WO1986003011A1

WO1986003011A1 - Measurement of total iron binding capacity

Info

Publication number: WO1986003011A1
Application number: PCT/AU1985/000264
Authority: WO
Inventors: Ross Thomas Starr
Original assignee: Ross Thomas Starr
Priority date: 1984-11-07
Filing date: 1985-10-31
Publication date: 1986-05-22
Also published as: US4886642A; EP0231191A1; US4735904A; EP0231191A4

Abstract

An apparatus for use in the measurement of total iron binding capacity of blood serum or plasma comprises a container having a removable closure and containing a measured quantity of dried iron-saturating substance, which is adhered to the inner surface of the container or of the closure and a measured quantity of dried alumina. The invention further provides a simple, rapid and inexpensive method for measurement of total iron binding capacity using said apparatus.

Description

MEASUREMENT OF TOTAL IRON BINDING CAPACITY This invention relates to the measurement of total iron binding capacity in serum.

Iron is carried in the blood plasma by a specific carrier protein called transferrin. This is a protein of molecular weight 76,000 - 80,000, which has two sites each capable of binding one iron atom. The total amount of transferrin present determines the total iron binding capacity (T.I.B.C.) of the serum.

The estimation of the T.I.B.C. is an important clinical procedure, with an established role in the diagnosis of such conditions as iron deficiency anaemia and haemochromatosis, and in the monitoring of therapeutic procedures. In iron deficiency states, T.I.B.C. is elevated; in iron overload conditions T.I.B.C. is depressed. Techniques which are currently used for 5 determination of T.I.B.C. rely on the saturation of transferrin and removal of excess iron from the serum with an adsorbent such as magnesium carbonate (Ramsay, W.N.M.: Clin. Chim. Acta 2_ 221 (1957)) or an ion exchange resin (Peters, T., Giovanello T.J., Apt, L. and Ross, J.R.; J. Lab. Clin. Med. _8

10 274 (1956)). Other methods employ direct measurement of the excess iron after saturation, and calculation of unsaturated iron-binding capacity (Williams, H.L., and Conrad, M.E.; J. Lab. Clin. Med. 61_ 171 (1966); O'Malley, J.A., Hassan, A., Shiley, J. , and Traynor, H.; Clin. Chemistry ]__ 92 (1970)).

15 The most commonly used magnesium carbonate method is subject to error owing to the inclusion of non-transferrin bound iron in the supernatant solution (Ramsay, W.N.M.; J. Clin. Pathol. 2_6 691 (1973)).

An improved method using an alumina column, which

20. was faster and simpler than the magnesium carbonate method while offering improved accuracy, was previously disclosed by the present inventor (Clin. Chemistry 2_6 156 (1980)).

The object of the present invention is to provide improvements in the measurement of T.I.B.C.

25 There is provided in accordance with the invention a tube closed at one end, having a removable closure, the tube containing a measured quantity of a dried iron-saturating substance, which is adhered to the inner surface of the base of the tube, and a measured quantity of dried alumina

30 (aluminium oxide, Al_0,).

Preferably the tube and closure are made of plastics.

Preferably the neck of the plastic tube is threaded to receive a plastics screw cap. The screw cap is most

35 preferably large enough to contain the quantity of alumina. Preferably the tube is made of polystyrene, most preferably of transparent polystyrene. Preferably the cap is made of polyethylene.

The dried iron-saturating substance is preferably a complex salt of ferric iron.

The alumina is preferably basic chromatographic grade alumina, most preferably Brock ann grade II.

Preferably the tube is designed to fit sample carriers of automatic analysers such that their sample probes will sample from above the level occupied by alumina in the tube. According to a further aspect of the invention, there is provided a method of performing the measurement of T.I.B.C. using the apparatus described hereinabove comprising pouring alumina out of the tube into the cap, adding a solvent, for example water, to the tube to dissolve the iron-saturating substance, and adding the sample of serum to the tube; then after a brief period of incubation to allow the iron to bind to transferrin in the serum sample, replacing the alumina in the tube, mixing the tube to allow binding Of the unbound iron to the alumina, and after allowing the alumina to settle, ' measuring the iron content of the supernatant by any suitable means, for example by automatic continuous flow analyser or by atomic absorption spectroscopy.

