Manuscript Paper2
Contents
1. enzymatic reactions involved in the assay 3 00 ml of glucose reagent Trinder is added to each of the thirteen test tubes to carry out the reactions Equal concentrations of the glucose reagent are added to ensure standardization of the glucose samples and enough reagents are used to complete the enzymatic reactions Glucose oxidase converts glucose into gluconic acid and hydrogen peroxide The hydrogen peroxide in the presence of 4 Aminoantipyrine and p Hydorxybenzene Sulfonate will oxidize to form a quinoneimine product via the horseradish peroxidase enzyme The quinoneimine dye is used to determine the proportionality of the concentration of glucose in the sample The buffer stabilizers and fillers are additional components added to the mixture to maintain the appropriate pH to carry out the oxidation reaction and maintain the stability of the enzyme and products formed Glucose standards come in six different concentrations 25 50 75 100 150 and 200 mg dL of glucose Each standard consists of 0 1 M sodium phosphate buffer with pH 7 0 The buffer is used to maintain the pH at appropriate levels to undergo the enzymatic reaction 40 uL of deionized water blank varying standard solutions or unknown sample are added at one minute intervals to the appropriately labeled test tubes 1 through 9 Figure 3 Table 1 The test tubes are allowed to incubate for ten minutes to allow sufficient time for the reagents to react before the absorbance A2. 8 Unknown 2 0 332 82 38 x glucose concentration Tube 9 Unknown 1 0 507 117 38 m slope 0 005 Tube 10 Unknown 2 0 119 39 78 Tube 11 Unknown 1 0 162 48 38 Tube 12 Unknown 2 0 367 89 38 Tube 13 Unknown 1 0 522 120 38 slope m 0 005 y intercept b 0 0799 Table 6 shows the values of the glucose concentrations of the two unknown samples derived from both Method 1 and 2 Although the values are not exact when compared they do fall within a relative range of each other with a slight margin of error Table 6 Glucose Concentrations of Unknowns From Method 1 and Method 2 Tube Glucose mg dL Temperature Celsius Absorbance 1 Blank 20 55 0 2 25 0 20 55 0 089 3 50 0 20 55 0 17 4 75 0 20 55 0 252 5 100 0 20 55 0 401 6 150 0 20 55 0 698 7 200 0 20 55 0 938 Method 1 Method 2 8 Unknown2 20 55 0 332 85 77 82 38 9 Unknowni 20 55 0 507 130 99 117 38 10 Unknown2 0 0 119 30 74 39 78 11 Unknowni 0 0 162 41 85 48 38 12 Unknown2 37 5 0 367 94 82 89 38 13 Unknowni 37 5 0 522 134 86 120 38 14 Glucometer Lab There are numerous commercially available glucometers available in today s market that may carry out similar functions but they also have variation in testing and the specific components used to measure the glucose readings The glucometer lab looks at these differences and determines whether certain glucometers are better than others and examines the overall design and biochemistry behind them Tests results are given in
3. Table 7 and Table 8 Verify that the instruments are working correctly and correctly coded with the appropriate test strips before testing can be done The Accu Chek Advantage glucometer use a specific test strip that only works for that particular brand The time and date of testing are input into the monitor before coding can be done with the test strip by turning on the power button and getting to the display board containing the time date and other information pertaining to the glucometer A display check to see if the meter is ready to be tested is performed by coding it with the test strip The test strip contains a code Code 139 that matches the code to be used on that particular glucometer model ensuring that further testing can take place A test strip is inserted in the glucometer according to the manufacturer s direction A blinking drop of blood appears on the display monitor indicating that the instrument is ready to be used and that the test strip is compatible with that particular glucometer The OneTouch Ultra glucometer is coded by matching the code on the meter display with the code on the testing strip vial code associated with it to ensure compatibility In order to perform this test the meter needs to be turned off A OneTouch Ultra test strip is 17 inserted into the appropriate slot to turn on the meter and various segments of the display are shown A code number appears on the display for three seconds and this
4. code is matched with the code on the strip vial Once the system check is done the meter is ready to use Also the display monitor should also have an image of a bolded hypen and mg dL indicating that the instrument is ready Perform the appropriate glucose reading using the glucose standards control solution provided by the manufacturer to determine the accuracy and precision of the system Prior to measuring the glucose mixture using the glucometer both the Accu Chek and One Touch Ultra glucometers a display check is performed to ensure that proper controls are taken A test strip is inserted right side up into the glucometer and upside down and results recorded Test 0 Since no glucose has been added to the strip the display does not indicate a numerical value but shows the blinking drop of blood need to add to the control solution The blinking drop of blood does not appear when the strip is inserted upside down because it s not properly inserted for the system to work Two drops 20 uL of the glucose standard solution are placed on the finger and transferred to the test strip that has been properly inserted into the glucometer Test 1 A glucose reading is obtained with the given amount of control solution on the glucometer as 271 mg dL 15 04 mmol L for the Accu Check monitor and 138 mg dL 7 66 mmol L for the One Touch Ultra Once the results of the readings have been recorded each of the test strip is removed
5. need to be taken into considerations as well as the many residing factors that may influence the final results such as patient health status and environmental conditions 21 ACKNOWLEDGEMENTS I wish to thank the members of my group involved in the experiments for their assistance and contributions to the lab I would also like to thank Dr Yan Zhang Dr Martin Safo and the TA s for their clinical assistance with the labs 22 REFERENCES 1 Mayo Clinic staff 2012 January 25 Diabetes lt http www mayoclinic com health diabetes DS01121 gt 2 PubMed Health Reviewed by A D A M Editorial Team David Zieve MD MHA and David R Eltz 2011 August 31 Diabetes lt http www ncbi nlm nih gov pubmedhealth PMH0002194 gt 3 Safo M Glucometers Lab Spring 2012 VCU School of Pharmacy Richmond VA 4 The New York Times Reviewed by American Accreditation HealthCare Commission Inc 2010 May 10 Diabetes lt http health nytimes com health guides disease diabetes overview html gt 5 Zhang Y Glucose Glucose Oxidase Lab Spring 2012 VCU School of Pharmacy Richmond VA 22
