Pleural Fluid
-Urine
-Serum

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15% lower than serum or plasma

Whole blood

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Unhemolyzed Venous plasma/serum

Serum

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Separation of liquid portion

• Serum be separated within 30 minutes
• Serum w/o bacterial contamination & w/o leukocytosis (^WBC) = up to 90 minutes delay
• Plasma must be separated from the cellular fraction (even with Na fluoride)

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Separation of liquid portion
• Serum be separated within____

30 minutes

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Separation of liquid portion

• Serum w/o bacterial contamination & w/o leukocytosis (^WBC) = up to_____ delay

90 minutes

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Separation of liquid portion

• Plasma must be separated from the______ (even with Na fluoride)

cellular fraction

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is the process in which glucose is broken down to produce energy.

It produces two molecules of pyruvate, ATP, NADH and water.

The process takes place in the cytoplasm of a cell and does not require oxygen.

Glycolysis

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______ decreases serum glucose by approximately 5% to 7% in 1 hour (5 to 10 mg/L) in normal uncentrifuged coagulated blood at room temperature.

Glycolysis

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Glycolysis decreases serum glucose by approximately _____ to ____ in 1 hour (___ to ___mg/L) in normal uncentrifuged coagulated blood at room temperature.

5% to 7%

5 to 10 mg/L

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Specimen for Glucose

In separated , nonhemolyzed sterile serum, stable as long as ___ hours at 25°C an up to___ hours at 4°C

8 hrs

72hrs

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Specimen consideration

Storage of samples

•Refrigerated (____)
–serum or plasma: stable up to____ hrs

–Whole blood:____mg Na fluoride per mL of whole blood (___hrs)

2-8ºC

48 hrs

2mg

48 hrs

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GLUCOSE METHODOLOGIES

CHEMICAL Method

ENZYMATIC Method

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CHEMICAL METHOD

Oxidation reduction method
Condensation method

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Oxidation Reduction Method (2)

AC
AF

Alkaline Copper Reduction Method

Alkaline Ferric Reduction Method

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Alkaline Copper Reduction Method

a. Folin Wu Method
b. Nelson Somogyi Method
c. Neocuproine Method
d. Benedict’s Method

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(Modification of Folin Wu)

Benedict’s Method

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Alkaline Ferric Reduction Method

Hagedorn Jensen

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CONDENSATION METHOD

Dubowski Method

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Reduction of cupric ions to cuprous ions forming cuprous oxide in hot alkaline solution by glucose.

Alkaline Copper Reduction Method

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Alkaline Copper Reduction Method

Principle:

Reduction of cupric ions to cuprous ions forming________ in _______ solution by glucose.

cuprous oxide

* hot alkaline solution*

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Alkaline Copper Reduction Method

Cuprous Ions + Phosphomolybdate

Phosphomolybdic Acid or Phosphomolybdenum Blue

Folin Wu Method

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Alkaline Copper Reduction Method

Cuprous Ions + Arsenomolybdate

Arsenomolybdic Acid or Arsenomolybdenum Blue

Nelson Somogyi Method

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Alkaline Copper Reduction Method

(2,9 Dimethyl 1,10 Phenantroline Hydrochloride)

Cuprous Ions + Neocuproine

Cuprous-Neocuproine Complex (Yellow or Yellow Orange)

Neocuproine Method

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Alkaline Copper Reduction Method used for detection and quantitation of ***reducing substances*** in body fluids like blood and urine.

Benedict’s Method (Modification of Folin-Wu)

stabilizing agent in Benedict’s method

citrate or tartrate

Folin Wu Method Product

Phosphomolybdic Acid or Phosphomolybdenum Blue

Nelson Somogyi Method Product

Arsenomolybdic Acid or Arsenomolybdenum Blue

Neocuproine Method Product

Cuprous-Neocuproine Complex (Yellow or Yellow Orange)

Benedict’s None

Blue solution

Benedict’s Traces of reducing sugar

Green / yellow ppt

Benedict’s Moderate

Orange red ppt

Benedict’s Large amount of reducing sugar

Brick-red ppt

OXIDATION REDUCTION METHOD Alkaline Ferric Reduction Method by ______

(Hagedorn Jensen)

It involves reduction of a yellow ferricyanide to a colorless ferrocyanide by glucose (Inverse Colorimetry)

Alkaline Ferric Reduction Method

Alkaline Ferric Reduction Method It involves reduction of a yellow ferricyanide to a ______ by glucose

colorless ferrocyanide

(Inverse Colorimetry)

Alkaline Ferric Reduction Method

CHEMICAL METHOD •condensation of glucose with a primary aromatic amine in glacial acetic acid, forming an equilibrium mixture of a glycosylamine and the corresponding Schiff base

