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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5535_Библиотеки_им_академика_М_И_Перельмана.pdf
996
the gastrointestinal tract and enters circulation, it is taken up by cells for energy. Glucose, the
major and simplest carbohydrate in blood, is used as the primary energy source by all cells
and especially by the red cells which cannot consume any other form of carbohydrate, the
energy package of the biological world.
During period of high blood glucose levels, glucose molecules enter red blood cells and
bind to haemoglobin, forming HbA1c (glycated haemoglobin). The amount of HbA1c is
proportional to the amount of glucose in circulation. Since average life span of red blood cells
is approximately 120 days, the level of HbA1c is related to the average amount of glucose it is
exposed to in the blood for that period of time. In reality the HbA1c more closely represents
levels over the previous 2–4 weeks.
As the fasting glucose level gives the patient an idea of the lowest level of glucose on
the daily basis, the physician reads the HbA1c value forjudging how tightly the patient has
controlled the blood glucose level over a period of about a month. Recent studies have shown
that HbA1c is the single best test for evaluating the risk of damage to nerves and the small
blood vessels of the eyes and kidneys. This damage leads to the complications of diabetes,
such as blindness and kidney failure. Clinical trials have shown that reducing the HbA1c
level in diabetics and maintaining it below 7% will prevent the development of, or further
progress of, complications from diabetes. It is true for both type I and type II diabetes.
The traditional methods of HbA1c determination are HPLC, TLC and electrophoresis.
These are available only in reference laboratories. Point of care analysis HbA1c is a recent
innovation and has proved to be very helpful to diabetic patients. Results are obtained within
few minutes. Additionally, some home glucose meters calculate the HbA1c from the average
of the glucose values in memory.
Medical Laboratory Technology: Volume 3
Manual Procedure
Laboratory determination of HbA1c may use a latex agglutination reaction in which a
monoclonal antibody is used as an epitope which recognizes glucose bound to HbA1c. The
measurement, however, will require an immunophotometer.
Preliminaries
1. Take necessary precautions for self-protection (gloves, laboratory coat, hand sanitization etc.).
2. Arrange the following:
•Sample dilution kit pouch
•Test cartridge pouch
• Specimen–capillary puncture or well-mixed heparinized blood sample (used in
haematology)
•Gauze pad, biohazard waste container
•Quality control materials
Procedure
1. Verify the test ordered and specimen received.
2. Bring samples, reagents, and monitors, at room temperature.
3. Wash hands and apply gloves.
4. Assemble the necessary equipment before starting the test.
5. Verify the kit has not expired.
6. Prior to the performance of the test, verify whether a quality control (QC) test needs to
be run with the control specimen (provided by the manufacturer). If so, complete that
test before the patient’s test is performed. The results must be in conformity with the
manufacturer’s directions.
7. Verify that all lot numbers match on the monitor, the sample dilution kit pouch, and the
test cartridge pouch.
8. Open the sample dilution pouch and remove the dilution device.
Routine Biochemical Test Procedures
9. Perform the capillary puncture if you are working in a physician’s oce or mix the
whole blood (heparinized) sample to prepare for the next step.
10. Add a 5-mL blood sample to blood collection device.
11. Plug blood collection device into the sampler body. Push rmly so that there is no gap
on insertion.
(Note If a gap is present, the sample will not combine with the dilution solution inside
the sampler body, and the results will be invalid).
12. Thoroughly mix/shake the sample (6-8 times) with the dilution solution.
13. Set sample assembly on the table top as the test cartridge is prepared.
14. Tear open the test cartridge pouch and ensure that the code number on the cartridge
matches the code number printed on the instrument.
15. Insert the test cartridge into the monitor until it is seated rmly and an audible click is
heard.
16. Verify that the instrument screen display reads WAIT.
17. When the instrument screen displays SAMPLE (or SMPL), pick up the sampler device
and remove the base piece, exposing the plunger.
18. Deliver the sample by pressing the plunger gently and rmly into the corresponding
sample application area on the cartridge. Remove aer 1 s.
19. The instrument screen will display a countdown from 5 min to 0. Do not move the
monitor during this time.
20. The results will display as percentage on the screen. Record results aer comparing
with the chart provided by the manufacturer.
21. Dispose o the sample dilution, cartridge, and test cartridge, as bio-hazardous trash
and disinfect work area.