6. stored as energy because their pancreas may not be producing enough insulin or their cells do not respond to the insulin produced leading to insulin resistance 2 There are currently three major types of diabetes Type 1 Diabetes Type 2 Diabetes and Gestationa Diabetes Type 1 diabetes also known as juvenile onset diabetes occurs mostly in children but can affect any age group 1 This particular type requires daily injections of insulin because the body makes little or no insulin of its own Type 2 diabetes results from insulin resistance body rejecting the insulin the pancreas produces and occurs most often in adulthood affecting mostly obese patients Gestational diabetes is high blood sugar that develops during pregnancy in women who normally don t have diabetes 2 Diabetes is becoming more prevalent in today s society especially with the increased obesity rates so patient monitoring is important to ensure that proper measurements and treatments are taken to protect the patients Various blood tests are used for screening and to confirm the diagnosis for diabetes especially for patients with blood levels higher than 200 mg dL 2 Three types of blood tests can be done to measure blood glucose levels each with its own standards of measurement and target ranges to confirm the diagnosis for diabetes They include the fasting blood glucose test Hemoglobin AIC test and the oral glucose tolerance test Patients who are at high risk for
7. uses only 5uL a drop of the glucose test solution instead of the normal 20 uL a 75 decrease in the volume of blood The glucose readings for both the Accu Check and One Touch Ultra monitors are not exactly the same as the other six readings that use the full drop but are still very close only a slight difference This indicates that both of the glucometers are able to detect the small amount of the samples and give an appropriate reading of each Having a larger volume of blood or control solution 20 uL 2 drops ensures that a reading can be obtained and produce consistent results but these 19 glucometers are manufactured in such a way that even a small volume of blood or solution can still be detected and produce similar results User variation may affect the proper use of the glucometers as well as the accuracy of the results Manufacturers recommend that test strips be stored in tightly capped containers away from extreme temperatures and only use glucose control solutions that are specific for that particular brand of glucometer Thus exposing the strips to air could decrease their effectiveness and cause inaccurate glucose measurements Test strips that have been left exposed for 24 hours are tested with the Accu Chek and OneTouch Ultra glucose standard solutions 20 uL and results recorded Test 5 The glucose readings increase significantly for both of the glucometers putting the Accu Chek glucose reading in
8. with the American Diabetes Association consensus statement Both of the glucometers are designed in such a way that they are easy to use and handled by the users When users first use these instruments they need to set up the date and time and make sure that the strip codes match the glucometers codes A code check ensures that the test strip is compatible with a particular brand of glucometer and produce accurate readings The system does a pretty good job in letting the patient know if the strip and the monitor aren t compatible by showing the incomplete symbols on the monitor screen However if the patients doesn t read the manual beforehand they would probably have a difficult time figuring out what the symbols mean and what steps are necessary to perform the system and code checks Therefore it s important to read the user manual first before doing the initial testing to figure out the meaning of the symbols and how to calibrate the glucometer efficiently Both the Accu Chek and One Touch require an initial setup such as IR the time date and display check to make sure that all the necessary components are ready to go before further testing can take place The One Touch Ultra glucometer requires a system check in which users have to put in the code of the test strip prior to doing the testing to ensure compatibility between the strip and the monitor itself The standard curve for the glucose oxidase assay is entered into the glucose m
9. 0564 6 882928273 6 549883357 6 993943245 Table 9 Results of the Readings Using the Manufacturer s Directions and Comparison With Other Groups Accu Chek Advantage Glucose mg dL Glucese mmol L Average 254 3333333 14 11740396 Test Strips 1 42 3 n 3 Std Dev 15 63116545 0 867646699 CV 6 145937 6 145937 Glucose mg dl Glucose mmol L Average 293 57 16 29144716 Test Strips 1 82 3 8 9 10 n 6 Std Dev 45 08991018 2 502827561 HCV 15 36283 15 36283 Table 10 Results of the Readings Using the Manufacturers Directons and Comparison With Other Groups One Touch Ultra Glucose mg dL Glucese mmol L Average 136 6666667 7 586023097 Test Strips 1 42 3 n 3 Std Dev 2 309401077 0 128189048 CV 1 689805666 1 689805666 Glucose mg dL Glucese mmol L Average 129 6656567 7 197470694 Test Strips 1 42 3 8 9 10 n 6 Std Dev 2 309401077 0 457277485 CV 1 781029108 6 353308044 DISCUSSION The proper measurement of glucose in biological fluids is an important component in diagnosing diabetes because it examines the abnormal carbohydrate metabolism in the body Diabetes is characterized by abnormally high concentrations of glucose in physiological 22 fluids due to the body s inability to break down the glucose to useable forms of energy 5 Many commercial glucometers are currently available in the market to measure blood glucose levels but there are sign