Preferred embodiments of the invention will now be described by way of example only with reference to the accompanying drawings in which:

Figure 1 represents an exploded view of a free-standing sample tube having a screw cap, containing a measured amount of alumina powder, and having a measured amount of an iron-saturating substance bonded to the interior surface of the tube;

Figure 2 represents an exploded view of free-standing sample tube as in Figure 1 but having a slidably fitting cap; Figure 3 represents an exploded view of a free-standing sample tube as in Figure 1, but having a screw cap with a solid central projection extending to a hollow opening, to the interior surface of which is bonded a measured amount of an iron-saturating substance.

According to one embodiment of the invention there is provided a free-standing sample tube as shown in Figure 1 comprising a polyethylene cap 1 which is adapted to screw onto the threaded neck 2 of a polystyrene tube 3 which is closed at one end, and has a rim 4 forming a base which enables the tube to be free-standing. The tube contains a measured amount of washed and dried chromatographic-grade alumina powder 5, and a measured amount of an iron-saturating substance 6 bonded to the base of the interior surface of the tube 3. According to one preferred embodiment there is provided a free-standing sample tube, comprising a polyethylene cap 1 which is adapted to fit snugly within the neck 2 of a polystyrene tube 3 which is closed at one end, and has a rim 4 forming a base which enables the tube to be free-standing. The tube contains a measured amount of washed . and dried chromatographic-grade alumina powder 5, and a measured amount of an iron-saturating substance 6 bonded to the- base of the interior surface of the tube 3.

The polyethylene cap defines a hemispherical enclosure large enough to contain the alumina and constructed so that the internal diameter of the cap is identical to that of the base. The cap is designed to fit into the base such that there is a continuous, unbroken internal surface.

The tube preferably is 44 mm high (not including the cap), has an outside diameter of 15. mm at the top and 12.5 mm at the base, and has a working capacity of 2 ml. More preferably the tube is 40 mm high.

Preferably 0.4 g of alumina is used.

In a preferred embodiment, the iron-saturating substance is attached to the base of the tube 3 by adding 10 ul of a solution of FeCl₃ at a concentration of 500 g Fe /l in 0.1 mole/1 citric acid containing 0.2% sodium azide and hheeaattiinngg the tube in an oven at 70° until dry (approximately 3 hours) .

In a specific example, the test is performed as follows:-

1. Invert the tube and remove the cap so that the alumina is retained in the cap.

2. Add 1 ml. of water to the tube and wait two minutes or longer to dissolve the dried iron saturating substance which is attached to the bottom of the tube.

3. Add 0.5 ml. of specimen (serum) and stand three minutes or longer to allow the iron to bind to the carrier protein transferrin. 4. Pour the alumina back into the tube and then replace the cap. 5. Place the capped tube onto a laboratory rotator and mix by constant inversion for ten minutes or longer. 6. Remove the tube from the rotator and stand minute or longer and either transfer a portion of the supernatant to another sample container or use the tube itself as a sample container. (The tube may be used as the sample container on some automatic instruments e.g. the

Technicon auto-analysers commonly used in hospitals. 7. Measure the iron content of the supernatant. According to another preferred embodiment, there is provided a free standing sample tube as shown in figure 3, comprising a polyethylene cap 1 which is adapted to screw onto the threaded neck 2 of a polystyrene tube 3 which is closed at one end and has a rim 4 forming a base which enables the tube to be free standing. The tube contains a measured amount of washed and dried chromatographic-grade alumina powder 5. The cap 1 contains a solid central projection which extends to a hollow opening, which may for example be hemispherical. A measured amount of an iron-saturating substance 6 is bonded to the interior surface of the opening. This cap design allows the test to be performed without decanting the alumina. The central projection to which the iron-saturating substance is bonded restricts contact of alumina with the iron-saturating substance, thereby allowing iron to dissolve and bind to transferrin before it is bound to alumina.

The tube preferably is 44 mm high (not including the cap), has an outside diameter of 15 mm at the top and 12.5 mm at the base and has a working capacity of 2 ml. The central projection has an external diameter of 7 mm and extends 11 mm into the tube when the cap is screwed on. . The hollow opening of the projection is able to contain 10 ul of solution.

