Poster

Poster RESULTS

This poster will be about patient (Mr B)
Dipstick
You do not need to include this dipstick results in the just you can refer to them in the discussion if necessary.
GLUCOSE Positive
• 111
• 20
• 200 GLUCOSE Positive
• 14
• 2.5
• 250
BILIROBIN Positive 1.5 BILIROBIN Positive
KETTONES • 0.15
• 15
Positive KETONES Negative
SPECIFIC GRAVITY Positive
1.005 SPECIFIC GRAVITY Positive
1.005 mmole/L
BLOOD Negative BLOOD negative
PH Positive
5 PH Positive
6
PROTEIN Negative PROTEIN Positive
• 3
• 300 plus
UROBILINOGEN Positive normal
• 3.2
• 0.2 UROBILINOGEN Positive normal
• 3.2
• 0.2
NITRATE positive NITRATE positive
LEUCOCYTES Positive
• LEU/U LEUCOCYTES LEU
positive
PH 3.92 PH 5.02

Biuret assay
1 5 0
2 4 1
3 3 2
4 2 3
5 1 4
6 0 5
QC 2ml 3ml

Results
standards absorbance
1 0.320
2 0.272
3 0.169
4 0.152
5 0.123
6 0.080
QC 0.662

Sample for Mr B
READING1 0.594
READING 0.596
READING 0.614
The correct result for protein biuret
10 0 10
8 28 5
4 6 4
2 8 2
0 10 0
Concentration Absorbance
1 0.362
2 0.290
3 0.225
4 0.180
5 0.117
6 0.037
QC 0.33
This picture should be included in the biuret section

Glucose oxidase assay
Concentration of glucose Mole stock Water volume
0 0 10
2 2 8
4 4 8
6 6 4
8 8 2
10 10 0

Result

Concentration absorbance
0 0.035
2 0.045
4 0.046
6 0.47
8 0.47
10 0.051

Investigation of Assay Characteristics and Designing a Diagnostic Strategy in Biomedical Science

Introduction

Aims of the project:
• To develop an understanding of performance characteristics of biomedical laboratory assays and design experiments to determine these.
• To determine an appropriate course of biomedical laboratory investigation for a patient given a set of clinical details
• To plan and carry out experiments efficiently and accurately and provide evidence that you have done so
• To report and explain your findings

To achieve these aims there are two parts to the project, which you will work in pairs to complete:
• Validating an assay; perform a method evaluation of the glucose oxidase method for determination of glucose
• Analysis of samples from two patients in order to answer questions about their clinical status. You will select the tests you wish to carry out on each patient
from the range available.
These sections don’t have to be carried out in any particular order, in fact you need to plan your own experiments around the availability of equipment, see table
below. You need to check which lab you will be in on each day and plan your work accordingly.
Lab Activity
Multipurpose lab e.g. 834, 826 or 841 Glucose oxidase assay, Biuret assay, dipsticks, pH measurement, creatinine measurement, urea measurement, TLC (TLC only
available in 841/826).
Analytical lab – 821 Analysis of patient samples using FES (FES instruments are only available in 821).
Glucose oxidase assay, Biuret assay, dipsticks, pH measurement, creatinine measurement, urea measurement.

There are a number of methods in this booklet that will be available to you. The methods are guidelines only. You will need to work out what samples you will run, how
you will handle/process your samples, what standards you need to make etc.

Equipment and techniques that are available are below.
Please note that not every test is relevant for each patient. Methods are on the following pages.
• Serum glucose assay – spectrophotometric Glucose Oxidase.
• Urine glucose assay – dipstick.
• Serum protein assay – spectrophotometric Biuret assay.
• Urine protein assay – dipstick.
• pH measurement – dipstick on urine samples / pH meter for serum.
• Urine ketones – dipstick.
• Serum creatinine – QuantiChromTM Creatinine Assay Kit.
• Plasma sodium and potassium by FES.
• Serum urea measurement – QuantiChromTM Urea Assay Kit.
• TLC for drugs

