WO2003052390A1

WO2003052390A1 - Simultaneous analysis of blood samples and estimation of blood (hemoglobin) content

Info

Publication number: WO2003052390A1
Application number: PCT/GR2002/000066
Authority: WO
Inventors: J. George. Reclos; J. Christine-Arghyro. Hatzidakis
Original assignee: Reclos J George; Hatzidakis J Christine-Arghyro
Priority date: 2001-12-17
Filing date: 2002-12-11
Publication date: 2003-06-26
Also published as: AU2002358917A1; BR0207836A; GR1004071B

Abstract

This method refers to the Simultaneous analysis of blood samples and estimation of blood (hemoglobin) content and relates to the quantitative or qualitative determination of analytes which exist in blood, said method comprising (a) the preparation of a sample for analysis; (b) a kinetic or not measurement (photometric, fluorimetric, mass measurement, chromatographic, enzymatic or other suitable method for the determination of the analyte) expressed in selected units or physicochemical parameters; said assay being characterised by (c) a photometric measurement of the sample at any wavelength in the range of 400-420 nm or 535-555 nm or 565-580 nm; (d) repetition of steps (b) and (c) described above upon a standard or control; and (e) calculating the blood content of the sample by solving the following ODSa/λ2 x Bst≡ Bsa ODStλ2 wherein ODsa is the optical density for the sample measured once at a particular wavelength (λ2), and ODst is the optical density of the standard standard or control or sample with known blood content measured once at that particular wavelength (λ2), λ2 is the wavelength at which a reading of Haemoglobin contained can be taken, Bst is the blood content of the standard, control or any examined sample expressed as % Hematocrite or any other unit which expresses blood content as stated by the manufacturer of the standard or control for the assay or the person who performed the blood analysis, Bsa is the blood content expressed as % Hematocrite or any other unit expressing blood content the standard, control or examined sample is expressed at.

Description

SIMULTANEOUS ANALYSIS OF BLOOD SAMPLES AND ESTIMATION OF BLOOD

(HEMOGLOBIN) CONTENT

The invention relates to a novel method for the simultaneous evaluation of blood content, usually but not always expressed as Hemoglobin (Hb) content in whole blood samples or eluants from dried bloodspots (DBS) and in particular is suitable for clinical chemistry analyses or screening for deficiencies such as Glucose-6- Phospate Dehydrogenase deficiency in newboms. This method is applicable for all analyses in which the analyte in question is or can be expressed in relation to the blood content present in the sample tested. This method is particularly useful for the evaluation of the content of analytes which are related to red blood cells being on or in them. This method is absolutely necessary when the comparison of analyte content which are related to red blood cells being either on or in them between different sample is desired.

Glucose-6-Phosphate dehydrogenase (G-6-PD) is a cytoplasmic enzyme, which is distributed in all cells and especially in red blood cells from which it is isolated and measured by elution. It catalyses the first step in the hexose monophosphate pathway producing NADPH as shown below.

G6PD

Reductive biσsynthetic reactions.

The main metabolic role of G-6-PD in red cells is the defense against oxidizing agents, epitomized by hydrogen peroxide. NADPH, a product of the G-6-PD reaction, is both the hydrogen donor for regeneration of reduced glutathione and a ligand for catalase (see text). GSSGR = glutathione reductase; GSHPX = glutathione pεroxidase; G-6-P = glucose-6-phosphate; 6PG = phosphogluconate.

This coenzyme is required as a hydrogen donor for reactions of various biochemical pathways as well as for the stability of catalase and the preservation and regeneration of the reduced form of glutathione. Catalase and glutathione are both essential for the detoxification of hydrogen peroxide, therefore the defence of the cells against H₂O₂ is heavily dependent on G-6-PD. The red cells are very sensitive to oxidative damage and lack other NADPH producing enzymes. The defence against oxidising agents, (epitomised by H₂O₂) is mainly realised by glutathione, which converts H₂O₂ to H₂0 stoichiometrically via glutathione peroxidase. NADPH is the hydrogen donor for the regeneration of reduced glutathione. An alternative pathway for H₂O₂ detoxification is via catalase, but this route is regarded as ineffective under normal conditions because of the lower affinity of catalase for H₂O₂ compared to that of glutathione peroxidase.