10. 2136403 18 87254526 19 03906772 17 48485811 Table 8 Results of the One Touch Ultra Tests Glucometer Model Accu Chek Advantage Control Solution No 92380 Accu Chek Comfort Curve Level 2 HI Control 4 mi Expires 08 26 11 Strip Test Glucose Sample Special Conditions 0 o Strip rightside up so o Strip upside down ut 20 pl none ut 20 pl reread after 60s 2 20 pl none 3 20 pl none 4 Spl none 5 20ul exposed 24 6 20ul different control 7 20ul maltose spiked 8 20 wl from other group 9 20ul from other group o 20ul from other group Glucometer Model One Touch Ultra by Lifespan Control Solution No 9A2A51 Expires 08 26 11 Strip Test Glucose Sample 0 0 0 0 1 20 ul 1 20 ul 2 20 ul 3 20 ul 4 Sul 5 20 ul 6 20 ul 7 20 ul 8 20 ul 9 20 ul 10 20 ul Special Conditions Strip rightside up Strip upside down none reread after 60s none none none exposed 24 h different control maltose spiked from other group from other group from other group 2 Glucometer No Test Strip Lot No glucose mg dL n a 138 Error 137 139 347 139 124 118 126 Other Observations Code 139 match Get a strip symbol with blinking drop of blood Asks for strip No blinking drop of blood SLDC14DGT Expires 5 12 3078441 Control Range 102 136 mg dL glucose mmol L n a n a 7 660033078 n a 7 660033078 7 438003134 7 604525592 7 715540564 19 26109767 7 71554
11. Test Strips Blood glucose meter Blood Glucose Meter and Test Strips The Breakdown On Glucose Enzymatic Oxidation of Glucose Via Glucose Oxidase and the Use of Glucometers in the Measurement of Blood Glucose Concentrations Lauren Flynt Alida Huynh Jessica Lam Rachel Doo Lim and Kristen Nuckles Virginia Commonwealth University School of Pharmacy 410 North 12th Street Richmond VA 23298 0581 ABSTRACT Diabetes is a rising epidemic in the U S affecting millions of people The use of glucometers has become a beneficial aid to these patients to help them monitor their condition on a continuous basis However there s a plethora of commercially available glucometers in the market that have similar functions but also slight differences between them depending on the testing conditions Thus it s important to know how these instruments work and what these differences are in order to make the proper recommendations to patients Two separate experiments are done to examine how glucose oxidase functions within a given glucometer and how certain experimental and instrumental variations may affect the glucose readings at the end The results of the experiments indicate that temperature could affect the final glucose reading so it s one factor that needs to be taken into consideration when doing the tests Additionally the improper use of the test strips and not following the manufacturers recommendations can drastically affect the
12. ar glucometer are that it s difficult to handle since it s so small so an older person would have trouble handling it and that a coding system putting in the actual code is required before to testing can begin to ensure compatibility It s also important to take to consideration that various factors could affect the accuracy of the glucometer such as environmental temperature and humidity which can degrade the enzymes and make them ineffective in the measurement of glucose Certain medical conditions can interfere with the glucose readings such as dialysis A patient receiving EXTRANEAL icodextrin peritoneal dialysis solution causes the metabolism of icodextrin to maltose in the body in vivo Thus their maltose levels can 2N increase significantly in the body and cause elevated glucose readings if the proper glucometer is not used Patients who are receiving this treatment should use the One Touch Ultra glucometer because the instrument uses an enzyme that s glucose specific so the maltose will not interfere with the readings and produce elevated glucose results Accu Chek glucometers are non specific so they will consider the maltose as glucose in the body and provide inaccurate glucose readings Today s market is filled with hundreds of glucometer types that all do pretty much the same thing measure a person s blood glucose level oftentimes as part of their diabetes regimen However there are slight variations between them that
13. ation value of each in relations to the average values According to the results the average concentration values of Unknown are consistently greater than the values of Unknown 2 at all three temperatures Table 4 provides a better picture showing the relationship between temperatures and the calculated values of the two Unknowns in question as well as the absorbance values for each 14 Table 4 Glucose Concentrations of Unknowns Using Method 1 Tube Glucose mg dL Temperature Celsius Absorbance 1 Blank 20 55 0 2 25 0 20 55 0 089 3 50 0 20 55 0 17 4 75 0 20 55 0 252 5 100 0 20 55 0 401 6 150 0 20 55 0 698 z 200 0 20 55 0 938 Method 1 8 Unknown2 20 55 0 332 85 77 9 Unknowni 20 55 0 507 130 99 10 Unknown2 0 0 119 30 74 11 Unknowni 0 0 162 41 85 12 Unknown2 37 5 0 367 94 82 13 Unknowni 37 5 0 522 134 86 The use of a linear regression to determine the concentrations of the unknowns Method 2 A graphical method is another useful way to determine the concentrations of the unknowns by providing a linear regression line of the data to show the relationship between glucose concentration and absorbance The x axis shows the glucose concentrations mg dL of the six standards of measurement with varying concentrations of glucose Test tube 1 7 at room temperature The y axis indicates the absorbance values of the standards that are measured with the spectrophotometer in the experiment The values of the standard glucose conc
14. be used for specific brands that they are specified for 3 An evaluation of the effect of a strip that s been exposed to air for 24 hours and how using different glucose control solutions on different brands of glucometers can affect glucose measurements and the accuracy of the reading The One Touch glucose standard solution is used for the Accu Chek Advantage and the Accu Chek Advantage is used for the OneTouch Ultra test to determine whether or not glucometers require specific control solutions in order to work Glucometer Enzyme Type Affecting Glucose Reading Some glucometers are glucose specific while others are not which can lead to false elevated readings of glucose Glucometers that use GOD glucose oxidase to carry out the enzymatic reactions are normally specific for glucose while those that use glucose dehydrogenase are non specific for glucose and cannot differentiate between glucose and other sugars in the sample leading to possible false readings Two drops 20 u of the spiked maltose control solution instead of the normal glucose solution is used to determine whether or not the glucometers would give an elevated or normal glucose reading An elevated reading above the acceptable range would indicate that the glucometer cannot differentiate between the various sugars in the sample and would not be appropriate to use for patients under dialysis 11 RESULTS Glucose Oxidase Lab Determine if temperature differ
