WO1998056719A2 - Control standards for clinical chemistry assays - Google Patents

Abstract

Stabilized compositions for use in clinical chemistry assays are disclosed. The composition is stable in the liquid form. The composition minimizes the use of human derived starting materials and uses recombinant thermophilic enzymes as a substitute for native enzymes commonly used in chemistry controls.

Description

CHEMISTRY CONTROL

Field of Invention The present invention relates generally to clinical chemistry. In particular, it relates to a stable liquid clinical chemistry control matrix for analytes .

Background of the Invention

The determination of the presence or concentration of certain constituents or analytes is useful in the diagnosis of disease and physical well- being. Compositions which behave similarly to how constituents present in human bodily fluids (e.g. blood, spinal fluid or urine) behave are used in clinical laboratories. These compositions assist in the determination of whether the clinical instrumentation and procedures used by the laboratory to measure the constituents are accurate. These compositions are also used to calibrate the clinical devices which measure the amount of constituent in a sample. These compositions will be referred to hereinafter as control compositions. There are many requirements that an ideal control composition should meet. First, it is clear that the analytes' concentration in a control composition should not change substantially over time (e.g. - be stable) . In addition, it is important that the analyte or analyte analog present in the control composition behave similarly to the corresponding analyte present in a patient ' s bodily fluid. Also, the control composition should accommodate as wide a variety of analytes as possible. It is also important that the control function on the majority of instruments and procedures available to determine each analyte. Further, liquid controls are preferred over frozen or lyophilized control compositions.

Often, however, lyophilization or "freeze- drying" processes assist in stabilizing analytes. U.S. 3,466,249 and U.S. 3,629,142 both discuss freeze-dried blood products for use as reference standards . Improved lyophilized serum-based compositions and method have been disclosed including using alkylene polyols to reconstitute the product (U.S. 4,121,905 and 4,189,401), or addition of amine chlorides (U.S. 4,324,685). However, the reconstitution process leads to irreproducibility and error. Liquid based products remove the irreproducibility and error that arises from reconstitution.

The analytes in the control compositions must mimic the analytes in normal human serum or urine. This is important to ensure that results read off a standard curve generated with the control composition are accurate when comparing the results to the actual biological milieu.

Many previous liquid control compositions include human or animal serum or urine to mimic the patient sample, however this provides very little stability to the composition and the preparation of the control composition is often accompanied by cumbersome procedures. For instance U.S. 3,682,835 discloses a liquid blood serum control sample. The blood serum must be treated with a strong acid cation exchange resin to reduce certain cation levels. Then the resin treated serum is dried under vacuum. Next fat solvents such as Freon are used to extract lipoproteins . Further, it is disclosed that such control standard is only stable for up to three months at about 4° C. U.S. 4,324687 discloses a blood control comprising aqueous red blood cells which have been stabilized with treatment with aldehyde and saline.

Some liquid chemistry control products are commercially available. They include Dade® Moni-

Trol® Liquid Chemistry Control, a serum based product sold by Dade International Inc.. That product, when unopened and stored at 2-8° C, is stable for sixty (60) days from the date of receipt by the laboratory and when opened is stable for fourteen (14) days when stored tightly capped at 2-8°C. Another liquid based product is Liquichek™ Chemistry Control, a human serum based product sold by Bio-Rad Corp. To retain stability that product, when unopened, must be stored frozen at -10° to -20° C and when opened is stable for fifteen days when stored tightly capped at 2-8°C.

Other problems are associated with the use of serum or urine in control compositions . Because of the variability of the source, serum and urine pools add an inconsistency or irreproducibility to the product. Moreover, use of normal or processed human serum or urine presents health issues to both clinicians and manufacturers. Thus, a matrix which lowers health risks is also highly desirable. In addition, the use of serum or urine in a control adds turbidity to the product. Thus, many methods have been devised to overcome this disadvantage. For instance, U.S. 4,438,202 provides a method for improving optical quality of serum using acid treatment, U.S. 4,626,511 discloses adding lipase, non-ionic surfactants and cylodextrins to blood, serum or plasma and U.S. 4,716,119 discloses adding such compositions as methanol, sodium acetate, triethanolammonium acetate and others to the serum to overcome problems of turbidity. Non-serum containing controls for certain analytes or classes of analytes have been disclosed. However, the number of analytes in these controls is limited. One reason is that some analytes in a control composition may require a different set of conditions to remain stable over time and to behave similarly to the corresponding analyte in bodily fluid than other analytes. For instance U.S. 4,289,649 discloses an alcohol-free lipid standard comprising water, a lipid, a lipid-solubilizing non- ionic detergent and ionic detergents. U.S. 4,677,075 discloses a urobilinogen control which includes concentrating urobilinogen in ethylene glycol and diethylamine, diluting the concentrated urobilinogen with ethylene glycol in combination with a small amount of protein, preferably adding a reducing agent a chelating agent, anti- fungal agents and antibiotics, filling the control in ampoules, purging with nitrogen and heat sealing. U.S. 4,935,373 discloses a reference liquid for quality control for electrodes which measure calcium and pH. The control comprises a Goods Buffer, water, a source of calcium ion and a salt. U.S. 5,028,542 discloses a non-serum based control for glucose including glucose, water, and polystyrene sulphonate . Thus, often many different base matrices need to be manufactured for different analytes or classes of analytes.

It would be desirable if a base matrix could be prepared wherein such base matrix imparted stability to a wide variety of analytes. In addition, it would be useful to have a base material that could accommodate and stabilize many different analytes in the liquid state and preferably contains minimal amounts of human derived analytes or ingredients . In addition to the difficulties of the base matrix being able to stabilize many different analytes, particularly in the liquid state, an additional difficulty is presented by the wide variety of methods and instruments that are used in clinical laboratories. It is desirable that the control material function in the majority of methods and instruments that are used in the laboratory. Thus, increasing the number of instruments or procedures that the control composition can be used with increases the value of the control, but increase the difficulty of formulation.

In many diagnostic assays, non-specific binding of the analyte to the test surface (e.g. solid support such as test tubes, paper, slides, etc.) must be minimized in order to keep calibration accurate and eliminate any risks of "discrepant" results.

Thus, the non-specific binding of the analyte in a matrix must be minimized and must be the same as the non-specific binding of samples. It is also important that during storage, the analytes do not appreciably bind to the storage container.

There are instances when an analyte analogue may be more desirable than the actual analyte. If an analyte analogue is used instead of the analyte, the binding of the analogue must mimic the binding of the analyte. Particular care must be used when selecting analogues for proteins because the binding of the analogue must mimic that of the protein. Thus, any conformational dependence of the protein for the binding site must be maintained in the analogue. In addition, stability of the analogue should be the same or greater than that of the analyte. Again, the stability of the protein analyte may be related to its conformation. Finally, if an analogue is substituted for an analyte, it is desirable that the analogue be more readily available or be more stable than the analyte or be from a non-human source. Summary of the Invention

The present invention is a defined base material useful as a control matrix for containing and stabilizing analytes or analyte analogs . The base material or matrix comprises a stabilizing protein, a means for maintaining a low protease activity, a means for maintaining the viscosity, a buffer to maintain the pH at 5-8, and a means for substantially obtaining and maintaining anoxia and/or antioxidants. The base material may contain primary amino acids or multi-peptides in excess of normal patient concentration, a means for maintaining the osmolality, anti-microbial agents and anti-fungal agents and other stabilizers specific for certain analytes .

