MLSC 3054 Clinical Fluid Analysis

Charity Accurso, Ph.D.

Chapter 7

Most frequent chem analysis performed on

urine
 Included in all physical exams to try to catch
diabetes – >50% undiagnosed
 Early diagnosis through blood and urine glucose
tests improves prognosis
 Home tests for diabetics to monitor for problems
leading to complications

 Almost all glucose that is filtered by glomerulus is

reabsorbed in PCT
 Minute amts in urine
 If hyperglycemic – tubular reabsorption stops, and
glucose appears in urine
 Body has enough
 Renal threshold : 160-180mg/dL
 Normal individuals – may have glucosuria following
high glucose meal
 Best specimens in controlled conditions

Charity Accurso, PhD 1

MLSC 3054 Clinical Fluid Analysis

 Gestational diabetes – 6th month of pregnancy

 Hormones secreted by placenta block insulin action
 Hyperglycemic and glucosuria

 Other causes of hyperglycemia associated with hormonal

function
 Pancreatitis, pancreatic cancer, acromegaly, Cushing’s syndrome,
hyperthyroidism, pheochromocytoma
 Hormones in opposition to insulin (glucagon, epinephrine, cortisol,
thryoxine, GH) increase leading to hyperglycemia and glucosuria
 Glucosuria in absence of hyperglycemia – reabsorption process
compromised – renal glucosuria
 End-stage renal disease, osteomalacia, Fanconi’s syndrome

 Two methods:
 Glucose oxidase – specific for glucose
 Copper reduction – glucose and other reducing substances

 Reagent strips – glucose oxidase

 Double enzymatic reaction
 Glucose oxidase and peroxidase
 Step 1: glucose oxidase catalyzes rxn between glucose and room air to
produce gluconic acid and peroxide
 Step 2: peroxidase catalyzes rxn between peroxide and chromogen to form
oxidized colored compound
 Proportional to amt of glucose

 Chromogens:
 Potassium iodide – green to brown
 Tetramethylbenzidine – yellow to green

 Terms: Negative, trace, 1+, 2+, 3+, 4+

 Quantitative term ranges: 100 mg/dL – 2 g/dL; 0.1% - 2%

 Specificity – glucose only

Charity Accurso, PhD 2

MLSC 3054 Clinical Fluid Analysis

 Interferences  False Negatives:

 False Positives:  Strong reducing reagents such
 Strong oxidizing reagents as ascorbic acid
(bleach, H2O2)  Aspirin
 Homogentisic acid
 Reduce sensitivity of test
 Moderately high ketones
 Reactivity varies with  Very elevated specific gravity
temperature – high SG  Unpreserved spec or at room
and low temp dec temp for long periods of time
sensitivity

 Benedict’s reaction
 Cupric ions + glucose or other reducing substance in the
presence of heat and alkali yield cuprous oxide (red) and
cuprous hydroxide (yellow)
 Clinitest (Benedict’s copper reduction)
 Tablet contains copper sulfate, sodium carbonate, sodium
citrate and sodium hydroxide
 Heat produced after addition of water to tablet

 Specificity
 Reacts with glucose, galactose, lactose and fructose
 Galactosemia in infants
 False positives
 Salicylates
 Penicillin
 Ascorbic acid
 Cephalosporins
 False negative: bacteria infection
 Watch for “pass through” phenomenon

 Sensitivity – 200 mg/dL

Charity Accurso, PhD 3

MLSC 3054 Clinical Fluid Analysis

 Clinitest not as sensitive as glucose oxidase

 1+ strip reading and neg Clinitest – not a concern

 +++ Strip and neg Clinitest – concern that strong

oxidizing agent present
 Neg strip w/ pos Clinitest
 Presence of other reducing sugars
 Galactose (most significant), fructose, pentose, lactose
 Galactose – inborn error of metabolism
 Screening newborns using Clinitest

 3 intermediates of fat metabolism

 Acetone, acetoacetic acid, beta-hydroxybutyric acid

 Appear in urine when carbs are not available for

energy and body stores of fat must be metabolized
 Fat or anaerobic glucose metabolism
 From acetyl CoA, then ketones in the liver
 20% acetoacetic acid (diabetic)
 78% beta-hydroxybutyric acid
 2% acetone

Charity Accurso, PhD 4

MLSC 3054 Clinical Fluid Analysis

 Reference range: Negative  Ketonuria:

