Test Tube No. 1 2 3 4 5 Calibrator dilution No. D1 D2 D3 D4 D5 Saline volume - 300 μL 300 μL 300 μL 300 μL Calibrator volume 300 μL 300 μL 300 μL 300 μL 300 μL

 

5. Total quantity of voided specimens should be over 80%

of intake (i.e. over 1200 mL).

Interpret as in concentration test above. This vasopressin

test gives reliable results in the presence of cardiac edema

or ascites. A further, application of the test is in diabetes

insipidus, where urine concentration is normal after giving

vasopressin but not after fluid restriction.

Conditions that Impair Concentrating Ability

Renal Diseases:

¾ Pyelonephritis

¾ Acute or chronic glomerular failure

¾ Nephrogenic diabetes insipidus

¾ Renal tubular acidosis.

Metabolic Disturbances:

¾ Osmotic diuresis (especially diabetes mellitus)

¾ Hypokalemia

¾ Hypercalcemia (especially hyperparathyroidism)

¾ Lithium use

¾ Ethanol use

¾ Prolonged overhydration

¾ Severe hypoproteinemia.

Systemic Diseases Affecting Renal Medulla:

¾ Multiple myeloma

¾ Amyloidosis

¾ Sickle cell anemia or trait.

PHENOL RED TEST

Principle

This is a measure of tubular excretion. Phenol red test

(PSP) is removed from peritubular capillary blood by the

renal tubules and excreted into the urine.

Intravenous Method

Make sure that no residual volume remains in the bladder

and evacuation is complete. At the beginning of the test,

the bladder should not be empty for it is necessary that the

patient be able to void for the 15 minutes collection.

1. The patient should drink 2 glasses of water and

additional water during the test to ensure a urine

volume sufficient to permit collection of urine

specimen at the stated time. Larger urine volumes

reduce error resulting from incomplete bladder

emptying.

2. Inject 1 mL of dye (6 mg) intravenously. Collect the

total volume of urine voided at 15, 30 and 60 minutes

after injection of the dye. Determine the PSP content

of each specimen.

Interpretation

% PSP excretion

15 min 30 min 60 min Total

Normal 15–27+ 12–20 13–20 55–60

Renal

insufficiency

< 15 < 12 < 12 < 40

Ureteral Catheterization Method (Cystoscopy)

The method can be used to study function of a single

kidney at a time. Have the 2 ureteral catheters dripping

into separate test tubes containing dilute NaOH and inject

106 Concise Book of Medical Laboratory Technology: Methods and Interpretations 1 mL of PSP IV. Normally, the dye appears within 3 minutes,

as indicated by pink coloration in the tubes.

Phenol Red Test for Residual Urine

(Modified PSP test to serve as a measure of residual urine).

Let the patient empty his bladder. Give IV 1 mL of PSP

(6 mg of dye). The water intake here is limited to 200 mL

or less during each of the 2 subsequent half-hour periods;

this is necessary because the rapid filling of the bladder in

the presence of uretheral obstruction may result in a loss of

bladder tone and a consequent increase in residual urine.

Collect all urine that the patient can pass half an hour and

1 hour after the injection of the dye.

Normally, the PSP excretion is 50–60% in the first halfhour, plus another 10–15% during the second half-hour.

Interpretation

First specimen (½ hour) Second specimen (1 hour)

Normal 50–60% 10–15%

Residual (a) 15% 25%

Urine (b) 35% 25%

Present (c) 25% 25%

If the initial half-hour excretion is less than, equal to,

or only slightly more than the second half-hour excretion,

residual urine must be present. In case the excretion curve

is flat and the morning specific gravity low, catheterization

should be done after collection of the second half-hour

specimen to confirm the presence or absence of residual

urine, since under these circumstances one cannot

distinguish between severely depressed renal function

and a large residual volume of urine. If the specific gravity

of the morning urine is high, renal insufficiency is unlikely

and a flat excretion curve is a reliable index of residual

urine volume.

A rough estimate of residual urine volume can be

calculated from the following formula:

Vol(½ h) (60 PSP)

PSP

(‰ h)

(‰ h)

− = mL residual urine

Vol(½ h) = Volume of first half-hour specimen.

PSP(½ h) = % of PSP recovered in first specimen.

60 = Expected normal PSP excretion in the

first half-hour.

(Values of the second half-hour are not used in this

calculation).