G-6-PD deficiency is the most commonly known enzymopathy with around 400 million people affected worldwide. The prevalence ranges from 5% to 25% in the endemic areas such as Africa, the Middle East, Asia, the Mediterranean and Papua New Guinea but the highest incidence of 65% is found in Kurdish Jews. Clinical manifestations associated with G-6-PD deficiency include:

1). Drug induced hemolysis - certain antimalarials, sulphonamides, sulfones and other drugs or chemicals are associated with significant hemolysis in subjects;

2). Infection induced hemolysis - numerous bacterial, viral and rickettsial infections have precipitated hemolysis, but the most important are infectious hepatitis, pneumonia and typhoid fever;

3). Favism - sudden onset of acute haemolytic anaemia within 24 to 48 hours of ingesting fava beans;

4). Neonatal jaundice - jaundice usually appears by 1 to 4 days of age

5). Chronic nonsperocytic haemolytic anaemia.

Such deficiencies have usually been measured by semi-quantitative assays such as those described by Beutler et al where he measured the enzyme activity in dried bloodspots (DBS). The principle of the methods described by Beutler is to take a sample by hole punch from the DBS, elute it with a haemolysing solution and incubate the sample from this with a reaction mixture at 37°C. Subsequently adding an acid solution to the mixture stops the reaction and an aliquot of said reaction mixture is then spotted onto filtration paper.

The NADPH formed by the above reaction is then visualised using a fluorimetric method by looking at the samples under ultra-violet light in a dark room. This gives a semi-quantitative interpretation, comparing the fluorescence of the sample with controls and the results are classified normal, intermediate or deficient. It should however be noted that such a test must only discriminate between normal and abnormal. Therefore if a limit is chosen (i.e. anything lower than 3.5 U/g Hb is to be regarded as not normal) the test only needs to make sure that it will not give results when the activity is lower than that (acceptance tests), or alternatively, will give results if the activity is lower than that (rejection tests). They do not give results in the whole range. Other problems with this method include that there is frequently interference from other enzymes producing NADPH, the method can only work at temperatures of 37°C and spontaneous fluorescence can cause misclassification of the sample.

Some commercial companies have modified the above method for use with automated fluorimetric plate readers ( Isolab, Labsystems ). Such instruments produce a quantitative result by measuring the amount of fluorescence and calculating the enzyme activity from this. There are also problems with these automated fluorimetric plate readers as they only work at temperatures of 37°C, there is a problem of interference from other enzymes producing NADPH, spontaneous fluorescence can cause misclassification of the sample and the quantitative result obtained therefrom is not reliable because it is influenced by the amount of haemoglobin eluted from the DBS. This can lead to misclassification of the sample.

The object of the present invention is to obviate or mitigate the problems of these prior art methods to provide an improved method capable of producing reliable results which can be used for analysing both DBS and whole blood samples.

According to the present invention there is provided a quantitative assay for an analyte of interest in blood samples, comprising

(a) the preparation of a sample for analysis;

(b) a kinetic photometric measurement of the sample, being periodic measurements recorded under a controlled temperature, at a selected wavelength in the range of about 330-360nm; said assay being characterised by

(c) a photometric measurement of the sample at a wavelength in the range of 400- 420nm or 535-555nm or 565-580nm;

(d) conducting steps equivalent to (b) and (c) above upon a control; and (e) calculating the result of the analysis by solving the following δODsλl/min/δODcλl/min x Control Value = Sample Value (Activity in U/g Hb) ODsλ2/ODcλ2 wherein δODs is the change in optical density (per minute) for the sample, λl is the wavelength at which optical density is measured under kinetic mode, δODc is the change in optical density (per minute) of a control measured under kinetic mode conditions at a particular wavelength (λl), ODs is the optical density for the sample measured once at a particular wavelength (λ2), and ODc is the optical density of the control measured once at that particular wavelength (λ2), λ2 is the wavelength at which a reading of Heamoglobin contained can be taken, the control value being determined by the change in optical density (per minute) of the control measured under kinetic mode at λl divided by the optical density of the control at λ2 i.e. δODcλl/mim/ODcλ2.