Background Information:
Method Evaluation
Assessing how well an assay performs and whether it is suitable for the analysis required is a fundamental requirement in a biomedical laboratory. For many analytes
there are several techniques or methods that could be used to determine the concentration and there may be a wide range of commercial kits available to choose from. It
is important to be able to determine which is the most suitable for the needs of the laboratory. The performance characteristics of an assay are an important factor in
making this decision together with other issues such as cost and availability.
However, even once a kit has been purchased it may be necessary to perform an ‘in-house’ evaluation to ensure it does what the manufacturer states. Also, if an assay
has been developed within the laboratory it must also be evaluated.
For a typical hospital laboratory assay there are several characteristics of importance. It is clearly important that the assay gives the correct answer. This is
accuracy. For analytes such as blood glucose, potassium and sodium as well as assays for drugs and hormones treatment may well depend on the concentrations in the
plasma. As this may be adjusted according to the result it is clearly important that the result is correct.
Another key characteristic is precision, in biomedical laboratories this is also often referred to as imprecision. This is determining how reproducible the results
are. This is important if a patient is being monitored on a regular basis. Acceptable imprecision is dependent on the analyte in question. It is not appropriate to
have an analytical variation that is greater than the expected clinical variation, for example potassium should only be allowed to vary 0.5mmol/L as its clinical range
is narrow.
An assay may be precise without being accurate and vice versa.
Internal quality control samples (QC) are run each time the assay is performed to ensure that there is accuracy. These control samples contain the analytes in known
concentrations and these must be checked each time to ensure they are giving appropriate results. Over time the variation in QC results can be used to check on overall
imprecision.
Laboratories also run External Quality Assurance (EQA) samples. These are sent to the laboratory by an external organisation such as UKNEQAS. The samples are analysed
in exactly the same way a patient sample would be and the laboratory returns the results to the sending organisation. The laboratory does not know the correct results
until they get a report back detailing how well they have performed on this test. This is an independent way to check that laboratories are giving the correct results.
You will analyse 3 EQA samples once you are happy your glucose assay is working correctly. You must report these results when you present your project.
For many analytes, drugs and other species where measuring a low or trace concentration is necessary it is important to know exactly how low the assay can measure.
This is analytical sensitivity. It is also necessary to know the linear range of the assay i.e. how high can be measured before dilution becomes necessary. Sometimes
clinically it is acceptable to report a value as > or < a set cut-off value but many times an absolute value is required.
The analytical specificity of the assay gives information on whether the assay measures only the analyte of interest. Sometimes specificity is not achieved and it the
responsibility of the biomedical scientist to know what other substances may have interfered with the assay.
Note that it is analytical sensitivity and specificity referred to here; clinical sensitivity and specificity are different measures and will be covered elsewhere in
the course.

Analysis of Clinical Cases
In biomedical science a key skill is the ability to take a given set of information and decide on a course of action or experiments in order to come to some conclusion
about the scenario described. In this project clinical case scenarios will be used. This is of particular relevance to biomedical science where knowledge of the
patient and their background can be useful in interpreting laboratory results. Whilst it may be the patient’s doctor requesting the initial tests it will often be a
biomedical scientist’s role to assist and advise the medical staff on what might be appropriate tests to carry out.

Patient information
Case details:
Patient Mr A is a 9 year old male. He has been drowsy and tired for a few weeks and today was unable to get out of bad and has begun vomiting. He has been brought into
hospital, now barely conscious. His respiration is deep, his blood pressure is slightly low and he has cold extremities. The junior doctor in A& E wants your advice on
the best strategy for laboratory testing for this patient. His parents have noticed that he has recently been drinking more water and also passing lots of urine. They
are also concerned as they found some white powder in A’s room. They have brought the powder with them.
Patient Mrs B is a 67 year old female. She is a known type 1 diabetic and has come in for her routine screening. She attends regularly every 3 months. She has kidney
function and liver function tests carried out as well as assessment of her glycaemic control by measurement of glycated haemoglobin. The results from some of these
tests are presented in the non-laboratory investigation section of the project. At her latest visit she reports feeling tired and the doctor notices she is rather
pale, her blood pressure is also slightly raised. The house officer at the outpatient clinic is concerned and would like to discuss how to investigate further.
The questions you need to answer from your practical and analytical work are:
What condition can you diagnose for patient Mr A?
Is there cause for concern with Mrs B?
Method Evaluation
Design experiments to determine accuracy, imprecision, analytical sensitivity and specificity
Glucose Oxidase Assay
Glucose is not easy to analyse. It has no chromophore and is often found in association with other very similar molecules. For this reason an indirect method of
analysis has been developed which uses the specificity of an enzyme reactions to generate a coloured molecule which may be conveniently measured by spectrophotometry.
The enzyme glucose oxidase (GOD) is used to oxidise glucose to gluconic acid and hydrogen peroxide. The hydrogen peroxide formed is then used to oxidise colourless
ABTS to a coloured product in a reaction catalysed by peroxidase. The amount of coloured product is proportional to the original concentration of glucose in the
sample.
GOD
Glucose + O2 + H2O ———————-> gluconate + H2O2