22. Remove gloves and sanitize hands.
23. Document your results immediately in record book and in patients’ request slip.
997
Automated procedure
The automated system commonly available for analysing HbA1c is Anion Analyzer and
others. This instrument uses boronate anity assay for the determination of percentage of
HbA1c in human whole blood. All reagents are contained in the disposable test cartridge
which also has an integrated sampling device taking only 1.5 mL of whole blood from a
nger prick. The blood sample is automatically diluted and mixed with a liquid that releases
haemoglobin from the erythrocytes. The haemoglobin precipitates. The sample mixture
is transferred to a blue boronic acid conjugate, which binds to glycated haemoglobin. The
reaction mixture is soaked through a lter membrane and all precipitated haemoglobin
remains on the membrane. All measurements and Calculations are performed by the
analyzer and percentage HbA1c is displayed on the screen. The analyzer system is easy to
use, gives reliable and accurate results. The analysis time is about 3 min and measuring range
is 4–15% HbA1c. The system is therefore particularly suitable for use in diabetic clinics.
Diabetic Association has provided guidance regarding the frequency of this test and the
interpretation of the result. A non-diabetic patient will have HbA1c levels below 6%. For
diabetic patients, the goal is to remain below 7%, as it is understood that there may be spikes
in the blood glucose that are unavoidable. As a part of the management of diabetes, those
who have unstable blood sugars should have heir HbA1c tested quarterly; those who appear
to have good glycaemic control should be checked at least two times per year. In addition, the
recommendation includes performance of an annual lipid prole and urine micro-albumin
testing to screen for renal damage. Micro-albumin testing will be discussed later in this
chapter.
998
Medical Laboratory Technology: Volume 3
serum ProteIn
Serum protein consists of albumin and globulin. Serum protein analysis can help diagnose
liver disorders, nutritional deciency of protein, renal failure and lymphoproliferative
disorders. Decrease in total protein values is associated with liver disorders (cirrhosis,
nephrotic syndrome), malnutrition, and neoplastic disease. Increased total protein values
may be diagnostic for multiple myeloma, and conditions associated with high globulin
concentration (autoimmune and lymphoproliferative disorders).
Total Protein in Serum
The total protein test measures the total amount of two classes of proteins found in the uid
portion of your blood. These are albumin and globulin. Proteins are important parts of all
cells and tissues. Albumin helps prevent uid from leaking out of blood vessels. Globulins
are an important part of the immune system.
Clinical signicance
This test is oen done to diagnose nutritional problems, kidney disease, or liver disease.
If total protein is abnormal, more tests will need to be done to look for the exact cause of the
problem.
Normal range (adult)
Total protein: 6.0–8.0 g/dL
Albumin fraction: 3.5–5.0 g/dL
Globulin fraction: 2.3–3.5 g/dL
Normal value ranges may vary slightly among dierent laboratories.
Specimen
Serum or plasma; specimen is collected by routine procedure.
Biuret method
Principle
Biuret method, modied by Lowry, for determining total proteins in serum, has been used
for decades. The procedure is simple and sensitive. It is based on two chemical reactions. The
rst is the biuret reaction, in which the alkaline cupric tartrate reagent complexes with the
peptide bonds of the protein. In the second reaction, the phenol reagent (Folin and Ciocalteu’s
phenol reagent) is reduced to a purple colour compound. Absorbance of the coloured
solution is read at 540 nm (range 530–560 nm). The protein concentration is determined from
a calibration curve or compared with a standard. The following description is based on the
technical bulletin of Sigma, manufacturer of laboratory kits.
Interfering substances: Haemolyzed serum or plasma samples should not be used in this
testing procedure. Lipaemia has no eect on the total protein values but grossly turbid serum
should not be used. If necessary, treat the serum with isopropanol, discard the solvent and
use the serum for protein assay.
The following account is taken from the brochure of Sigma kit in order to illustrate the
procedure. There are many other manufacturers who oer similar kits for the local market.
Caution Read and comprehend the procedure given in the insert before performing the test.
Reagents (provided with the kit)
1. Lowry reagent, powder 5 × 2 g
2. 0.15% Deoxycholate (DOC) solution 20 mL
3. Folin and Ciocalteu’s Phenol reagent, 1 bole
4. Protein standard, 5 × 1 vial—2 mg of BSA (Bovine serum albumin) per vial
Routine Biochemical Test Procedures
Handle all chemicals carefully. Consider all chemicals poisonous. Specimens should be
considered as potential biohazards.