Condensation Method ***Ortho-toluidine (Dubowski Method)***

CHEMICAL METHOD B. Condensation Method ***Ortho-toluidine (Dubowski Method)*** •Procedure: –Glucose in a PFF (3% TCA) reacts to O-Toluidine in hot______ solution will yield a_____ colored compound with maximum absorbance at______nm

acidic GREEN 630 nm

ENZYMATIC METHODS

1. GLUCOSE OXIDASE METHOD 2. HEXOKINASE METHOD 3. GLUCOSE DEHYDROGENASE METHOD 4. DEXTROSTICS (cellular strip) 5. INTERSTITIAL GLUCOSE MEASURING DEVICE

1. GLUCOSE OXIDASE METHOD (2)

a. Colorimetric Glucose Oxidase Method (Saifer Gerstenfield Method) b. Polarographic Glucose Oxidase

+**Colorimetric Glucose Oxidase Method*** (Saifer Gernstenfield Method) 1. Increased levels of (3) can cause ***falsely decreased values*** as a result of these substances being oxidized by peroxidase, which then prevents the oxidation and detection of the chromogen. 2. Strong oxidizing substances, such as____, can cause ***falsely increased values***

uric acid, bilirubin, and ascorbic acid bleach

GLUCOSE OXIDASE METHOD Measures rate of oxygen consumption which is proportional to glucose concentration.

***Polarographic Glucose Oxidase***

Polarographic Glucose Oxidase ______ in the reagent catalyzes the oxidation of glucose by oxygen under first order conditions, forming hydrogen peroxide. Quantitated by the consumption of oxygen on an_______

Glucose oxidase oxygen-sensing electrode

Polarographic Glucose Oxidase _______is prevented from re-forming oxygen by adding molybdate, iodide, catalase and ethanol.

Hydrogen peroxide

ENZYMATIC METHODS ***MOST SPECIFIC GLUCOSE METHOD; REFERENCE METHOD*** Less interference Plasma collected using heparin, EDTA, flouride, oxalate or citrate may be used for this test. Other samples; Urine, CSF and serous fluids

HEXOKINASE METHOD

ENZYMATIC METHODS HEXOKINASE METHOD Gross hemolysis and extremely elevated bilirubin may cause a _____ in results.

false decrease

ENZYMATIC METHODS Glucose is reduced to produce a chromophore that is measured spectrophotometrically or an electrical current ***NADH = glucose concentration*** Close agreement with hexokinase procedures ________ is added to shorten the time

GLUCOSE DEHYDROGENASE METHOD Mutarotose

ENZYMATIC METHODS ***GLUCOSE DEHYDROGENASE METHOD*** Glucose is reduced to produce a______ that is measured spectrophotometrically or an electrical current NADH =______ Close agreement with hexokinase procedures _____is added to shorten the time

chromophore glucose concentration Mutarotose

ENZYMATIC METHODS An enzyme-impregnated strip used with a small portable electronic colour-measuring device for convenient estimation of the blood sugar levels by diabetics.

DEXTROSTICS (cellular strip)

ENZYMATIC METHODS ***DEXTROSTICS (cellular strip)*** An________ used with a small portable electronic colour-measuring device for convenient estimation of the blood sugar levels by diabetics.

enzyme-impregnated strip

It should not be used to diagnose diabetes or hypoglycemic disorders.

DEXTROSTICS (cellular strip)

ENZYMATIC METHODS -used for cobtinuous monitoring of glucose levels in people with diabetes

INTERSTITIAL GLUCOSE MEASURING DEVICE

ENZYMATIC METHODS -uses electrochemical methods to automatically and frequently measure glucose levels in the interstitial fluid of dermis or subcutaneous fat tissue.

INTERSTITIAL GLUCOSE MEASURING DEVICE

ENZYMATIC METHODS INTERSTITIAL GLUCOSE MEASURING DEVICE -used for continuous monitoring of glucose levels in people with____ -uses______ methods to automatically and frequently measure glucose levels in the ______of dermis or subcutaneous fat tissue.

diabetes electrochemical interstitial fluid

Laboratory Test for Glucose (7)

1.Random plasma glucose 2.Fasting plasma glucose 3. Tolerance test 4. HbA1c 5. Fructosamine 6. Urine Microalbumin 7. Ketone testing

LABORATORY TESTS - specimens collected ***anytime of the day*** - usually done in a ***glucometer*** - ***NO NORMAL VALUES***

Random Plasma Glucose

Random plasma glucose Formerly known as

random blood sugar (RBS)

Random Plasma Glucose - specimens collected_____ - usually done in a_____ - normal values?