peroxidase
H2O2 + ABTS ————————> coloured complex + H2O
Reagents / Equipment
Reagent Hazard
Glucose No hazard
Glucose Oxidase (GOD-PERID) reagent
QC sample (Glucose, Low): 2.0 mmol L-1
(1 x 200 mL bottle for class to share) No hazard
QC sample (Glucose, High): 4.5 mmol
(1 x 200 mL bottle for class to share) No hazard
EQA samples (x3) values unknown No hazard
Spectrophotometer
Linear range not stated but known to be less than 10mmol L-1.
Method
1. Prepare a suitable set of standards.
2. Add 25 µl of each your standards to a separate labelled tube. Remember to also prepare a blank.
3. Add 1.0 mL glucose oxidase reagent solution and mix thoroughly.
4. Incubate at room temperature for 25 minutes.
5. Read on a spectrophotometer at 570 nm. Ask the lab demonstrator if you are unsure how to do this.
6. When you have finished tidy your bench area. Pour assay solutions down the sink, Rinse glassware and put in the red trolley.
7. Place used plastic tubes, pipette tips and plastic graduated pipettes in the waste bag on your bench.

REMEMBER TO RUN A QUALITY CONTROL SAMPLE EACH TIME YOU RUN A QUANTITATIVE ASSAY. YOU SHOULD CHECK THIS IS THE CORRECT RESULT BEFORE CONTINUING.
FOR THIS ASSAY YOU MUST ALSO RUN THE 3 EQA SAMPLES AT THE SAME TIME AS ANALYSING THE PATIENT SAMPLES AND REPORT YOUR RESULTS WHEN YOU PRESENT THE PROJECT.
Protein Biuret Assay
The Biuret reaction is a method used for the determination of protein in a sample. The term Biuret refers to the chemical product which occurs when urea is heated to
180C¬. This can react with Cu2+ in alkaline solution to form a violet complex. This reaction also occurs with the peptide bonds of protein giving the same violet
complex at 544 nm, thus allowing the determination of proteins colorimetrically.

Reagents / equipment
Reagent Hazard
Bovine serum albumin No hazard
Biuret reagent
QC sample Protein: 70 g/L No hazard

Spectrophotometer

Biuret assay method
1. Prepare a suitable set of standards from the Bovine serum albumin to cover the range of the instrument.
2. Add 9.0 mL Biuret reagent to 1.0 mL of each of the protein standards (in separate, labelled tubes), mix well and allow to stand at room temperature for 15
minutes. At the same time, add 9.0 mL Biuret reagent to 1.0 mL of your samples, mix well and allow to stand at room temperature for 15 minutes. You should also
prepare a blank sample (i.e. use 1.0 mL water in place of your sample or the standards).
3. Read your samples on the spectrophotometer at 544 nm. As your demonstrator if you are unsure how to do this.
4. When you have finished pour your assay solutions down the sink and rinse the tubes. Place the glass ware in the red trolley at the front of the class.
5. Place your empty cuvettes, plastic tubes, pipette tips and plastic graduated pipettes in the waste bag on your bench.
Measurement of sodium and potassium using Flame Emission Spectroscopy (FES).
You have previously used FES in the Bioanalytical Techniques practical class. FES is almost entirely restricted to the visible region of the spectrum and to elements
which are excited by relatively low temperature flames. Because the intensity of emission is dependent not only on concentration but also on flame temperature it is
essential that a calibration curve is performed at the same time as every analysis. You should therefore prepare all of your standards and samples for each element
before you start to use any of the instruments.
We have two types of instrument that are capable of FES: Flame Photometers (FP) and Atomic Absorption/Emission Spectrometers (AAS/AES). You will only run your analyses
on one type of instrument, however, since you will not know which instrument you will use until the day of the class you need design appropriate standards for both
types of instrument before you come to the lab.

