WO2001081930A2

WO2001081930A2 - Reagent and method for "serum iron" assay in plasma

Info

Publication number: WO2001081930A2
Application number: PCT/US2001/012927
Authority: WO
Inventors: Yen Yue
Original assignee: Dade Behring Inc.
Priority date: 2000-04-26
Filing date: 2001-04-20
Publication date: 2001-11-01
Also published as: WO2001081930A3

Abstract

A method and composition for the assay (determination) of iron and the total iron binding capacity in plasma is disclosed. Tris or other suitable buffer is added to the assay mixture in order to determine the amount of iron or the total iron binding capacity in plasma.

Description

Reagent and Method for "Serum Iron" Assay in Plasma

BACKGROUND OF THE INVENTION

This invention relates to a composition and process for measuring iron. The invention also provides a method for the determination of the total iron binding capacity in either serum or plasma. Iron exists in the body mainly as hemoglobin in red blood cells and in the liver, the spleen, the bone marrow, and the like. About two-thirds of the body's iron is found in the hemoglobin (~2.5g). The total weight of iron in a human body is about 4 grams. The body loses about one to 2 milligrams of iron per day. Therefore, the balance can be maintained by absorbing about one to 2 milligrams of iron from food every day. The average American diet provides 10 to 15 mg of iron daily. Normally, the body is under very tight control to absorb 1 to 2 mg of iron from food to maintain the balance. If too little iron is taken in, an anemic condition may occur. If too much is taken in, an iron overload condition may occur.

The Approximate Iron distribution in the body is as follows: Hemoglobin: -2.5 g

Myoglobin: -130 mg Labile pool: ~80 mg Tissue: ~8 mg (as co-enzyme)

Iron storage: as ferritin and Hemosiderrin: -800 mg for men and 0 to 200 mg for women.

Plasma: ~ 2.5 mg Transferrin is the principal plasma protein for transport of iron. Its concentration correlates with the total iron-binding capacity (TIBC) of serum. Transferrin is a betal globulin of 75,000-80,000 Dalton. The iron in serum/plasma is physiologically bound to transferrin. The amount of iron bound to transferrin is about four milligrams. Transferrin has the capacity to bind two iron atoms per molecule in the ferric (trivalent) form. The main function of transferrin is to transport and transfer iron from the mucous membrane of the small intestine to the red blood cell precursors in bone marrow, where it is incorporated into hemoglobin. These cells then enter the circulation as mature red blood cells, where they remain nearly 4 months before they become metabolically "worn out" and engulfed by phagocytes. The iron is released from hemoglobin and returns to plasma transferrin being re-used for making hemoglobin, thus completing one cycle and beginning another. Of the total amount of transferrin time about one-third is bound to iron while about two thirds is not bound to iron and there is a substantial unsaturated plasma iron binding capacity. An unexpected influx of iron can be handled easily.

In the field of clinical diagnostics, the amount of iron bound to transferrin is commonly referred to as the serum iron. There is essentially no difference between plasma and serum iron. Serum is usually used for the iron assay for reasons of technical capability. Normally the amount of iron in serum/plasma (not including hemoglobin) is about 100 micrograms per 100 milliliters of blood. The amount of transferrin not bound to iron is referred to as the unsaturated iron-binding capacity (UIBC) in serum. The UIBC is nonnally about 200 microgram per 100 milliliter of blood. The total amount of transferrin present in blood represents the total iron binding capacity. Normally the total amount of transferrin is about 300 micrograms per 100 milliliters of blood. The unsaturated iron binding capacity (UIBC) can be calculated as the difference between the total iron binding capacity (TIBC) and the serum iron.

The determination of serum iron is one of the most frequently performed diagnostic trace element tests. Evaluation of iron (IRN), total iron binding capacity (TIBC), and the calculated ratio of iron concentration and total iron binding capacity [(IRN/TIBC)*100 =%Sat] are useful for the differential diagnosis of anemia and iron overload and monitoring the treatment of certain disease conditions. For example, in iron deficiency anemia, normal or low blood iron, increased TIBC and decreased % Sat is typical. In iron overload, high blood iron (>150 ug/dl) and high %Sat>50% (normal about 33%) and low or normal TIBC will occur. Thus, the measurement of both serum IRN and TIBC provide more detailed information and thus greater significance. Serum iron levels reflect the rate of production and destruction of red blood cells. Further, the unsaturated iron binding capacity relate to the metabolism of hemoglobin and its measurement is indispensable for a differential diagnosis of anemic persons. The tests also used to monitoring pregnancy, Hepatitis, acute inflammation (respiratory), abscess immunization, myocardial infarction and chronic inflammation or malignancy.