CLEARANCE TESTS

By these tests, the capacity of the kidneys to clear waste

products or foreign materials (inulin, etc.) from the

blood into the urine is found. From the determination of

blood concentration of the test material and the quantity

eliminated in the urine, “clearance” can be calculated in

terms of millimeters of blood cleared per unit time.

Creatinine Clearance

Creatinine is filtered through the glomerulus. Under

ordinary circumstances, the clearance of endogenous

creatinine approximates the glomerular filtration rate. The

clearance formula is:

Clearance = UV

P

 UV

where, U = mg% creatinine in urine

 P = mg% creatinine in plasma

 V = mL of urine excreted per minute or per

24 hours.

Methods

1. Twenty-four hours endogenous creatinine clearance.

The entire volume of 24 hours period is collected. A

blood sample is withdrawn during the forenoon of the

day of the test. Creatinine concentrations in plasma/

serum and urine are found and the volume of the urine

is measured and the clearance estimated as per the

formula given.

2. Two to six hours clearance periods may be used.

Fasting state is preferred for the brief clearance period.

A 2 to 6 hours urine collection is completed and a

blood sample withdrawn at about the midpoint of the

urine collection period. Creatinine concentrations are

estimated in urine and plasma and the urine volume

is found and clearance estimated.

Interpretation

Normal values for men and women corrected to 1.73 sq m

body surface area range from 140–180 liters/24 hours

(100–150 mL/minute). To correct clearance to standard

1.73 sq m body surface area

Clearance observed ×

1.73 sq m

Estimated surface area

 = Corrected clearance.

(This test is superior to urea clearance).

Renal Function and its Evaluation 107

Creatinine Clearance Test

Normal Range

110–150 mL/min (males)

105–132 mL/min (females)

Effect of Age on Normal Function

Ages 50–75, subtract 5 mL for each 5-year interval.

Age 75 and above, subtract 8 mL for each 5-year interval.

Artefacts that Lower Calculated Figure

¾ Incomplete urine collection

¾ Bacterial multiplication in collecting vessel

¾ Ketones, barbiturates, PSP in urine at higher levels than

is plasma.

Causes for Reduced Creatinine Clearance

Acute: Shock, hypovolemia, nephrotoxic chemicals, acute

glomerulonephritis, malignant hypertension, eclampsia.

Chronic: Glomerulonephritis, pyelonephritis, hypertensive

nephrosclerosis, polycystic kidneys.

PRINCIPLES OF PRECISE TESTS OF

RENAL FUNCTION (TABLE 6.1)

Clearance of inulin or endogenous creatinine and of

iodopyracet (Diodrast) or PAH helps differentiate diseases

of glomeruli and tubules.

Glomerular Filtration Rate (GFR)

Inulin, a polysaccharide is eliminated exclusively through

the glomeruli, is neither excreted nor absorbed by the

tubules. Inulin clearance is therefore, a measure of the

glomerular filtration rate. Other radioactive labeled

substances can be used. Normal in adults is 100–130 mL/

minute per 1.73 sqm of body surface.

Renal Plasma Flow (RPF)

Para-aminohippurate (PAH), iodopyracet and 131I or 125I

labeled sodium iodohippurate at low concentration in

plasma are cleared almost completely by filtration and

tubular secretion as the plasma flows through kidney.

If, for example, at a plasma PAH concentration of 1 mg%,

6 mg PAH appear, in urine per minute, 600 mL of plasma

must be passing the kidneys per minute. The normal range

is 500–800 mL of plasma per minute per 1.73 sq m of body

surface. Varying with the hematocrit, this indicates a flow

through the kidneys of 1000–1500 mL of whole blood per

minute or almost one-third of the cardiac output at rest.

Filtration Fraction

Ratio of volume of glomerular filtration to the volume of

plasma from which the filtrate was obtained is expressed

TABLE 6.1: Renal function tests at a glance

Determination Normal values

Phenolsulfonphthalein (PSP, Phenol red) 1 mL IV 15 min 35% (28–51)\

30 min 17% (13–24) 55–60%

60 min 12% (9–17)

120 min 6% (3–10)

Clearance tests

Inulin clearance

Iothalamate 131I

Endogenous creatinine clearance

Glomerular filtration rate

-do-

-doCorrected to 1.73 sq m SA

Male: 110–150 mL/min

Female: 105–132 mL/min

Iodohippurate 131I

PAH clearance

Renal plasma flow Male: 560–830 mL/min

Female: 490–700 mL/min

Filtration fraction GFR

FF

PRF

Male: 17–21%

Female: 17–23%

Urea clearance (Cu) Standard: 40–65 mL/min

Maximal: 60–100 mL/min

Maximal glucose reabsorptive capacity TmG Male: 300–450 mg/min

Female: 250–350 mg/min

Maximal iodopyracet capacity TmD Male: 43–59 mg/min

Female: 33–51 mg/min

Maximal PAH excretory capacity Tm PAH 80–90 mg/min.