15. containing surface of the meters to determine the intensity of the color change photometric measurement The intensity of the color change is proportional to the concentration of glucose in the blood sample The newer generation of glucometers incorporates an electrochemical cell within the device to measure the integrated current produced by the glucose oxidation reaction electrochemical measurement 3 D glucose in the blood sample is oxidized to gluconolactone by the enzyme Glucose Oxidase GOD found in the test strip The reduced enzyme is then re oxidized by ferrocyanide ion causing a change in the electrical current that indicates the amount of glucose concentration in the sample Figure 2 Most glucometers use glucose oxidase as the primary oxidizing enzyme however some glucometers use strips with different enzymes such as glucose dehydrogenase that carry out the same principle 3 HOCH HOCH HOCH a oH H2 HOCH OH Hoo emote i E n o OH Glucose OH oH 2 oxidase H202 OH A ko 1 HOL H Oxidase g TOR OH H H y i OH a Glucose Gluconolactone h gow K4Fe CN o Anpa l le o Change in Current 202 pai SS Gini CigHipFe Constant Voltage HAN CH OH Figure 2 Glucose Oxidation Figure 1 Electrochemical Reaction Within Glucometers Glucose oxidation reaction is dependent on various variables such as enzyme concentration glucose concentration cofactor concentration time pH and temperature 5 Temp
16. diabetes should take precaution and get screened annually especially if they are overweight BMI greater than 25 for adults and have other risk factors i e family history heart disease high cholesterol levels etc 2 Self testing is an important part of controlling diabetes by allowing patients to monitor their blood sugar levels at home and taking the necessary steps to avoid further complications with the condition Many commercially available glucometers are available to patients to help monitor their sugar levels and determine the appropriate care for the existing condition such as diet exercise and how much medications insulin and glucose lowering pills to take 4 Careful monitoring using glucometers ensures that the patients blood sugar levels remain within the target range and provides important information about trends in blood sugar levels as part of insulin therapy or treatment plan Most modern hand held finger stick glucometers work through enzymatic oxidation of glucose by glucose oxidase The first generation glucometers contain a photometric sensor to measure the current of the glucose oxidation reaction D glucose is oxidized to gluconic acid and hydrogen peroxide via glucose oxidase The hydrogen peroxide reacts with aminoantipyrine and p hydroxysulfonate in the presence of peroxidase to form a highly colored quinoneimine product Figure 1 5 The meters measure the amount of light reflected or absorbed by the dye
17. ences affect enzymatic reactions of glucose glucose oxidation Varying concentrations of glucose are used to calibrate the standard curve in order to determine the concentrations of the two unknowns given Unknown 1 and Unknown 2 Equal volumes 0 04 ml of each of the six standard glucose concentrations and appropriate unknown solutions Unknown 1 and Unknown 2 are added to the designated test tubes at 20 55 C room temperature and allowed to react with the other reagents for 10 minutes After the incubation time the absorbance of all the test tubes are measured by the spectrophotometer and the results are given in Table 2 Table 2 Absorbance Results Tube Glucose mg dL 1 Blank 2 25 0 3 50 0 4 75 0 5 100 0 6 150 0 7 200 0 8 Unknown2 9 Unknowni 10 Unknown2 11 Unknowni 12 Unknown2 13 Unknowni 0 04 ml of the each of the unknowns are added to the corresponding test tubes at appropriate temperatures 0 C and 37 C to determine the effects of temperature on the oxidation of glucose Again the reagents are allowed to incubate for 10 minutes before the absorbance is measured Table 2 Temperature Celsius 20 55 20 55 20 55 20 55 20 55 20 55 20 55 20 55 20 55 0 0 37 5 37 5 17 Absorbance 0 089 0 17 0 252 0 401 0 698 0 938 0 332 0 507 0 119 0 162 0 367 0 522 Standardization of the glucose curve to estimate the concentration of the unknown Method 1 The standard concentra
18. entrations and their corresponding absorbance values are graphed accordingly to form a linear regression Graph 1 From the linear regression an equation of the line is given as well as the R value that accounts for the discrepancy or variability of the data along the line Determining Glucose Concentration of Unknowns y 0 005x 0 0799 0 9 R 0 9907 0 8 07 T06 0 5 2 r 5 m 5 Vertical Value Axis Major Gridlines Unknown 0 4 iaaii a E 03 Linear Unknown 0 2 0 1 Q 0 0 0 50 0 100 0 150 0 200 0 250 0 Glucose concentration mg dl Figure 4 Linear Regression Line of Standard Solutions and Absorbance Values 14 The given equation y 0 005x 0 0799 with y being the absorbance value and x being the glucose concentration can be used to calculate the glucose concentrations of the two unknown samples according to their absorbance values Table 5 that are given from the spectrophotometer readings Similar to the results from Method 1 the glucose concentrations for Unknown 1 and 2 are not consistent with each other which may be due to the differences in temperatures However it can be inferred from the given data that Unknown 2 has a lower glucose concentration than Unknown 1 which is consistent across all three temperatures Table 5 Glucose Concentrations of Unknowns Using Linear Regression Method 2 y 0 005x 0 0799 Absorbance Glucose concentration mg dL y absorbance Tube
19. erature and the verification of the calibration of the instrument are the only two variables that most users have control of when using a commercial glucometer The glucose oxidase lab examines the chemistry behind the use of glucometers specifically at the effects of temperature on the enzymatic reaction of glucose and how a standard curve calibration of varying glucose concentrations can quantitate unknown glucose concentrations in the sample A second experiment looks at the functioning component of the glucometer including its accuracy and precision user variation and differences in the design of the glucometers or test strips that may glucose reading 3 It s important to know the chemistry behind how glucometers work in order to determine which ones are appropriate to use and recommend to patients with diabetes for self monitoring purposes The labs provide a better understanding and evaluation of glucometers currently available in the market to become more familiar with the instruments and to know the appropriate similarities and differences between each one EXPERIMENTAL Glucose Oxidase Lab Reagents Glucose Trinder reagent consists of 4 Aminoantipyrine 0 5 mmol L p Hydroxybenzene Sulfonate 20 mmol L Glucose oxidase from Aspergillus niger 15 000 U L Horseradish peroxidase 10 000 U L Buffer pH 7 0 0 1 Stabilizers and Fillers The Trinder reagent is a composite of all the components necessary to carry out the
