UA 2: CHEMICAL EXAMINATION OF URINE (PROTEIN, GLUCOSE) Flashcards
1
Q
T/F:
only a few low molecular weight proteins are filtered out by the glomerulus
A
TRUE
- small percentage should be found in urine; usually albumin
- a (+) urine protein result requires additional testing to determine whether the protein represents a normal or pathologic condition
- most LMW proteins are readily absorbed (eg. hemoglobin, myoglobin)
2
Q
the major protein in the urine
A
Albumin
- others present in urine: serum microglobulin, proteins produced in the genitourinary tract
3
Q
clinical proteinuria
A
- > /= 30mg/dL CHON
- causes can be grouped based on the origin of the CHONs:
a. Pre-renal
b. Renal
c. Post-renal
4
Q
proteinuria caused by conditions prior to reaching the kidneys
A
Pre-renal proteinuria
- frequently transient
- not indicative of renal disease
- happens when the filtration of CHONs exceeds the normal reabsorptive capacity of renal tubules
- result: overflow of CHONs in urine (incl. LMW CHONs, acute phase reactants, Bence-Jones protein)
5
Q
proteins can be added to a urine specimen as it passes through the lower urinary tract
A
Post-renal proteinuria
- CHONs added include:
- exudates (from bacterial & fungal infections, inflammation)
- blood (as a result of injury & menstrual contamination)
- prostatic fluid
- spermatozoa
6
Q
Bence-Jones protein
A
- found in patients with multiple myeloma (MM)
- a proliferative disorder of the plasma cells
- serum contains markedly high levels of monoclonal Ig light chains
7
Q
determination of Bence-Jones protein
A
- Boil
- Filter
- Cool down to 60 or 40C
- Observe for turbidity
- - turbid = positive for Bence-Jones protein
- - clear = negative for Bence-Jones protein
8
Q
proteinuria associated with true renal disease
A
Renal proteinuria
- may be the result of either:
- glomerular damage = affects filtration (up to 4g/day)
- tubular damage = affects reabsorption (markedly increased CHONs in urine)
9
Q
renal proteinuria caused by impaired filtration
A
- Glomerular proteinuria
- Microalbuminuria
- Orthostatic proteinuria
10
Q
the glomerular membrane is damaged, resulting in impaired selective filtration
A
Glomerular proteinuria
- when selective filtration is impaired, high amounts of serum proteins, WBCs & RBCs pass through the membrane and are excreted in urine
11
Q
major causes of glomerular proteinuria
A
- exposure to abnormal substances
- eg. amyloid material, toxic substances, immune complexes (LE, streptococcal glomerulonephritis) - increased pressure from blood
- may override the selective rotation of the glomerulus
- reversible
- does not necessarily mean glomerular damage
- caused by strenuous exercise, dehydration, hypertension - pregnancy
- glomerular proteinuria occurs during the latter months
- may indicate a “pre-eclamptic state”
- should be considered by the physician in conjunction with other clinical symptoms to determine if the condition exists - other causes of benign proteinuria
- transient (diminishes over time)
- eg. high fever, exposure to colds
12
Q
common occurrence in patients with Type I & II DM
A
Microalbuminuria
- in TI & II DM patients, development of diabetic nephropathy is common
- detection can predict the onset of renal complications
13
Q
preventing the onset of renal complications
A
stabilizing blood glucose & control hypertension
14
Q
T/F:
microalbuminuria is associated with increased risk of CVD
A
TRUE
- CVD = cardiovascular disease
15
Q
detection of microalbuminuria
A
Measure albumin quantitatively
- 24h specimen
- significant results:
- 30-300mg albumin/24h
- albumin excretion rate = 20-200ug/minute
16
Q
postural proteinuria
A
Orthostatic proteinuria
- long periods spent in a vertical position is believed to cause pressure in the renal vein & override filtration –> benign proteinuria
- common in young adults
- disappears when horizontal position is assumed
17
Q
detection of orthostatic proteinuria
A
- patients are requested to empty their bladder to before going to bed
- collect the first morning specimen
- collect a 2nd specimen hours after
- patient should be in a vertical position - test for CHONs