anytime of the day glucometer NO NORMAL VALUES

Principles: Electrochemistry (Amperometry) Glucose oxidase

Random plasma glucose

LABORATORY TESTS - specimens collected after 8-10 hours fasting (new guideline) >7.0 mmol/L or >126 mg/dL (DM)

Fasting Plasma Glucose

Fasting Plasma Glucose - formerly known as

fasting blood sugar (FBS)

Fasting Plasma Glucose - specimens collected after ____hrs fasting (new guideline) >___ mmol/L or >___ mg/dL (DM)

8-10 hours >7.0 mmol/L or >126 mg/dL (DM)

LABORATORY TESTS 1. Fasting sample + urine 2. 75 grams glucose load is orally taken within 15 minutes 3. 1-hr sample + urine 4. 2-hr sample + urine

Oral Glucose Tolerance Test

LABORATORY TESTS Glucose Tolerance Test -Diagnosis of GDM • Fasting: >___ mmol/L (>___ mg/dL) • 1 h: >___ mmol/L (>___ mg/dL) • 2 h: >___ mg/dL (>___ mmol/L)

• Fasting: >5.1 mmol/L (>92 mg/dL) • 1 h: >10.0 mmol/L (>180 mg/dL) • 2 h: >153 mg/dL (>8.5 mmol/L)

LABORATORY TESTS -H6A1c "Hemoglobin A that is irreversibly glycosylated at one or both N-terminal valines of the B-chains of the tetrameric hemoglobin molecule" (International Federation of Chemistry Working Group on HBA1c)

Glycated/Glycosylated Hemoglobin

Now the preferred test to assess ***glycemic control*** Widely used marker of ***chronic hyperglycemia*** (reflecting average blood glucose levels over a 2-to 3-month period of time)

Glycated/Glycosylated Hemoglobin

Glycated/Glycosylated Hemoglobin Now the preferred test to assess_____ control Widely used marker of_____ (reflecting average blood glucose levels over a 2-to 3-month period of time)

glycemic control chronic hyperglycemia

LABORATORY TESTS Glycated/Glycosylated Hemoglobin -METHODS: 5

• Affinity Chromatography • HPLC • Electrophoresis • Spectrophotometry • 2-step Non-enzymatic Method

Specimen requirement for HBA1c is????

Whole blood

HBAc1 Anticoagulant of choice:

EDTA

LABORATORY TESTS • Most widely used to assess ***short-term (3-6 week) glycemic control*** • Most useful if the HBA1c is unreliable due to ***hemoglobinopathy or hemolysis*** • Not ideal: serum albumin level is < 3 g/dL or when serum albumin turnover is accelerated (cirrhosis)

Fructosamine

***Fructosamine*** • Most widely used to assess short-term (3-6 week)_____ • Most useful if the HBA1c is_____ due to_____ or ____ • Not ideal:_______ level is < 3 g/dL or when ______ turnover is accelerated (cirrhosis)

glycemic control unreliable/ hemoglobinopathy or hemolysis serum albumin

LABORATORY TESTS • test to detect very small levels of protein (albumin) in urine - To detect early diabetic nephropathy (kidney damage)

Urine Microalbumin

***Urine Microalbumin*** • test to detect very small levels of____ in urine - To detect early_____

protein (albumin) diabetic nephropathy (kidney damage)

LABORATORY TESTS • Beta-HBA, acetoacetic acid, and acetone • To detect ketosis in DM type???

Ketone Testing DM T1

Ketone testing methods (4)

• METHODS: - Electrochemistry - chromatography - Electrophoresis - Colorimetric methods

Ketone Testing • METHODS: • use of ferric chloride reacted with acetoacetic acid to produce a red color

Gerhardt's

Ketone Testing • METHODS: • reacts with acetoacetic acid in an alkaline pH to form a purple color • urine reagent strip test and Acetest tablets

Sodium Nitroprusside

Ketone Testing • METHODS: • to detect either 3-b-hydroxybutvric acid or acetoacetic acid

3-hydroxybutyrate dehydrogenase

LABORATORY TESTS 2 hours following regular meal

2-Hour Postprandial Glucose

LABORATORY TESTS -75 grams glucose load - Blood collection after 2 hours • 200 mg/dL - DM

2-Hour Postprandial Glucose (more standardized)

DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS • Random Plasma Glucose

>200 mg/dL (11.1 mmol/L)

DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS Fasting Plasma Glucose

>126 mg/dL (7.0 mmol/L)

DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS 2-hour Plasma Glucose OGTT

>200 mg/dL (11.1 mmol/L)

DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS Н6А1с

>6.5%