WO2006111741A1

WO2006111741A1 - A method of measuring the glomerular filtration rate of a human or animal patient, a self-use kit for providing blood samples for use in measuring the glomerular filtration rate of a patient, and a method of collecting timed samples of capillary blood from a patient

Info

Publication number: WO2006111741A1
Application number: PCT/GB2006/001426
Authority: WO
Inventors: Richard Henry Sammons; David John Newman
Original assignee: Epsom & St Helier University Hospitals Nhs Trust; Newman, Jane
Priority date: 2005-04-18
Filing date: 2006-04-18
Publication date: 2006-10-26
Also published as: GB2440081B; GB2466737A; GB0721595D0; GB0507838D0; GB201005968D0; GB2466737B; GB2440081A

Abstract

A method of measuring glomerular filtration rate in a human or animal patient comprising: (i) intravenously administering a known amount of filtration marker to the patient at a known time; (ii) taking a sample of capillary blood from the patient at a known time after administration and absorbing a droplet of blood on an absorbent substrate to form a blood spot on the substrate; (iii) causing or allowing the blood spot to dry on the substrate; (iv) removing a portion of said substrate, said portion having a predetermined, substantially constant volume, and being substantially uniformly impregnated with the blood spot; (v) extracting an analyte from the portion; (vi) quantitating the amount of said filtration marker in said analyte; and (vii) determining the glomerular filtration rate of the patient from the quantitated amount of said filtration marker.

Description

A method of measuring the glomerular filtration rate of a human or animal patient, a self-use kit for providing blood samples for use in measuring the glomerular filtration rate of a patient, and a method of collecting timed samples of capillary blood from a patient

The present invention relates to a method of measuring the glomerular filtration rate of a human or animal patient. The present invention also comprehends a method for collecting timed samples of capillary blood for use in measuring the glomerular filtration rate of a patient, and a self-use kit for use in such a method.

Glomerular filtration rate ("GFR") is a valuable parameter in the determination of renal function. A low or decreasing GFR is a good index of chronic kidney disease, e.g. the progression of diabetic nephropathy. Additionally, to avoid potential drug toxicity, the estimation of GFR in clinical practice allows the proper dosing of drugs excreted by glomerular filtration.

However, clinical practice reference GFR procedures are rarely used, and in large scale research studies a great deal of effort and experience is required which is a considerable disincentive to using GFR as an end-point. Most clinical practice is therefore conducted without recourse to an accurate measure of GFR, and relies on estimates of GFR given by serum creatinine and creatinine clearance which are inaccurate and insensitive owing to differences in production rates between individuals as well as extensive and variable tubular handling by the kidney tubules, and/or inappropriate urine collection. The National Kidney Foundation Disease Outcome Quality Initiative (NKF/DOQI) guidelines recommend that GFR should be estimated from predictive equations such as Cockcroft-Gault^ for

1 Cockcroft, et al: Prediction of creatinine clearance from serum creatine. Nephron, 1976; 16: 31-41 adults (Schwartz^ and Counahan-Barrattβ for children) that take into account serum creatinine concentration and some or all of the patient's age, gender, race, and body size. When such demographic information is unavailable, the use of another endogenous marker for GFR such as Cystatin C has been proposed. It has been shown that plasma levels of Cystatin C are as good an indicator of GFR as serum creatinine concentrations. (Simonsen, et al, 1985^). Cystatin C is eliminated almost entirely through glomerular filtration (Tenstad, et al, 1996^), and is produced at a substantially constant rate by all nucleated cells. Immunoturbidimetry methods for determining GFR based on plasma Cystatin C levels are less prone to interference from haemolysis and icterus, and can be fully automated (Newman, et al, 1995^). All this recommends Cystatin C as better marker than creatinine, especially for the early detection of renal damage.

GFR can be measured by different methods, and with different filtration markers. Regarded as the "gold standard" reference method, the use of Inulin is laborious, requiring pump infusion and the collection of multiple urine and blood

2 Schwartz, et al: The use of plasma creatinine concentration for estimating glomerular filtration rate in infants, children, and adolescents. Pedriatr. Clin. North. Am., 1987 Jun; 34(3): 571-90

3 Counahan, et al: Estimation of glomerular filtration rate from plasma creatinine concentration in children. Arch. Dis. Child., 1976 Nov;51(l l):875-8

4 Simonsen, et al: The blood serum concentration of cystatin C (gamma-trace) as a measure of the glomerular filtration rate. Scand. J. Clin. Lab. Invest., 1985 Apr; 45(2):97-101

5 Tenstad, et al: Renal handling of radiolabeled human cystatin C in the rat. Scand. J. Clin. Lab. Tnvest., 1996 Aug; 56(5): 409-14

6 Newman, et al: Serum cystatin C measured by automated immunoassay: a more sensitive marker of changes in GFR than serum creatinine. Kidney Int., 1995 Jan; 47(1): 312-8 samples, and is therefore not widely available. Cr-EDTA has been proposed as the marker of choice, but as a radiolabeled tracer it is not optimal in certain patient groups.

The use of Iohexol clearance for determining GFR has been disclosed, for example, by Nilsson-Ehle? and Brown, et al, 1991^, the contents are which are incorporated herein by reference. Iohexol clearance has been compared with Cr-

EDTA clearance (Krutzen et al, 1984⁹), with 99mTc-DTPA (Houlihan, et al, 1999Iu₅ tøg contents of which are incorporated herein by reference), and with Inulin clearance in adults (Brown, 1991) and in children (Lindblad, et al, 199411), and has been found to be similar. Its low acute toxicity (Salvesen,

1983l2)₅ easy handling, and stability recommend Iohexol clearance as a reference method.

7 Nilsson-Ehle, P: Iohexol clearance for the determination of glomerular filtration rate: 15 years' experience in clinical practice. EJIFCC, vol. 13, no 2: http://www.ifcc.org/eiifcc/voll3no2/130201005n.htm

8 Brown, et al: Iohexol clearance for the determination of glomerular filtration rate in clinical practice: evidence for a new gold standard. J. Urol, 1991 Sep; 146(3): 675-9

9 Krutzen, et al: Plasma clearance of a new contrast agent, iohexol: a method for the assessment of glomerular filtration rate. J. Lab. Clin. Med., 1984 Dec; 104(6): 955-61

10 Houlihan C, et a A comparison of the plasma disappearance of Iohexol and 99mTc-DTPA for the measurement of glomerular filtration rate (GFR) in diabetes. Aust. NZ J. Med., 1999; 29(5): 693-700

H Lindblad, et al: Comparative evaluation of iohexol and inulin clearance for glomerular filtration rate determinations. Acta Paediatr., 1994 Apr; 83(4): 418-22

12 Salveson: Acute intravenous toxicity of iohexol in the mouse and in the rat. Acta Radiol. Suppl., 1980; 362: 73-5 GFR is generally measured by giving a patient an intra-venous injection of a chosen tracer at a known time into the antecubital vein of one arm, and then following the disappearance of the tracer from the blood by measuring the serum concentration of the tracer remaining in one or more blood samples taken from the patient by venipuncture of the antecubital vein of the contralateral arm at known times after administration of the tracer. The rate of clearance can be calculated and used to determine GFR as long as the timing of the blood sample(s) is accurately recorded. At least two samples should be taken over a period of at least three hours after injection, but preferably four samples are drawn between three and four hours after injection. According to Jacobsson,

198313, the contents of which are incorporated herein by reference, however, the clearance of Iohexol can be accurately estimated in most subjects from a single sample drawn three or four hours after injection.