- result for O. proteinuria:
- - first morning spx = (-)
- - 2nd spx = (+)
18
Q
renal proteinuria caused by tubular damage
A
- other LMW CHONs that are usually reabsorbed are present
- causes:
- exposure to toxic substances & heavy metals
- severe viral infections
- Fanconi syndrome
19
Q
methods of protein determination
A
- reagent strip
- SSA precipitation test
- micral test
- immunodip
- albumin:creatinine ratio
20
Q
reagent strip method principle & mode of measurement
A
Principle:
protein error of indicators (pH indicators)
- pH indicators have a characteristic color at certain pH levels
- color when there is no reaction: yellow
Mode of measurement:
colorimetric
- at constant acid pH (3.0), proteins in urine cause a changed color in the indicator resulting to an end color of blue to green
– end color depends on CHON concentration in urine
21
Q
reading time for reagent strip method
A
60 seconds
*reagent strip method is more sensitive to albumin since it has more amino group
22
Q
reagent strip brands most commonly used
A
- Multistix
- reagent: Tetrabromophenol blue
- sensitivity: 15-30mg/dL albumin - Chemstrip
- reagent: 3’,3”,5’,5”-tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein
- sensitivity: 6mg/dL albumin
23
Q
interference/s to reagent strip method
A
Change in pH
- the method highly depends on the pH indicators
- main reaction interference would be any condition that changes the pH of the
24
Q
sources of error in reagent strip method
A
- highly buffered alkaline urine
- pH is set at 3.0 (acidic) - prolonged contact with the reagent pad & urine
- causes bleaching out of the reagent
25
causes false positive reagent strip result
- highly buffered alkaline urine
- prolonged contact with urine
- contamination with quarternary ammonium compounds (QAC), detergents, antiseptics
- specimens with high SG
- highly pigmented urine
- - interferes with color reaction, not with the pH
26
causes false negative reagent strip result
- presence of CHONs other than albumin
| - microalbuminuria
27
Identify this test:
principle: cold precipitation
proteins detected: all forms
specimen required: centrifuged urine
SSA precipitation test
- SSA = sulfosalicylic acid
- centrifuged urine is used to remove any extraneous contamination
28
procedure for SSA precipitation test
1. add 3mL of 3% SSA reagent to 3mL centrifuged urine
2. mix by inversion
3. observe for cloudiness
29
reporting of SSA turbidity
Negative
- less than 6mg/dL
- no increase in turbidity
Trace
- 6-30mg/dL
- noticeable turbidity
+ (1+)
- 30-100mg/dL
- distinct turbidty, NO GRANULATION
++ (2+)
- 100-200mg/dL
- turbidity, granulation, NO FLOCCULATION
+++ (3+)
- 200-400mg/dL
- turbidity, granulation, flocculation
++++ (4+)
- >400mg/dL
- clumps of protein
30
a new method for microalbuminuria testing that is specific for albumin
Micral test
- patient at risk for renal disease can be monitored using random or 1st morning specimens
Manufacturer:
Roache Diagnostics
Principle:
EIA
Sensitivity:
0-10mg/dL (0-100mg/L)
Specificity:
albumin
Specimen:
1st morning urine
31
application time, reading time & end color of micral test
Application time:
5 seconds
Reading time:
1 minute
Color range:
white to red
End color:
red
32
compositions of the micral test reagent strip
1. Zone 1
- conjugate matrix fleece
- reagent gold-labeled anti-human albumin-enzyme conjugate
- enzyme: B-galactosidase
2. Zone 2
- capture matrix fleece
- human serum albumin
3. Detection pad
- substrate: chlorophenol red galactoside
33
micral test procedure
1. dip the strip into the urine
- dip up to a level marked on the strip
- hold for 5 seconds
2. allow excess urine to drain
- place strip on a neon absorbent surface OR across the top of the collection cup
- wait for 1 minute
3. compare the color of the reaction pad on the strip with the color scale on the test strip vial
34
identify the test:
principle: immunochromography
specimen: first morning
specificity: albumin
Immunodip
- manufactured by Sekisui Diagnostic
- reagents: blue latex particles coated with anti-human albumin
35
rationale of immunodip
Migration of bound and unbound particles