The defined base material may be utilized to prepare controls containing a wide variety of analytes. For instance a chemistry control including analytes such as acetaminophen, acid phosphatase, albumin, bilirubin, cholesterol, total CK, Folic acid, glucose, lactate, magnesium, sodium, theophylline, TSH, and vitamin B-12 may be prepared. The chemistry control has minimal amounts of human derived material .

In addition, the present invention includes the use of certain stabilized analytes or analyte analogues or classes of stabilized analytes or analyte analogues in a chemistry control and methods to stabilize particular analytes.

The resulting solution is stable at 2-8° for extended periods in the liquid state, but can also be stored frozen or can be lyophilized if appropriate fillers are included. Brief Description of Figures

Figures 1-19 show the stability of various analytes in a liquid chemistry control.

Figure 1 is Liquid Chemistry Control, the Analyte is Acetaminophen, Level 1, the Temperature is 4°C. The information is as follows:

Y- inter X-coeff Rsq Pass/Fail 10% 41.447748 (0.04679) 0.524105

Day Value %Recov Y Predict Days to failure

99.9% 99.86 213.7

99.9% 99.72

99.6% 99.39

99.2% 99.02

95.8% 97.75

98.4% 97.05

Figure 2 is Liquid Chemistry Control, the Analyte is Albumin, Level 1, the Temperature is 4°C. The information is as follows :

Y- inter X-coeff Rsq Pass/Fail 10% 2.8845059 (0.04661) 0.358503

Day Value %Recov Y Predict Davs to failure

100.5% 99.86 214.6

97.1% 99.72

100.5% 99.39

100.5% 99.02

97.1% 97.76

97.1% 97.06

Figure 3 is Liquid Chemistry Control, the Analyte is Alkaline Phosphatase, Level 1, the Temperature is 4°C. The information is as follows:

Y- inter X-coeff Rsq Pass/Fail 10% 138.00941 (0.03320) 0.021779

Da-. Value %Recov Y Predict Days to failure

102 2% 99.90 301.2

100 7% 99.80

101 4% 99.57

95 .6% 99.30

89 .8% 98.41

105 .1% 97.91

Figure 4 is Liquid Chemistry Control, the Analyte is ALT, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 67.881022 (0.14623) 0.829669

Day Value %Recov Y Predict Davs to failure

97.2% 99.56 68.4

100.2% 99.12

98.7% 98.10

98.7% 96.93

91.3% 92.98

91.3% 90.79

Figure 5 is Liquid Chemistry Control, the Analyte is Amylase, Level 1, the Temperature is 4°C. The information is as follows :

Y-inter X-coeff Rsσ Pass/Fail 10% 224.38864 (0.01832) 0.127877

Day Value %Recov Y Predict Davs to failure

98.0% 99.95 545.7

101.6% 99.89

99.8% 99.76

99.8% 99.62

99.4% 99.12

98.5% 98.85

Figure 6 is Liquid Chemistry Control, the Analyte is AST Native, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 65.961573 (0.41119) 0.977155

Day Value %Recov Y Predict Days to failure

100.1% 98.77 24.3

95.5% 97.53

94.0% 94.65

92.5% 91.36

81.9% 80.26

72.8% 74.09

Figure 7 is Liquid Chemistry Control, the Analyte is Bilirubin, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 2.3301872 (0.10674) 0.182079

Day Value %Recov Y Predict Days to failure

97.8% 100.00 93.7

96.1% 100.43

103.9% 100.75

103.9% 101.49

106.0% 102.24

101.7% 104.48

Figure 8 is Liquid Chemistry Control, the Analyte is Carbamazepine , Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 6.619842 (0.06287) 0.057875

Day Value %Recov Y Predict Davs to failure

104.8% 100.19 159.1

98.0% 100.38

98.3% 100.82

97.1% 101.32

113.3% 103.02

98.0% 103.96

Figure 9 is Liquid Chemistry Control, the Analyte is Cholesterol, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 238.16424 (0.01087) 0.059484

Day Value %Recov Y Predict Days to failure

98.7% 99.97 920.3

100.4% 99.93

99.5% 99.86

101.6% 99.77

99.1% 99.48

99.1% 99.32

Figure 10 is Liquid Chemistry Control, the Analyte is C02 , Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Pass/Fail 10% 21.499305 (0.04518) 0.019859

Day Value %Recov Y Predict Davs to failure

92.6% 99.86 221.3

104.2% 99.73

106.0% 99.41

92.1% 99.05

107.4% 97.83

90.7% 97.15

Figure 11 is Liquid Chemistry Control, the Analyte is Creatinine, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 2.6844947 (0.02587) 0.043065

Day Value %Recov Y Predict Days to failure

104.3% 99.92 386.5

96.9% 99.84

100.6% 99.66

96.9% 99.46

96.9% 98.76

100.6% 98.37

Figure 12 is Liquid Chemistry Control, the Analyte is Ethosuximide, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 34.555183 (0.01381) 0.002447

Day Value %Recov Y Predict Days to failure

110.2% 99.96 724.0

91.7% 99.92

99.4% 99.82

96.7% 99.71

100.5% 99.13

Figure 13 is Liquid Chemistry Control, the Analyte is GGT, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 66.560834 (0.04258) 0.349808

Day Value %Recov Y Predict Davs to failure

97.7% 99.87 234.8

100.7% 99.74

100.7% 99.45

99.2% 99.11

99.2% 97.96

96.2% 97.32

Figure 14 is Liquid Chemistry Control, the Analyte is Glucose, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 150.15046 0.18206 0.908094

Day Value %Recov Y Predict Davs to failure

101.2% 100.55 54.9

102.6% 101.09

101.9% 102.37

101.2% 103.82

109.2% 108.74

111.9% 111.47

Figure 15 is Liquid Chemistry Control, the Analyte is Iron, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 103.17242 (0.00022) 0.000003

Day Value %Recov Y Predict Days to failure

97.9% 100.00 46045.9

97.9% 100.00

100.8% 100.00

105.6% 100.00

97.9% 99.99

99.8% 99.99

Figure 16 is Liquid Chemistry Control, the Analyte is lactic acid, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 20% 1.0568676 0.15901 0.210060

Day Value %Recov Y Predict Davs to failure

104.1% 100.48 125.8

94.6% 100.95

94.6% 102.07

113.5% 103.34

113.5% 107.63

104.1% 110.02

Figure 17 is Liquid Chemistry Control, the Analyte is LDH, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 315.63097 (0.03042) 0.301952

Day Value .Recov Y Predict Days to failure

99.8% 99.91 328.7

98.2% 99.82

101.4% 99.60

99.8% 99.36

97.9% 98.54

98.2% 98.08

Figure 18 is Liquid Chemistry Control, the Analyte is lipase, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 964.12648 (0.03486) 0.009699

Day Value .Recov Y Predict Davs to failure

92.5% 99.90 286.9

96.6% 99.79

98.8% 99.55

112.5% 99.27

105.3% 98.33

88.9% 97.80

Figure 19 is Liquid Chemistry Control, the Analyte is NAPA, Level 1, the Temperature is 4°C. The information is as follows:

Y-inter X-coeff Rsq Pass/Fail 10% 4.8585628 (0.11122) 0.147388

Day Value %Recov Y Predict Davs to failure

95.7% 99.67 89.9

94.7% 99.33

109.9% 98.55

96.1% 97.66

91.8% 92.99

Detailed Description of the Invention

The present invention is a defined liquid base material useful as a control matrix for containing and stabilizing analytes or analyte analogs comprising, stabilizing protein, a means for maintaining a low protease activity, a means for substantially obtaining and maintaining anoxia, antioxidants, a means for maintaining the viscosity, and a buffer to maintain the pH at 5-8.