In the clinical laboratory, the amount of iron measured in serum can be done by known methods. In one method, serum iron is assayed by adding a serum sample to a reagent buffered at an acid pH. At this acid pH, ferric ion dissociates from transferrin. The reagent includes a reducing agent, which aids in the dissociation process and reduces ferric ion to ferrous ion. A chromogenic reagent is then added and the chromogen complexes with ferrous iron to form a colored complex. The colored complex is measured spectrophotometrically. There are also known methods to determine the unsaturated binding capacity of serum. Again, the unsaturated iron binding capacity (UIBC) is the difference between the TIBC and the serum iron and is normally about 200 microgram per 100 milliliter of blood. UIBC represent the free iron-binding sites available. It can be measured by adding known amount of excess iron to the serum sample at neutral pH. Added iron binds to unbound transferrin. The amount of iron remaining unbound after sample addition is measured photometrically and the difference in the original known amount less the remaining amount represents the UIBC.

Another method to determine UIBC comprises adding an alkaline buffer solution to serum, adding a known amount of iron to the mixture to bind all unbound transferrin to the added iron, and colorimetrically determining the residual iron to measure the unsaturated iron-binding capacity [the Schade method: Proc. Soc. Exp. Biol. & Med., 87, 443-448 (1954)]. More in detail, the Schade method comprises adding 1.0 ml of serum to 2.0 ml of a 1.0 M tris buffer solution (pH=8.1), allowing the resulting mixture to stand for 5 minutes, adding 1.0 ml of an ammonium ferrous sulfate solution (3.51 mg/dl) containing 0.5% by weight of ascorbic acid to the resulting mixture, measuring absorbance at 535 nm by using a photoelectric colorimeter (blank), adding a drop of 0.5% bathophenanthroline sulfonic acid-disodium salt for color formation, allowing to stand at 25 degrees Celsius for 10 minutes or more, and measuring absorbance at 535 nm.

There are problems with the Schade method. Iron is released from transferrin at pH 4.0 or lower while the bonding between iron and transferrin is strong and stable at the alkaline side. However, when a chelate color-forming reagent such as bathophenanthroline is added, dissociation of transferrin and iron begins to take place at about pH 7.0 or lower. However, when the pH is 7.5 or higher, no dissociation of transferrin and iron takes place even if bathophenanthroline is present. Therefore, according to the Schade method, it is essential to conduct the measurement at pH of 7.5 or higher so that only the residual iron is measured. On the other hand, the color-forming reaction rate is maximum at about pH 5.0 in the case of using bathophenanthroline as a color-forming reagent. The color-forming rate is slowed down when the pH is either higher or lower than that pH (about 5.0). When bathophenanthroline is used as a color-forming reagent in the Schade method, it is necessary to make the pH of the solution about 7.5 to 8.5 considering the stability of serum iron. However, when pH=8.1 is selected in practice, it takes about 10 minutes for the color formation. Thus, the length of the color- formation in the Schade method makes it difficult to use an auto analyzer, although the Schade method is a very simple method without using centrifugal separation, removal of proteins and complicated procedures.

Another method to measure iron and unsaturated iron binding capacity is disclosed in U.S. 5,420,008. The methods require the use of the enzyme aconitase. A pH between 5-9 and optimally 6-8 is maintained to bind iron to aconitase. The buffers to maintain the basic pH include PIPES buffer, tris buffer, Triethylnolamine buffer, glycine buffer, GTA buffer, and phosphate buffer. The method to measure unsaturated iron binding capacity requires a stable, alkaline, excess iron solution of known concentration to fully saturate the transferrin with iron. U.S. 5,420,008 states that this is a difficult step because the intrinsic properties of iron ion are such that the ion can exist under acid pH conditions, such as in an aqueous solution of nitric acid or hydrochloric acid. However in slightly acid to alkaline condition iron ion forms iron hydroxide, causing sedimentation or adsorption and therefore iron cannot exist stably at a pH which is favorable for the binding or iron to transferrin, for example, a pH of preferably 8-9. However, it is noted a somewhat stable bivalent iron compound (ammonium iron (II) sulfate or ferrous chloride) rather than a trivalent iron compound, is used in a Tris buffer solution in the co-presence of a reducing reagent such as ascorbic acid for stabilization. After all of the transferrin has iron bound thereto, the unbound iron is measured by binding it to aconitase. Although the TIBC is most clinicians' preferred method, the UIBC has been easier to automate than the TIBC. As discussed above, the total iron binding capacity (TIBC) is the maximum amount of iron that can be bound. The iron binding capacity of serum is accounted for almost entirely by transferrin. TIBC can be measured by adding excess iron to saturate all the transferrin iron binding sites. The unbound iron is removed by adsorption to an alumina column or precipitated by magnesium carbonate. In either approach, centrifugation is required to separate the unbound iron from the saturated transferrin. The eluant from the alumina column or the supernatant from magnesium carbonate treatment containing the transferrin-iron complex is then collected in a cup. Free iron remains absorbed on the column or in the precipitate. Then the saturated transferrin in the eluant or supernatant is assayed in an iron measuring system as discussed above for the determination of serum iron. That iron value measured represents the maximum iron the transferrin can bind. The removal of unbound iron requiring adsorption and separation makes it difficult to automate the assay. Hachiro Yamanishi et al, disclose an automated TIBC method (Clin Chem 1997; 43:2413-7). In this method, excess iron is added at an alkaline pH so that it binds with transferrin. Next, the indicator ferrozine is added to complex with and eliminate unbound iron. The pH is made acidic to dissociate iron from transferrin. The dissociated iron is reduced and reacts with the ferrozine. The increase in absorbance at 570 nm is measured. However, in this method HEDTA must be added as a calibrator to generate a calibration factor to provide an accurate result. Traditional serum iron assays have been problematic for several reasons. First, serum contains chromatic components such as hemoglobin, which significantly absorb light at the measuring absorption wavelength of the iron-chromogen colored complex. This problem is generally solved by forming a complex that absorbs at a wavelength at which the chromatic components of serum do not interfere. Another problem as discussed above is the importance of understanding the binding of iron and transferrin at different pH values. Another problem is that turbidity due to lipemia in serum interferes with spectral measurements of serum iron, as do serum proteins. These problems can be solved by obtaining a serum blank measurement to correct for differences in serum turbidity and/or masking reagents such as citric acid (Garcic). Addition of DMSO is thought to lower the interference caused by lipemic samples. Denney et al. disclosed the use of DMSO in a serum iron assay to speed up the assay and to diminish turbidity due to lipemia and /or protein precipitation. The percent of DMSO employed in the serum iron assay is 20%. (Demiey ET al., U.S. Pat. No. 4,224,034 and Outcalt ET al., U.S. Pat. No. 4,154,929). In addition, it has been suggested that the addition of certain mixtures of non-ionic and anionic detergents improve the turbidity due to lipemic samples. Recently US 5,763,281 suggested the use of a particular detergent (a fatty alcohol polyglycol ether) and urea or a urea derivative to correct for lipemic samples.

