Laboratory Analysis of Glucose

Why is Plasma that is used routinely not whole blood

Whole Blood 15% lower due to cellular dilution

Glucose Pre-analytical Comments

Refrigerate and separate serum/plasma from cells within 1 hr

Unpreserved whole blood is unstable at room due to on-going cellular glycolysis

Glycolytic inhibitors

Grey-top (Sodium-Fluoride) tube used for whole blood as it inhibits glycolysis

GLUCOSE IN CSF

Ref range 50-80 mg/dL

CSF levels approx. 60% of plasma levels

Assay promptly as cells in CSF may consume glucose

Low levels suggestive of bacterial meningitis

Enzymatic colorimetric

Glucose oxidase

Hexokinase

GLUCOSE OXIDASE (GO) METHOD

Color Rxn: Trinder reaction (measure peroxide)

Falsely low with high levels of ascorbic acid (Vit C),
uric acid, bilirubin (they scavenge peroxide)

Method incorporated into qualitative urine dipstick
methods

No other sugars will react other than glucose

HEXOKINASE METHOD

The consequent increase in absorbance at 340 nm is directly proportional to glucose concentration.

Very few Interferences (unaffected by Vit C)

OXYGEN RATE METHOD

In use on all Beckman-Coulter instruments; uses glucose oxidase reaction

Measure rate of disappearance of O2 using an O2
measuring electrode

Non-photometric method: pigments in plasma -no effect

URINARY GLUCOSE

Glucose is excreted in the urine when serum glucose levels exceed ≈180 mg/dL
(so‐called ‘Renal Threshold’)

Urine screening insensitive for diagnosis

Renal threshold is variable across patients

ANCILLARY TESTS FOR DIABETES MONITORING/DIAGNOSIS

Hemoglobin A1c(Glycoslylated Hgb)

Microalbumin

Ketone analysis

Insulin Immunoassay

C-Peptide

Antibody testing - autoimmunity

Glycolsylation

Addition of a glucose residue to amino groups of proteins

GLYCOSYLATED PROTEINS

Results in increased negative charge (useful for electrophoresis and chromatography)

GLYCOSYLATED HEMOGLOBIN

Principle circulating form of Hb is HbA (97%)

2α + 2β chains

Electrophoresis reveals additional subfractions due to
glycosylation (A1a, A1b, A1c )

A1c

Major sub-fraction (60-80% of population)

Glucose residue

Added to the N-terminal valine residue of the β‐chain of Hemoglobin A

MONITORING HBA1C IN DIABETICS

Preferred measure is HbA1c subfraction

Normal: 4-6%

Remember A1c >= 6.5%

Primary goal of therapy

HbA1c value <7%

1% change in A1c

35 mg/dL change in average plasma glucose

6%=135; 7%=170; 10%=275 mg/dL

CATION-EXCHANGE CHROMATOGRAPHY

Separation utilizing differences in ionic interactions between cation exchange on column resin and hemoglobin components in patient sample

IMMUNOASSAYS (VARYING FORMATS)

Direct detection with a HgbA1c specific antibody

Agglutination reaction that can be measured via spectrophotometry (more HgbA1c, the less agglutination)

Can also be done with total hemoglobin at the same time

LIMITATIONS OF HGBA1C METHODS

Altered RBC metabolism (increased RBC turnover)

Hemolytic diseases, more “young” RBC’s

Hemoglobinopathies may cause interference
(HbS, HbC, HbF and others)

Immunoassay interferences

Can’t be used in children yet

Fructosamine or glycosylated albumin may be used as alternative

Estimated Average Glucose

28.7 x A1c – 46.7

URINARY ALBUMIN (MICROALBUMINURIA)

Diabetics are high risk of developing renal disease

High albuminuria: urine dipstick > 300 mg/24 hrs

Microalbuminuria (aka moderately increased albuminuria) 30-300 mg/24 hrs

Assess urine albumin using an automated urinalysis analyzer (confirm with

nephelometry or spectrophotometry.

URINARY ALBUMIN SPECIMEN OPTIONS

Random urine
Ratio to creatinine, (report as: mg
albumin/mg creatinine)

First morning specimen preferred

24-hr Urine Collection, mg/24 hrs

Assess annually (if negative)

Ketone bodies

Acetoacetate, acetone & β‐hydroxybutyrate formed from acetyl‐CoA when the supply of TCA‐cycle intermediates is low

β‐hydroxybutyrate

Preferred analyte for measurement of “ketones” because it’s often present in the highest concentration (78%)

Ketones is present in

Diabetes or glycogen storage disease, prolonged fasting or vomiting and if on ketogenic diet

QUANTITATIVE Β-HYDROXYBUTYRATE (PLASMA BHB)

Enzymatic method with β-hydroxybutyrate dehydrogenase --- 340 nm on automated chemistry analyzers

Point-of-Care Ketone meters also perform BHB using special test strips (incorporates a 2nd reaction)

INSULIN IMMUNOASSAY

Primarily used to assess patients with hypoglycemia (looking or β-cell function) & newly diagnosed Type-2 Diabetes

Occasionally used to assess insulin-secreting pancreatic tumors (insulinomas)

Interference from

Exogenous insulin (Not useful in diabetics receiving exogenous insulin)

Endogenous insulin auto-antibodies which develop in diabetics

C-PEPTIDE

Connecting Peptide (C-peptide) in combination with insulin makes up the molecule PROINSULIN

C-peptide is biologically NACTIVE and not found in exogenous insulin sources (i.e. animal insulin used to treat Type I Diabetics)

C-PEPTIDE IMMUNOASSAY

Allows assessment of endogenous insulin secretion in diabetics receiving insulin

C-PEPTIDE IMMUNOASSAY is used primarily for

To assess Hypoglycemia

Also used to assess insulin-secreting pancreatic tumors (asses β‐cell function)

When insulin is high due too much administered (hypoglycemia)

C-peptide levels are low

TYPE 1 DIABETES AUTOANTIBODY TESTING

Islet cell autoantibodies -difficult assay

Glutamic Acid Decarboxylase (GAD) Antibodies

Insulin autoantibodies—present in 90% of children who develop D.M before age 5

Insulinoma-associated antigens

Zinc transporter (ZnT8)