Reconstitute the reagents according to manufacturer’s direction.
• The Lowry reagent solution is prepared by adding 40 mL of water to a bole of Lowry
reagent powder. Mix well by inverting to completely dissolve the contents. Do not shake
so as to minimize foaming.
• The Folin and Ciocalteu’s phenol reagent working solution is prepared by transferring
the Folin and Ciocalteu’s phenol reagent (18 mL) to the amber glass bole provided for
the working solution. Rinse the Folin and Ciocalteu’s phenol reagent bole with 10 mL
of water and add the rinse solution to the working solution bole. Add an additional
80 mL of water to the working solution bole and mix well. Store the Folin and Ciocalteu’s phenol reagent working solution at room temperature.
• A protein standard solution (400 µg/mL) is prepared by adding an appropriate volume
of water (5 mL) to the vial. Read the instructions carefully. The exact protein content
of the protein standard vial may be found on a lot-specic Certificate of Analysis. Swirl
gently to completely dissolve the contents.
Storage and Stability
• Store the Lowry reagent solution and the Folin and Ciocalteu’s phenol reagent at room
temperature. Do not refrigerate.
• Store the protein standard solution in a refrigerator (2–8°C) or freezer (below 0°C). The
solution is stable for at least 3 months when stored refrigerated. Discard the protein
standard solution if turbidity develops.
999
Procedure
A. Protein determination without protein precipitation
1. Set up three test tubes (19 × l50 mm), marked blank (B), standard (S) and test (T). If there
are more specimens, mark them as T1, T2, etc.
2. Add 1.0 mL of water in tube marked B, 1 mL of standard (400 ug/mL or 40 mg/dL) in
tube marked S and 1 mL of test specimen in the tube marked T.
3. Add 1.0 mL of the Lowry reagent solution to all the tubes.
4. Mix well by hand or Vortex.
5. Allow solutions to stand at room temperature for 20 min.
6. With rapid and immediate mixing, add 0.5 mL of the Folin and Ciocalteu’s phenol
reagent working solution to each tube.
7. Allow colour to develop for 30 min.
Note If your cuvee requires more than 2.5 mL volume, add an appropriate volume of
water to each tube before reading. Treat standard, blank, and sample/test tubes identically. Then transfer solutions to cuvees.
8. Zero the instrument (colorimeter/spectrophotometer) with the blank in order to make
all measurements of absorbance against the blank.
9. Measure the absorbance of the standards (AS) and test specimen (AT) at a wavelength
540 nm (range 530–560 nm).
10. Absorbance reading is good for 30 min.
Note The absorbance reading is proportional to the amount of protein present.
Calculation
Concentration of protein in specimen (mg/dL) = AT/AS × 40
Concentration of protein in specimen (µg/mL) = AT/AS × 400
1000
Medical Laboratory Technology: Volume 3
Serum Albumin
Albumin is the major protein in serum (about 60%). It is synthesized in liver. The other
protein is globulin. Antibodies are all globulins. Albumin plays important physiological
roles, including maintenance of colloid osmotic pressure and binding of key substances such
as long-chain fay acids, bile acids, bilirubin, haematin, calcium, and magnesium. It has
antioxidant and anticoagulant eects, acts as a carrier for nutritional factors and drugs, and
is an eective plasma pH buer.
Clinical significance
Decreased levels of albumin in serum may be found in certain conditions such as cirrhosis of
the liver and other liver disorders. Loss of albumin due to kidney disorder such as nephritic
syndrome will also lead to a decline of serum albumin level as also with malnutrition,
malignancy, and chronic protracted conditions like ulcerative colitis. Serum albumin is a
reliable prognostic indicator for morbidity and mortality, and protein-losing enteropathies.
High level of albumin may be associated with dehydration.
Normal range
3.5–5.0 g/dL
Specimen
Serum or plasma; use fresh samples and store in refrigerator until use.