A suitable working range of the FP instruments is: 0 to 10 mmol.L–1 for sodium and 0 to 5 mmol.L–1for potassium. A suitable working range of AAS/AES instruments in
the emission mode is: 0 to 1 mmol.L-1 for sodium and 0 to 0.5 mmol L-1for potassium. This is summarised in the table below:

Instrument Working range of instruments:
For Sodium For Potassium
Flame Photometers (FP) 0 to 10 mmol.L–1 0 to 5 mmol.L–1
Atomic Absorption/Emission Spectrometers (AAS/AES) in AES mode. 0 to 1 mmol.L-1 0 to 0.5 mmol L-1

Reagents
Reagent Hazard
Sodium chloride (powder) No hazard
Potassium chloride (powder) No hazard
QC sample (sodium): 140 mmol L-1 sodium
(1 x 200 mL bottle for class to share) No hazard
QC sample (potassium): 4.5 mmol L-1 potassium
(1 x 200 mL bottle for class to share) No hazard
Ultrapure Deionised water (for making dilutions) No hazard
Method
1. Prepare suitable sets of standards using the sodium and potassium provided. Ensure you make mixed standards (i.e. they should contain potassium and sodium).
Ensure the standards are appropriate for the type of instrument that you will use. It is recommended that you prepare 100 mL of each standard. Remember to rinse all
glassware with ultrapure deionised water before use. You should also dilute the samples at this point if you have planned to do so.

SAFETY NOTE – The chimneys of the Atomic Absorption/Emission and Flame Emission spectrometers are very hot!
DO NOT TOUCH OR PLACE HANDS OVER THE TOP OF THE FLAMES.

2. Ask for a demonstration of the instrument if you are unsure how to use it. Remember to ensure that the instrument is set up correctly for the element that you
want to measure. If you need to set up the instrument, follow the instruction sheet next to the instrument. This will involve selecting the element you want to
quantify, setting the gain using your most concentrated standard, and setting the zero using your blank. When the instrument is set up, ensure that the aspirating tube
is back in the distilled water before you begin your analysis.
3. Aspirate all the standard solutions, working from the lowest concentration to the highest, noting the reading from each in your lab book. Aspirate with
deionised water between each standard to avoid crystallisation of salts on the burner which would adversely affect your results.
4. Aspirate your samples, diluting any that give a reading in excess of the highest standard. Ideally sample readings should be roughly in the middle of the
range of standards.
5. Check your calibration by aspirating and recording the standards again straight away.
6. Reset the instrument and take readings for the second electrolyte of interest, following steps 3 to 6, above.
Before you leave the instrument ensure the aspirating tube is in de-ionised water.
7. Empty all volumetric flasks and beakers into the sink, rinse with plenty of water and place in the red trolley.
8. Place all used glass serological pipettes in the pipette boots at the end of the benches. Any plastic serological pipettes used should be disposed in the large
incineration bins.

Urinalysis Dipstick Screening
The dipsticks that you will use are able to detect several diagnostically important analytes simultaneously. They consist of a plastic strip with reagent pads
attached. The reagent pads have been cleverly designed to produce a standardised colour change to indicate the level of each analyte within the sample.
Reagents
Dipsticks
Patient Urine sample

Method
1. Remove one dipstick from the bottle and replace the cap immediately.
2. Briefly immerse the dipstick in the patient’s sample. As you remove the dipstick from the sample you should draw the edge of the dipstick along the rim of the
tube to remove excess urine.
3. Hold the dipstick horizontally for exactly 60 seconds, then compare the colours of the reagent pads with the corresponding colours on the chart provided with
the bottle.