The problem with this kind of timed measurement is that it requires the patient to remain in an outpatient clinic or surgery for several hours. This is inconvenient to the patient and to the clinical staff who must remain in attendance to collect the blood samples. These limitations have severely reduced the usage of this important kidney function test on grounds of inconvenience and expense. Harvey, 200214 h_as disclosed the use of filter paper to collect blood samples for diagnostic applications. A blood spot dried onto filter paper as a collection matrix is easily shipped through the mail, handled safely, and may provide an extremely stable analyte enjoinment and convenient method of storage. Neonatal blood samples collected on filter paper have been used for PKU

13 Jacobsson, L: A method for the calculation of renal clearance based on a single plasma sample. J. Clin. Physiol., 1983; 3: 297-305

14 Harvey, MA: The use of filter paper to collect blood samples for diagnostic applications, http://www.acefesa.es/bio/iso/papel/papel.doc testing of newborns since 1963. (Guthrie, 1963^), Since then, blood collected onto filter paper has been used to acquire samples for testing of thyroid hormones, antibodies against infectious diseases, therapeutic drug monitoring, drugs of abuse, haemoglobinopathies, and circulating lead levels. However, in order to be suitable for use in a reliable, practical diagnostic test, the collection matrix, such as filter paper, must be capable of collecting a dried specimen stably and reproducibly, and must not bind the analyte of interest or contribute interferences to the assay. Further, analyte must be easily and reproducibly extracted from the collection matrix. An object of the present invention is to provide an improved method of measuring GFR in an human or animal patient.

In particular it is an object of the present invention to provide a reliable, reproducible method of measuring GFR which is more convenient for patients and medical staff than the periodic collection of serum samples. Another object of the present invention is to provide an improved method of collecting timed samples of capillary blood from a patient.

Yet another object of the present invention is to provide apparatus, such as a self use kit, to facilitate the collection of timed samples of capillary blood from a patient. A particular obj ect of the present invention is the provision of apparatus, such as a self-use kit, which is convenient to use and which facilitates the collection of accurately timed samples of blood from a patient.

A further object of the present invention is to provide an improved method of diagnosing chronic kidney disease. Further objects and advantages of the present invention will be apparent from the following disclosure thereof.

15 Guthrie, R, et a A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics, 1963; 32(3): 338-343 In accordance with one aspect of the present invention therefore there is provided a method of measuring glomerular filtration rate in a human or animal patient comprising:

(i) intravenously administering a known amount of filtration marker to the patient at a known time;

(ii) taking a sample of capillary blood from the patient at a known time after administration and absorbing a droplet of blood on an absorbent substrate to form a blood spot on the substrate;

(iii) causing or allowing the blood spot to dry on the substrate; (iv) removing a portion of said substrate, said portion having a predetermined, substantially constant volume, and being substantially uniformly impregnated with the blood spot;

(v) extracting an analyte from the portion;

(vi) quantitating the amount of said filtration marker in said analyte; and (vii) determining the glomerular filtration rate of the patient from the quantitated amount of said filtration marker.

In another aspect, the present invention provides an ex vivo method of determining the glomerular filtration rate of a human or animal patient comprising: (a) providing one or more pieces of absorbent substrate, the or each piece of absorbent substrate comprising a dried blood spot consisting of a sample of capillary blood obtained from the patient at a known time after intravenous injection of the patient with a known amount of a filtration marker;

(b) removing a portion of the or each piece of absorbent substrate, said portion having a predetermined, substantially constant volume, and being substantially uniformly impregnated with the dried blood spot;

(c) extracting an analyte from the or each portion;

(d) quantitating the amount of filtration marker in the or each analyte; and

(e) calculating the glomerular filtration rate of the patient from the quantitated amount(s) of filtration marker. The absorbent substrate may be any substrate that is capable of absorbing and holding a blood sample within its structure. Preferably the absorbent substrate is at least one filter paper.

Capillary sampling is simple, effective and may significantly reduce the time and costs of performing plasma clearance GFR measurements. It has been found that blood samples comprising a filtration marker are stably and reproducibly absorbed by an absorbent substrate such as a filter paper, and filtration marker-containing analytes can be easily and reproducibly extracted from an absorbent substrate for diagnostic analysis. The absorption of blood onto an absorbent substrate in the form of a blood spot which is dried for handling, and the subsequent extraction of an analyte from the absorbent substrate does not measurably interfere with the determination of GFR. Standard linear regression analysis has found good agreement between prior art methods of measuring GFR by determining the concentration of the filtration marker in serum samples, and the methods of the present invention; Bland Altaian analysis has shown that there is no significant bias between them.

Accordingly the method of the present invention may make GFR measurement more accessible for clinical practice, and large scale epidemiological studies feasible with central laboratory analyses. In some embodiments, steps (ii) and (iii) may be repeated periodically at known times after administration to obtain a plurality of blood samples on different, respective pieces of absorbent substrate; a portion of each piece of absorbent substrate may be removed, and an analyte extracted from each portion; the amount of filtration marker in each sample may be quantitated; thereafter the glomerular filtration rate of the patient may be determined from the quantitated amounts and the corresponding known times.

Advantageously, steps (ii) - (iii) may be carried out at a first location, and the piece or pieces of absorbent substrate are then conveyed to a second location where steps (iv) - (vii) are performed. The first location may be the patient's home, or any other location that is convenient for the patient, and the second location is typically a clinic or analytical laboratory.

Said piece or pieces of absorbent substrate may be conveyed from said first location to said second location by post. Paired comparisons (t-test) have shown that is no significant difference between GFRs determined from blood spots on filter paper that is retained at one location, and blood spots which are shipped through the mail. Thus, in accordance with a particular aspect of the present invention, a patient may be released from a clinic or surgery after having been injected with intravenous filtration marker as a tracer for measuring kidney function - and after a suitable short post-injection period to ensure that the patient suffers no adverse reaction to the injection -, and the patient may then return home, or to any other convenient location, where the sample or samples of capillary blood may be collected on the absorbent substrate for posting to the clinic or surgery, or to a different location, such for example as a central testing laboratory, for analysis. Not only is this more convenient for the patient, but it may also be more convenient for the medical staff at the clinic who are not needed to attend on the patient for the period of the test.