Bromocresol purple (BCP) method
Principle
Albumin selectively binds with bromocresol purple (BCP) dye or bromocresol green (BCG)
in a suitable buer to form respective coloured complexes with bromocresol. The intensity of
the colour is measured as absorbance at 610 nm. The absorbance is directly proportional to
the albumin concentration in the sample. We will focus on BCP although the method is same
as in BCG.
The use of bromocresol purple (BCP) albumin assay kit, provided by various manufactur-
ers, has simplied the assay in diagnostic laboratories. The following account is taken from
the technical bulletin published by Sigma-Aldrich. It is designed to measure albumin directly
without any pre-treatment of samples, such as serum, plasma or urine. The optimized formulation substantially reduces interference by other substances (lipids/other proteins) in the raw
samples. It may also be used to measure eects of drugs and other compounds on albumin
metabolism.
The kit may be used for cuvee or multi-well plate assays. The multi-well plate assay uses
samples as small as 5 mL and can be readily automated.
Reagents
These reagents are supplied in the kits:
BCP Reagent 50 mL
Albumin standard (5 g/dL) 1 mL
Instructions for handling the reagents
• Use double distilled water for dilutions.
• Bring reagent to room temperature and shake before use.
• Do not use outdated kit.
• The kit is stored at room temperature or in a refrigerator (2–8°C).
• Store the albumin standard at –20°C. Frozen standards must be thawed completely
before use.
• All reagents must be considered as biohazards.
Routine Biochemical Test Procedures
1001
Supplies needed other than kit
• 96-well plate assay: 96-well at-boom plate—It is recommended to use clear boom
plates for colorimetric assays.
• Spectrophotometric multi-well plate reader.
• For cuvee assay: Cuvees suitable for reading absorbance at 610 nm.
• Spectrophotometer suitable for reading absorbance at 610 nm.
• Automatic micropipeor.
Procedure
The procedure involves addition of a single working reagent (BCP) and 5-minute incubation.
The optimized formulation has greatly enhanced reagent and signal stability.
96-well plate assay
Follow instructions given by the manufacturer.
1. Transfer 20 µL of blank (B), standards (S), and test specimen (T) to appropriate wells.
2. Add 200 µL of BCP reagent and tap lightly to mix. Avoid bubbles.
3. Let stand for 5 min at room temperature (incubation).
4. Measure absorbance at 610 nm (range 590–630 nm).
Calculations
Concentration of albumin (g/dL) = DAT/DAS × 5
Where,
DAS = Dierence of absorbance between standard and blank
DAT = Dierence of absorbance between test and blank
5 = Concentration of albumin in standard (5 g/dL)
Note If the absorbance of a specimen is higher than the absorbance of standard, dilute the
specimen with double-distilled water (ultrapure) and repeat the assay.
Cuvee Assay
1. Transfer 100 µL of water in tube marked B (blank), 100 µL of albumin standard (5 g/dL)
in tube marked S, and 100 µL test specimen in rube marked T.
2. Add 1000 µL (1 mL) of BCP reagent in all and tap lightly to mix.
3. Wait for 5 min (incubation at room temperature).
4. Transfer incubated mixtures to dierent cuvees B, S and T. (Note Matched cuvees
recommended).
5. Zero the instrument (colorimeter/spectrophotometer) with blank (cuvee B).
6. Read absorbance of the standard (cuvee S) at 610 nm.
7. Read absorbance of the test specimen (cuvee T) at 610 nm.
Calculation
Concentration of albumin in serum specimen (g/dL) = AT/AS × 5
Where,
A
A
= Absorbance of test specimen
T
= Absorbance of standard
S
5 = Albumin concentration in standard in g/dL
Conversion factors for albumin: 0.1 g/dL= 15 mM = 0.1% = 1000 ppm
Note If absorbance (at 610 nm) of a test specimen (T) is higher than the standard (S), dilute
the sample with double distilled water (ultrapure) and repeat the assay.
1002
Medical Laboratory Technology: Volume 3
Preparation of Standard Curve
Bring the reagent to room temperature and shake before use. Dilute Bovine Serum Albumin
Standard or BSA (5 g/dL) in double distilled water.