108 Concise Book of Medical Laboratory Technology: Methods and Interpretations as GFR/RPF. This ratio is called the filtration fraction. The

normal filtration fraction is:

 120 mL/min

600 mL

 = 0.2

MAXIMAL TUBULAR CAPACITY (TM)

At high concentrations of iodopyracet, iodohippurate,

or PAH in plasma, the excretory capacity of the renal

tubule is exceeded. By measuring the amount of test

material excreted under these conditions and correcting

for the amount of test material simultaneously filtered

through the glomerulus, the maximal excretory capacity

of the tubule is obtained. Tm for reabsorption of glucose,

amino acids, etc. can be determined similarly, although

in this instance the amount filtered minus the amount

excreted per unit of time equals the maximal reabsorptive

capacity.

Cystatin C

Quantitative Turbidimetric Immunoassay for

Estimation of Cystatin C in Human Serum

(Turbidimetry as a technology is given in Turbidimetric

Assays in the one of the following/subsequent chapters)

Summary

Cystatin C, a non-glycosylated, low molecular weight

(13 kDa) protein belongs to the family of cysteine protease

inhibitors. Cystatin C is produced at a constant rate by nearly

every nucleated cell in the human body. Its biochemical

characteristics allow its free filtration in the glomerulus.

Subsequently it is reabsorbed and almost completely

catabolized in the proximal tubule. Practically no Cystatin

C returns to the blood. Therefore, Cystatin C concentration

in human blood is closely related to Glomerular filtration

rate (GFR). An increased Cystatin C concentration in

human blood may indicate a reduced GFR, which may

be due to renal diseases. The production of Cystatin C in

the body is not influenced by renal condition, increased

protein catabolism or dietetic factors. Moreover, it does not

change with age or muscle mass like creatinine does. Serum

Cystatin C is therefore proposed to be a ideal endogenous

marker of glomerular filtration rate (GFR) especially in

patients with moderate to severe renal impairment. Studies

have demonstrated that Cystatin C is the most suitable

marker of moderately impaired renal function.

Quantia-Cystatin C is an immunoturbidimetric assay

useful for quantitative measurement of Cystatin C in

human serum/plasma.

Reagents

1. Quantia-Cystatin C Activation Buffer (R1): Ready to

use buffer solution.

2. Quantia-Cystatin C Latex Reagent (R2): Purified

immunoglobulin fraction that is directed against

Cystatin C which is covalently linked to uniform

suspension of polystyrene latex particles.

3. Quantia-Cystatin C Calibrator: Ready to use human

pool serum containing Cystatin C equivalent to

the stated amount on mg/L basis. An International

Cystatin C calibrator is being prepared by a working

group formed by the IFCC/EU. Quantia-Cystatin C

calibrator is traceable to a standard that has been

validated against the coming IFCC standard, by the

university of Lund, a leading partner in the working

group, The Quantia-Cystatin C calibrator is already

standardized against the International Cystatin C

calibrator.

Each batch of reagents undergoes rigorous quality

control at various stages of manufacture for its specificity,

sensitivity, and performance.

Reagent Storage and Stability

1. Store the reagent at 2–8°C. Do not freeze.

2. The shelf life of the reagent, activation buffer and the

calibrator is as per the expiry date mentioned on the

respective vial labels.

Onboard Stability on Automated Analyzer

In appropriate bottles the Quantia-Cystatin C activation

buffer and Quantia-Cystatin C latex reagent are stable for

9 weeks when stored at 2–8°C onboard the analyzer.

Principle

Quantia-Cystatin C is a turbidimetric immunoassay for

the quantitative determination of Cystatin C in human

serum and is based on the principle of agglutination

reaction. The test specimen is mixed with Cystatin C latex

reagent (R2) and activation buffer (R1) and allowed to

react. Presence of Cystatin C in the test specimen results

in the formation of an insoluble complex producing a

turbidity, which is measured at wavelength of 546 nm. The

extent of turbidity corresponds to the concentration of

Cystatin C in the specimen.

Note:

1. In vitro diagnostic reagent for laboratory and

professional use only. Not for medicinal use.