20. erature gets too high it can denature the enzyme and prevent it from breaking down the glucose in the reaction so less product would be produced as a result Temperature is an important element that needs to be taken into account when using a glucometer to prevent getting any inaccurate results or readings Test strips should not be placed in extreme temperatures because the enzymes can become denatured or ineffective when measuring the blood glucose Thus all test strips should be placed in a tightly sealed container at room temperature to maintain their effectiveness The second method uses a linear regression line to figure out the glucose concentration of the unknown samples The linear regression line shows the relationship between glucose concentration using the standard solutions and absorbance measured by the 25 spectrophotometer The equation of the linear regression line can be used to determine how much glucose is in the unknown samples by their absorbance values Although this is an appropriate method to use to figure out the glucose concentrations it s also important to consider the variability of the data along the line and account for any discrepancy that might occur The results for this particular experiment have very consistent numbers so the variability is not as great with a R value of 0 9907 close to 1 Hence there should be reduced discrepancy of the final glucose concentrations of the two unknown samples Like the fi
21. es 08 26 2011 with a range of 286 388 mg dL The One Touch Ultra s control solution lot no 9A2A51 has an expiration date of 3 2011 Two drops 20 uL of the glucose standard solution is transferred to the finger and then applied to the test strip siphoning LAB Multiple glucose readings are taken with the test strips being placed right side up and upside down to accurately reflect that patients may not follow the manufacturers directions exactly The correct placement of the test strips inside the glucometers ensures accurate glucose readings and to make sure that the results are consistent with patients base values Additional readings on additional strips are taken with the same glucose stand solution to determine the precision of the readings and whether they are within acceptable range as indicated on the test strip codes For one strip only 5 uL 1 2 a drop of glucose test solution is applied to the strip to see if a small volume of blood would still reflect a reading or if a minimum volume is needed before an accurate measurement can be taken 1N Variables That May Impact Glucose Readings Certain variables such as patient negligence and not following the manufacturers recommendations can lead to inaccurate glucose readings and measurements Manufacturers recommend that test strips be stored in tight containers at room temperature and away from extreme heat or cold and that glucose control solutions should only
22. f the test strips to air and light destroy the enzymes on the test strips so they become ineffective and produce inaccurate results and bad data Surprisingly the glucose readings are still within the acceptable range of the control solution within 2 SD However this can be attributed to the expired products providing inconsistent and inaccurate results or that the enzymes on the strips haven t been fully denatured yet Additionally when only 5 uL of the volume of solution is used the results are still consistent and within 2 SD o the first reading and within the acceptable range some if error is taken into account However when a different control is used the glucose reading increase significantly to go outside the acceptable range for the One Touch Ultra but still within range for the Accu Chek system The lab also examines the effect of the control solutions recommended for use by the manufacturers to determine the precision and accuracy of the glucometer readings whether they are within the acceptable range set up by the manufacturers The acceptable range for each control solution is found in the bottle lab indicating whether the testing is more high HI or low LO levels of glucose The results for the Accu Chek Advantage glucometer tend to be below the acceptable range 286 388 mg dL for that particular HI high control solution This is probably due to the fact that both the control solutions and test strips are expired which can yie
23. from the glucometer and reinserted after one minute 60 seconds and results recorded This test determines whether patients can read an old test strip once it has been 1R removed from the glucometer or that a fresh strip is required for a new reading In both cases the monitor indicates an ERROR message for both of the glucometers meaning that the strips cannot be used once they have been removed from the insert Two additional tests similar to Test 1 using 20 uL of the glucose standard solution are done Test 2 and Test 3 to determine if the results are consistent with each other precision test and if the results fall within the acceptable range established by the manufacturers for that particular control solution The acceptable range for the Accu Chek control solution is 286 388 mg dL and 20 600 mg dL for the One Touch Ultra control solution The glucose readings for the Accu Check using 20 uL of the control solution are below the acceptable range has a more narrow range The readings are for the One Touch Ultra are within the appropriate range has a much wider range Certain glucometers require a minimum volume of blood in order to work and produce a reading Sometimes patient do not use enough blood which can interfere with the readings or give inaccurate results that are not reflective of the actual glucose levels in the body Thus it s important to use enough blood in order to ensure a proper reading on the glucometers Test 5