Standard # Amount of Albumin
standard µL (5 g/dL)
1 100 0 5.0 (1 × undiluted)
2 80 20 4.0 (1.2 × diluted)
3 60 40 3.0 (1.7 × diluted)
4 40 60 2.0 (2.5 × diluted)
5 30 70 1.5 (3.3 × diluted)
6 20 80 1.0 (5 × diluted)
7 10 90 0.5 (10 diluted)
8 (blank) 0 100 0 (No standard)
Amount of water added (in µL) to make
up a total volume of 100 µL
Final concentration of
Standard (g/dL)
Note
• Diluted standard may be stored at –20°C for future use.
• The calibration curve must be drawn with every new batch of reagent and must be
made for each individual colorimeter used.
• Calibration curve must be linear (straight line).
• If the specimen does not fall within the linear scale, the specimen should be diluted with
saline and in nal calculation multiply the result by dilution factor.
Additional information
• Bilirubin does not seriously interfere with the test.
• If a slight precipitate is formed in the colour reagent during storage, lter it before use.
The reagent does not lose its sensitivity.
• Saline is not used in diluting the specimen. The addition of electrolytes decreases the
absorbance. Neither bilirubin nor succinate buer seems to interfere.
• In case of low serum albumin levels, the results tend to be high owing to the aachment
of dye to other proteins. The technician should be aware of this problem and the physician should be informed.
Calculation of Globulin Concentration of Serum
Globulin concentration in serum is indirectly determined by subtracting the albumin
concentration from the total protein concentration. The normal globulin concentration in
serum is in the range of 2.3–3.5 g/dL.
Myoglobin
Myoglobin is a protein that is commonly found in the heart and skeletal muscles. The only
time that myoglobin is found in the bloodstream is when injury to a muscle has occurred.
In particular, injury to the heart muscle results in the release of myoglobin. Thus, when
detected through a blood test, the presence of myoglobin is clinically signicant. Currently
developed serum myoglobin blood test is relatively easy to follow that measures the levels of
myoglobin in the blood stream.
Clinical significance
This test is typically ordered when it is suspected that a patient is having a heart aack. Most
of the time, heart aacks are obvious. However, in some cases, a heart aack is not outwardly
clear. Serum myoglobin can be elevated in cases of inammatory and degenerative muscle
diseases and following muscle trauma.
Routine Biochemical Test Procedures
Serum myoglobin test has, for the most part, been replaced by the troponin level test
to provide a positive diagnosis of a heart aack. This is because troponin levels are more
specic to heart damage than myoglobin levels. Additionally, troponin levels will stay higher
than myoglobin levels for longer when there is a heart aack. However, serum myoglobin
is still used in some cases. The test is commonly ordered in conjunction with other tests for
cardiac problems. This is why it is called one of the biomarkers. Other tests associated with
the “biomarker” include troponin, creatine kinase (CK), and creatine kinase-MB (CK-MB).
These will be discussed at appropriate places. Negative results obtained from a myoglobin
test can be used to rule out a heart aack. However, positive results do not conrm that a
heart aack has indeed occurred. In order to diagnose a heart aack, troponin levels must
also be measured.
The serum myoglobin test may also be ordered following the diagnosis of a heart aack.
Once damage to the heart muscle has been conrmed, values obtained from the serum
myoglobin test can help physicians estimate the amount of muscle damage that has occurred.
Serum myoglobin may also be ordered if the patient has symptoms of kidney disease or
kidney failure.
Normal range
0–85 ng/mL
Specimen
Serum, plasma (EDTA or heparin anticoagulated)
1003
ELISA Kit for determination of Myoglobin
The ELISA kit for myoglobin contains 96-well strip plate coated with anti-myoglobin. The
minimum detectable value is 0.30 ng/mL. The kit has high sensitivity and excellent specicity
for the detection of myoglobin. No signicant cross-reactivity or interference between
myoglobin and analogues is noticed. The kit has high linearity and stability.
Principle of assay
The test principle applied in this kit is sandwich enzyme immunoassay which is shown
in Figure 33.3. The microtitre plates (provided in the kit) are pre-coated with an antibody
specic to myoglobin. This is the solid phase. Standards or test samples are then added
for capturing the analyte or antigen (myoglobin). Following incubation, myoglobin (antigen)
sticks to the antibody and is not washed away in the following step. Aer wash, a second
antibody to myoglobin and conjugated to Horseradish Peroxidase (HRP) is added to each
microplate well (detection reagent A). This is then allowed to re-incubate at 37°C. Aer
incubation, TMB substrate solution (detection reagent B) is added and re-incubated. Only
those wells that contain myoglobin, biotin-conjugated antibody and enzyme-conjugatedantibody will exhibit a change in colour. The enzyme-substrate reaction is terminated by the
addition of sulphuric acid solution and the colour change is measured spectrophotometrically
at a wavelength of 450 nm ± 10 nm. The concentration of myoglobin in the samples is then
determined by comparing the absorbance of the samples to the standard.