The present invention may contain a means for maintaining the osmolality, primary amino acids or multipeptides in excess of normal patient concentration, anti-fungal and anti-microbial agents and other stabilizers specific for certain analytes.

The stabilizing proteins may be one or a combination of albumin, globulins, ovalbumin, caesin or gelatin. Since albumin and total amount of proteins are often measured for routine chemistry tests, it is preferred to include a protein in addition to albumin to create a differential in the measurement between total protein and albumin. It has been found that it is most preferred to include binding globulins such as IgG as the stabilizing proteins besides albumin. Surprisingly it was found that the use of binding globulin not only provides the differential for measurement, it also enhances the stability of certain native enzymes such as native prostatic acid phosphatase. It is noted that the same effect is not seen for recombinant acid phosphatase. See Table 1 which shows the stability of recombinant acid phosphatase (rAcP) and native prostatic acid phosphatase (PAcP) in the base material described in Example 1 (excluding cholesterol and cyclodextrin) with and without IgG. The IgG is tested from multiple sources. Results were obtained on a Dade Dimension® Analyzer, available from Dade International Inc.

Effect of IgG on rAcP and PAcP at 4C° in Base Material

% Recovery rAcP O Time Day 1 Day 4 Day 7 Day 14

BSA (4.51 u/L) 100 100 100 104 105

BSA/lgG (lntergen)(4.23 u/L) 100 31 0 0 0

BSA/lgG (Desert Biol.)(6.12 u/L) 100 100 97 76 58

BSA/lgG (Bioresource) (5.78 u/L) 100 61 16 0 0

Recovery

PAcP O Time Day 1 Day 4 Day 7 Day 14 Day 21 Day 28

BSA (5.27 u/L) 100 88 88 89 92 95 98 BSA/lgG (lntergen)(5.46 u/L) 100 89 89 87 90 91 103

BSA/lgG (Desert Biol.)(4.60 u/L) 100 90 88 91 98 97 120 BSA/lgG (Bioresource)(4.41u/L) 100 92 91 91 93 103 104

Ef fect of IgG on rAcP and PAcP at 25° C in Base Material

% Recovery rAcP O Time Day 1 Day 4 Day 7 Day 14 Day 21

BSA (4.51 u/L) 100 94 85 72 58 33

BSA/lgG (lntergen)(4.23 u/L) 100 0 0 0 0 0

BSA/lgG (Desert Biol.)(6.12 u/L) 100 82 0 0 0 0

BSA/lgG (Bioresource)(5J8 u/L) 100 0 0 0 0 0

PAcP O Time Day 1 Day 4 Day 7 Day 14 Day 21

BSA (5.27 u/L) 100 67 46 48 50 29

BSA/lgG (lntergen)(5.46 u/L) 100 84 84 89 93 105

BSA/lgG (Desert Biol.)(4.60 u/L) 100 88 85 79 87 56

BSA/lgG (Bioresource)(4.41 u/L) 100 94 97 112 134 256 TABLE 1 The source of the stabilizing protein is irrelevant although recombinant or bovine proteins are preferred. Preferably the stabilizing protein is substantially protease free.

The concentration of the stabilizing protein may be anywhere from between 1% to over 12%. If concentrations over 12% are employed salts should be added to improve the filterability of the resulting solution. The amount of stabilizing protein in a control that mimics low levels of total protein and/or albumin is necessarily lower than those controls that mimic normal and high amounts of total protein and/or albumin. It is preferred that the solution have low protease activity. Protease activity can be minimized by both physical methods such as pH treatment or chemical methods such as addition of specific protease inhibitors. Protease inhibitors such as aprotinin and "Protease Inhibitor" (Sigma) are effective. They may be added and may be used at the manufacturer's recommended concentration. Examples of other protease inhibitors include benzidine, (2S, 3R) -3 -Amino-2 -hydroxy- 5- methylhexanoyl] -Val-Val-Asp (Amastatin-Sigma) ,

[2S,3R] -3-Amino-2-hydroxy-4- [4-nitrophenyl] -butanoyl- L-leucine, Antipain, [2S, 3R] -3 -Amino-2 -hydroxy-5 - methylhexanoyl] -Val-Val-Asp (Epiamastatin-Sigma) , ( [2R,3R] -3-Amino-2-hydroxy-4-phenylbutanoyl) -L- leucine (Epibestatin-Sigma) , Foroxymithine, Acetyl- Leu-Leu-Arg-al (Leupeptin-Sigma) , 4-Amino-3 -hydroxy- 6 -methyl-heptanoic acid, 4 -Amino-3 -hydroxy-6- methylheptanoic acid, N- (α-Rhamnopyranosyloxy- hydroxyphosphinyl) -Leu-Trp and phenyl methane sulfonyl fluoride (PMSF) . It is most preferred that the means to provide a substantially protease free solution is to use substantially protease free proteins or recombinant analytes. Since albumin is included in the most preferred embodiment of the present invention, it is most preferable to use albumin that is substantially protease free. If IgG is also included it should also be substantially protease free.

Protease activity may be measured by certain assays such as the Peptag® assay. These assays have picomolar sensitivity to proteases. It is desirable that the control composition have less than picomolar levels of protease activity.

A means for adjusting the viscosity must be utilized because certain analytes such as purified proteins tend to increase the viscosity. The viscosity may be adjusted by adding certain high molecular weight compounds. Compounds that can be used to adjust viscosity include low molecular weight compounds such as sugars and glycols . Certain detergents such as Tween-20 and Tween-40 and the octylglucosides are useful to adjust viscosity. The Brij class of detergents is to be avoided if a control is to be used on certain of the dry film chemistry technologies . In addition palmitates and nonphenols are also to be avoided. Preferably the means to adjust viscosity is Tween-20.

Viscosity is often adjusted qualitatively, although it can be measured. The compound used to adjust the viscosity may be added in the range from 0.01% to 2%, and most preferably at about 0.10% to 1.0%. For instance, Tween-20 may be added in the range from 0.05-0.5%, and most preferably is added at 0.1%. A means for substantially obtaining and maintaining anoxia is required. Anoxia is substantially obtained and maintained by degassing the matrix, adding biocatylitic oxygen reducing agents and/or by adding other oxygen scavengers. Roughly, 8 ppm oxygen is saturation. The oxygen in the control should be less than 1 ppm, and is preferably less than 0.1 ppm.

Oxygen reducing agents remove dissolve oxygen. Biocatylitic oxygen reducing agents are prepared from microorganism cell membrane extracts. The crude extract contains lactate oxidase complex. Glucose oxidase and catalase were also found to produce oxygen-free solutions. Jacobson, et al . , Partial Purification of an Oxygen Scavenging Cell Membrane Fraction for Use in Anaerobic Biochemical Reactions, Biotechnology and Applied Biochemistry, 9 , 368-379 (1987) .