US 5,763,281 states that serum or plasma can be used in the claimed method of using urea or a urea derivative and a fatty alcohol polyglycol ether.

Many diagnostic tests run serum and/or plasma samples. Plasma is a component of blood that is generally free of blood cells, but, unlike serum, still contains the clotting proteins. In many cases, the laboratory needs to run a plasma instead of serum as a sample type when fast results are needed, (called "star" in clinical lab). This is because plasma can be obtained more quickly than serum. To obtain plasma a blood sample is centrifuged for 10 to 15 minutes. Then the plasma can be run in a diagnostic test. On the other hand, for serum sample, after a blood sample is drawn, thirty to sixty minutes is needed to let blood clot. Then the sample must be centrifuged before it can be run. In the case of the IRN and TIBC tests, it is also important that plasma compares to serum because dialysis patients often have serum similar to plasma since these patients are often on heparin medication. Their serums are not sufficiently clotted because of the heparin in their system. This group of patients tends to develop severe anemia during the dialysis treatment so their blood IRN and TIBC need to be continuously monitored. The colorimetrically measurement used in many auto analyzer for IRN and TIBC methods tends to give erroneously high results to dialysis patients because of the serum plasma discrepancy. Therefore, a false diagnose can occur. Thus, many laboratories would like to have the ability to measure the amount of iron in plasma. In order to be meaningful that measurement in plasma must correlate with what the measurement would be if the sample were a serum sample. However, the clotting proteins (particularly fibrinogen) cause turbidity in the sample that is unrelated to the turbidity caused by lipemic samples. This turbidity is not corrected by serum blank measurements or the addition of detergents. The NCCLS guideline for the Determination of Serum Iron provides a procedure so that both serum and plasma can be used. A precipitating-reducing reagent is made by dissolving 5 g of L-ascorbic acid in 200 mL 50% (w/v) trichloroacetic acid. Then 33 mL of 6 M hydrochloric acid is added. The volume of the solution is adjusted to one liter using deionized water. In this method two milliliters of the precipitating-reducing reagent are combined and mixed with two milliliters of serum or heparinized plasma. The tubes are centrifuged at about 1500g for 15 minutes. The supernatant is removed and is assayed by the NCCLS procedure. This procedure is time-consuming and difficult to automate. Another disadvantage of this method is very strong acidic reagent, trichloroacetic acid was used, which is caustic and corrosive. As discussed above, Hachiro Yamanishi et, al, disclose an automated TIBC method (Clin Chem 1997; 43:2413-7), however, because of the chemistry used in the method only serum can be used in that system. Additional compositions and methods are needed to use plasma samples in the determination of serum iron particularly in automated systems.