Use of a pH meter.
Calibrate the pH meter using the standard pH solutions provided:
• Switch on by pressing I/O key.
• Remove cap from electrode with a turning motion. Ensure you only take off the black cap. The glass bulb should still be protected by plastic.
• Place electrode in buffer solution pH 7.0.
• Press CAL key. Display will show CAL1.
• Adjust to pH 7.0 using ▲▼ keys.
• Press the CAL key again. The display will show CAL2.
• Remove electrode and rinse with deionised water.
• Place electrode in second buffer solution (pH 4.0 or pH 10 depending on the range you need).
• Adjust to the desired pH using ▲▼ keys.
• Press the CAL key. The display will return to pH mode for sample measurement.
Thin Layer Chromatography
Thin-Layer Chromatography (TLC) is used in clinical biochemistry for the quick and simple analysis of various substances in urine, serum and occasionally faeces
including sugars, amino acids and drugs. It is also widely used in forensic studies, in pharmaceutical analysis, in organic chemistry and biochemistry, and in many
other fields.
Reagents
Reagent Hazard
Unknown white powder brought by Mr A’s parents Treat as toxic
Ethanol (methylated spirit: denatured with 3-6% methanol)
1% (w/v) solution of Aspirin in ethanol
1% (w/v) solution of Paracetamol in ethanol
1% (w/v) solution of Ibuprofen in ethanol
TLC developing solvent

(hexane : ethyl acetate : ethanol : glacial acetic acid
in the ratio 1 : 1 : 0.1 : 0.02 respectively)

Method
1. Place approximately one quarter of the powder into a 1.5 mL microcentrifuge tube. Add 0.5 mL ethanol and mix to dissolve.
2. Using a pencil (NOT A PEN) and a ruler, prepare the plate for spotting as shown below. Take care not to press too hard and damage the stationary phase.

3. Using a separate spotting capillary tube for each sample, fill the tube by dipping it in the sample and then touch it to the plate on the vertical dash
assigned for that sample. The smaller the spot the better the analysis will be, and must be no larger than 1-2 mm in diameter.

4. Take your TLC plate to a TLC tank (these must be kept in a fume hood due to the hazards of the developing solvent). Pour a depth of 0.5 cm of developing
solvent into the TLC tank, ensuring that the sample spots will not be below the solvent line.
5. Work with another group to carefully place two TLC plates into the tank and then close the lid.
6. Once the solvent has risen to within 1-2 cm of the top of the plates, the plates can be removed from the developing tank. IMMEDIATELY mark the solvent line
with a pencil. Allow the solvent to completely evaporate in the fume hood.
7. Examine both plates under a UV lamp and outline the spots seen with a pencil. The Rf value can then be calculated for each spot and the components of the
unknown samples discovered.
Measuring Serum Creatinine
Creatinine is the most commonly measured marker of renal function. It is derived from creatine and phosphocreatine which are both found in muscle fibres. It is useful
as a renal marker because it is freely filtered at the glomerulus and there is no reabsorption. However a very small amount of creatinine found in the urine (< 10% of
the total) is secreted by the tubules. Serum creatinine is affected by a range of factors including gender, age, muscle mass as well as some drugs. However, as serum
creatinine is largely a function of muscle mass it remains fairly constant in an individual on a day to day basis. An increase in the serum creatinine is indicative of
impaired renal function but it should not be used in isolation to assess function as it can remain within the reference range even though significant renal function
has been lost.
Clearance of creatinine is often measured as an indicator of renal function, since it is released and excreted at a constant rate. To do this accurately requires a
timed urine collection which can be difficult to obtain. Also because of the tubular secretion of creatinine the GFR estimated in this way tend to be an overestimate
sometimes by as much as 40%.
The relationship between serum creatinine and GFR is made non-linear by several variables but these can be corrected for and several equations have been derived which
allow for this calculation of an estimated glomerular filtration rate. There is some data to analyse using these equations in the non-laboratory investigations
section.
Creatinine is usually measured by the Jaffe reaction and an improved version of this is utilised in the kit. This reaction uses picric acid to react with creatinine in
the samples and gives a red colour which can be read at 492 nm.

Reagents:
Reagent Hazard
Creatinine QuantiChrom™ Assay Kit
Contains low concentrations of hazardous substances – handle with care
Kit Content : 500 tests in 96-well plates
Reagent A: 50 mL (contains 0.1-2 % sodium hydroxide)
Reagent B: 50 mL (contains 0.1-1 % picric acid)
Creatinine Standard: 1 mL 50 mg/dL Not hazardous
Quality control sample Creatinine 100 µmol/L Not hazardous

Safety:
• Reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents.
• You should wear gloves and safety glasses at all times.
• Any spillages should be reported to the teaching staff and should be cleaned up and the area disinfected.