For assays not expressing results in relation to Hemoglobin the said invention provides a quantitative assay for an analyte of interest in blood samples, comprising (a) the preparation of a sample for analysis;

(b) a photometric measurement of the sample, being measurements recorded at a selected wavelength ; said assay being characterised by

(c) a photometric measurement of the sample at a wavelength in the range of 400- 420 nm or 535-545 nm or 565-580 nm;

(d) conducting steps equivalent to (b) and (c) above upon a standard or control; and

(e) calculating the Hb content of the sample by solving the following ODSaλ2 x Bst = Bsa

ODStλ2 wherein ODsa is the optical density for the sample measured once at a particular wavelength (λ2), and ODst is the optical density of the standard or control or sample with known blood content measured once at that particular wavelength (λ2), λ2 is the wavelength at which a reading of Haemoglobin contained can be taken, Bst is the blood content of the standard, control or any examined sample expressed as % Hematocrite or any other unit which expresses blood content as stated by the manufacturer of the standard or control for the assay or the person who performed the blood analysis, Bsa is the blood content expressed as % Hematocrite or any other unit expressing blood content the standard, control or examined sample is expressed at.

Preferably the sample preparation is by a pre-incubation of a sample at a selected temperature, usually within the range of from about 20°C to about 40°C, preferably at a temperature of about 22-25°C, or about 25-30°C, or about 37°C, for a period dependent upon the incubation temperature and typically not exceeding about 60 minutes at temperatures around 22-25°C, about 45 minutes for temperatures around 25-30°C and about 20 minutes for a temperature of about 37°C.

The invention has particular utility for analytes of interest to be found in dried blood stains (DBS) but is surprisingly effective also for whole blood, including red blood cells in solution e.g. saline. The assay is particularly useful in quantitative measurement of enzyme activity in such samples, and enables assessment of the haemoglobin content of these samples and provides results in terms of Units per gram haemoglobin. The assay is also useful in the quantitative measurement of any analyte in such samples and enables assessment of the haemogolobin content of these samples and provides results in % hematocrite or any other unit the standards are calibrated by with reference to their haemoglobin content. Samples with known blood content expressed in any relevant unit (% Hematocrit, number of red blood cells / volume, grams hemoglobin etc.) can be used instead of the standards for the Hb (Blood content) Normalization.

The sample preparation may be such that the sample is obtained from a dried blood stain on a physical support or carrier such as filter paper which can be hole punched to obtain a suitable sample "spot", this spot may be then incubated with haemolysing solution, and an aliquot of that may subsequently be incubated with substrate, coenzyme and oxidising agent. Elution buffer, Lysis buffer and dilution buffer may be present in the sample when presented for analysis.

The method for the measurement of the enzymatic activity in such samples will now be further described below with reference to the examples described below. In this example, our invention allows for the simultaneous measurement of enzymatic activity and blood content which is then expressed in grams Hemoglobin.

The present invention provides a method for the quantification of enzyme activity in eluates from DBS on filter paper or from whole blood samples in microtiter plates. In particular the method includes an assay utilising G-6-PD, which in the presence of NADP, catalyses the oxidation of G-6-P to 6-phosphogluconate. The NADPH produced is measured colorimetrically at 340nm in kinetic mode and the results are calculated by evaluating the increase in optical density (OD) per minute (slope) for unknowns against the slope for a Standard with known G-6-PD activity. However, it will be understood by those in the art that such a method as described can also be used to measure many other such deficiencies and its application is not solely limited to measuring G-6-PD deficiencies.

The first step is carried out by taking a sample from a DBS on filter paper using a hole punch and incubating this spot with a haemolysing solution., a sample of which is then incubated with a reaction mixture. Alternatively whole blood samples can be measured in micro titre plates by addition of the reagents to the wells in the micro titre plate which contain the whole blood.

The reaction mixture contains:

Substrate - Glucose-6-Phosphate

Coenzyme - β-NADP

Oxidising agent - L-Glutathione

Dilution Buffer - Phosphate buffered saline rated at pH 7.9 with the addition of

Na₂HPO₄ (Sodium Monohydrogen Phosphate) and Sodium Azide as preservative (0.12% w/v)). Elution buffer - Hypotonic solution which is a mixture of 0.01M Na₂HPO₄ and

0.01M NaH₂PO₄ rated at a pH of 7.15 and containing sodium azide as a preservative (0.12%w/v).

A pre-incubation of the sample in the reagent mixture is then allowed. In assays performed at 22-25°C the incubation time is 10 minutes, at 22-30°C it is 5 minutes, and at 37°C it is 2 minutes. The incubation period is then followed by photometric measurements at 340nm at 1 minute intervals up to a maximum of 25 measurements after which the reaction rate is expressed in mOD/min.