One such biocatylitic oxygen reducing agent, prepared from e. coli is EC Oxyrase® oxygen reducing agent available from Oxyrase, Inc. The cell extract is filtered to obtain a suspension of 0.2 microns or less. The commercially available material has an activity greater than 30 units/mL. See also, U.S. 4,476,224; U.S. 4,996,073 and U.S. 5,240,853. If any biocatylitic oxygen reducing agents are used, they should be highly purified and should not interfere with the determination of any analytes. The preferred means to substantially obtain and maintain anoxia is by degassing the resultant solution. In addition, antioxidants are required if certain analytes such as bilirubin are included. The antioxidants that are useful include ascorbate, phenol, glutathione, vitamin E, BHT, and BHA. The most preferred antioxidant is ascorbate, added as ascorbic acid. The concentration of ascorbic acid is preferentially 0.25 mM to 1.25 mM. The buffers may generally be any of the Good's buffer that function in the pH range of 5 to 8. Of these buffers, the buffers that are preferred function are in the pH range of 6-8. The concentration of buffer is between 10 mM to 200 mM. It is preferred to keep the buffer concentration lower - in the range of 20-100 mM. The preferred buffer is bis Tris propane.

The means for maintaining and adjusting the osmolality should maintain the osmolality for a chemistry control preferably between about 100-600 mOsm/kg, more preferably between about 150-500 mOsm/kg, and most preferable between about 200-400 mOsm/kg. Albumin, casein, salts and sugars all tend to increase the osmolality. Thus, the osmolality may need to be adjusted, maintained and regulated. If necessary, the osmolality is maintained by adding one or more of such components as dextrose polymers, such as isomaltodextrin, starch, hydroxyethyl starch, collidon (polyvinyl pyrollidone) , straight chain polysaccharides or other high molecular weight complexes .

The selection of osmolites can be limited by the analytes and test method that a laboratory will use to determine the analyte . For instance sorbitol and mannitol often interfere with the determination of common analytes . The amount of osmolites to be added can vary depending upon the type of osmolite added and the osmolarity of the initial solution. The osmolarity may be measured using standard methods until the desired level is achieved.

It has been found that certain of the compounds used to adjust osmolality also adjust viscosity. These compounds are such high molecular weight compounds like maltodextrin and collidon (PVP) . Using the same compound to adjust osmolality and viscosity simplifies the manufacturing process.

A means to retard deamination of the composition may be included. Thus, primary amino acids and multipeptides are added in excess of the amounts found in a normal patient, which is less than about 0.5%. Examples of these primary amino acids include glycine, lysine, glyclyglycine, polylysine, serine, alanine, leucine, valine, threonine, arginine, and isoleucine. The amount of primary amino acid in the composition may be from 0.2 to 5%, preferably from 0.5 to 2%.

Serum may be included if desired, but in the most preferred embodiments serum is omitted because of the problems associated with serum.

Anti-microbial and anti-fungal agents are added to prevent growth and may include those commonly found in the prior art at the concentrations found in the prior art . In a preferred embodiment, the prepared base matrix is substantially free of human derived products or materials.

A chemistry control should have many of the analytes listed in Table 2. Generally controls are found at more than one level. Three levels are preferred, representing clinically abnormal or sub- therapeutic low levels of analytes, clinically normal or therapeutic levels of analytes, and clinically high levels of analytes . Some examples of what the levels can be are also found in Table 2. Prior to filling it is preferable to sterile filter each solution. It is convenient to fill small vials (e.g. 2-30 mLs) with the different concentration levels of each control . It should be understood that apparent amounts of an analyte may vary depending upon the method and/or instrumentation used by the laboratory. In addition, when possible it is preferred that the analyte or its equivalent is derived from a non-human source .

ANALYTE UNITS LEVEL 1 LEVEL 2 LEVEL 3

ACETAMINOPHEN μg/mL 15-25 35-55 125-175

ACID PHOSPHATASE U/L 0.9-1.3 5-7.5 3.0-4.5

ALBUMIN g/dL 3.5-4.5 1.5-2.5 5-6

ALKALINE PHOSPHATASE U/L 70-100 300-400 200-275

ALT (PT) U/L 25-40 80-110 175-225

AMIKACIN μg/mL 3-8 20-30 35-45

AMYLASE U/L 60-80 250-350 450-550

AST (OT) U/L 25-41 110-130 180-220

BICARBONATE mmol/L 22-30 12-18 35-45

BILIRUBIN, DIRECT mg/dL 0.1-0.3 0.6-0.7 0.4-0.5

BILIRUBIN, TOTAL mg/dL 0.5-0.11 5.4-6.4 3.5-4.5

CALCIUM mg/dL 8.5-10.5 11.5-13.5 5.4-7.4

CARBAMAZEPINE ug/ L 0.5-1.5 3-5 16-20

CHLORIDE mmol/L 98-108 85-95 115-125

CHOLESTEROL mg/dL 95-125 235-265 135-165

CHOLESTEROL, HDL mg/dL 60-70 23-29 14-20

CHOLINESTERASE U/mL 11-15 5-7 3-5

COPPER ug/mL 0.9-1.3 2.5-3.5 0.5-0.9

CORTISOL ug/dL 9-15 20-30 1-3

CK U/L 161-201 460-560 312-372

CK-MB U/L 2-4 60-80 40-50

CREATININ mg/dL 0.7-1.1 3.5-6.5 0.4-0.6

DIGOXIN ng/ml 1.1-1.5 0.7-0.9 2.2-2.8

ETHOSUXIMIDE ug/mL 30-40 70-80 120-130

FERRITIN ng/mL 120-160 30-50 500-700

FOLIC ACID ng/mL 8-12 1.1-1.11 5-7

GEN AMICIN ug/mL 5.0-9.0 1.0-2.0 3-5

GGT U/L 15-25 125-145 80-100

GLUCOSE mg/dL 75-95 225-275 47-67

HCG mlU/mL 4-8 30-40 350-450

IRON ug/dL 80-100 180-240 24-34

IRON BINDING ug/dL 302-402 200-250 N/A

CAPACITY

LACTATE mmol/L 1.0-2.0 4-6 2.8-3.8 ANALYTE UNITS LEVEL 1 LEVEL 2 LEVEL 3

LACTATE U/L 130-170 370-470 251-311

DEHYDROGENASE (LDH)