24. glucose readings Many factors environmental experimental individual etc may affect the glucose measurements when working with these glucometers so special attention need to given to reduce the effects of these conditions Proper counseling is also important to ensure that patients understand how to perform these tests at home and be aware of certain external factors that may affect the results INTRODUCTION Glucose is an essential component of the body s metabolism because it is a source of energy for the body s many functioning systems such as muscle cells and brain tissues Although glucose is a fundamental element in the body too much of it can lead to serious health problems such as diabetes mellitus a group of diseases that affect how the body uses blood glucose Diabetes mellitus is a health condition that affects more than 20 million Americans with over 40 million Americans having pre diabetes early Type 2 diabetes 2 The lifelong chronic disease is characterized by unusually high concentrations of glucose in the blood due to abnormal metabolism of sugars in the body 5 Diabetes is often caused by having too little insulin or resistance to the existing insulin in the body Insulin is a hormone produced by the pancreas to control blood sugars by moving glucose from the bloodstream into fat muscle and liver cells where it is used as fuel for the body 2 Diabetic patients have trouble moving sugars into their cells to be
25. gs Thus all the sugars are assumed to be glucose by the glucometer that can lead to a higher reading of glucose and inappropriate dosing by the patient For test 7 a spiked maltose control solution is used instead of the manufacturers control glucose solution to see if the glucometer is able to distinguish between the glucose and other sugars Accu Chek Advantage uses GDH PQQ as its main enzyme so it s not glucose specific while One Touch Ultra uses GOD glucose oxidase and is more specific for glucose This is reflected in the glucose readings for the two instruments The Accu Chek Advantage has a much lower reading 249 mg dL for the glucose below the range than expected using the maltose spiked solution while the reading with the One Touch Ultra glucometer yields a more appropriate result 139 mg dL that s consistent with the other previous readings Test 8 9 and 10 compares the results with other groups working with the same meter type to determine the consistency and precision of the data 71 Table 7 Results of the Accu Chek Advantage Tests Glucometer No Test Strip Lot No glucose mg dL n a 271 Error 240 252 270 328 249 343 315 8503364095 Expires 12 31 05 Accu Chek Comfort Curve for Whole Blood Used for Accu Chek Advantage meter and Acos Chek Complete Meter 50 pack glucose mmol L n a n a 15 04252873 n a 13 32179666 13 98788649 14 98702124 18 20645543 16 37470839 13 8
26. ic equation varying concentrations of glucose standards and absorbance values to estimate the concentrations of the unknowns It s also important to take into account the effect of temperature on the final results and the discrepancy between the values At room temperature Unknown is 2A calculated to be 130 99 mg dL and 85 77 mg dL for Unknown 2 At 0 C the concentrations decrease significantly with Unknown 1 being 41 85 mg dL and 30 74 mg dL for Unknown 2 At 37 5 C the value for Unknown 1 is 134 86 mg dL and 94 82 mg dL for Unknown 2 The glucose concentrations are not consistent due to the varying of temperatures in which the reactions take place At low temperatures the calculated glucose concentrations of the unknown samples are much lower than at normal temperatures due to the fact that the glucose oxidation reaction might be slower and take longer for the enzyme to fully react with the other reagents At low temperatures the enzymes may not react as fast to break down the glucose and form the highly colored quinoneimine product so the solution is more lightly colored The intensity of the colored product formed is directly proportional to the concentration of the glucose in the sample At high temperatures however the glucose concentrations of the unknowns are much higher because more glucose is reacted with the glucose oxidase enzyme speeding up so more of the quinoeimine product is produced darker color However if the temp
27. ificant variations in each that need to be taken into account Glucometers work by way of enzymatic oxidation of glucose through a variety of enzyme types such as glucose oxidase or glucose dehydrogenase Different enzymes process sugars in the body differently some are more glucose specific than others hence appropriate glucometers are needed depending on the user s medical condition The labs examine the enzymatic variables and factors that may affect enzymatic oxidation of glucose and the variations in the different glucometer types Most enzymatic reactions such as the oxidation of glucose are dependent on numerous enzymatic variables such as enzyme concentration glucose concentration cofactor concentration time pH and temperature 5 When using the commercial glucometers the only variables that the users have much control over are the temperature and verification of the calibration of the instrument The glucose oxidase lab looks at how these variables specifically the temperature and the standard curve calibration can be used to determine the glucose concentrations of a sample and the degree of the concentrations high or low Two different methods are used in the experiment to determine the glucose concentrations of the two unknown samples given The first method uses a standard curve calibration while the second method uses a linear regression to determine the appropriate concentrations The calibration of the standard curve uses a specif
28. ld inaccurate results The test strips have been expired since 12 31 05 and the control solution expired last year 08 26 11 However the manufacturers recommend that all control solutions and test strips that are expired or opened more than 3 months ago need to be discarded because they can compromise the glucose measurements and put them out of range Groups that have their control solutions and test strips not yet expired have more consistent results that are within the acceptable range Test 8 9 10 27 The One Touch Ultra glucometer also has expired control solutions 3 11 but their test strips are valid until 5 12 of this year However the values of the glucose readings are still above the normal range Thus it s important to use control solutions and test strips that haven t yet expired to do the measurements to ensure accuracy and precision For the Accu Chek glucometer the mean value of the expected range for the glucose control solution is 337 mg dL and 119 mg dL for the One Touch Ultra solution The American Diabetes Association allows the glucose measurements using the control solution to be within 10 of the range 257 4 426 8 mg dL for Accu Chek and 91 8 149 6 mg dL for the One Touch Ultra and within 15 of the reference value Based on the glucose readings and the 10 error acceptance both the readings for the Accu Chek and One Touch Ultra instruments would be within the appropriate range and would be consistent