- migration is controlled by the size of the particles
- a blue band is first formed by the unbound particles
- bound particles continue to migrate up the strip to form a second blue band
Therefore,
Top blue band = bound particles
Bottom blue band = unbound particles
36
procedure for immunodip
1. place the test device in the urine sample
- wait for 3 minutes
- urine level must be above the mark on the test device
2. read results after 3 minutes
- compare the color intensity of the bands against the manufacturer's color chart
37
interpret:
top band = present, faint
bottom band = present, dark
NEGATIVE
| - presence of <1.2mg albumin/dL
38
interpret:
top band = present
bottom band = present
band colors equal
BORDERLINE
| - 1.2-1.8mg albumin/dL
39
interpret:
top band = present, dark
bottom band = present, faint
POSITIVE
| - 2.0-8.0mg albumin/dL
40
interpret:
top band = absent
bottom band = present
INVALID
| - no bands present = also invalid
41
done by comparing albumin excretion to the consistent excretion rate of creatinine
Albumin:Creatinine ratio
- estimation of 24h microalbumin excretion
- albumin reading can be corrected for over-hydration & dehydration in a random sample
- method: reagent strip
- - Clinitek Microalbumin reagent strips
- - Multistix Pro reagent strips
42
A:C ratio:
reagent strip reaction for albumin
Principle:
dye-binding reaction
Indicator:
bis(3'3"-diiodo-4'4"-dihydroxy-5'5"-dinitrophhenyl)-3,4,5,6-tetrabromosulphonphthalen (DIDNTB)
Sensitivity:
8-15mg/dL (80-150mg/L)
43
interference/s in the reagent strip reaction for albumin
- visibly bloody urine
- abnormally colored urine
*interferences cause change of color in the reagent pad
44
other compositions of the reagent strip for albumin
1. polymethyl vinyl
- increases specificity of the reagent pad to albumin by decreasing the nonspecific binding of polyamino acids to the albumin pad
2. bis-(heptapropylene glycol) carbonate
- treatment with said substance prevents reaction interference by highly buffered alkaline urine
45
A:C ratio:
reagent strip reaction for creatinine
Principle:
pseudo-peroxidase activity of copper-creatinine complexes
Reagents:
- CuSO4
- 3,3',5,5'-tetramethyl-benzidine (TMB)
- diisopropyl-benzene-dihydroxyperoxide (DBDH)
Normal Value:
10-300mg/dL
46
interference/s in the reagent strip reaction for creatinine
- visibly bloody urine
- abnormally colored urine
- intake of gastric acid-reducing medication (cimetidine (Tagamet))
47
T/F:
| urine contains large amounts of glucose
FALSE
- only MINUTE AMOUNTS are present in urine
- glucose in the blood is readily filtered by the glomerulus
- most of the glucose is reabsorbed by ACTIVE TRANSPORT
- renal threshold: 160-180mg/dL
- - represents the blood level of glucose at which tubular reabsorption stops
48
the body produces (insulin, glucagon) in response to increased blood glucose
Insulin
- converts glucose to glycogen
* HI = High blood glucose, Insulin released
* GLow = Glucagon is released when blood glucose is Low
49
opposing hormones
- hormones that work in opposition to insulin
- break down glycogen to glucose by glycogenolysis, resulting in an excess of glucose excreted in urine
- hormones include:
- - glucagon
- - epinephrine
- - cortisol
- - tyrosine
- - growth hormone
- occurs in disorders affecting hormonal functions eg:
- - pancreatitis
- - acromegaly
- - Gaucher's syndrome
- - hyperthyroidism
- - pheochhromocytome
- - thyrotoxicosis
50
effect of severe stress in urine glucose levels
- body releases epinephrine
- breaks down glycogen to glucose by glycogenolysis
- blocks the secretion of insulin --> excess glucose excreted
51
gestational diabetes
- occurs during pregnancy & disappears after delivery
- hyperglycemia & glycosuria occurs normally around the 6th month of pregnancy
- women with this condition are prone to developing TII DM
52
macrosomia
- when excess glucose presented to the the baby is stored as fat
- the baby is fat; baby is at risk for obesity & diabetes
- some of the excess glucose enters the placenta
- - insulin cannot enter the placenta, so glucose cannot be converted to glycogen
53
happens when reabsorption of glucose by the renal tubules is compromised
