Pus Absent Parasites Absent Fat Colorless, neutral fat (18%) and fatty acid crystals and soaps Undigested None to small amount food, meat fibers, starch, trypsin Eggs and segments of

 

Note: The calibrator values are lot dependent. Always

check the calibrator dilution concentrations when a new

calibration is performed. The calibration curve is usually

stable for 4 weeks.

Semiautomated Analyzer

Assay conditions:

Wavelength 546 nm

Reaction temperature 37°C

Cuvette 1 cm path length

Test Procedure for Preparation of Calibration Curve

1. Zero the instrument with distilled water.

2. Pipette 400 μL of Quantia-Cystatin C Activation buffer

(R1) and 75 μL of Quantia-Cystatin C Latex reagent

(R2) in the measuring cuvette. Mix well and incubate

for 5 minutes at 37°C.

3. Add 5 μL of calibrator (D1), mix gently and start the

stopwatch simultaneously.

4. Read absorbance (A1), exactly at 10 seconds, and

absorbance (A2) again at the end of exactly 280

seconds.

5. Repeat steps No. 2–4 for each selected diluted

calibrator (D2 - D5) for preparing calibration curve.

6. Calculate DA (A2 - A1) for each selected calibrator

(D1 - D5). Plot a graph of DA versus concentration of

Cystatin C on the graph paper provided with the kit.

7. The calibration curve so obtained is valid only for the

same lot of Quantia-Cystatin C reagents.

Test Procedure for the Determination of Cystatin C in

Specimen:

1. Follow steps 2 – 4 as mentioned in the above procedure

for calibration curve on Semiautomatic analyzers

using the test specimen in place of the calibrator.

2. Calculate DA (A2-A1) for the test specimen.

Calculations

Cystatin C concentration in mg/L can be obtained as

mentioned below:

The concentration of Cystatin C (mg/L) can be determined directly by interpolating DA of the test specimen

from the calibration curve. If the DA of the test specimen

is greater than DA of the highest standard concentration

(D1) then the test has to be rerun by carrying our dilution

of test specimen such as 1:2, 1:4, etc. Interpolate the DA

of the diluted test specimen on the calibration curve and

obtain the cystatin C concentration. Multiply the obtained

Cystatin C concentration “C” with the dilution factor of the

test specimen to determine the Cystatin C concentration

in neat specimen.

Automated Analyzer

General Application Parameter Set up (Table 6.2)

A defined application for the Quantia-Cystatin C

immunoturbidimetric assay must be installed in

accordance with the general instrument settings given

below. For instructions refer the respective instrument

manual.

TABLE 6.2: Suggested instrument applications

Parameters Suggested applications

Filter 546

Quantia-Cystatin C activation

buffer (R1) volume

220 μL

Sample volume 3 μL

Incubation time before addition of R2 120 seconds

Quantia-Cystatin C

latex reagent (R2) volume

45 μL

Read absorbance A1 immediately after mixing reagents and sample

and absorbance A2 at the end of 5 minutes.

Note: Applications suitable for Olympus AU 400/600/2700,

Synchron LX 20, Hitachi 902/911/917/Vitros 5.1/ Modular

P/Architect/Advia 1650 &1800/Cobas c501/Pentra 400 can

be made available on request.

Test Procedure for Preparation of Calibration Curve

Perform the calibration as per the instrument protocol

given by the manufacturer.

Test Procedure for the Determination of Cystatin C in

Specimen:

When a valid calibration has been performed and the

controls are within the expected range (provided in

assay value sheet), specimens can be measured. Check

that sufficient amount of sample is present as per the

requirement of the instrument protocol.

Calculations

The results are automatically calculated by the analyzer

and presented in mg/L.

For GFR Prediction Calculation:

For calculation of GFR from Cystatin C values measured

with Quantia-Cystatin C assay the following prediction

equation is recommended using mg/L as the unit factor.

GFR [mL/min/1.73 m2

] = 79.901

CystatinC (mg /L)

 1.4389

GFR can be calculated with the GFR calculator available

on our website www.tulipgroup.com.

Renal Function and its Evaluation 111

Quality Control

The calibration of Quantia-Cystatin C must be validated

with Quantia-Cystatin C Control set.

Specific Performance Characteristics

The performance characteristics mentioned further

have been validated using Quantia-Cystatin C on Abbott

Architect ci8200 and Quantiamate.

Measuring Range

The Quantia-Cystatin C assay has been designed to measure

Cystatin C concentration in the range of 0.5–8.0 mg/L. The

exact range is dependent on the calibrator value, which is lot

specific. The Quantia-Cystatin C assay is linear within the

measuring range.

Detection Limit

0.33 mg/L. The detection limit represents the lowest

measurable Cystatin C concentration that can be

distinguished from zero.

Prozone Limit

No prozone effect was observed in concentration less than

16 mg/L of Cystatin C.