In accordance with the present invention therefore the patient need only remain in the clinic or surgery for a short time, thereby freeing up clinical staff and significantly reducing the costs and inconvenience of the test, and making the measurement of GFR a practical epidemiological tool which may offers significant advantages to pharmaceutical companies, for example, who may wish to study the effects of novel agents on renal function, since fewer subjects would be required (as the test is more sensitive and specific than others in current usage) with associated reduced recruitment times.

Preferably the step of taking a sample from the patient's skin is by pricking a suitable part of the patient's skin.

The glomerular filtration rate may be determined by calculating the blood concentration of the filtration marker in the or each sample by reference to the volume of blood absorbed by the respective portion of absorbent substrate, using the filtration marker blood concentration to calculate the plasma concentration of filtration marker in the or each sample, and thereafter calculating the glomerular filtration rate from the plasma concentration of filtration marker at the or each of said known times. Preferably the plasma concentration of filtration marker may be calculated from the blood concentration by means of the haematocrit correction

In some embodiments, the glomerular filtration rate may be calculated from the plasma concentration of filtration marker at the or each known time by a single compartment analysis with the Brochner-Mortensen^ correction, as taught by Pucci, et al., 200117, the contents of which are incorporated herein by reference.

In some embodiments, a single sample may be taken at a known time after administration, preferably at least three hours after administration. The GFR may be calculated according to the method disclosed by Jacobsson, 1983 or Boijsen, et al, 1988¹⁸.

Preferably, two or more samples of blood are taken after administration within a period of at least three hours, and typically three to five samples of blood may be taken over a period of three to five hours, for example four hours. For example, three samples may be taken at intervals of 120, 180 and 240 minutes after filtration marker injection.

1° Brochner-Mortensen: A simple method for the determination of glomerular filtration rate. Scand. J. Clin. Lab. Invest., 1972 Nov; 30(3): 271-4

17 Pucci, et al: Iohexol as a marker of glomerular filtration rate in patients with diabetes: comparison of multiple and simplified sampling protocols. Diabet. Med. 2001 Feb; 18(2): 116-20

18 Boijsen, et al: Glomerular filtration rate estimated from a single plasma sample after contrast-enhanced radiological examinations. CHn. Physiol, 1988 Jun; 8(3): Said analyte may be extracted from the or each portion using any suitable reagent which deproteinises the sample and acts as a solvent for the filtration marker for extracting the filtration marker from the absorbent substrate matrix. Preferably, when Iohexol is used as the filtration marker perchloric acid may be used, but alternative reagents include acetonitrile.

The reagent/analyte mixture may be vortexed, and then centrifuged to separate the solution from solid precipitant.

The amount of filtration marker in the or each analyte may be quantitated by any suitable method known to those skilled in the art. Reverse phase HPLC is a standard method for measuring a wide range of chemicals, in biological laboratories and in industry, which works by separating out the constituents of a mixture according to their different affinities for a mobile phase which is past under pressure over a solid phase, typically packed in a column. There are a number of possible alternatives to HPLC, but most may be prohibitively expensive. A practical alternative to HPLC may be capillary electrophoresis which uses an electric charge to separate the constituents of a mixture.

In some embodiments, the or each portion of absorbent substrate removed from the or each piece may have an area of about 20-40mm2, preferably about

30mm2. The portion may be removed as a punch, for example a substantially circular punch having a diameter of about 6.0 - 6.5mm, e.g. 6.3mm. Thus, the or each portion may contain about 7.5 to 15μl of blood, preferably about 1 lμl.

In a different aspect of the present invention there is provided a self-use kit for providing blood samples for use in measuring the glomerular filtration rate of a patient, said kit comprising: a container that is adapted to be sent by post; one or more pieces of absorbent substrate of a quality and consistency suitable for the collection of blood spots for diagnostic analysis; and a timer comprising a user-operable actuator, recording means for recording the time the or each time the actuator is operated, and means for extracting said time or times from the timer; whereby a patient obtains one or more timed samples of capillary blood after being intravenously injected at a known time with a filtration marker, collecting a blood spot on the or a respective one of said pieces of absorbent substrate, and actuating said actuator upon obtaining the or each sample to record the time of the or each sample; whereafter the piece or pieces of absorbent substrate and the timer may be packaged in the container and sent by post to a laboratory for analysis of the timed blood samples to determine the rate of clearance of the filtration marker.

Preferably the absorbent substrate is any material that is capable of absorbing the blood sample and holding it in its structure. More preferably the absorbent substrate is at least one filter paper.

Thus the present invention also comprehends apparatus to facilitate the collection at a first location of a timed capillary blood sample or samples by a patient for use in the methods of the present invention for determining glomerular filtration rate. The patient is given an intravenous injection of a filtration marker at a medical clinic, surgery or the like, and is then allowed to leave the clinic or surgery and travel to the first location, which may typically be their home, having been instructed to take one or more timed capillary blood samples using the kit of the present invention. Said sample or samples of capillary blood may be obtained by pricking a suitable part of the patient's body, e.g. on a finger or toe, with a sterile lancet or the like and dripping the resulting blood onto the piece or pieces of filter paper provided in the kit, on which piece or pieces of filter paper the time or times of sampling may be recorded in writing. The kit of the invention comprises a timer that independently records the time(s). The capillary sample or samples may be air-dried (thus preventing the postage of any liquid biological samples) and when all have been taken the container, including the timer and samples may be re-sealed and posted to a second location, which may the same as the clinic or surgery or different, for analysis.

Conveniently said kit may further comprise one or more sterile lancet devices for use in pricking the patient's skin. Preferably, the or each lancet device may comprise an automatic lancet device comprising a lancet, and selectively operable means for rapidly urging said lancet forwards for pricking the skin. In accordance with yet another aspect of the present invention there is provided a self-use kit for providing blood samples for use in measuring the glomerular filtration rate of a patient, said kit comprising a robust container that is adapted to be sent by post, said container containing: one or more pieces of absorbent substrate removably disposed within the container, said piece or pieces being of a quality and consistency suitable for the collection of blood spots for diagnostic analysis; a blood withdrawing means for withdrawing a sample of blood from the patient said means being removably disposed within the container; a holder removably disposed within the container for said blood withdrawing means, said holder comprising a gripping portion for releasably gripping a part of the patient's body from which blood is to be withdrawn, thereby to attach the holder stably to the patient's body, and to squeeze said part of the patient's body, thereby to encourage the formation of a droplet of blood for collection on said absorbent substrate to obtain a sample of capillary blood, and a holding portion for holding releasably the blood withdrawing means stably in juxtaposition with said part of the patient's skin; and a timer comprising a user-operable actuating device, a recorder device for recording the time the or each time the actuator is operated, said recorder device comprising a solid state memory device for storing the time of the or each blood sample, and being configured for automatically recording the time of each operation of said actuating device, and a device for extracting said time or times from the timer.