LIDOCAINE ug/mL 2.5-3.5 1.0-2.0 5.6-7.6

LIPASE U/L 110-150 590-790 387-537

LITHIUM mmol/L 0.6-1.0 1.5-2.5 1.3-1.7

MAGNESIUM mg/dL 1.9-2.3 4.0-6.0 1.2-1.6

NAPA ug/mL 5-9 8-12 12-16

PHENOBARBITAL ug/mL 23-27 39-45 31-37

PHENYTOIN ug/mL 12-18 20-30 3-5

PHOSPHORUS , INOR. mg/dL 3.0-4.0 6-8 1.8-2.8

POTASSIUM mmol/L 3.8-4.8 6-8 2.3-3.3

PRIMIDONE mg/mL 7-11 12-18 2.5-5.5

PROCAINAMIDE ug/mL 5-9 10.5-14.5 3-5

PROTEIN, TOTAL g/dL 5.8-7.8 3.5-5.5 >8.6

QUINIDINE ug/mL 2.6-4.6 5.5-7.5 0.9-1.9

SALICYLATE mg/dL 5.5-9.5 19-29 50-70

SODIUM mmol/L 135-145 110-120 150-160

TRANSFERRIN mg/dL

T3 UPTAKE (TU) % 27-37 44-64 15-21

T3 , TOTAL ng/dL 1.3-1.9 4.0-5.0 <1.0

T4 , TOTAL ug/dL 6.5-10.5 12.5-18.5 2.5-5.5

THEOPHYLLINE ug/mL 12-18 20-30 8-12

TOBRAMYCIN ug/mL 5.5-7.5 1.0-2.0 7.5-9.5

TRIGLYCERIDE mg/dL 110-130 210-250 140-170

TSH ulU/ml 0.31-0.33 7-9 10-14

UREA mg/dL 10-16 45-55 65-75

URIC ACID mg/dL 3-5 8-12 5.7-7.7

VALPROIC ACID ug/mL 80-90 125-145 30-40

VACOMYCIN ug/mL 32-38 5-9 40-46

VITAMIN B12 pg/mL 100-200 300-500 900-1100

Table 2 In addition, when the control includes an analyte such as cholesterol or other steroids such as lipids or lipophiles, additives or methods which solubilize the lipophiles must be utilized. For example, sugar polymers such as chondroitin sulfate or cyclodextrins may be added. For instance, cholesterol inhabits that cavity of dimethyl beta cyclodextrin to render the cholesterol soluble at diagnostically significant levels. Alternatively, certain lipophiles such as LDL cholesterol may be covalently coupled to large synthetic molecules such as Starburst dendrimers to aid in solubilization and facilitate separation by HDL/LDL techniques.

Additional agents may be incorporated to provide even further enhanced stability. For instance, reducing agents may stabilize certain enzymes such as creatine kinase; substrate co-factors such as AMP add stability to total CK, while zinc ion stabilizes ALP. Table 3 shows the effect of 0.25 mM Zn²* on ALP and on other enzymes in the control base material. The base material is as described in Example 1 but only including BSA and bis Tris propane (BSA/BTP) . Results were obtained on a Dade Dimension® Analyzer, available from Dade International Inc..

Effect of Zn on ALP and other Enzymes in Base

Material at 37 °C

Recovery

Base Alone 0 Time Day 3 Day 7

ALP (129 u/L) 100 24 13 ALT (67 u/L) 100 85 75

ALP (126 u/L) 100 24 17 AST (76 u/L) 100 93 90

ALP (129 u/L) 100 22 15 CK (190 u/L) 100 81 66

ALP (132 u/L) 100 27 14 LDH (220 u/L) 100 94 99

ALP (133 u/L) 100 27 10 GGT (63 u/L) 100 101 97

ALP (126 u/L) 100 25 14 AMY (229 u/L) 100 90 97

Recovery

Base/Zinc at 0.25 mM 0 Time Day 3 Day 7

ALP (124 u/L) 100 95 94

ALP (122 u/L) 100 96 93 ALT (61 u/L) 100 87 82

ALP (125 u/L) 100 96 88 AST (83 u/L) 100 100 94

ALP (127 u/L) 100 95 87 CK (283 u/L) 100 80 70

ALP (128 u/L) 100 103 102 LDH (263 u/L) 100 99 102

ALP (130 u/L) 100 95 87 GGT (98 u/L) 100 97 96

ALP (128 u/L) 100 93 88 AMY (207 u/L) 100 94 100

TABLE 3

As shown in Table 3, any additional stability enhancers should not corrupt the measurement of other analytes. For instance, AMP should not be used to stabilize total CK when ALP is another analyte because the AMP acts as a substrate for ALP.

In a preferred embodiment, creatine kinase is pre- incubated with creatine to form a stock solution. The stock solution is then spiked into BSA/BTP . In this procedure, a 100 mM solution of creatine is prepared. Creatine kinase (.84 mLs) stock solution at 1 unit/mL (available from Scripps) is diluted with the 100 mM creatine solution (.16 mL) to provide about 16 mM creatine with the creatine kinase.

If creatinine is an analyte in the final control, the creatine should be added with the creatine kinase to form a stock and then added to the base material . When the creatine kinase and creatine are added separately to the base material the creatine may be converted to creatinine. Thus, when creatinine is an analyte, creatine should be first combined with creatine kinase. The stability using ^recovery is shown in Table 4. The effect of creatine on creatinine measurement is shown in Table 5. Results were obtained on a Dade Dimension® Analyzer, available from Dade International Inc..

% Recovery

Sample and Day Day Day Day Day Day Conditions 0 2 5 7 14 21

4C

1. CK in Base (200 u/L) 100 99 98 95 89 88 2. Separate addition of CK and 100 97 96 94 88 87 creatine in base (309 u/L)

CK + creatine 100 100 97 100 100 96 added as a stock solution to base (113 u/L)

25C

CK in Base (200 u/L) 100 87 81 80 74 54

Separate addition of CK and 100 88 81 80 73 58 creatine in base (309 u/L)

CK + creatine 100 94 89 89 81 80 added as a stock solution to base (113 u/L)

37C 1. CK in Base (200 u/L) 100 76 59 53 37 25

2. Separate addition of CK and 100 76 58 54 39 31 creatine in base (309 u/L) 3. CK + creatine 100 83 65 57 38 33 added as a stock solution to base (113 u/L)

TABLE 4 Creatinine Recoveries mg/dL

Sample and Day Day Day Day Day Day Conditions 0 2 5 7 14 21

4C 1. CK in Base 0.1 0.1 0.1 0.1 0 0.1

2. Separate addition of CK and 0.3 0.4 0.4 0.4 0.3 0.6 creatine in base 3. CK + creatine 0.1 0.1 0.1 0.1 0.1 0 added as a stock solution to base

25C

1. CK in Base 0.1 0.1 0.1 0.2 0.2 0.2

2. Separate addition of CK and 0.3 1.0 1.7 2.3 4.5 6.5 creatine in base

3. CK + creatine 0.1 0.1 0.1 0.2 0.3 0.3 added as a stock solution to base 37C

CK in Base 0.1 0.2 0.1 0.1 0.2 0.1

2. Separate addition of CK and 0.3 3 6.9 9.7 19.5 31.5 creatine in base

3. CK + creatine 0.1 0.1 0.1 0.1 0.1 0.2 added as a stock solution to base

TABLE 5

It has been found that native enzymes may be substituted with certain extremophilic enzymes, particularly thermophilic enzymes. Extremophilic enzymes are obtained from micro-organisms that function in extreme environments, such as extreme temperatures, extreme pH, or extreme salinity. The enzymes generally perform optimally under the extreme conditions. The microorganisms are found, for instance, in hot springs, freezing waters, sulfur rich geothermal springs, highly saline waters, or extremely acidic or basic conditions. Most of the microorganisms are from Archaea or Bacteria.

U.S. 5,354,676 describes an NAD dependent dehydrogenase (lactate dehydrogenase) from Bacillus stearothermophilus; U.S. 5,306,629 describes an enzyme that converts ADP to ATP (Ppase) derived from Bacillus or Thermo genus; U.S. 5,304,723 describes a maltopentose forming amylase derived from Bacillus licheniformis and U.S. 5,244,798 describes a reagent composition for the enzymatic determination of blood triglycerides comprising a thermostable lipoprotein lipase obtained from Streptomyces 7825. The latter states that the LPL can be preserved for an extended period of time as compared to conventionally available LPL preparations. These microorganisms have also been used in nucleic acid amplification methods and DNA sequencing methods. See, for instance, U.S. 5,527,670 and 5,614,364.