Renal glycosuria
- caused by diseases:
- - end stage renal disease
- - cystinosis
- - Fanconi syndrome
- renal threshold may also be lowered in pregnancy
- - BUT reabsorption is not impaired
54
methods of urine glucose determination
1. reagent strip
| 2. copper reduction test
55
identify the test:
Principle: double sequential enzymatic reaction
Specificity: glucose
Reagent strip
- "glucose oxidase reaction"
- an indirect method; does not directly measure glucose
- - glucose oxidase catalyzes a reaction between glucose & room air, producing gluconic acid & peroxide
- - peroxidase catalyzes the reaction between the peroxide & a chromogen, forming an oxidized colored compound that is directly proportional to glucose concentration
56
reagents used in the reagent strip for glucose
- glucose oxidase
- peroxidase
- chromogen (depends on the brand)
- - Multstix: potassium iodide (KI)
- - Chemstrip: TMB
- buffer
57
end color of glucose reagent strip
a. Multistix
(+) = green
(-) = brown
b. Chemstrip
(+) = yellow
(-) = green
58
reading & interpretation of reagent strip for glucose
a. Qualitative
- negative, trace, +, ++, +++, ++++
b. Quantitative
- 100mg/dL (.1%) to 2g/dL (2%)
59
T/F:
| reagent strips for glucose provide semi-quantitative measurements
TRUE
| - only an estimate of the amount of glucose in the urine
60
possible cause/s of false positive or false high glucose results
containers contaminated with peroxide or strong oxidizing reagents
61
possible cause/s of false negative or false low glucose results
- presence of strong reducing agents (eg. ascorbic acid)
- increased ketone, SG
- decreased temperature
- bacterial degradation of glucose
62
what to do to minimize interference from ascorbic acid?
incorporate strips with additional chemicals (eg. iodate) to oxidize ascorbic acid
63
these also cause false positive or high results
Increased ketone, SG & low temperature
- high levels of ketones are usually accompanied by marked glycosuria
- high SG & low temp. may decrease the sensitivity of the test
64
the greatest source of false negative error
Bacterial degradation of glucose
| - as a result of allowing the specimen to remain unpreserved at room temperature for extended periods
65
Benedict's test
Copper reduction test
- can also be in tablet form (Clinitest)
Principle:
CuSO4 is reduced to Cu2O by reducing substances in the presence of alkali & heat
Reagents:
- Benedict's solution
- - copper sulfate
- - sodium carbonate
- - sodium citrate (buffer)
- sodium hydroxide (in Clinitest tablet)
66
Clinitest procedure
1. add 5 drops of urine into a thick glass test tube placed in a rack
2. add 10 drops of distilled H2O
3. drop 1 Clinitest tablet & observe reaction until cessation of boiling
4. wait for 15 seconds after conclusion of effervescence (bubbles)
5. shake tube gently
6. compare results to the color chart
- ranges from blue (negative) to orange (positive)
- report results in mg/dL or %
67
"pass through" phenomenon
- occurs at high glucose levels
- the color produced pass through the orange or red stage quickly & returns to green-brown --> (-) result may be reported
68
sensitivity of Clinitest
200mg/dL
| - cannot be used as confirmatory test for glucose
69
interference/s for glucose
- other reducing sugars (galactose, lactose, fructose, maltose, pentoses)
- ascorbic acid
- certain drug metabolites
- antibiotics (cephalosporins)
70
storage & handling of Clinitest tablets
- should be stored in tightly closed packages
- tablets are very hygroscopic
- - tend to absorb moisture from the air
- - a strong, blue color in unused tablets signifies deterioration due to moisture & vigorous tablet fizzing
71
clinical significance of copper reduction tests in children
- usually performed on children up to 2 years old
- screening for galactosemia
- - galactose in newborns = "inborn error of metabolism"
- - lack of galactose-1-phosphate uridyl transferase
- - galactose breakdown is prevented --> complications --> death
72
T/F:
| the appearance of other reducing sugars is usually of medical significance
TRUE
| - lactose is frequently found in the urine of nursing mothers