Preferably the blood withdrawing means comprises one or more lancet devices as described herein, for use in pricking the patient's skin. The lancet device may comprise a lancet, a selectively operable firing mechanism for rapidly urging the lancet forward for pricking the skin. Thus, in accordance with yet another aspect of the present invention there is provided a method of collecting timed samples of capillary blood from a patient who has been intravenously injected at a known time with a known amount of filtration marker for determining the patient's glomerular filtration rate, said method comprising: providing said patient with a kit comprising a robust container that is capable of being sent by post, said container containing one or more pieces of absorbent substrate of a quality and consistency suitable for the collection of blood spots for diagnostic analysis, a means for withdrawing blood form the patient, and a timer comprising a user-operable actuator, recording means for recording the time the or each time the actuator is operated, and means for extracting said time or times from the timer; instructing the patient to use said means for withdrawing blood and said one or more pieces of absorbent substrate to collect periodically one or more samples of capillary blood as blood spots on said absorbent substrate, to operate the actuator of the timer the or each time a sample is obtained, to allow the blood spot or spots to dry, to replace the piece or pieces of absorbent substrate in the container, and thereafter to transmit the container to a laboratory; and receiving the container at the laboratory, removing the piece or pieces of absorbent substrate carrying the blood spots, and extracting from the timer the time of the or each blood sample after filtration marker administration.

Advantageously the timer may be integrated into the container.

In some embodiments, said time of the or each blood sample may be extracted by connecting the timer to a computer and uploading the time or times from the recording means.

The kit of the present invention may further comprise a holder for the or each lancet device; said holder may comprise means for releasably gripping a part of the patient's body thereby to attach the holder stably to the patient's body, and means for holding said lancet device stably in juxtaposition with a part of the patient's skin which is to be pricked to obtain a sample of capillary blood. Said holding means may be adapted to hold releasably said lancet device, whereby the holder can be used with a plurality of lancet devices in succession. Said gripping means may be adapted for releasably gripping a patient's finger or toe. Preferably said gripping means may be adapted to squeeze the part of the patient's skin that is to be pricked with the lancet device, thereby to encourage the formation of a droplet of blood for collection on filter paper.

In some embodiments said holder may comprise a holder in accordance with International patent application published as WO-A-2004064637, the contents of which are incorporated herein by reference. In some embodiments, said holder may be stored within the container.

Suitably, said timer may comprise a solid state memory device for storing the time of the or each blood sample. The timer may be configured to record automatically the time of each operation of said actuator.

The timer may be able to record the exact time the sample is taken from the patient. Preferably, the timer may be configured to instantaneously record the exact time when a blood sample is withdrawn from the patient. The timer may thus be able to automatically record the time when sampling occurs upon blood being withdrawn from the patient. The timer may be integrated into the lancet device such that upon blood being withdrawn from the patient by pricking his skin with the lancet, the timer is automatically and instantaneously capable of detecting the withdrawn blood and records the time. In another aspect, the timer may be integrated in to the filter paper such that when the patient blots the withdrawn blood on to the filter paper the timer is automatically and instantaneously capable of detecting the withdrawn blood and records the time. The timer located within the lancet or filter paper may record the time by detecting the withdrawn blood directly or by detecting a signal generated by the filter paper or lancet upon contact with withdrawn blood.

Said extracting means comprise means for displaying the time of the or each sample. In some embodiments, the extracting means may comprise means for connecting the timer to a computer for uploading the recorded time or times from the timer to the computer.

In yet another aspect of the present invention there is provided a self-use kit for providing blood samples for use in measuring the glomerular filtration rate of a patient, said kit comprising a robust container that is adapted to be sent by post, said container containing: one or more pieces of filter paper removably disposed within the container, said piece or pieces being of a quality and consistency suitable for the collection of blood spots for diagnostic analysis; one or more automatic lancet devices removably disposed within the container for use in pricking the patient's skin, the or each lancet device comprising a lancet, and a selectively operable firing mechanism for rapidly urging said lancet forwards for pricking the skin; a holder removably disposed within the container for the or each automatic lancet device, said holder comprising a gripping portion for releasably gripping a part of the patient's body that is to be pricked with the lancet device, thereby to attach the holder stably to the patient's body, and to squeeze said part of the patient's body, thereby to encourage the formation of a droplet of blood for collection on filter paper to obtain a sample of capillary blood, and a holding portion for holding releasably the or one of the lancet devices stably in juxtaposition with said part of the patient's skin; and a timer comprising a user-operable actuating device, a recorder device for recording the time the or each time the actuator is operated, said recorder device comprising a solid state memory device for storing the time of the or each blood sample, and being configured for automatically recording the time of each operation of said actuating device, and a device for extracting said time or times from the timer.

In yet another aspect of the present invention there is provided a method of diagnosing chronic kidney disease in a human or animal subject, said method comprising measuring the glomerular filtration rate of said subject according to any of the methods of the present invention and comparing said glomerular filtration rate of said subject with a predetermined glomerular filtration rate of a healthy individual, wherein a deviation in said glomerular filtration rate of said subject from the glomerular filtration rate of said healthy individual is indicative of chronic kidney disease.

Preferably, a decrease in the glomerular filtration rate of the subject compared to the healthy individual is indicative of diabetic nephropathy.

The methods or self use kit of the of the present invention require the step of intravenously administering an amount of filtration marker to the patient. Preferably, the filtration marker is biologically inert, freely filtered by the kidney and does not undergo metabolism, tubular secretion or absorption. Suitable filtration markers include non-radioactive contrast agents. Other suitable filtration markers include ethylendiaminetetraacetic acid (EDTA) and diethylenetriaminepentacetate acid (DTPA). EDTA or DTPA may be radio labelled with a radioactive species. Preferably, the marker is not radioactively labelled and is isothalamate or Iohexol. Most preferably, the filtration marker is Iohexol.

Following is a description by way of example only with reference to the accompanying drawings of embodiments of the present invention. In the drawings:

FIG. 1 is a graph showing the correlation between GFRs determined in accordance with the method of the present invention, and a prior art method of analysing plasma obtained from blood samples.

FIG. 2 is a histogram showing the recovery of Iohexol from plasma spots, and blood spots.

FIG. 3 is a perspective view of a self-use kit for collecting timed capillary blood samples in accordance with the present invention, with the lid removed for clarity.