Often, because of the environment that is most suitable for these microorganisms, these microorganisms are difficult to culture in the laboratory. Thus, if sufficient material is available to determine the sequences, recombinant techniques may be used to prepare these enzymes. See Chemical and Engineering News, December 18, 1995, Enzymes From Microorganisms in Extreme Environments .

Technology has been developed to obtain large libraries able to produce a variety of enzymes, including recombinant thermophilic enzymes. Specifically, Recombinant Biocatalysis Inc. developed a process to hasten the development. In general, DNA is extracted and purified from biomass of these microorganisms (generally procaryotes) . The DNA is cut into fragments for cloning. The fragments are about the average gene size. The clones are catalogued and archived in libraries before being expressed. The clones are usually expressed in Escherichia coli. The expression products from several clones are pooled and then screened for enzyme activity using robotic screening. Clones that express enzyme activity are sequenced. The enzyme may be optimized by preparing random mutations over several generations. This mutagenesis can alter the enzymes so they will perform better or under altered conditions and still retain the stability under what would generally be considered adverse conditions. See Chemical & Engineering News, October 14, 1996: pp.31-33. Proposed uses of these enzymes include DNA Fingerprinting, oil production, therapeutics, diagnostics, pulp and paper and animal feed improvement. The selection of the enzyme is generally related to the conditions under which the reaction will occur. For instance, applications that require reactions at high temperature would benefit from thermophilic enzymes. Currently, kit libraries are commercially available from Recombinant Biocatalysis for lipases, glycosidases aminotransferases , phosphatases , and cellulases. Each library contains 3 to 10 enzymes of a specific type. If an enzyme appears suitable for a technique, it can be optimized using mutagenesis techniques. The substitution of the recombinant thermophilic enzymes for corresponding native or recombinant enzymes may add stability to any of the controls/control matrices used in the art. A thermophilic enzyme functions optimally at higher temperatures . A thermostable enzyme is stable at higher temperatures .

In particular, it was found that a clone from the phosphatase library, PHO-05, a recombinant thermophilic enzyme, can be substituted for PAP with increased stability and a clone from the aminotransferase library, AMN-03, a recombinant thermophilic enzyme can be substituted for AST even though in normal usage the control is not subjected to the high optimal temperatures, but instead is stored at 4°C and used at room temperatures . Table 1 shows a comparison between thermophilic rAcP and PAcP. Table 6 shows a comparison between rAST and AST. Pilots 1, 2A and 2B are prepared similarly to Examples 1-3. Pilot 2 contained only rAST and BSA/BTP. Other thermophilic enzymes can be screened for stability by preparing pilots such as Pilot 2 in Table 6. Results were obtained on a Dade Dimension® Analyzer, available from Dade International Inc.. Recombinant thermophilic enzymes that show comparable or better stability to native enzymes can be substituted for those native enzymes. Thus, it can be concluded that the recombinant thermophilic enzymes can be substituted for native enzymes. It should be understood that the clones that produce the recombinant thermophilic enzymes do not necessarily have the same amino acid sequence of the native enzymes. Instead, the clones yield enzymes that provide activity that is similar to a native enzyme.

% Recovery rAST 0 Time Day 4 Day ' 7 Day 14 Day 21

Pilot 1 4°C 100 112 95 100 101

(65 u/L) 25°C 100 103 94 69 30 rAST 0 Time Day 1 Day 7 Day 13

Pilot 2 4°C 100 99 108 96

(90 u/L) 37°C 100 106 112 106

%Recovery

AST 0 Time Day 1 Day 7 Day 13

Pilot 2A 4°C 100 90 64 13

(162 u/L) 25°C 100 83 14 17

AST 0 Time Day 1 Day 7 Day 13

Pilot 2B 4°C 100 96 92 54

(160 u/L) 25°C 100 86 31 12

TABLE 6

It has also been found that lactoferrin and iron binding proteins from milk powder can be used as a substitute for transferrin to simulate total iron binding capacity and resultant unsaturated iron binding capacity. Lactoferrin is preferred over transferrin because lactoferrin is not of human origin. Lactoferrin is readily available in recombinant form and from mild extract. The use of lactoferrin for transferrin has been demonstrated. Lactoferrin at various concentrations is dissolved in base material . The iron and iron binding capacity are determined for each sample. Results were obtained on a Dade Dimension® Analyzer, available from Dade International Inc.. See Table 7. % Lactoferrin Iron μg/dL Iron Binding Capacity

0% 87 103 0.25% 87 163 0.50% 87 229

Table 7

Example 1

Preparation of a stock buffer solution.

To prepare one liter of a mid-level chemistry control about 5.65 grams of bis Tris propane, about 2.2 grams of substantially protease free bovine serum albumin, about 1.5 grams of sodium chloride, about 2.3 grams of substantially protease free IgG, (if recombinant thermophilic acid phosphatase will be utilized, it is preferred to omit the IgG) about 4.0 grams of beta cyclodextrin, about 1 ml of Tween-20, about 2.5 grams of cholesterol are combined all at a pH of about 7.6 with sufficient distilled, deionized water. The resulting stock buffer solution is filtered through a 0.22 micron filter and the volume is measured.

Example 2

Preparation of Analyte Stock Solutions.

Next, stock solutions of analytes are prepared. A 25 mg/mL phenytoin stock solution is prepared by dissolving 25 mgs of phenytoin in 1 mL of methanol. If the solution is not clear, sodium hydroxide may be added dropwise until the solution clears. A 4 mg/mL theophylline solution is prepared by adding 25 mgs of theophylline to 6.25 mLs of purified water. A 5 mg/mL quinidine solution is prepared by adding 6.5 mgs of quinidine to 1.3 mLs of methanol . A 10 mg/mL carbamazepine stock solution is prepared by adding 4 mgs of carbamazepine to 0.4 mLs of methanol . A ferric chloride solution is prepared by adding 10 mgs of ferric chloride to 1 mL of purified water. A stock solution of gamma glutamyl transferase (GGT) is prepared by adding 1,000 units of GGT to 1 mL of stock buffer solution. A stock solution of lactate dehydrogenase (LDH) is prepared by adding 4,000 units of LDH to 1 mL of a buffer containing only BSA/bis Tris propane (BSA/BTP buffer) in the amounts described in Example 1. A stock solution of prostatic acid phosphatase (PAcP) is prepared by adding 100 units of ACP to 1 mL of BSA/BTP buffer. A stock solution of alkaline phosphatase (ALP) is prepared by adding 2000 units of ACP to 1 mL of BSA/BTP buffer. A stock solution of amylase is prepared by adding 3000 units of amylase to 1 mL of BSA/BTP buffer. A stock solution of creatine kinase (CK) is prepared by adding 2000 units of CK to 1 mL of BSA/BTP buffer. Creatine may be added as described herein above. A stock solution of lipase is prepared by adding 6000 units of lipase to 1 mL of BSA/BTP. A stock solution of ALT/GT is prepared by adding 1000 units of ALT/GT to 1 mL of BSA/BTP buffer. A stock solution of cortisol is prepared by dissolving 50 mgs of hydrocortisone in 50 mL of ethanol. This is further diluted 1:10 in phosphosaline buffer. A stock solution of digoxin is prepared by dissolving 0.060 grams of digoxin in 200 mLs of 95% ethanol. A stock solution of thyroxine is prepared by wetting 12 mgs of L-thyroxine with 1 N NaOH and adding 10 mLs of methanol. A stock solution of hTSH is prepared by dissolving 2 IU of hTSH in 2 mL of 0.1% bovine serum albumin, preferably substantially protease free. A stock solution of hCG is prepared by dissolving 1000 IU in 1 mL of purified water. Thyroid binding globulin is supplied as a frozen liquid at about 5 mg/mL in a 0.05 M phosphate buffer at pH 7.5 containing 0.15 M NaCl. A stock solution of total bilirubin is prepared by dissolving 0.06 grams of bilirubin in about 6 mLs of 0.1 N NaOH. Both bilirubin and the resulting stock solutions should be protected from light. Alpha monoglycerides (available from ABITEC Corporation) comprising primarily caprylic acid side chains are assayed using standard methods for triglycerides. Stock solutions of other analytes may be prepared using procedures known in the art .