Reagents
The kit provides all necessary reagents for the test and instruction manual. Strictly follow the
instruction manual while performing the test.
• Pre-coated, ready to use 96-well strip plate
• Plate sealer
• Standard
• Detection reagents A and B
• Substrate
• Wash buer (30 × concentrate)
1004
Medical Laboratory Technology: Volume 3
Figure 33.3 Sandwich enzyme immunoassay
• Standard diluent
• Assay diluents
• Stop solution
Other supplies
• Automatic micropipeors
• Well reader
Procedure
1. Prepare all reagents according to manufacturer’s instructions.
2. Label test samples (T1 T2, etc.) and standards (S) on the plate.
3. Bring everything to room temperature.
4. Add 100 µL standard (S) and test sample (T) to respective well. For blank, use 100 µL of
double distilled water.
5. Incubate for 2 h at 37°C.
6. Aspirate and add 100 µL prepared Detection Reagent A.
7. Incubate for 1 h at 37°C.
8. Aspirate and wash 3 times.
Routine Biochemical Test Procedures
9. Add 100 µL prepared Detection Reagent B.
10. Incubate 30 min at 37°C.
11. Wash and aspirate 5 times.
12. Add 90 µL substrate solution (colour developer).
13. Incubate 15–25 min at 37°C.
14. Add 50 µL Stop Solution (sulphuric acid).
15. Read absorbance at 450 nm immediately.
1005
Troponin
Troponin is another protein present in serum. The normal value of serum troponin is less
than 0.01 ng/mL. It consists of a complex of three regulatory proteins (troponin C, troponin I,
and troponin T) that is integral to muscle contraction in skeletal muscle and cardiac muscle,
but not smooth muscle. An increased level of the cardiac protein isoform of troponin circulating in the blood has been shown to be a biomarker of heart disorders.
Clinical significance
Physicians request for troponin assay in the diagnosis of acute myocardial infarction (AMI).
Raised troponin levels indicate cardiac muscle cell death as the enzyme is released into the
blood upon injury to the heart.Its release paern is similar to CK-MB (4–6 h aer the onset
of AMI). However, CK-MB levels return to normal aer 36–48 h, while levels of troponin I
remain elevated for upto 6–10 days. The level of troponin I is very low in normal healthy
people, and is not detected in patients with skeletal muscle injury. Therefore, troponin I is a
specic marker for the diagnosis of AMI.
Normal values
<0.01 ng/mL
Values > or = 0.01 ng/mL have been shown to have prognostic value.
Specimen collection and preparation
Freshly collected serum is recommended. If delay is expected, store the specimen in refrigerator. Allow the sample to reach room temperature before proceeding. Sodium azide (0.1%)
may be used as a preservative without aecting the test results. If it is used, dilution factor of
the serum should be noted.
Laboratory assay
The Rapid Troponin I test is described here which is based on immune-chromatography
(sandwich immunoassay). It is a one-step test designed for a qualitative determination of
cardiac troponin I in human serum specimens. It is very helpful in the diagnosis of myocardian
infarction under emergency situation in remote areas of developing countries. It is available
from rapid diagnostics as a test strip.
Principle
Rapid Troponin I test is based on sandwich immunoassay. The manufacturer provides a
solid pad on which there is a spot with colour-producing antibody. When the serum moves
through the solid pad through capillary action, c-troponin, if present, reacts with the anti-c
troponin conjugate (antibody) present on the pad and appears as a coloured band on the test
region of the pad. This happens only when the level of c-troponin in the serum is more than
1.5 ng/mL (diagnostic level). Thus, a positive reaction is the formation of a pink to purple
colour, otherwise the spot remains colourless (normal serum). The pad also provides a control
area. If there is no c-troponin I in the sample, the area will remain colourless. The sample,
however, continues to move to the control area by capillary action and forms a pink to purple
colour on the “control” spot. This conrms the test and the results are considered valid.