Method:
Measuring Serum Creatinine
Equilibrate reagents to room temperature prior to use.
1. Dilute standard to 2 mg/dL by mixing 5 µl of 50 mg/dL standard stock and 120 µl distilled water. Transfer 30 µl of diluted standard into 96 well plate.
2. Load 30 µl of each sample into a 96 well plate.
3. Work out how much working reagent you need and prepare it by mixing equal amounts of reagent A and B.
4. Load 200 µl of working reagent to each used well, quickly. Tap plate lightly to mix.
5. Incubate the plate at room temperature for 1 minute then measure the absorbance at 492 nm using a plate reader. Incubate for a further 4 minutes at room
temperature before measuring the absorbance again.
Calculation
Creatinine concentration of the sample is calculated as

= ODsample5min – ODsample1min x concentration of standard (mg/dL)
ODstandard5min – ODstandard1 min
Conversions: 1mg/dL creatinine equals 88.4 µM, 0.001% or 10 ppm.
Measuring Serum Urea
Urea is the main end product of protein metabolism in the body. Hepatic metabolism of amino acids forms ammonia which is rapidly converted to urea (ammonia is toxic).
Urea is excreted via the kidneys. It is filtered at the glomerulus but is then about 40-60% reabsorbed. Serum urea forms part of the urea and electrolytes profile.
Whilst impaired glomerular function will affect the urea measurement, it can also be affected by the hydration status of the individual. The BioAssay Systems’ urea
assay kit used here is designed to measure urea directly in biological samples without any pretreatment. The improved Jung method utilizes a chromogenic reagent that
forms a coloured complex specifically with urea. The intensity of the colour, measured at 492 nm, is directly proportional to the urea concentration in the sample.

Reagents:
Reagent Hazard
Urea QuantiChrom™ Assay Kit
Contains low concentrations of hazardous substances – handle with care
Kit Content : 500 tests in 96-well plates
Reagent A: 50 mL (contains 10% sulphuric acid and 0.4% o-phthalaldehyde)
Reagent B: 50 mL (contains 22% sulphuric acid and 0.8% boric acid)
Urea Standard: 1 mL 50 mg/dL Not hazardous
Quality control sample Urea 5.5 µmol/L Not hazardous

Safety:
• Reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents.
• You should wear gloves and safety glasses at all times.
• Any spillages should be reported to the teaching staff and should be cleaned up and the area disinfected.

Method: Measuring serum urea
1. Equilibrate samples to room temperature prior to use.
2. Add 5 µl of each sample and standard into a 96 well plate.
3. Work out how much working reagent you need and prepare it by mixing equal amounts of reagent A and B. Use this reagent within 20 minutes of mixing.
4. Using a multi-channel pipette add 200 µl of this Working Reagent to all the used wells and tap the plate lightly to mix.
5. Incubate the plate for 20 minutes at room temperature (incubation time can be increased if samples have low urea levels).
6. Measure the absorbance at 492 nm using a well plate reader.

Calculation
Urea concentration (mg/dL) of the sample is calculated as

= ODsample – ODblank x n x concentration of standard (mg/dL)
ODstandard – ODblank

n = dilution factor i.e. 1 for serum sample which has been assayed directly.

Conversions: 1mg/dL urea equals 167 µM, 0.001% or 10 ppm.
Non-laboratory investigation
The estimated glomerular filtration rate or eGFR is becoming a widely used measure of glomerular function and allows calculation based on the serum creatinine levels.
There are various equations which will calculate this, one of the more commonly used ones in the UK is the Modification of Diet in Renal Disease study group formula.
This is usually referred to as the MDRD equation:
eGFR = 32788 x serum creatinine -1.154 x Age -0.203 x [ if Black 1.212] x [if female 0.742] There are other equations available such as the Cockcroft-Gault although this is more commonly used in the USA.
Serum creatinine levels for Mrs B’s last 4 clinical visits are provided. Age 67
12 months ago 90 µmol/L
9 months ago 95 µmol/L
6 months ago 120 µmol/L
3 months ago 140 µmol/L

Mrs B’s diabetes has also been monitored over this time period by measuring her glycated haemoglobin
12 months ago 4.8 %
9 months ago 5.0 %
6 months ago 7.3 %
3 months ago 8.5 %

FOR YOUR ASSIGNMENTS TO BE DONE AT A CHEAPER PRICE PLACE THIS ORDER OR A SIMILAR ORDER WITH US NOW