Example 3

Preparation of a chemistry control containing analytes .

Next, about 2.5 grams of glucose, about 0.05 grams of creatinine, about 0.1 grams of uric acid, about 1.1 grams of urea, about 0.4 grams of magnesium chloride-6H20 , about 0.07 grams of lithium carbonate, about 0.1 mL of ferric chloride stock solution, about 1.25 grams of sodium bicarbonate, about 0.35 grams of calcium chloride, about 2 mL of a stock solution of monoglycerides (sufficient to provide normal range of triglycerides), about 0.5 mL of 200 mM ammonia stock solution, about 6 mL of 12 mg/mL phosphoric acid, about 0.45 grams of lactic acid, about 0.5 grams of potassium chloride, about 0.4 mLs of carbamazepine stock solution, about 1 mL of phenytoin stock, about 6.25 mLs of theophylline stock, about 0.15 mLs of valproic acid, about 1.5 mLs of cortisol stock solution, about 1.3 mLs of quinidine stock solution, about 0.2 mLs of thyroxine solution, about 0.13 mLs of hTSH stock solution, about 0.05 mLs of hCG stock solution, about 0.8 mLs of ferritin stock, about 0.1 mLs of 5000 ng/mL Vitamin B12 stock solution, about 2 mL of TBG stock solution, about 0.055 mLs of 100 units/mL of acid phosphatase solution, about 0.2 mLs of 2000 units/mL of alkaline phosphatase solution, about 0.1 mLs of 3000 units/mL of amylase stock, about 43 mgs of 28 units/mg of glutamate oxalacetic transaminase, (AST/GOT) stock, about 0.25 mLs of 2000 units/mL of creatine kinase solution, about 0.13 mLs of 1000 units/mL of gamma glutamyl transpeptidase (GGT) stock solution, about 0.1 mL of 4000 units/mL of lactic dehydrogenase chicken heart (CLDH) solution, about 0.12 mLs of 6000 units/mL of lipase solution, about 0.1 mL of 1000 units/mL of ALT/GPT solution, are combined into about 900 mLs of the stock buffer solution with gentle stirring. It is preferred that the solutions be kept at 10°C or less during the preparation.

In addition to adding sodium chloride, sodium ion and chloride ions may be adjusted by adding 5N sodium hydroxide and 5N hydrochloric acid respectively to obtain clinically significant levels. During any adjustment, the pH must be maintained between 6.0 and 7.65. Any other adjustments to levels of analytes may be made by adding the additional quantity of analyte or analyte stock solution. In addition, the levels of analytes may be varied in multiple stock solutions to provide multiple controls containing different levels of analytes.

The final solution is brought up to about IL and degassed. A Liqui-cel apparatus may be used to degas the solution. The final solution is stored under nitrogen at about 4°C.

About 5 mL of the bilirubin stock solution and 0.132 g/L ascorbic acid are added in the absence of oxygen to the degassed final stock solution. The resulting final chemistry control solution is filled in appropriate vials.

Example 4

Use of recombinant thermophilic AcP

Examples 1-3 are repeated except IgG is .omitted and rAcP is substituted for PAcP.

Example 5

Preparation of Low and High Control Levels For a Low-level control, examples 1-3 are repeated except that the amounts of analyte are added in sub-therapeutic/ low clinical ranges. For a High-level control, examples 1-3 are repeated except that the amounts of analyte are added in post-therapeutic/high clinical ranges.

Example 6 Open stability of analytes in a chemistry control.

The stability of a control prepared similarly to the control described in Example 1-3 is prepared but with the analytes listed in Table 8. On Day 0, vials are equilibrated to room temperature opened and sampled and tested. Then the opened vial is stored capped at 4°C. On each of Day 1, 2, 3, and 4 the open vial is uncapped again, retested, and compared with a vial that has not been opened but has also been stored at 4°C. Open, reference and normalized results are shown in Table 8. All analytes are evaluated on the Dade Dimension® Analyzer unless otherwise specified.

Open Vial Stability: 2-8°C

Days

Constituents Level 1 method 0 1 2 3 4

Free T4 (TDX) as falls Abbott 3.03 3.42 3.39 3.43 3.31

Quinidine (TDX) 4 Abbott 4.45 4.33 4.51 4.85 4.39

Valproic Acid (TDX) 90 Abbott 68.3 67.6 65.7 65.4 72.5

Cortisol (TDX) 15 Abbott 15.8 18.1 16.2 16.5 16.5 hTSH (IMX) 2 Abbott 1.05 1.27 1.27 1.15 1.2

Albumin 4 dim 2.9 2.9 2.8 2.8 2.9

Alk Phos 150 dim 126 129 139 131 133

ALT 60 dim 60 60 57 60 62

Amylase 222 dim 219 220 215 219 218 rAST 60 dim 60 56 56 54 61

Calcium 10.5 dim 10.6 10.4 10.5 10.5 10.4

Cholesterol 240 dim 237 236 240 235 237

CK 100 dim 57 56 60 56 53

Creatinine 2.5 dim 2.6 2.6 2.5 2.7 2.6

GGT 70 dim 63 62 61 64 64

Glucose 145 dim 153 150 150 151 155

Iron 130 dim 103 99 101 100 100

Lactic Acid 2 dim 1.2 1.4 1.2 1.2 1.1

LDH 300 dim 294 293 282 292 297

Lipase 1000 dim 854 1042 828 1087 1086

Magnesium 2.4 dim 2.3 2.2 2.3 2.3 2.3

Phosphorus 3.9 dim 4.4 4.3 4.2 4.3 4.3

Total Protein 6.5 dim 7.1 7 6.9 7 7

Urea nitrogen 30 dim 35 35 33 34 34

Uric Acid 7.5 dim 7.2 7.1 7.2 6.9 6.4

Sodium 142 dim 154 153 151 151 153

Potassium 4.5 dim 5.5 5.4 5.3 5.3 5.4

Chloride dim 141 143 138 128 144

CO2 30 dim 19.6 19.4 20.4 19.6 16.1

Total T4 9 dim 10.7 12.1 11.2 11.5 10.4

Digoxin 2 dim 0.14 0.07 0.13 0.07 0.11

Theophylline 20 dim 13.9 13.9 13.6 13.8 14.2

Phenytoin 15 dim 13.4 13.7 13.2 7.5 13.8

Carbamazepine 8 dim 7.6 7.5 7.4 7.5 7.4

T3 UPtake 42 dim 44 43 43 43 44

Lithium 1 Flame

Ferritin 80 Stratus 52.5 50.5 52.9 56.4 51 hCG 50 Stratus 38.6 37.3 35.8 35.6 33.7