 Diabetic ketoacidosis
 Starvation, low carb- or low-
 Clinical reasons cal diets, fasting,
 Inability to metabolize carbs  Metabolic acidosis
 Inc loss of carbs – vomiting  Toxic states accompanied
 Inadequate carb intake – by vomiting and diarrhea
starvation or malabsorption  Alcoholism
 Pregnancy
 Frequent strenuous exercise

 Basic test Principle:

 Ketones in an alkaline medium react with sodium
nitroprusside to produce a purple color
 Limitations
 Specificity : Acetoacetic acid ONLY
 Sensitivity: 5-10 mg/dl acetoacetic acid

 Terms
 Negative, small, moderate, large
 Negative, 1+, 2+, 3+

 Interferences
 False Positives: False negative
 High amounts of L-dopa  Loss of labile ketones from
metabolites specimen
 Large amounts of phenylketones
 8-hydroxyquinoline
 Dyes or pigmented urines
 High specific gravity (Trace)
 High pH (Trace)
 Moisture

Charity Accurso, PhD 5

MLSC 3054 Clinical Fluid Analysis

 Confirmatory tests
 Acetest – figure 7.6
 Reacts with acetone and acetoacetic acid
 Sodium nitroprusside and glycine
 Sensitivity
 5-10 mg/dl acetoacetic acid
 20-25 mg/dl acetone

 Can be early indication of liver disease

 Appears long before development of jaundice
 Extrahepatic (Common bile duct) – obstruction – cannot
be trs to intestines
 Gallstones
 Carcinoma
 Liver inflammation
 Intrahepatic (Liver damage) – leakage of bilirubin
 Hepatitis
 Cirrhosis

 Use in combination with urobilinogen

 Bile duct obstruction
 Bilirubin - +++
 Urobilinogen – Normal
 Liver damage
 Bilirubin - +/-
 Urobilinogen - ++
 Hemolytic disease
 Bilirubin – Negative
 Urobilinogen - +++

Charity Accurso, PhD 6

MLSC 3054 Clinical Fluid Analysis

 Basic test Principle

 Couples with diazonium salt in an acid medium

 Limitations
 Sensitivity: 0.4-0.8 mg/dl
 Specificity: Specific for bilirubin

 Terms
 Neg, small, mod, large
 Neg, 1+, 2+, 3+

 Foam test: yellow foam after shaking

 Interferences
 False Positives
 Urine pigments
 Highly colored metabolites of drugs
 False Negatives
 Sensitivity is reduced by high levels of:
 Ascorbic acid
 Nitrites
 Exposure to light – bilirubin is unstable

 Ictotest – Confirmatory test

 Principle: Diazo – same method as reagent strip
 Lower sensitivity: .05-.10 mg/dl
 Figure 7.9

Charity Accurso, PhD 7

MLSC 3054 Clinical Fluid Analysis

 Bile pigment resulting from breakdown of hemoglobin

 Produced in intestine from reduction of bilirubin by intestinal
bacteria
 Small amt - <1 mg/dL – normally found in urine

 Reference range: 0.2-1.0 mg/dl

 Increased urinary urobilinogen
 Liver disease
 Cirrhosis
 Hepatitis
 Hemolytic disorders
 Decreased urinary urobilinogen
 Infants
 Adults with decreased intestinal normal flora
 Obstructive liver disease

 Basic test principle

 Azo-coupling - Chemstrip
 Modified Ehrlich reaction - Multistix

 Limitations
 Sensitivity:  0.2 mg/dl
 Specificity: refer to interferences

Charity Accurso, PhD 8

MLSC 3054 Clinical Fluid Analysis

Interferences
 False Positives:  False Negatives:
 Porphobilinogens,  Formalin
5HIAA  Improper storage
 Sulfonamides  Excessive nitrites
 p-aminosalicylic acid
 Excessively warm urine

 Confirmatory tests
 Hoesch test
 Watson-Schwarz test
 Ehrlich’s test

 Vitamin C
 Dietary presence; supplementation
 Water soluble

 Strong reducing substance that affects many

reactions on some reagent strips
 H2O2; diazonium salt

 Table 7.13 – limitations

Charity Accurso, PhD 9

MLSC 3054 Clinical Fluid Analysis

 Based on ascorbic acid reducing dye in reagent

pad
 Blue to orange color change

 False positives – free-sulfhydral meds

 Will be specified by laboratory

 Table 7.14

Charity Accurso, PhD 10