The second step involves a single reading of the same reaction mixture at 400- 420nm or 535-555nm or 565-580nm. This reading is representative of the amount of haemoglobin eluted from the filter paper or the whole blood sample. This measurement is preferably taken after the elution stage and before the addition of the reagent mixture. Alternatively a sample of the same eluate can be mixed with the Dilution Buffer using the same volumes as in step one, followed by a single reading at 400-420nm or 535-555nm or 565-580nm.

The result of the first step ( mOD/min ) is divided by the result of the second step (OD) and multiplied by a FACTOR X to express the final test result in Units per gram Haemoglobin. The Factor is calculated using a standardised solution with known Enzyme Activity and Haemoglobin content.

Procedure

The samples are collected from the patient by standard procedures. The reagents are warmed up slowly to 18-25°C prior to use. 14ml of the oxidizing agent is mixed with 14ml of dilution buffer, 14ml of substrate is mixed with 14ml of dilution buffer and 3.5ml of coenzyme is mixed with 3.5ml of dilution buffer. The reaction mixture is prepared by mixing equal volumes of the above dilutions. Approximately 25μl of each component is required for each test.

Procedure for carrying out the test with micro titration plates

1. Punch blood spots approximately 3/16 inch in diameter in U bottomed micro titre plate. Use position Al as Normal Control and use 2 wells for the Intermediate and Deficient Controls.

2. Add 75μl of Elution buffer to each well.

3. Place micro titre plate on orbital shaker for 30 minutes at room temperature.

4. During the elution prepare Reagent Mixture. (If performed at 30°C the reagent and a flat bottomed micro titre plate need to be warmed to 30°C).

5. Transfer 15μl of the eluate from each well to the corresponding well of the new flat bottom microtiter plate.

6. Add 75μl of the reagent mixture to each well.

7. Place the plate on an orbital shaker for 1 minute.

8. Place the plate in the incubator and wait until (a)10 minutes have passed after reagent mixture has been added for temperature range 22-25°C or (b) 5 minutes have passed after reagent mixture has been added for temperature range 25-30°C.

9. Read the plate in a plate reader at 340nm for 15 minutes with 60 second intervals. 10. After the readings are completed, change the program of the reader to a single reading at 405nm and read the plate (containing the same mixture) again.

Procedure for carrying out the test with Cobas Mira (37°C)

1. Punch blood spots 2 by 1/8 inch in a sample cup, leave caps open.

2. Add 150μl of elution buffer to each well.

3. Place sample rack on an orbital shaker for 30 minutes.

4. During the elution, reconstitute reagents if needed and prepare reagent mixture (1 test uses 150μl of reagent mixture).

5. Close sample cups and place sample rack on the instrument.

6. Load reagent mix.

7. Insert empty cuvettes, and run the test.

Procedure for carrying out the test with whole blood specimens

1. Pipette lOμl of whole blood into a sample cup

2. Add 150μl of Elution buffer

3. Close caps and mix gently

4. Load reagent mix

5. Insert empty cuvettes, and run test.

Hb Protocol Cobas Mira

Run the test using the same sample as used for the G-6-PD activity assay. There is sufficient sample in the cup for a second test. The reagent mix becomes the dilution buffer in this case and instrument settings should be set at 405nm to run the test.

The G-6-PD activity of the sample is directly comparable to that of the control (OD control 340nm/min). Since the control's activity is already given in Units/g Hb (U/g Hb) the sample's activity can readily be expressed in the same units. The equation which gives results in U/g Hb is given below.

OD_samp|_e34_0nm/min / δOD_COntrol340nm/min X Control Value = Sample Activity in U/g Hb

OD_Sample405nm / OD_contro|405nm

(OD sample at 405nm (whereby 405 can be substituted by any wavelength in the range 400-420nm or 535-555nm or 565-580nm)) A kit is also provided for carrying out the method which generally comprises 2 rubber stoppered glass vials securable with an aluminium crisp seal, which are labelled accordingly. Also included are 50ml of elution buffer and 37,5 ml of lyophilised reagent mixture which contains substrate, oxidising agent and coenzyme. This size of kit enables the researcher to carry out approximately 500 tests.