Objects of the Invention

Some of the objects of the invention include providing a composition and process to eliminate the serum and plasma discrepancy that exists in the colorimetric methods of IRN and TIBC methods. Another object of the invention is to provide a composition and process for a fully automated TIBC method used for both serum and heparinized plasma as sample type. Another object of the invention is to provide an iron and TIBC assay that does not require a sample blank to subtract interference from samples. SUMMARY OF THE INVENTION

The present invention is a method to determine iron and TIBC using either serum or plasma. This invention is characterized by the use of a special reagent mixture as a buffer solution to eliminate the serum/plasma discrepancy, which exists in photometric IRN and TIBC method. In the method of the present invention for iron (IRN), a serum or plasma sample is first combined with a buffer solution at about pH 8.5. Next, the pH is lowered to a pH which allows all the iron bound to transferrin to be released (pH about less than 5). The released iron is measured by addition of a chromogen. In the present method of the invention for automated TIBC, a serum or plasma sample is combined with a buffer solution at about pH 8.5 and a large excess of Fe III ions (ferric ions). The transferrin not already bound to iron binds to added iron. Since the Fe III is added in excess, some remains in solution. Next, an excess of chromogen that reacts with the remaining Fe III is added. A blank measurement of the sample is taken on the photometer. Next, the pH is lowered to less than pH 5 to cause all of the iron bound to transferrin to be released. The released iron is measured by addition of the excess chromogen that correlates to the total iron binding capacity. The invention provides fully automated methods using a novel reagent for the assay of serum iron and TIBC with both serum and plasma as appropriate sample types.

The present invention overcomes the discrepancy in results of serum and plasma samples. It has been found that in the IRN and TIBC methods the clotting proteins in the plasma precipitate causing turbidity and incorrect (generally high) results. The precipitation occurs upon addition of the acidic buffer to the plasma sample during the reaction(s). However, the acidic buffer is required so that bound iron may be released from transferrin. An alkaline pH buffer added prior to the addition of the acid buffer prevents the plasma protein precipitation and allows for matched serum and plasma results in both IRN and TIBC methods. The methods of the present invention use human transferrin as the calibrator material.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses a diagnostic kit for the assay of iron comprising a first composition comprised of an alkaline buffered solution and a second composition comprised of a chromogenic indicator that reacts with iron to produce in serum or plasma a detectable change when measured in a photometer and a reducing agent to reduce Fe III to Fe II and a third composition comprising an acidic buffer. The second composition may be combined with the first or third composition. Any of the solutions may contain as an additive surfactants, such as Triton X, alkylammonium halides and polyhydroxyalkylene ethers to assist in the prevention of protein precipitation; reagents to minimize potential copper ion interference such as thiourea; and/or microbial inhibitors. In one embodiment, the reducing agent may be supplied as a tablet and the acid buffer added to it prior to use.

The first composition of the IRN kit method is a buffer having a pH of greater than 7 to a pH less than the pH that Fe (OH) does not precipitate, generally about 10. Preferably, the pH is at about 8-9 and most preferably the pH is about 8.6+/- 0.2. This alkaline reagent, preferably of TRIS buffer, could be any other buffer which will not complex with iron. Examples include Bis-Tris propane, HEPES, PIPES, Tricine Triethanolamine (TEA), GTA buffer, Diethylnolamine (DEA) and others.

The buffer reagent should yield a final concentration in the assay of above 0.1 M, however 0.2 M is used most often. For convenience in dilution and ease of manufacture the buffer concentration in the first composition should be at about 5 - 50 times of the final concentration (e.g. .5 M - 10 M). A molarity of 1 - 3 M is convenient for most dilution purposes.

The second composition comprises a solution of an indicator, a reducing agent to reduce Fe III to Fe II and an acetate buffer at an acidic pH. The second composition may also include an agent to block copper ion interference and detergents to aid with the interference from lipemic serum samples and to aid in preventing the precipitation of proteins, particularly serum proteins. In alternative embodiments of the kit the indicator may be packaged separately from both the reducing agent and the acidic buffer. In yet another embodiment the acidic buffered solution may be packaged (with or without agents that block copper interference and/or detergents) as a separate composition from the composition comprising the indicator and/or reducing agent.

The indicator is an agent that reacts with the Fe II ion to provide a detectable signal. It is preferably a colorimetric molecule. One preferred indicator is the Ferene® Indicator, (3 - 2 - pyridyl) - 5,6 - bis - 2 - (5 - furyl sulfonic acid) - 1,2,4 - triazine, disodium salt. This indicator complexes with Fe II to provide a blue complex that can be measured with a photometer. Other indicators are also known from the literature and include Ferrozine®, chromazurol B, chromazurol S, Sodium bathophenanthroline (SBS), and others. Ferene® or Ferrozine® are preferred due to the fast reaction kinetics and high sensitivity (High molar extinction coefficient) with these chromogens. The indicator must be provided in excess of the maximum amount of iron that would be expected in any sample. The preferred amount as used in the assay is greater than about 0.40 mM. In the composition the indicator should have a convenient concentration for dilution purposes and for ease of manufacture, that is 10 - 1000 times the concentration required in the assay, conveniently about 4 mM to 1000 mM, preferably 200 mM to 600 mM. Most conveniently, it is about 40 mM. The reducing agent must be of a type and at a concentration to reduce substantially all of the Fe III to Fe II. The reducing agent is commonly ascorbic acid but other reducing agents have been used in the field and include any reducing reagent commonly used in an IRN method, such as ascorbic acid, thioglycolic acid, thiomalate, cystein, 2- mercaptoethanol, reduced glutathion, dithionite, hydroxylamine, and others. The concentration used in the assay is in large excess compared to the amount of ferric ion that needs to be reduced. Generally the concentration is greater than about 15 mM. Commonly it is about 10 - 50 mM as used in the assay. The reducing agent in the composition is about 10 - 100 times the concentration that is required in the assay format - that is about 100 mM - 5 M. Conveniently it may be about 300 - 500 mM.