Comparative Example 1 After informed consent, eighty-two subjects comprising twenty-two healthy volunteers and sixty patient volunteers were included in the study. Their respective heights and weights were recorded. For the healthy volunteers (nine male and thirteen female) the median age was 41 years (range 21-62 years). All healthy volunteers had normal levels of serum creatinine. The patients (with differing degrees of renal impairment, and a wide range of GFRs) volunteering for the study (thirty-four male and twenty-six female) were selected on the basis of their serum creatinine levels as follows;

Normal: <130 μmol/1 S-creatinine Median: 130-300 μmol/1 S-creatinine

High: >300μmol/l S-creatinine

All subjects had a restricted diet (low protein intake and non-caffeinated drinks, etc.) for about six hours before the test.

Iohexol stock solution (647mg I/ml, available under the trade name Omnipaque® 300 from Mycomed Amersham pic, Buckinghamshire, UK) was diluted into mobile phase (acetonitrile 3.5%, pH adjusted to 2.5 with concentrated orthophosphoric acid) to provide two Iohexol standards having final concentrations of 129 μg/ml and 294 μg/ml Iohexol. The Iohexol standards were stored at 4⁰C. From the same Iohexol stock solution, three quality control plasma samples were also prepared to give final concentrations of 20.2, 40.4 and 80.8 μg/ml and stored at -70⁰C.

The subjects attended a location where the following procedure was carried out. A zero-time blood sample, and an additional EDTA blood sample (for haematocrit analyses) were collected from each subject prior to the intravenous injection of 5 ml of Iohexol into the anticubital vein of one arm, and the exact time of the injection was recorded. The cannula was flushed with 10 ml of 0.9% saline. The subjects were supervised for 15 minutes for possible adverse reactions, and were then allowed to leave the location between sampling if they wished.

After injection of Iohexol, blood samples were collected at about 120, 180, and 240 minutes by separate venipunctures of the antecubital vein of the contralateral arm. The exact collection times were recorded. Serum was separated from the blood samples and stored at 4⁰C at the location for analysis the next day.

56μl of each of the serum samples, standards, and quality controls were then treated with 850μl of perchloric acid to precipitate serum proteins. The samples were vortexed for 3 mins., ultrasonicated for 15 mins., and then, after 30 mins. on the bench, spun at 1400Og for 10 mins.

Iohexol was quantitated from serum by automated reverse phase high- pressure liquid chromatography (HPLC) using external calibration under the following conditions: Column: Nucleosil® 120, particle size 5μm, with a 5cm guard column packed with the same material intermediate to the injector

Pump: 1.5ml/min

Detector wavelength: 254nm

Range 0.05 Running time: 10 min

The volume of one injection was lOOμl.

Iohexol elutes from the column as two peaks; the height of the second peak, which elutes at 6.5 mins., was used for the calculation of GFR. (The first peak can also be used for calculation of GFR). Iohexol clearance was calculated according to a one-compartment model

(plus Brochner-Mortensen correction) by plotting Iohexol concentration (C) against time (t) after injection, calculating a "best fit" straight line for the three time-points, and determining the slope (k) and intercept (Ct=o) °f said line. According to the one compartment model, the clearance of Iohexol after bolus injection is described by a one-exponential function. The best fit line was calculated by the least squares method, although other, equivalent methods known to those skilled in the art could be used.

In the present example, GFR values were calculated by entering the measured Iohexol concentrations and times of sample collection into a software spreadsheet programme (Microsoft Excel®) in which the following formulae were employed (in which 12, J2, and K2 were the spreadsheet cells containing the measured Iohexol concentrations for the collection times stored in cells F2, G2, H2 respectively): k = LINEST ((LN (I2:K2)), F2:H2)

The value of k was stored in cell L2.

C_t=o = EXP (LN(K2)+(L2*F2))+EXP(LN(J2)+(L2*G2)))/3

Clearance = Dose in (mg)*k/Ct=o

The value of said clearance was stored at cell N2. Corrected clearance - (0.990778*N2)-0.001218*N2^Λ2

(LINEST returns an array that describes a straight line that best fits the data, calculated according to the least squares method. EXP raises e to the power of a given number. LN returns the natural log of a given number. ^Λ Raises a given number to the power of another given number. * Multiplies a given number by another given number).

The corrected clearance figures were further adjusted for the standardised body surface area according to Du Bois, et ah, 1989*9 at 1.73 cm^. (Ref). Inventive Example 2

At the same intervals as taking blood samples from each patient by venipuncture in Comparative Example 1, samples of capillary blood were also

19 Du Bois, et at A formula to estimate the approximate surface area if height and weight be known. 1916. Nutrition, 1989 Sep-Oct; 5(5): 303-11: discussion 312-3 collected as blood spots on filter paper (Schleicher & Schuell® Grade 903-) by a finger-prick method. The exact collection times were recorded, and the blood spots were air-dried and retained at the location for analysis within 48 hrs.

A 6.3mm punch of each blood spot (containing 11.2 μl of blood) was treated with 170μl 5% perchloric acid to extract an analyte from the filter paper, which analyte was then vortexed, ultrasonicated, and spun as before. The concentration of Iohexol in each analyte was determined by HPLC as described in Comparative Example 1.

The concentrations of Iohexol in the samples of capillary blood were then calculated assuming a fixed volume of blood per mm^ of filter paper, and the corresponding serum concentrations of Iohexol (S-Iohexol) were calculated using the haematocrit correction formula: [S-Iohexol]= [Iohexol]/(l-ht).

The haematocrit correction formula is a standard method for correcting the concentration of substances found in plasma to the corresponding concentration for whole blood.

A single compartment analysis (plus Brochner-Mortensen correction) of the S-Iohexol concentrations of the three capillary blood samples at the recorded collection times was then used to calculate the GFR as described above in Comparative Example 1.

Standard linear regression was used to compare the techniques of

Comparative Example 1 and Inventive Example 2, and Bland Altman^O analyses were also carried out. As shown in FIG. 1, for the eighty-two GFR measurements, performed in a mixture of healthy volunteers and patients with differing degrees of renal impairment, the standard linear regression analyses showed good

20 Bland, et al: Applying the right statistics: analyses of measurement studies. Ultrasound Obstet. Gynecol., 2003 JuI; 22(1): 85-93 agreement between the two techniques (r²=0.953); the Bland Altman analyses showed no concentration dependent bias.

Inventive Example 3

At the same time as collecting the capillary blood spots for retention at the location and analysis the next day in accordance with Inventive Example 2, second blood spots on filter paper were collected and air-dried as before. The second blood spots were then securely packaged, self-addressed, and posted back to the location for analysis the following day at the same time as the blood spots of Inventive Example 2 which were retained at the location. The second blood spots were thus passed through the mail service.

Upon receipt of the second blood spots, a 6.3mm punch of each blood spot was treated with perchloric acid as per Inventive Example 2 to extract an analyte from the filter paper. The analytes were then further treated and analysed as described in Inventive Example 2 to calculate the GFR. (The blood spots obtained in Inventive Example 2 were analysed at the same time).

The results of Inventive Examples 2 and 3 were analysed by paired comparisons (t-test), which showed no significant difference between the two measurements.