EXAMPLES

The Kit described above was used to demonstrate the effectiveness of the second reading on a Cobas Mira Plus ( Roche Diagnostics ). The experiment was set up to mimic the effect of incomplete elution on the measurement of enzyme activity. According to the assay protocol described above 2 spots of 3.2mm must be punched from the sample and inserted into the sample cup. This was done using whole blood samples of 3 human donors spotted and dried onto filtration paper. The instrument was programmed for the second reading by repeating the assay from the same sample, only with a single reading at 405nm instead of the kinetic reading at 340nm. To mimic the effect of incomplete elution the same samples were used but only one spot was punched and inserted into the sample cup.

The results are shown below in Table 1:

Table 1

mOD/ min 405nm

340nm

Units 2 spot 1 spot % 1 spot 2 % spot

MMRC02 N 19.42 9.98 51.4% 1.81 3.28 55.3%

MMRC02 N 23.18 11.2 48.3% 1.92 3.81 50.5%

USA 1 8.29 4.2 50.7% 1.04 2.07 50.2%

USA 1 7.76 4.71 60.7% 1.12 1.97 56.8%

USA 2 12.98 5.98 46.1% 1.16 2.48 46.9%

USA 2 13.05 6.07 46.5% 1.14 2.40 47.7% Ratio Norm alised

2 spot 1 spot 1 spot 2 spot %

MMRC02 N 1.000 1.810 18.06 19.42 93.0 %

MMRC02 N 1.162 1.705 19.10 19.96 95.7

%

USA 1 0.631 3.154 13.25 13.14 100. 8%

USA 1 0.601 2.932 13.81 12.92 106. 9%

USA 2 0.756 2.823 16.88 17.17 98.3 %

USA 2 0.732 2.866 17.40 17.84 97.6

%

It can be seen that :

1) The result of measurement at 405nm is proportional to the amount of blood eluted from the sample.

2) The results can be normalised by relating the 405nm results to a reference sample

3) Any wavelength in the range 420-400nm or 535-555nm or 565-580nm will work in the same aspect.

Verifying an alternative wavelength.

It has been established that the optimum wavelength to measure the Hb content is at 414 or 417nm. However, this would limit the usefulness of the proposed assay to the labs using automated analyzers or spectrophotometers because the microplate readers have a limited range of optical filters available. One such filter being the 405nm optical filter. It was examined whether this particular wavelength, though not optimal, is Hb specific and yields a high enough signal. The first thing was to make sure that the rest of the chemicals did not absorb in the 380-460 area nor do the products of the kinetic reaction. Firstly the spectrum of a normally lyzed whole blood sample, using the Elution Buffer contained in the kit was obtained, before and after the addition of the Reagent Mixture and the first spectrum was subtracted from the second (Figure 1). It is clear that the reagents absorb only at wavelengths below 360nm. The elution buffer gave an almost identical spectrum to deionized water (data not shown here).

The results shown in Figure 3 demonstrate the suitability of the 405nm optical filter for an accurate determination of the Hemoglobin content of the samples. The dried blood spots are far more difficult to be evaluated because the blood actually eluted greatly varies from sample to sample. The same sample may give a 10% difference when examined under the same experimental conditions. On top of that, the blood initially spotted and dried on each disk is not the same. The situation is further complicated in the "punching" stage where not everyone punches at the center of the spot or, even worse, not all the disk cut is covered with blood. Thus, apart from the normal differences between samples which are due to parameters like hematocrite or average Hb content per erythrocyte, many other parameters that have to do with the assay itself magnify them. This is clearly shown in Figure 3. Twenty five dried blood spot samples from a randomly selected microplate are shown in this Figure. It can be seen that the Hb content of the spots varies from 0.076 at 405nm (0.107 at 417nm) to 0,528 at 405nm (0.731 at 417nm). The average OD at 405nm is 0.307 + 0,126 (0.442 ± 0.179 at 417nm). However, the ratio of the ODs (405nm / 417nm) is amazingly stable (light gray bars) under these varying conditions (0.693 + 0.031). A total of 2.000 neonates have been examined and OD values ranging from 0.040 to 0.610 at 405nm have been found. However the average ratio (n=2000) was almost identical to the one mentioned earlier (0.701 + 0.0309). This is an indication that the reading at 405nm can substitute the reading at the optimal wavelength (414nm or 417nm).