As an alternative to combining the ascorbic acid with the second composition, the ascorbic acid may be supplied in tableted form and reconstituted with water or buffers or either composition 1 or 2 prior to use or even packaged separately. Alternatively, the reducing agent and the indicator may be supplied with the first composition. The second composition may include an acidic buffer solution. Preferably, however, the acidic buffer is packaged separately as a third composition, thereby causing less exposure to the sample. The preferred buffer is an acetate buffered system. The pH of this composition or the second composition, if it contains the acidic buffer should be less than 5.5, preferably about less than or equal to 4.5 so that substantially all of the iron is released from transferrin. The acidic buffer concentration must be at least one time higher than the alkaline buffer concentration. The acidic buffer must not complex with iron. The acidic buffer must be strong enough to lower the reaction mixture pH from alkaline to acidic levels causing transferrin to release all the bound iron. However, the acid should not be too strong to precipitate the plasma proteins. Many buffers can lower the pH about 4 to 5 and will not complex with iron for instance, acetate buffer, GTA buffer, oxalic acid buffer, succinate buffer, and others, preferably acetate buffer pH 3 to 5. Buffers, such as dicarboxylic acid, tricarboxylic acids (e.g. citric acid), ethlenediamineteraacetic acid, and phosphate that will complex with iron should be avoided.

The concentration of the acidic buffer as used in the assay should be about 0.2 - 2.0 M and is preferably about .4 - .8, most preferably about 0,5 M. The acidic buffer as packaged in the composition can be conveniently about 1 - 10 M. If agents to reduce copper ion such as thiourea are to be included, the final concentration of such agent as used in the assay has been found effective at about 30 - 50 mM. Thus, as included in the composition, the concentration should be about 100 mM to 1 M or more. Detergents such as Triton X are effective at final assay concentrations of about 0.5 - 1.5%. Concentrations in the compositions then are conveniently about 3 - 10%. If the acidic buffer is not included in the second composition, it may be included in a separate composition.

Thus the diagnostic kit comprises a first composition comprising an alkaline buffer and a second composition comprising an indicator, a reducing agent and an acidic buffer and may contain detergents and agents that minimize copper ion interference. Alternative embodiments include a diagnostic kit comprising a first composition of an alkaline buffer, an indicator and a reducing agent and a second composition comprising an acid buffer and a diagnostic kit comprising a first composition comprising an alkaline buffer, a second composition comprising an indicator and reducing agent and a third composition comprising an acidic buffer. Any may include detergents, anti-microbials, preservatives or agents that minimize interference. In any embodiment the reducing agent may be provided separately in liquid or tablet format. Similarly, the indicator may be provided separately.

Table 1: General requirement of IRN Diagnostic Kit

Another embodiment includes the use of an indicator to Fe III. In that case a reducing agent is unnecessary, but it is important to free the Fe III from transferrin at a pH that will not cause ferric hydroxide to precipitate. Again, iron is released from transferrin at low pH values.

The performance of a total iron binding capacity test, requires that the diagnostic kit comprise a saturated solution of Fe (III), for instance a saturated solution (about greater than or equal to .35 mM) of ferric chloride to provide a Fe (III) solution of about .02mM. The Fe (HI) may be supplied in a solution at pH less than about 7. One preferred solution is ferric chloride in citric acid.

Table 2: General requirement of TIBC Diagnostic Kit

The method to assay for iron comprises adding to a sample of serum or plasma, first a solution comprising an alkaline buffer of preferentially pH 8 - 9. Next a solution (or solutions) of an indicator and a reducing agent is added. Next, an acidic solution is added.

The reactions for the iron method are as follows: alkaline buffer

Transferrin - Fe III Sample at alkaline pH (of the Sample) reducing agent

Indicator + Sample → Indicator - Fe (II)

(alkaline pH) acidic buffer (color change) pH < 4.5

The reactants may be added at different steps, except that the alkaline buffer must be added first. For instance the indicator can be included with the alkaline buffer as can the reducing agent. It is preferred to add the acidic reagent such that the sample is exposed to the acidic conditions for the shortest amount of time.