The iron-saturating substance is attached to the interior of the hollow opening by adding 10 ul of a solution of FeCl, at a concentration of 500 mg Fe /I in 0.45 mole/1 ^* citric acid containing 1% glycerol to the inverted caps and heating at 70 C in an oven until dry (approximately three hours). The concentration of citric acid (0.45 mole/1) is chosen to promote optimum binding or iron to transferrin -in the presence of alumina. The glycerol is added to enhance bonding of the iron-saturating substance to polyethylene.

Preferably 0.4 g of alumina is used.

In a specific example the test is performed as follows:-

1. Remove the cap containing the iron-saturating substance.

2. To the tube containing the alumina add 1 ml of water followed by 0.5 mis of serum.

3. Replace cap and proceed with steps 5 to 7 as before. The present invention represents a considerable improvement compared to the prior art with respect to the cost of materials, stability of reagents, speed and convenience of carrying out the procedure, and reliability of results. The cost of materials for the present invention is less than one third the cost for the column technique. The prepared tubes are stable for at least six months at room temperature, compared to the stability of the stock FeCl.. solution of the present inventor's earlier publication (Clin. Chemistry 2__ 156 (1980)) which was three months at 4°C. The working solution in that case was stable for one week only. The procedure of the present invention can be performed in one minute (excluding the incubation period) , compared to 1 minutes for the earlier procedure.

Furthermore, a considerable amount of labour is avoided by the provision of the reagents in pre-packaged form, eliminating the need for washing and drying and weighing or dispensing of alumina and preparation and dispensing of iron solution for the column method.

Quality control for the procedure can be enhanced by the pre-testing of batches of alumina before addition to the tubes. This overcomes the problem of the occasional batch of alumina which binds iron inconsistently, leading to errors and further expense.

Thus the use of the "one-tube" technique of the present invention results in cost savings in both materials and labour. The procedure of the present invention is not subject to error resulting from lipaemic, icteric or hae olysed serum samples. Preliminary studies indicate that the procedure is applicable to plasma samples as well as to serum samples. It will be clearly understood that the invention in its general aspects is not limited to the specific details referred to hereinabove.

Claims

1. An apparatus for use in the measurement of total iron binding capacity of blood serum or plasma, comprising a container having a removable closure and containing a measured quantity of dried iron-saturation substance, which is adhered to the inner surface of the container or of the 'closure and a measured quantity of dried alumina.

2. An apparatus according to Claim 1, comprising a tube closed at one end, having a removable closure remote from said closed end, the tube containing a measured quantity of dried iron-saturating substance, which is adhered to the inner surface of the tube or the closure, and a measured quantity of dried alumina.

3. An apparatus according to Claim 1 comprising a tube closed at one end, having a removable closure, the tube containing a measured quantity of a dried iron-saturating substance, which is adhered to the inner surface of the base of the tube, and a measured quantity of dried alumina.

4. An apparatus according to Claim 2 or Claim 3 in which the tube and closure are made of plastics.

5. An apparatus according to Claim 4 in which the neck of the plastics tube is threaded to receive a plastics screw cap.

6. An apparatus according to Claim 5 in which the screw cap is large enough to contain the quantity of alumina.

7. An apparatus according to any of Claims 2 to 6 in which the tube is made of polystyrene. •

8. An apparatus according to any of Claims 5 to 7 in which the cap is made of polyethylene.

9. An apparatus according to any of the preceding claims in which the dried iron-saturating substance is a complex salt of ferric iron.

10. An apparatus according to any of the preceding claims in which the alumina is basic chromatographic grade alumina.

11. An apparatus according to any of the preceding claims in which the alumina is of Brockmann grade II.

12. An apparatus for use in the measurement of total iron binding capacity of blood serum or plasma, comprising a polyethylene cap which is adapted to screw on to the threaded neck of a polystyrene tube which is closed at one end, and having a rim forming a base which enables the tube to be free-standing, in which the tube contains a measured amount of washed and dried basic chro atographic grade alumina powder and a measured amount of an iron-saturating substance bonded to the base of the interior surface of the tube.