In order to optimize the method of the invention, recovery and precision experiments were performed as follows:

To test the recovery of Iohexol from blood, blood and plasma samples from five patients were spiked with 50μg/ml Iohexol and incubated for 0, 1, 2, 3, and 4 hours at 37⁰C. The samples were then spotted onto filter paper to form blood spots and plasma spots respectively, and treated and analysed as described in Comparative Example 2 to ascertain whether, and if so the extent to which, the red blood cells absorbed Iohexol. The concentration of Iohexol in whole blood and in plasma calculated by the haematocrit correction (HCT) was recorded. As shown in FIG. 2, the results showed that there was a 40% under-recovery of Iohexol from blood spots as compared with the plasma samples. In order to check the precision of the method of the present invention, the quality control plasma samples at 20.2, 40.4, and 80.8 μg/ml Iohexol were spotted onto filter paper to form plasma spots. Twenty spots were made at each reference concentration, and the concentration of Iohexol in the quality control plasma samples and plasma spots was then measured as described in Comparative Example 1 and Inventive Example 2 respectively. For each concentration, ten plasma spots were analysed the same day and ten spots were analysed after ten days ("between days"). The results are given in Tables 1 and 2 below:

Table 1: Within day

Plasma (n= 10)

20.2 μg/ml 40.4 μg/ml 80.8 μg/ml Mean

CV 3.4 0.3 4.2 2.7

Plasma spots (n=10)

CV 3.3 3.8 3.5 3.5

(CV = coefficient of variation)

Table 2: Between days

Plasma (n=10)

20.2 μg/ml 40.4 μg/ml 80.8 μg/ml Mean

CV 6.3 6.2 5.6 6.0

Plasma spots (n =10)

CV 8.1 6.2 4.8 6.4

Analyses of plasma and plasma spots showed mean CVs of 2.7 respectively 3.5 within day. Between days, the mean CV was 6 for plasma, and 6.4 for plasma spots.

FIG. 3 shows a self-use kit 10 in accordance with the present invention for use in collecting timed capillary blood samples. The kit 10 comprises a hinged two-part container 12 consisting of a receptacle portion 14 and a lid (not shown). In FIG. 3, the lid is removed for clarity, to show the contents of the container 12, but is hinged to the receptacle 14 at 16, 18 to allow the container to be opened to gain access to its contents. A securing means is provided (not shown) of any kind known to those skilled in the art to be suitable for securing the lid to the receptacle portion for preventing accidental opening of the container, for example when the container is in transit as described below.

Said container 12 is sufficiently robust to withstand transit through the mail, and may for example be moulded from a suitably tough synthetic resin material, such as a thermoplastic resin, e.g. polyethylene or polypropylene. The exterior of the container 12 is preferably adapted to carry a means (not shown) for addressing the container 12 for the purposes of mail delivery. For example the exterior surface of the container 12 may comprise a recessed region which is shaped to receive an address label and/or postage stamps. The receptacle portion 14 of the container 12 comprises a housing portion

20 that accommodates a timer device 30 as described in more detail below, and defines a compartment 22 adjacent the housing portion 20 that accommodates a plurality of items that are removable from the container 12.

Advantageously the compartment is formed with a plurality of integrally moulded formations 24 that are adapted to hold said items firmly in transit. In particular, said formations 24 are adapted to hold one or more (for example 3, 4 or 5) pieces of filter paper 41, one or more sterile lancet devices 42, a pencil (not shown), and a lancet holder 45.

Said piece or pieces of filter paper 41 are suitably of a grade adapted for the collection of blood samples, for example Schleicher & Schuell® Grade 903.

The or each lancet device 42 is preferably an automatic lancet device comprising a needle and a selectively operable "firing" mechanism for propelling the needle rapidly forwards to prick a person's skin to draw a blood droplet. Suitable automatic lancing devices are well known to those skilled in the art, for example the device described in US-A-5487748, the contents of which are incorporated herein by reference.

Said lancet holder 45 comprises a means 46 for releasably gripping a part of the patient's body thereby to attach the holder stably to the patient's body, and a means 47 for holding one of said lancet devices 42 stably in juxtaposition with a part of the patient's skin which is to be pricked to obtain a sample of capillary blood. Said holding means 47 is adapted to hold said lancet device 42 releasably, whereby the holder 45 can be used with each of the lancet devices 42 in succession, where more than one are provided in the container 12. Said gripping means 46 may be adapted for releasably gripping a patient's finger or toe.

Advantageously, the gripping means 46 may be adapted to squeeze the part of the patient's skin that is to be pricked with the lancet device 42, thereby to encourage the formation of a droplet of blood for collection on the or one of the pieces of filter paper 41. More particularly, said lancet holder 45 may comprise a holder in accordance with copending UK application no. 0301587.2, having two opposing jaw members 48,49, each jaw member comprising a generally half-cylindrical, member-engaging portion, wherein said jaw members define a generally cylindrical space 50 therebetween, which space is adapted to accommodate a member of a patient's body; two cooperating levers 51, 52, each lever having a proximal end that is joined to a respective one of the jaw members, and a distal end; a fulcrum portion 53 between said levers intermediate said proximal and distal ends, such that upon squeezing the distal ends of the levers together, the proximal ends are moved apart, thereby to open said jaw members; a compression spring means 54 between the distal ends of the two levers 51, 52 for squeezing said jaw members together for removably attaching the device stably to the member and for gently squeezing the member between said jaw members; said lancet holding means 47 positioned on one of said jaw members, said lancet holding means being configured for securely holding a lancing device 42 juxtaposed said member, with the needle directed towards said member for pricking the member; and a needle passage that extends through said one jaw member to allow said needle to pass therethrough into the space between said jaw members for pricking said member.

The timer device 30 is battery-operated, and the housing portion 20 includes a battery-compartment 21 which is adapted to accommodate one or more power cells needed to power the timer device 30. Said timer device comprises a clock device, a memory device, a user-operable actuator, and control means, which control means are configured to write to the memory device the time of the clock device every time the actuator is operated. Preferably said memory device comprises a solid state memory that cannot be erased by an accidental loss of power, and cannot be erased by a magnetic field that could be experienced during passage through a postal system. Said actuator may comprise a push button 32.

The timer device may include a display 33 for displaying times stored in the memory device and/or an output port (not shown) adapted for connection to an external computer (not shown) for uploading to the computer the stored times. Said control means may be configured to respond to an appropriate control signal from an external computer to cause the time or times recorded in the memory device to be displayed on said display 33 and/or to be transmitted to the external computer.

In some embodiments, the timer device may comprise an integrated data access device to allow access to the data stored in the memory device upon actuation of a key or the like.

The timer device 30 is configured so as to prevent the times recorded in the memory device from being deleted or changed by the patient.

The kit 10 according to the present invention is thus adapted to facilitate collecting one or more timed samples of capillary blood from a patient for analysis.