The method for TIBC is as follows:

Alkaline buffer Transferrin + excess Fe (III) -» Fe (III) - Transferrin + unbound Fe (III) Reducing agent

Unbound Fe (III) + excess Indicator -> Fe(II) - Indicator complex

(absorbs at certain nm)

Acidic buffer Fe(III)- Transferrin → Transferrin + Fe (III)

Reducing Agent Fe(III) + Indicator → Fe (II) - Indicator complex

(absorbs at certain nm)

Here the order of addition must be such that the alkaline buffer must be added prior to adding reagents that cause the dissociation of Fe III from transferrin. In addition, the dissociation of the iron from transferrin can be caused only after the excess iron is removed or blanked. In the method for TIBC the Fe III that did not bind to transferrin (since it was added in excess of the transferrin concentration) is reduced and reacted with the excess amount of indicator. The resulting color change is blanked (zeroed) on a photometer. Then the acidic buffer is added to cause dissociation of transferrin — Fe. The released iron reacts with the reducing reagent and iron indicator (which were added in excess at the step where the conditions were made alkaline) to provide and increase in absorption (increased color).

The alkaline and acidic buffers used in the present invention can be varied but it is critical that the combination must cause the plasma protein, particularly fibrinogen of the plasma samples to remain in solution. The combinations are readily discernible to those skilled in the art. Central to the invention is having a balanced buffer system in the IRN method. That is, prior to sample addition an alkaline or neutral pH buffer is added. This buffer must have the capability to stabilize plasma protein in acidic condition. Some buffers do not meet the criteria. As discussed above other buffers may be readily evaluated.

Example 1 The commercially available IRN method was evaluated on the Dimension® RxL

System (available from Dade Behring Inc.) and compared with the IRN method of the present invention. All procedures used in these examples were done on a Dimension®

RxL System. All the reactions were carried out at 37 Celsius. All the measurement was done bichromatically with 600 nm as primary wavelength and 700 nm as reference wavelength.

In the commercially available iron method the reaction principle is:

Ascorbic acid Acetate buffer ρH4.5 Transferrin-Fe (III)+Ferene® -» Transferrin (colorless) + Ferene®-Fe (II)

(blue absorbs at 600 nm)

Reaction sequences for Commercially Available Dimension® System Iron Method:

Table 3: Commercially available IRN Method Information

Read 2 minus Read 1 correspond to the serum IRN concentration.

The reagents in the commercially available iron method are shown in Table 4.

Table 4: Reagent Compositions

Procedure: Reagent 1 (Rl), the acetate buffer was added followed by 50ul of serum or plasma sample and a measurement (Read 1) was taken as a sample/reagent blank. After 3.5 minutes, reagent 2, which is iron chromogen was added. After 4 minutes, another measurement was taken (Read 2) and iron concentration correlated to read 2 minus read 1. The total reaction volume is brought to 400 μL with water. The kinetic results of a matched serum and plasma sample using the above parameters are shown in Figure 1. While the method is fast, it is evident that the plasma sample shows a discrepancy when compared to the serum sample.

Example 2

In the IRN method of the present invention the reaction principle is:

Tris buffer pH 8.6

Transferrin - Fe III Sample at alkaline pH (of the Sample)

Ascorbic acid Ferene® + Sample -* Transferrin (colorless)+

(alkaline pH) Acetate buffer pH 4.5 ÷Ferene® - Fe (II)

The Reaction Sequences for the IRN method of the present invention is presented in Table 5.

Table 5: IRN Method of the Present Invention Information

Read 2 minus Read 1 correspond to the IRN concentration.

The IRN reagent composition of the present invention is shown in Table 6.

Table 6: Reagent Composition

Procedure: Reagent 1 (Rl), Tris buffer, was added first, followed by 50 ul of serum or plasma sample After 1.5 min reagent 2(R2), which is iron chromogen was added. After 2 minutes a measurement (Read 1) was taken as a sample and reagent blank. Finally reagent 3, which is acetate buffer was added. After 1.5 min. another measurement was taken (Read 2). The iron concentration correlated to read 2-read 1. The total reaction volume is brought up to 450 μl using water. Unlike many commercial method, the IRN method does not need an additional sample blank measurement, because it uses the difference reading (R2 minus Rl) to determine IRN concentration and all the interference from the sample will be canceled out. The kinetics results of the same matched serum and plasma sample as in Example using the method of the present invention are shown in Figure 2. The serum and plasma match.

Example 3 The commercial IRN method was compared with the IRN method of the present invention using the parameters described in Example 1. Blood samples were freshly drawn from individuals. Serum and Lithium - heparin plasma samples were obtained from the individuals. The results of the mean of N equals five for each sample using the commercial method are presented below.

Table 7: Commercial IRN Serum and Plasma

The results for the iron method of the present invention are detailed below.