29. ments Glass test tubes pneumatic pipets are used to transfer the reagents into the proper test tubes parafilm to mix the reagents prior to measuring with the spectrophotometer Spectronic 21D measuring at 505 nm to formulate the standard curve calibration Additionally a heated block and ice bath are used to alter the temperatures in which the enzymatic reactions can undergo One of the variables that users of commercially manufactured glucometers have control over is temperature so it s important to see how different temperatures can affect the overall calibration curve of various unknown concentrations Glucometers Lab Glucometers A comparison between two different blood glucose monitoring systems that are commercially available in the market Accu Check Advantage Model No 8503364095 and One Touch Ultra Model No SLDC14DGT One Touch Ultra is specific to glucose because it uses glucose dye oxidoreductase GOD to carry out the oxidation reaction of glucose Accu Check Advantage on the other hand uses dehydrogenase instead of oxidase to oxidize glucose and is not glucose specific Certain glucometers like Accu Check Advantage cannot distinguish between glucose maltose or other sugars and are at greater risk of providing false glucose readings These glucometers usually indicate a higher than normal glucose reading because they can t differentiate between glucose and the other sugars in the blood sample Patients wh
30. ng warranty if registered easy to navigate website If I made a call to the 800 number to ask why I received two different 29 readings in a row and wanted to know which was right I was told that the glucometer might be having some glitches and precision issues I was told to get rid of that glucometer and get a new one to fix the problem I didn t think that was an appropriate answer because I thought they were trying to scam me to buy a product The operator didn t ask for more information and tried to help me figure out the problem instead tried to sell me a new product instead The disadvantage of this system is not it s restricted to the finger only and not other sites and the difficult to recall prior reading difficult to navigate the system and know which buttons to push as well as their poor support system However this system would be appropriate for the elderly who have poor vision and severe arthritis but not for dementia because of the poor memory system The monitor itself provides a good grip and the numbers are big and easy to read The One Touch Ultra glucometer is a slimmer model than the Accu Chek Advantage so would be appropriate for a young student to have on hand takes up less storage space The One Touch glucometer is easy to use and clean up sample can be taken from other sites and not just the finger easy to manage website that addresses FAQ s that patients have and good memory system The cons for this particul
31. o are receiving EXTRANEAL icodextrin peritoneal dialysis solution should not use glucometers that are non glucose specific because it can lead to falsely elevated glucose readings inaccurate insulin dosing and possible health problems such as hypoglycemia 3 Test Strip Each glucometer uses a specific test strip that only works for that particular brand Accu Chek Advantage test strips lot number is 548139 and expire 12 31 2005 The description on the container indicates the following Accu Chek Comfort Curve for Whole Blood Used for Accu Chek Advantage meter and Accu Chek Complete Meter The lot number for One Touch Ultra test strips is 3078441 and expires 5 2012 with a control range of 102 136 mg dL and expires 3 2011 Display and System Check Verify that the glucometer is working correctly by making sure the instrument is correctly coded with the appropriate test strip and that the display is working properly For the Accu Chek Advantage a blinking drop of blood on the monitor indicates that the glucometer is ready to use when the appropriate test strip is placed in the slot Accuracy and Precision of System Determine if the blood glucose reading is within acceptable range as stated on the glucose test strips by using the appropriate glucose standards for each brand of glucometer The control solution for Accu Chek Advantage is called Accu Chek Comfort Curve lot no 92380 with Level 2 HI control of 4 ml and expir
32. of each tube is read on the spectrophotometer Repeat the latter steps for the test tubes 10 through 13 for the ice bath and heated block allowing time for incubation before measuring the absorbance of each Deionized water blank is used as a filler and control for zero glucose concentration in the sample and is applied to the standard curve Figure 3 Test Tubes Containing the Reagents and Standard Solutions or Unknowns Table 1 Standard Solutions of Glucose or Unknown in Appropriate Test Tubes Tube Glucose mg dL Temperature Celsius 1 Blank 20 55 2 25 0 20 55 3 50 0 20 55 4 75 0 20 55 5 100 0 20 55 6 150 0 20 55 Z 200 0 20 55 8 Unknown2 20 55 9 Unknown1 20 55 10 Unknown2 0 11 Unknown1 0 12 Unknown2 37 5 13 Unknown1 37 5 Standard curve Varying concentrations of glucose are calibrated together to form the standard curve that is then used to determine the concentration of glucose of the unknown sample Unknown sample The unknown samples contain a specific concentration of glucose that s determined by forming a standard curve and matching the spectrometric output to the curve Two unknown samples are given in this lab each having its own glucose concentration and are tested in duplicates have two test tubes to ensure consistency The unknown samples are tested in three different temperatures room temperature 20 55 C 0 C and 37 5 C each running in duplicates for a total of six samples Other instru