For the measurement of GFR, a subject may be given an intravenous injection of a given amount of a suitable tracer such, for example, as Iohexol as described above. The time of injection is recorded accurately. The injection is given at a suitable location such, for example, as a medical clinic or surgery by or under the supervision of trained medical staff. After a short time to ensure that the subject does not suffer any adverse reaction to the injection, the subject is provided with a kit as hereinbefore described and allowed to leave the location. Typically the subject may return home.

At timed intervals as instructed the subject uses the kit 10 to collect timed samples of capillary blood by opening the container 12 removing the holder 45 from the compartment 22. The subject operates the levers 51, 52 of the holder 45 to open the j aws 48, 49 which the subj ect then places around one of their fingers or toes. The levers are released allowing the jaws to close around the finger or toe, and to squeeze the finger or toe gently to encourage blood flow towards the surface of the skin.

The or one of the lancing devices 42 is fitted to the holding portion 47 of the holder 45 and operated to cause the needle of the device to prick the subject's finger or toe. The holder 45 is then removed, and a drop of blood is dripped onto the or one of the pieces of filter paper 41. The subject operates the actuator 32 of the timer device 30 to record the accurate time of collection, and may also use the pencil to write the time on the piece of filter paper 41. The blood spot on the filter paper is then allowed to dry in the air, and the piece of filter paper is then replaced in the container 12.

Where instructed the subject takes further samples of blood as described above, and operates the actuator to record their respective times in the memory device of the timer device 30. After use the lancet devices 42 are thrown away.

When all the samples have been obtained and placed in the container 12, the container 12 is closed and secured and then sent to another location for analysis, which other location may be the same or different from the place where the injection was administered. The other location may for example be a central testing laboratory adapted to process large numbers of tests. The container may be sent to the other location by post.

At the other location, the container is opened and the timer device 30 operated to obtain the accurate times of the samples. The correspondence between the pieces of filter paper 41 and the recorded times will generally by evident from the measured amounts of tracer in the blood spots on the papers, but the handwritten times provided by the subject may assist in identifying which piece of filter paper corresponds to which time.

Advantageously the container 12 may be sufficiently robust to withstand multiple uses and postings, and may therefore be adapted for easy cleaning and sterilisation between successive uses.

Claims

1 A method of measuring glomerular filtration rate in a human or animal patient comprising:

(v) extracting an analyte from the portion;

(vi) quantitating the amount of said filtration marker_in said analyte; and (vii) determining the glomerular filtration rate of the patient from the quantitated amount of said filtration marker.

2 A method as claimed in claim 1 , characterised by repeating steps (ii) and (iii) periodically at known times after administration to obtain a plurality of blood samples on different, respective pieces of absorbent substrate, removing a portion of each piece of absorbent substrate, extracting analyte from each portion, quantitating the amount of filtration marker in each sample, and thereafter determining the glomerular filtration rate of the patient from the quantitated amounts and the corresponding known times.

3 A method as claimed in claim 1 or claim 2, wherein steps (ii) - (iii) are carried out at a first location, and the piece or pieces of absorbent substrate are then conveyed to a second location where steps (iv) - (vii) are performed.

4 A method as claimed in claim 3, wherein said piece or pieces of absorbent substrate are conveyed from said first location to said second location by post.

5 A method as claimed in any preceding claim, wherein the glomerular filtration rate is determined by calculating the blood concentration of said filtration marker in the or each sample by reference to the volume of blood absorbed by the respective portion of said absorbent substrate, using said filtration marker blood concentration to calculate the plasma concentration of filtration marker in the or each sample, and thereafter calculating the glomerular filtration rate from the plasma concentration of filtration marker at the or each of said known times.

6 A method as claimed in claim 5, wherein the plasma concentration of said filtration marker is calculated from the blood concentration by means of the haemocrit correction 7 A method as claimed in claim 5 or claim 6, wherein the glomerular filtration rate is calculated from the plasma concentration of said filtration marker at the or each known time by a single compartment analysis with the Brochner- Mortensen correction.

8 A method as claimed in any preceding claim, wherein a single sample is taken at a known time after administration, preferably at least three hours after administration.

9 A method as claimed in of claims 1 to 7, wherein two or more samples of blood are taken after administration within a period of at least three hours.

10 A method as claimed in claim 9, wherein three to five samples of blood are taken over a period of three to five hours.

11 A method as claimed in any preceding claim, wherein the analyte is extracted from the or each portion using perchloric acid or acetonitrile.

12 A method as claimed in any preceding claim, wherein said filtration marker in the or each analyte is quantitated by reverse phase HPLC or capillary electrophoresis.

13 A method as claimed in claim 12, wherein the or each analyte is vortex ed, ultrasonicated, and centrifuged prior to quantitation.

14 A method as claimed in any preceding claim, wherein the or each portion has an area of about 20-40mm2, preferably about 30mm2. 15 A method as claimed in any preceding claim, wherein the or each portion contains about 7.5 to 15μl of blood, preferably about l lμl. 16. A method as claimed in any preceding claim wherein said absorbent substrate is a filter paper. 17. A method as claimed in any preceding claim wherein said step of taking a sample of blood from a patient is by pricking a suitable part of the patient's skin

18. A method of measuring glomerular filtration rate in a human or animal patient substantially as hereinbefore described.

19. A self-use kit for providing blood samples for use in measuring the glomerular filtration rate of a patient, said kit comprising: a container that is adapted to be sent by post; one or more pieces of absorbent substrate of a quality and consistency suitable for the collection of blood spots for diagnostic analysis; and a timer comprising a user-operable actuator, recording means for recording the time the or each time the actuator is operated, and means for extracting said time or times from the timer; whereby a patient obtains one or more timed samples of capillary blood after being intravenously injected at a known time with a filtration marker, collecting a blood spot on the or a respective one of said pieces of absorbent substrate, and actuating said actuator upon obtaining the or each sample to record the time of the or each sample; whereafter the piece or pieces of absorbent substrate and the timer may be packaged in the container and sent by post to a laboratory for analysis of the timed blood samples to determine the rate of clearance of the filtration marker. 20. A self-use kit as claimed in claim 19, wherein said kit further comprises one or more lancet devices for use in pricking the patient's skin. 21. A self-use kit as claimed in claim 19 or 20, wherein the patient obtains one or more samples of blood by pricking a suitable part of his or her body with a said lancet. 22. A self-use kit as claimed in claim 19 to 21, wherein the or each lancet device comprises an automatic lancet device comprising a lancet, and selectively operable means for rapidly urging said lancet forwards for pricking the skin.

23. A self-use kit as claimed in claim 19 to 22, wherein the or each lancet device can be supplied within the container.

24. A self-use kit as claimed in any of claims 19 to 23, wherein said kit further comprises a holder for the or each lancet device, which holder comprises means for releasably gripping a part of the patient's body thereby to attach the holder stably to the patient's body, and means for holding said lancet device stably in juxtaposition with a part of the patient's skin which is to be pricked to obtain a sample of capillary blood.