2. All the reagents derived from human source have been

tested for HBsAg and HIV antibodies and are found

to be non-reactive. However, handle the material as if

infectious.

Renal Function and its Evaluation 109

3. Reagent contains sodium azide as preservative in

concentrations that is not characterized as dangerous.

Avoid contact with skin and mucosa. On disposal flush

with large quantities of water.

4. The reagent can be damaged due to microbial

contamination or on exposure to extreme temperatures.

It is recommended that the performance of the reagent

be periodically verified with Quantia-Cystatin C

control set (Cat. No. 108202005) .

5. Gently mix the Quantia-Cystatin C latex reagent (R2)

well before use to disperse the latex particles uniformly

to improve test performance.

6. As the reagents within lots have been matched,

reagents from different lots must not be interchanged.

7. Calibrators of different manufacturers must not be

used with Quantia-Cystatin C reagents.

8. The Quantia-Cystatin C reagents are not adaptable

for Nephelometric analyzers.

Specimen Collection and Preparation

No special preparation of the patient is required prior to

specimen collection by approved techniques.

EDTA/Heparinized plasma or serum should be used for

testing. Should a delay in testing occur, store the samples

at 2–8°C.

Interference

No interference was observed with hemoglobin 8 g/L,

bilirubin 420 mg/L, and triglycerides 12.5 mmol/mL.

Interference of RF does not take place with QuantiaCystatin C assay as it uses avian antibodies.

Reference Values

The reference values for Cystatin C (architect ci8200) was

determined to be 0.51–1.05 mg/L.

It is recommended that each laboratory must define its

own reference range for relevant population taking into

account all affecting factors.

Remarks

1. Usage of well-calibrated equipment and accessories

and procedures is critical for achieving correct results.

2. Markedly lipemic, hemolyzed, and contaminated

serum samples could produce nonspecific values.

3. It is recommended that results of the tests should be

correlated with clinical findings to arrive at the final

diagnosis.

4. Several Cystatin C based prediction equations for

calculation of GFR for adults and children have been

published. It should be noted that these formulas were

evaluated with different Cystatin C assays and may

reveal inaccurate GFR.

5. In contrast to creatinine concentration, Cystatin

C levels are lower in hypothyroid and higher in

hyperthyroid state as compared with the euthyroid

state. Therefore, thyroid function has to be considered

when Cystatin C is used as a marker of kidney function.

Warranty

This product is designed to perform as described on the

label and package insert. The manufacturer disclaims any

implied warranty of use and sale for any other purpose.

Samples can be stored for up to one week at 2–8°C,

provided they are not contaminated. Do not use hemolyzed,

icteric, or highly turbid sera. Turbid or particulate samples

must be clarified by centrifugation at 2000 rpm for 15

minutes prior to testing. Use the clear supernatant for

testing.

Additional Material Required

Analyzer, well-calibrated micropipettes, disposable tips,

isotonic saline, test-tube rack, Optically clean disposable/

glass semi-microcuvettes (for cuvette mode semiautoanalyzers).

Test Procedure

Method for preparation of Cystatin C calibration curve on

semiautomatic and automatic analyzer.

Bring reagents and samples to room temperature before

use.

The Quantia-Cystatin C calibrator is ready to use.

The concentration (S) of Cystatin C is mentioned on the

calibrator vial label and at the end of the package insert.

Use saline for preparing dilutions of the calibrator. In

analyzers, where onboard dilution of calibrator is possible,

follow instructions provided in the instrument manual. In

analyzers where onboard calibrator dilution is not possible

follow the procedure mentioned below:

Dilute the Quantia-Cystatin C calibrator serially as

mentioned below for the preparation of the calibration

curve.

Test Tube No. 1 2 3 4 5

Calibrator dilution No. D1 D2 D3 D4 D5

Saline volume - 300 μL 300 μL 300 μL 300 μL

Calibrator volume 300 μL 300 μL 300 μL 300 μL 300 μL

Concentration volume (S)

of Cystatin C in mg/L

S S/2 S/4 S/8 S/16

¾ At least five dilutions of the calibrator (D1-D5) covering

the measuring range (0.5–8.0 mg/L) must be used for

preparing the calibration curve

110 Concise Book of Medical Laboratory Technology: Methods and Interpretations ¾ During calibration on instrument with programming

facilities, increasing concentration of the standard

must be used for preparing the calibration curve.

¾ Check that sufficient amount of calibrator is present in

sample cup as per the requirement of the instrument

protocol during testing in automated analyzer.