33. onitoring device by way of using the control solution to determine if the glucose readings and measurements will fall within an acceptable range set up by the manufacturers them The control solutions test for high or low levels of glucose as form of control and standardization A comparison of the standard deviations and percent coefficient of variation YoCV of both of the glucometers indicates the variability of the data obtained from the experiment For the Accu Chek system the average glucose reading using one glucometer is 254 33 mg dL which is way below the acceptable range given by the manufacturer However the SD value is pretty high for this particular value 15 63 so there s a lot of variability in the data obtained However when using the data from other groups with the same glucose control solution the glucose average is much higher 293 5 mg dL and within the acceptable range however the SD has increased significant 45 09 so there s an increase in the variability of the data obtained However the standard deviation and CV for the One Touch are comparable when comparing the two instruments from the other groups The SD value is roughly around 2 31 a low value compared to the Accu Chek system meaning there s less variability in this system and the data is more acceptable in this case Different glucometers have their advantages and disadvantages For the Accu Chek system it is easy to use simple clean up procedure lo
34. rst method the values of the Unknown 1 and Unknown 2 are not consistent with each other due to the differences in temperatures However when the values of the concentrations are compared across Method 1 and 2 the values differ slightly but still within approximate range Although the exact concentrations cannot be known for the samples only the averages can be measured it can be concluded based on the results that Unknown 1 has a higher glucose concentration than Unknown 2 consistent across all three temperatures Again this second method emphasizes the importance of temperature and their effects on glucose readings that should be taken into account when exposing the test strips to high or low temperatures Test strips are highly sensitive to temperature and other factors that may reduce their effectiveness and ability to measure blood glucose in the body The accuracy of the glucose measurements is highly dependent on the proper functioning of the test strips and the glucometer that s associated with them Any variation in the glucometer or test strip such as using a different control solution exposing the strips to air for more than 24 hours or simply not using enough sample for testing can compromise the glucose results readings The glucometer lab looks at the various factors that may affect the sensitivity of the test strips and 2A user variations that may impact the use of the glucometers as well as the measurements Prior exposure o
35. tions of glucose can be calibrated appropriately according to their absorbance results to form a calibration curve which can then be used to figure out the concentrations of the unknown samples Each standard value can be used as a proportionality constant to estimate the unknown but these values need to be corrected for possible error in the evaluation of the unknown samples Glucose Concentration mg dL of Sample Asample Ablank Astandard Ablank X Concentration of Standard The latter equation involves the use of standard values for both the concentrations and absorbance of the standard solutions to figure out the concentrations of the unknowns Asample Absorbance value of the unknown given Ablank 0 if the spectrophotometer is zeroed with deionized water and reagent Astandard Absorbance value of the standard solution Each test tube 8 12 containing the unknowns has to be calculated with each of the six standard glucose solutions from the above equation and then averaged out It s important to note that since each standard concentration contains a small margin of error the value for the unknown concentration is different for each The average is taken to provide a standardization of the all the values received for that particular unknown in reference to all the standards used in the calculations Table 3 Standard deviation calculates the spread or dispersion of the overall data specifically pertaining to the mean or average val
36. to the control range set up by the manufacturer 286 388 mg dL The glucose reading for the One Touch Ultra however is slightly above the range 139 mg dL so it s considered invalid Using an inappropriate standard glucose solution that s not specifically for a certain brand of glucometer not recommended by the manufacturer can also produce inaccurate results A fresh test strip not exposed for 24 hours is tested using a different brand of control solution for each of the glucometers and the results recorded Test 6 The One Touch glucose standard solution is used for the Accu Chek Advantage monitor while the Accu Check stand solution is used for the One Touch glucometers When a different control solution is used to test the glucometers the results increase drastically 295 mg dL for the nn Accu Chek and 347 mg dL for the One Touch Ultra The use of a different control solution puts the glucose reading within the normal control range for the Accu Chek monitor but causes the One Touch instrument to be way above the normal range Some glucometers require a specific enzyme type in order to produce accurate results Some glucometers that use glucose oxidase enzyme in the test strip are highly sensitive and glucose specific Other glucometers that use glucose dehydrogenase are less sensitive because they cannot distinguish between glucose maltose and other sugars in the body which can provide falsely elevated glucose readin
37. ue A low standard deviation indicates that the data given is concentrated around the mean while a high SD implies greater spread of data Hence a low SD value would give a better picture of the 12 data and its distribution in terms of the average that s calculated for all the standards used in the calculation Table 3 Calibrated curve values and concentrations of unknown samples Method 1 Standard Concentrations Tube amp Unknown 2 Tube 9 Unknown 1 Tube 10 Unknown 2 Tube 11 Unknown 1 Tube 12 Unknown 2 Tube 13 Unknown 1 25 mg dL 93 258 142 416 33 427 45 506 103 09 146 629 Glucose concentration of sample mg dL 50 mg dl 97 647 149 118 35 47 647 107 941 153 529 75 mg dL 98 81 150 893 35 417 48 214 109 226 155 357 100 mg dL 150 mg dl 200 mg dL 82 793 126 434 29 676 40 399 91 522 130 175 71 347 108 954 25 573 34 814 78 868 112 178 70 789 108 102 25 373 34 542 78 252 111 3 Average 85 774 130 9862 30 74433 41 853567 94 81633 134 8613 Standard Deviation r 12 71605753 19 4191151 4 557992087 6 204667942 14 05656164 19 99331823 The averages of the two unknowns Unknown 1 and Unknown 2 are not exact and fall within a wide range according to the calculations This is the due to the margin of error of the standards that can yield some discrepancy with the concentrations as well as the standard devi
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