25. A self-use kit as claimed in claim 24, wherein said holding means are adapted to hold releasably said lancet device, whereby the holder can be used with a plurality of lancet devices in succession. 26. A self-use kit as claimed in claim 24 or claim 25, wherein said gripping means are adapted for releasably gripping a patient's finger or toe.

27. A self-use kit as claimed in claim 24, claim 25 or claim 26, wherein said gripping means are adapted to squeeze the part of the patient's skin that is to be pricked with the lancet device, thereby to encourage the formation of a droplet of blood for collection on said absorbent substrate.

28. A self-use kit as claimed in any of claims 24 to 27, wherein said holder can be stored within the container.

29. A self-use kit as claimed in any of claims 19 to 28, wherein said timer comprises a solid state memory device for storing the time of the or each blood sample.

30. A self-use kit as claimed in any of claims 19 to 29, wherein the timer is configured to record automatically the time of each operation of said actuator.

31. A self-use kit as claimed in any of claims 19 to 30, wherein said extracting means comprise means for displaying the time of the or each sample. 32. A self-use kit as claimed in any of claims 19 to 31, wherein the extracting means comprise means for connecting the timer to a computer for uploading the recorded time or times from the timer to the computer.

33. A self-use kit as claimed in any of claims 19 to 32, wherein said timer is integrated into said container.

34. A self-use kit as claimed in any of claims 19 to 33 wherein said absorbent substrate is a filter paper.

35. A self-use kit substantially as hereinbefore described with reference to and as shown in FIG. 3 of the drawings. 36. An ex vivo method of determining the glomerular filtration rate of a human or animal patient comprising:

(a) providing one or more pieces of absorbent substrate, the or each piece of absorbent substrate comprising a dried blood spot consisting of a sample of capillary blood obtained from the patient at a known time after intravenous injection of the patient with a known amount of a filtration marker;

(c) extracting an analyte from the or each portion; (d) quantitating the amount of filtration marker in the or each analyte; and

(e) calculating the glomerular filtration rate of the patient from the quantitated amount(s) of filtration marker.

37. An ex vivo method of claim 36 wherein said absorbent substrate is a filter paper. 38. A self-use kit for providing blood samples for use in measuring the glomerular filtration rate of a patient, said kit comprising a robust container that is adapted to be sent by post, said container containing: one or more pieces of absorbent substrate removably disposed within the container, said piece or pieces being of a quality and consistency suitable for the collection of blood spots for diagnostic analysis; a blood withdrawing means for withdrawing a sample of blood from the patient said means being removably disposed within the container; a holder removably disposed within the container for said blood withdrawing means, said holder comprising a gripping portion for releasably gripping a part of the patient's body from which blood is to be withdrawn, thereby to attach the holder stably to the patient's body, and to squeeze said part of the patient's body, thereby to encourage the formation of a droplet of blood for collection on said absorbent substrate to obtain a sample of capillary blood, and a holding portion for holding releasably the blood withdrawing means stably in juxtaposition with said part of the patient' s skin; and a timer comprising a user-operable actuating device, a recorder device for recording the time the or each time the actuator is operated, said recorder device comprising a solid state memory device for storing the time of the or each blood sample, and being configured for automatically recording the time of each operation of said actuating device, and a device for extracting said time or times from the timer.

39. A self use kit of claim 38 wherein said blood withdrawing means comprises one or more automatic lancet devices for use in pricking the patient's skin, the or each lancet device comprising a lancet, and a selectively operable firing mechanism for rapidly urging said lancet forwards for pricking the skin

40. A self-use kit as claimed in claim 38, wherein the extracting device comprise means for connecting the timer to a computer for uploading the recorded time or times from the timer to the computer.

41. A self-use kit as claimed in any of claims 38 to 40, wherein said timer is integrated into said container.

42. A method of collecting timed samples of capillary blood from a patient who has been intravenously injected at a known time with a known amount of filtration marker for determining the patient's glomerular filtration rate, said method comprising: providing said patient with a kit comprising a robust container that is capable of being sent by post, said container containing one or more pieces of absorbent substrate of a quality and consistency suitable for the collection of blood spots for diagnostic analysis, a means for withdrawing blood form the patient, and a timer comprising a user-operable actuator, recording means for recording the time the or each time the actuator is operated, and means for extracting said time or times from the timer; instructing the patient to use said means for withdrawing blood and said one or more pieces of absorbent substrate to collect periodically one or more samples of capillary blood as blood spots on said absorbent substrate, to operate the actuator of the timer the or each time a sample is obtained, to allow the blood spot or spots to dry, to replace the piece or pieces of absorbent substrate in the container, and thereafter to transmit the container to a laboratory; and receiving the container at the laboratory, removing the piece or pieces of absorbent substrate carrying the blood spots, and extracting from the timer the time of the or each blood sample after filtration marker administration.

43. A method as claimed in claim 42, wherein the timer is integrated into the container.

44. A method as claimed in claim 42 or claim 43, wherein said time of the or each blood sample is extracted by connecting the timer to a computer and uploading the time or times from the recording means.

45. A method as claimed in claim 42, claim 43 or claim 44, wherein said container is received at the laboratory by post.

46. A method as claimed in any of claims 42 to 45 wherein said absorbent substrate is a filter paper.

47. A method as claimed in any of claims 42 to 46 wherein said means for withdrawing blood is at least one lancet device for use in pricking the patient's skin. 48. A method or self use kit as claimed in any preceding claim wherein said filtration marker is biologically inert, freely filtered by the kidney and does not undergo metabolism, tubular secretion or absorption.

49. A method or self use kit as claimed in any preceding claim wherein said filtration markers is a non-radioactive contrast agent.

50. A method or self use kit as claimed in any preceding claim wherein said filtration markers include ethylendiaminetetraacetic acid (EDTA) and diethylenetriaminepentacetate acid (DTPA).

51. A method or self use kit as claimed in any preceding claim wherein said filtration marker is radiolabeled with a radioactive species.

52. A method or self use kit as claimed in any preceding claim wherein said filtration marker is isothalamate or Iohexol.

53. A method of diagnosing chronic kidney disease in a human or animal subject, said method comprising measuring the glomerular filtration rate of said subject according to any method as claimed in the previous claims and comparing said glomerular filtration rate of said subject with a predetermined glomerular filtration rate of a healthy individual, wherein a deviation in said glomerular filtration rate of said subject from the glomerular filtration rate of said healthy individual is indicative of chronic kidney disease. 54. A method of diagnosis according to claim 53 wherein a decrease in the glomerular filtration rate of the subject compared to the healthy individual is indicative of diabetic nephropathy.