13. An apparatus for use in the measurement of total iron binding capacity of blood serum or plasma, comprising a polyethylene cap which is adapted to fit snugly within the neck of a polystyrene tube which is closed at one end, and has a rim forming a base which enables the tube to be free-standing, in which the tube contains a measured amount of washed and dried basic chromatographic grade alumina powder, and a measured amount of an iron-saturating substance bonded to the base of the interior surface of the tube, and further characterized in that the polyethylene cap defines a hemispherical^' enclosure large enough to contain the alumina and constructed so that the internal diameter of the cap is identical to that of the base, the cap being designed to fit into the base such that there is a continuous, unbroken internal surface.

14. An apparatus for use in the measurement of total iron binding capacity of blood serum or plasma, comprising a polyethylene cap which is adapted to screw on to the threaded neck of a polystyrene tube which is closed at one end and has a rim forming a base which enables the tube to be free standing, in which the tube contains a measured amount of washed and dried basic chromatographic grade alumina powder, and further characterized in that the cap contains a solid central projection which extends to a hollow opening, which may for example be hemispherical, and in that a measured amount of an iron-saturating substance is bonded to the interior surface of the opening.

15. An apparatus according to any one of Claims 12 to 14, in which the tube is 40-45 mm high (not including the cap), has an outside diameter of 15 mm at the top and 12.5 mm at the base, and has a working capacity of 2ml.

16. An apparatus according to Claim 15 in which the tube is 40 mm high.

17. An apparatus according to any one of Claims 12 to 16 in which the amount of alumina is 0.4 g.

18. An apparatus according to Claim 12 or Claim 13 in which the iron-saturating substance is attached to the base of the tube by adding 10 ul of a solution of FeCl, at a concentration of 500 mg Fe 3+/l in 0.1 mole/1 citric acid containing 0.2% sodium azide and heating the tube in an oven at 70°C until dry.

19. An apparatus according to Claim 14 in which the central projection has an external diameter of 7 mm and extends 11 mm into the tube when the cap is screwed on, such that the hollow opening of the projection -is able to contain 10 ul of solution.

20. An apparatus according to Claim 14 in which iron-saturating substance is attached to the interior of the hollow opening by adding 10 ul of a solution of FeCl^ at a concentration of 500 mg Fe 3+/l in 0.45 mole/1 citric acid containing 1% glycerol to the inverted caps and heating at

70°C in an oven until dry.

21. An apparatus according to any of the preceding claims in which the container or tube will fit the sample carrier of an automatic analyser such that the sample probe of the analyser will sample from above the level of alumina in the tube.

22. A method for measurement of total iron binding capacity using the apparatus described hereinabove comprising the steps of

(a) pouring alumina out of the tube into the cap,

(b) adding a solvent, for example water, to the tube to dissolve the iron-saturating substance, te) adding the sample of serum or plasma to the tube,

(d) briefly incubating to allow the iron to bind to transferrin in the sample,

(e) replacing the alumina in the tube,

(f) mixing the tube to allow binding of the unbound iron to the alumina,

(g) allowing the alumina to settle,

(h) measuring the iron content of the supernatant.

23. A method according to Claim 22, comprising the steps of

(a) Inverting the tube and removing the caps so that the alumina is retained in the cap.

(b) Adding 1 ml . of water to the tube and waiting two minutes or longer to dissolve the dried iron saturating substance which is attached to the bottom of the tube.

(c) Adding 0.5 ml. of serum or plasma and standing three minutes or longer^* to allow the iron to bind to the carrier protein transferrin.

(d) Pouring the alumina back into the tube and then replacing the cap.

(e) Placing the capped tube onto a laboratory rotator and mixing by constant inversion for ten minutes or longer.

(f) Removing the tube from the rotator and standing 1/2 minute or longer and either

(i) transferring a portion of the supernatant to another sample container or

(ii) using the tube itself as a sample container.

(g) Measuring the iron content of the supernatant

24. A method for measurement of total iron binding capacity in which the apparatus of any one of Claims 14, 15, 19 or 20 is used, comprising the steps of

(a) Removing the cap containing the iron-saturating substance. (b) Adding solvent for example water to the tube containing the alumina followed by the sample of serum or plasma.

(c) Replacing the cap.

(d) Mixing the contents of the tube

(e) Allowing the alumina to settle ( (ff)) Measuring the iron content of the supernatant.

25. M Meetthods and apparatus substantially as hereinbefore described,