Vitamin B-12 700 Stratus 664.2 628.2 608 639 634

TABLE 8a Open Vial Stability: 2-8°C Reference (closed vial)

Days

Constituents Level 1 method 0 1 2 3 4

Free T4 (TDX) as falls Abbott 3.03 3.35 3.51 3.57 3.48

Quinidine (TDX) 4 Abbott 4.45 4.42 4.65 4.9 4.12

Valproic Acid (TDX) 90 Abbott 68.3 66.5 66.3 64.5 70.2

Cortisol (TDX) 15 Abbott 15.8 18.8 16.2 16.9 16.8 hTSH (IMX) 2 Abbott 1.05 1.21 1.25 1.18 1.15

Albumin 4 dim 2.9 2.9 2.9 2.8 2.7

Alk Phos 150 dim 126 136 140 130 129

ALT 60 dim 60 59 59 58 55

Amylase 222 dim 219 216 220 213 207 rAST 60 dim 60 56 56 55 55

Calcium 10.5 dim 10.6 10.5 10.5 10.3 9.9

Cholesterol 240 dim 237 242 238 235 225

CK 100 dim 57 59 54 57 55

Creatinine 2.5 dim 2.6 2.6 2.6 2.1 2.5

GGT 70 dim 63 65 64 64 58

Glucose 145 dim 153 149 153 155 145

Iron 130 dim 103 102 99 100 95

Lactic Acid 2 dim 1.2 1.2 1.1 1.2 1.1

LDH 300 dim 294 288 290 290 277

Lipase 1000 dim 854 1038 833 1042 1041

Magnesium 2.4 dim 2.3 2.2 2.2 2.2 2.1

Phosphorus 3.9 dim 4.4 4.3 4.4 4.3 4.1

Total Protein 6.5 dim 7.1 7 7 6.9 6.6

Urea nitrogen 30 dim 35 35 34 34 32

Uric Acid 7.5 dim 7.2 5.5 7 6.8 6.8

Sodium 142 dim 154 154 154 152 144

Potassium 4.5 dim 5.5 5.4 5.4 5.3 5.1

Chloride dim 141 147 143 135 135

CO2 30 dim 19.6 21.8 22 17.3 17.5

Total T4 9 dim 10.7 11.6 11.5 10.3 9.6

Digoxin 2 dim 0.14 0.08 0.09 0.1 0.14

Theophylline 20 dim 13.9 13.9 13.1 14.6 13.6

Phenytoin 15 dim 13.4 13.9 13.2 13.3 13

Carbamazepine 8 dim 7.6 7.9 7.5 7.9 7.5

T3 UPtake 42 dim 44 43 43 46 42

Lithium 1 Flame

Ferritin 80 Stratus 52.5 50.4 50.3 52.1 49.4 hCG 50 Stratus 38.6 36 36.4 33.4 34.6

Vitamin B-12 700 Stratus 664.2 633 660 650 589

TABLE 8b Open Vial Stability: Normalized Data represented as % of reference value

Days Constituents 0 1 2 3 4

Free T4 (Abbott TDX) 1.00 1.02 0.97 0.96 0.95

Quinidine (Abbott TDX) 1.00 0.98 0.97 0.99 1.07

Valproic Acid (Abbott TDX) 1.00 1.02 0.99 1.01 1.03

Cortisol (Abbott TDX) 1.00 0.96 1.00 0.98 0.98 hTSH (Abbott IMX) 1.00 1.05 1.02 0.97 1.04

Albumin 1.00 1.00 0.97 1.00 1.07

Alk Phos 1.00 0.95 0.99 1.01 1.03

ALT 1.00 1.02 0.97 1.03 1.13

Amylase 1.00 1.02 0.98 1.03 1.05 rAST 1.00 1.00 1.00 0.98 1.11

Calcium 1.00 0.99 1.00 1.02 1.05

Cholesterol 1.00 0.98 1.01 1.00 1.05

CK 1.00 0.95 1.11 0.98 0.96

Creatinine 1.00 1.00 0.96 1.00 1.04

GGT 1.00 0.95 0.95 1.00 1.10

Glucose 1.00 1.01 0.98 0.97 1.07

Iron 1.00 0.97 1.02 1.00 1.05

Lactic Acid 1.00 1.17 1.09 1.00 1.00

LDH 1.00 1.02 0.97 1.01 1.07

Lipase 1.00 1.00 0.99 1.04 1.04

Magnesium 1.00 1.00 1.05 1.05 1.10

Phosphorus 1.00 1.00 0.95 1.00 1.05

Total Protein 1.00 1.00 0.99 1.01 1.06

Urea nitrogen 1.00 1.00 0.97 1.00 1.06

Uric Acid 1.00 1.29 1.03 1.01 0.94

Sodium 1.00 0.99 0.98 0.99 1.06

Potassium 1.00 1.00 0.98 1.00 1.06

Chloride 1.00 0.97 0.97 0.95 1.07

CO2 1.00 0.89 0.93 1.13 0.92

Total T4 1.00 1.04 0.97 1.12 1.08

Digoxin 1.00 0.88 1.44 070 079

Theophylline 1.00 1.00 1.04 0.95 1.04

Phenytoin 1.00 0.99 1.00 0.56 1.06

Carbamazepine 1.00 0.95 0.99 0.95 0.99

T3 UPtake 1.00 1.00 1.00 0.93 1.05

Lithium (Flame) n/a n/a n/a n/a n/a

Ferritin (stratus) 1.00 1.00 1.05 1.08 1.03 hCG (Stratus) 1.00 1.04 0.98 1.07 0.97

Vitamin B-12 (stratus) 1.00 0.99 0.92 0.98 1.08

TABLE 8c Example 7

A low level control is prepared similarly as described in Examples 1-3. The stability of certain analytes considered to have the low stability is presented in Figures 1 to 19. The "Days to Failure" described in these figures represent a predictive model using a best fit curve.

Claims

We claim :

1. A composition for use as a standard in clinical chemistry assays comprising an aqueous buffered solution of lactoferrin wherein the lactoferrin provides total iron binding capacity and unsaturated iron binding capacity determination.

2. A base matrix useful for preparing a chemistry control for use in clinical chemistry assays comprising a buffer, a stabilizing protein, a means for obtaining and maintaining a low protease activity, a means for maintaining viscosity and a means for substantially obtaining and maintaining anoxia.

3. The base matrix of claim 2 further comprising cyclodextrin sufficient to solubilize a diagnostically significant amount of cholesterol.

4. The base matrix of claim 3 further comprising cholesterol and triglycerides.

5. The base matrix of claim 4 further comprising bilirubin or bilirubin salts and an antioxidant.

6. The base matrix of claim 5 wherein the antioxidant is ascorbic acid.

7. The base matrix of claim 5 wherein the anoxia is substantially obtained and maintained by degassing.

8. The base matrix of claim 7 wherein the oxygen content is less than 1 ppm.

9. The base matrix of claim 3 further comprising hormones of clinical interest, thyroid binding globulin, enzymes of clinical interest, therapeutic drugs, or therapeutic drug metabolites.

10. The base matrix of claim 9 further comprising electrolytes of clinical interest vitamins and metabolites of clinical interest.