Table 8

As can be seen from Table 8 the results from serum and plasma samples match.

Example 4

A method correlation of the commercial IRN method and the method of this invention was compared using 21 serum samples, calibrators and QC material. The assay range is form 0 to 996 μg/dl. This new iron method can be calibrated using the current commercial iron calibrator. A slope of 0.9936, intercept of 3.8 ug/dl and r square of 0.9978 were obtained in the comparison. Good correlation seen with the current method when serum samples are used as seen in Figure 3. Example 5

In the current commercially available (from Dade Behring) TIBC assay at a neutral pH excess iron is added to a transferrin serum sample. The excess iron is removed using an alumina column. A centrifuge step lets iron-transferrin pass out of the column while iron ion remains bound. The eluant is collected and evaluated in the commercial iron method discussed in Example 1. Serum/Plasma discrepancy is the same as in Examples 1 and 2.

With the TIBC of the present method (called the IBCT), the entire assay is automated on the analyzer. Instead of physically removing the excess iron from the solution, it is blanked by measuring its absorbance in the presence of a chromogen Ferene®. In addition, the first step is performed using an alkaline TRIS buffer. In the TIBC method of the present invention, in addition to the reagents for the new IRN method detailed in Table 6, an iron saturating solution was added to saturate all the transferrin iron binding sites. In this iron-saturating reagent, the iron must be in its ferric form and must be added in excess. The preferred ferric salt is ferric chloride. It is also preferred to use more than 1000 μg/dl (or 180 μM) of ferric ion. Since the ferric ion is unstable at alkaline pH values, a concentrated (about 20 times) ferric ion solution is prepared in very dilute (less than 10 mM) HC1 or other acid solution. Thus, when the iron saturating solution is added to the alkaline buffer (e.g. TRIS buffer) the reaction pH will not substantially change. All of the other reagents have the same requirements as set forth for the new IRN assay. Thus the reaction procedure for TIBC of the present invention is:

Excess ferric iron and the same alkaline pH Tris buffer were added to the serum or plasma to saturate all the transferrin iron binding sites, then a reducing reagent and color forming reagent were added and a photometric measurement was made (Read 1). This reading corresponded to the excess unbound iron. In the last step an acid buffer was added to release all the transferrin bound iron and the final photometric reading was made (Read 2). The absorbance increased after the addition of the acid buffer and corresponds to the total iron binding capacity (Read 2-Read 1),

Thus, instead of physically removing the excess iron from the solution (magnesium carbonate precipitation or alumina column), the excess iron is blanked by measuring its absorbance in the presence of a chromogen. In addition, no sample blank is needed because the difference reading (R2-R1) is used to determine TIBC, and all the interference from the sample will be cancelled out. With this new procedure no serum plasma discrepancy is observed.

In the procedure Reagent 1 and Reagent 2 were added first. Next 25 μL of serum or plasma were added. After 1.5 minutes Reagent 3 was added. Next a measurement was taken to blank out excess unbound iron. In the last step Reagent 4 was added. Then another measurement was taken and the TIBC concentration was correlated to the difference between Read 2 and Read 1. Nine matched serum and plasma samples were run and no serum/plasma discrepancy was noted. The kinetic results of the same matched serum and plasma samples as in Examples 1 and 2 using the TIBC method of the present invention is shown is Figure 4.

The TIBC reagent composition of the present invention is shown in Table 9.

Table 9

The Reaction Sequences for the TIBC method of the present invention is shown in Table 10.

Table 10: TIBC Method of the Present Invention Information In the TIBC method of the present invention the reaction principle is:

Alkaline buffer

Transferrin + excess Fe(III) Fe(III) - Transferrin + Fe(III)

Fe(III) + Ferene® + Ascorbic Acid -> Dehydroascorbic Acid + 2H⁺

+

Fe(II) - Ferene® complex

(absorbs at 600 nm) pH 4.5

Fe(III) - Transferrin Transferrin + Fe(IH)

2Fe(III) + Ferene® + Ascorbic Acid --> Dehydroascorbic Acid + 2H⁺

+

Fe(II) - Ferene® complex

(absorbs at 600 nm)

The results of the TIBC method of the present invention are set forth below.

Table 11

Example 6

The procedure described in Example 5 for the TIBC method of the present invention was compared using 74 matched serum samples and Li-Heparin or Na-Heparin plasma samples for the TIBC method of the present invention. The range tested was 270 to 540 μg/dl. No serum/plasma discrepancy was seen. Table 12 and Figures 5 and 6 show the summary of the results.

Table 12

Example 7

The procedure described in Example 5 for the commercial TIBC and the TIBC method of the present invention were compared using 137 serum samples. The method comparison is summarized in Table 13 and Figure 7.

Table 13

Claims

I claim:

1. A diagnostic kit for the measurement or determination of iron in serum or plasma comprising:

(a) a first composition comprising an alkaline buffer; (b) a second composition comprising a chromogenic indicator, a reducing agent and an acidic buffer.