Average healthy adults defecate from three times a day to

three times a week. Common pattern is once a day. The

stool tends to be soft and bulky on a diet high in vegetables

and small and dry on a diet high in meat. Two thirds of

the stool weight is attributable to its water content. The

normal brown color is of still undetermined origin. The

odor results from indole and skatole, produced by bacteria

from tryptophan.

Feces are composed of:

1. Waste residue of indigestible material in food.

2. Bile (pigments and salts).

3. Intestinal secretions, including mucus.

4. Leukocytes that migrate from the bloodstream.

5. Shed epithelial cells.

6. Large numbers of bacteria that make up to one-third

of total solids.

7. Inorganic material (10–20%) that is chiefly calcium

and phosphates.

8. Digested food (present in very small quantities).

SPECIMEN COLLECTION

A wide mouthed jar with a screw cap is good enough,

provided it is neat, clean, and without any extraneous

material in it. It should, however, never be overfilled

and should be opened slowly to release the gas that

accumulates frequently in it (if not done so, the contents

may be released explosively). Since, rectal evacuation is not

completely at will and feces passed correlate very poorly

with the food consumed; hence, collection should be done

over a period of 3 days. The accuracy of this method can be

enhanced somewhat by having the patient ingest carmine

dye (0.3 g) and charcoal (1 g) at the beginning and the end

of a collecting period, respectively, collecting the stools

from the beginning of the appearance of the dye to the

beginning of the appearance of the charcoal.

Be Careful

¾ Feces should be urine free when collected. Collect

the entire stool and transfer to another container by a

tongue blade. Deliver to the laboratory immediately

after collection

¾ Warm stools are best for detecting ova and parasites.

Do not refrigerate for ova and parasites

¾ Some coliform bacilli produce antibiotic substances

that destroy enteric pathogens

¾ Refrigerate stool if it cannot be examined immediately.

Never place a stool in an incubator

¾ A diarrheal stool will usually give good results

¾ A freshly passed stool is the specimen of choice

¾ Preferably, stool specimens should be collected before

antibiotic therapy is initiated and as early in the course

of disease as possible

¾ Only a small amount of stool is needed; the size of a

walnut. If mucus and blood are present, they should be

included in part of the specimen to be examined

¾ Do not use a stool that has been passed into the toilet

bowl or that has been contaminated with barium or

other X-ray medium

¾ Label all stool specimens with patient’s name, date,

and reason for examination/testing.

Interfering Factors

¾ Meat interferes with some tests and should usually be

omitted from the diet for 3 days before a test for blood

(not necessary for the guaiac me

(not necessary for the guaiac method)

7

Stool Examination

C H A P T E R

Stool Examination 113

¾ Stool specimens from patients receiving barium,

bismuth, oil, or antibiotics are not satisfactory

¾ Bismuth from paper towels and toilet tissues interferes

with tests.

Normal Values in Stool Analysis

These are listed in Table 7.1.

INSPECTION OF FECES

A simple inspection of feces may lead to a diagnosis

of parasitic infection, obstructive jaundice, diarrhea,

malabsorption, rectosigmoidal obstruction, dysentery or

ulcerative colitis or gastrointestinal tract bleeding.

Note the quantity, form, consistency and color of the

stool (Table 7.2).

Interfering Factors

1. Stool darkens on standing.

2. Color is influenced by diet, food dyes, certain foods,

and drugs.

a. Yellow to yellow green color occurs in the stool

of breastfed infants who lack normal intestinal

flora. It also occurs in sterilization of bowel by

antibiotic.

b. Green color occurs in diets high in chlorophyllrich vegetables and with use of the drug calomel.

c. Black or very dark brown color may be due to

drugs such as iron, charcoal, and bismuth, to

foods such as cherries, or to an unusually high

proportion of meat in the diet.

d. Light-colored stool with little odor may be due to

diets high in milk and low in meat.

TABLE 7.1: Normal values in stool analysis

Macroscopic Normal examination

Color Brown

Odor Varies with pH stool and depends upon

bacterial fermentation and putrefaction

Consistency Plastic; not unusual to see seeds and vegetable skins; soft and bulky in a high vegetable diet; small and dry in a high meat diet

Size and shape Formed

Gross blood Absent

Mucus Absent

Pus Absent

Parasites Absent

Fat Colorless, neutral fat (18%) and fatty acid

crystals and soaps

Undigested None to small amount food, meat

fibers, starch, trypsin

Eggs and segments of

parasites

Absent

Yeasts Absent

Leukocytes Absent

Chemical examination Normal

pH Neutral to weakly alkaline

Adult 7.0–7.5

Newborn 5.0–7.0

Bottle-fed infants Neutral to slightly alkaline pH of 7.0–8.0

Breastfed infants Slightly acidic

Occult blood Negative

Urobilinogen 50–300 mg/24 hr