2. The kit of claim 1 wherein the first and/or second composition further comprises an agent that reduces interference from copper ions and a surfactant.

3. The kit of claim 1 wherein the reducing agent is supplied separately in a third composition.

4. The kit of claim 1 wherein the indicator is supplied separately in a third composition.

5. The kit of claim 1 wherein the acidic buffer is supplied separately in a third composition.

6. The kit of claim 3 wherein the indicator is supplied separately in a fourth composition.

7. A diagnostic kit for the measurement or determination of iron in serum or plasma comprising:

(a) a first composition comprising an alkaline buffer and a chromogenic indicator and a reducing agent;

(b) a second composition comprising an acidic buffer.

8. The kit of claim 7 wherein the first and/or second composition further comprises an agent that reduces interference from copper ions and a surfactant.

9. The kit of claim 7 wherein the reducing agent is supplied separately in a third composition.

10. The kit of claim 7 wherein the indicator is supplied separately in a third composition.

11. A diagnostic kit for the measurement or determination of iron in serum or plasma comprising: (a) a first composition comprising an alkaline buffer and a chromogenic indicator and; (b) a second composition comprising an acidic buffer and a reducing agent.

12. The kit of claim 11 wherein the first and/or second composition further comprises an agent that reduces interference from copper ions and a surfactant.

13. The kit of claim 11 wherein the indicator is supplied separately in a third composition.

14. The kit of claim 11 wherein the reducing agent is supplied separately in a third composition.

15. A diagnostic kit for the automated determination of the total iron binding capacity in serum or plasma comprising: (a) a first composition comprising an alkaline buffer;

(b) a second composition comprising an excess ferric salt;

(c) a third composition comprising an indicator and a reducing agent; and

(d) a fourth composition comprising an acidic buffer solution.

16. The kit of claim 15 wherein the first and/or second composition further comprises an agent that reduces interference from copper ions and a surfactant.

17. The kit of claim 15 wherein the second composition is combined with the first composition.

18. The kit of claim 15 wherein the second and third compositions are combined with the first composition.

19. A method of assaying for the concentration of iron in a sample of serum or plasma the method comprising:

(a) combining said sample with an alkaline buffer, a chromogenic indicator for iron as Fe(II), and an agent that reduces Fe(III) to Fe(II); and

(b) next, adding an acidic buffer.

20. The method of claim 19 wherein the alkaline buffer is selected from the group consisting of TRIS, Bis-Tris propane, HEPES, PIPES, Tricine, Triethanolamine (TEA), GTA, and Diethylnolamine (DEA).

21. The method of claim 20 wherein the pH of alkaline buffer is from 7 to 10.

22. The method of claim 19 wherein the chromogenic indicator is selected from the group consisting of Ferrozine® indicator, chromazurol B, chromazurol S, Sodium bathophenanthroline (SBS), and Ferene® indicator.

23. The method of claim 19 wherein the agent that reduces Fe(ffl) to Fe(II) is selected from the group consisting of ascorbic acid, thioglycolic acid, thiomalate, cystein, 2-mercaptoethanol, reduced glutathion, dithionite, and hydroxylamine.

24. The method of claim 1 wherein the acidic buffer has the ability to lower the pH of the solution to about 4 to 5 and will not complex with iron.

25. The method of claim 19 wherein the acidic buffer is acetate buffer, GTA buffer, oxalic acid buffer, succinate buffer.

26. A method of assaying for the total iron binding capacity in a sample of serum or plasma the method comprising: (a) combining said sample with an alkaline buffer, an excess of Fe(III), a chromogenic indicator for iron as Fe(II), and an agent that reduces Fe(III) to Fe(II); and (b) next, adding an acidic buffer.

27. The method of claim 26 wherein the alkaline buffer is selected from the group consisting of TRIS, Bis-Tris propane, HEPES, PIPES, Tricine, Triethanolamine (TEA), GTA, and Diethylnolamine (DEA).

28. The method of claim 27 wherein the pH of alkaline buffer is from 7 to 10.

29. The method of claim 26 wherein the chrornogenic indicator is selected from the group consisting of Ferrozine® indicator, chromazurol B, chromazurol S, Sodium bathophenanthroline (SBS), and Ferene® indicator.

30. The method of claim 26 wherein the agent that reduces Fe(III) to Fe(II) is selected from the group consisting of ascorbic acid, thioglycolic acid, thiomalate, cystein, 2-mercaptoethanol, reduced glutathion, dithionite, and hydroxylamine.

31. The method of claim 26 wherein the acidic buffer has the ability to lower the pH of the solution to about 4 to 5 and will not complex with iron.

32. The method of claim 26 wherein the acidic buffer is acetate buffer, GTA buffer, oxalic acid buffer, succinate buffer.

33. The method of claim 26 wherein the Fe(III) is added as ferric chloride.