Results from 25 whole blood samples (a randomly selected microplate) are shown in Figure 4. The ODs ranged from 0.735 at 405nm (0.837 at 417nm) to 1,115 at 405nm (1.351 at 417nm). The average OD at 405nm (417nm) for these 25 samples was 0.962 + 0.103 (1.159 + 0.156), while the ratio of ODs (405nm / 417nm) was found 0.834 + 0.038. Even when whole blood was further diluted 1:2 and 1 :4 with normal saline, the ODs observed was proportionally lower but the ratio of the ODs (405nm / 417nm) remained the same (0.822 + 0.037 for an 1:2 dilution, n=25; 0.819 ± 0.038 for an 1 :4 dilution, n = 30; light bars). Dried blood spot samples (DBS)

Figure 3. DBS samples after the completion of the kinetic reaction.

Graph 4. Whole blood samples after the completion of the kinetic reaction

In Graph 5, the absorption of 25 dried blood spot samples and 25 whole blood samples at 405 and 417 nm are shown. It is evident that despite the different origin of the samples the 405 / 417 nm ratio is constant. In Tables 4 and 5 the values of those samples are shown in detail.

Graph 5. Graphic presentation of the results.

Dried blood spot samples show lower absorptions while whole blood samples show higher absorptions.

Normalization of Samples for Hb content. The activity of the G-6-PD enzyme in this assay is measured indirectly by the formation of β-NADPH following the oxidation of glucose-6-phosphate to 6-phosphogluconate. The NADPH produced is measured at 340nm in kinetic mode and the change of OD per minute is proportional to the G-6-PD activity of the sample. By definition, one International Unit of G-6-PD activity is the amount of G-6-PD activity which will convert lmmol of substrate per minute under the conditions (mainly temperature) specified in the assay. This activity is directly proportional to the number of erythrocytes actually lyzed during the elution process which can be expressed as grams of Hemoglobin in the sample. In order to have comparable results, the activity measured in each sample has to be calculated for the same amount of Hb irrespectively of the actual Hb contained in that sample. Since all comparisons are made against the controls, the Hb of the controls should become the basis of comparisons. We therefore divided all ODs measured at 405nm following the kinetic (OD_sampie405nm) by the OD at 405nm of the control (OD_COnt_roi405_nm)- The resulting values represent the "relative" Hb content of each sample as compared to the control. The activity measured in each sample (ΔOD_samp|_e34o_nm/nnin) is then divided by the "relative Hb" factor in order to get the activity of the sample which corresponds to the Hb content of the control. Expressing Results in Units / gram Hb. The G-6-PD activity of the sample is now directly comparable to that of the control (δOD_COn_troi₃₄0nm/nnin). Since the control's activity is already given in Units / g Hb (U/g Hb) the sample's activity can be readily expressed in the same units. Special care should be taken to use the controls which are rated at the temperature used for the assay. Thus, when using analyzers or microplate readers equipped with a heating device, any control can be used. When performing the assay at room temperature it is preferable to use controls which are rated at 24°C. The equation that gives results expressed in U/g Hb is given below.

OD_samp|_e340nm/min / δOD_Control340nm/^rnin X Control Value = Sample Activity in U/g Hb

OD sample405nm / OD control405nm

Results for verification. In order to verify that this equation will give accurate and reproducible results the following experiment was performed. A set of ten samples was prepared as shown in Table 2. Thirty sets of these samples were tested in the same day using the Single Test kit in order to simulate a multiple day screening condition (different Reagent Mixture for each set). One box (MMR010, Batch 01/98) was opened and used for each set.

Table 2 indicates Applicant's samples

Neonate #1 was classified as "Normal" during screening whereas Neonate #2 and #3 were classified as "deficient" (using the currently employed semi-quantitative Beutler method). All samples were compared to * Normal in column 2 and the assay was performed at 37°C. These results are presented in Table 3.

Table 3

Sample n- Nominal Value Mean + Standard

It is evident that this method has a very good accuracy and reproducibility and the results obtained from different sessions and/or days are directly comparable to each other.

Limitations of the method.

This method assumes that the optical properties of the samples are the same with the controls. Since the molecular extinction coefficient of the Beer Lambert Law (e) applies for a given compound this method can be used to directly express results in U/g Hb by comparing them to controls tested with the same Reagent Mixture and under the same conditions. Next day's microplates should be compared against controls tested with the Reagent Mixture used at that day. However results expressed in U/g Hb are directly comparable irrespectively of when they were obtained. Measurement at λ2 is preferably taken after the elution stage and before the addition of the reagent mixture.

Workable Wavelengths :

In order to determine which wavelengths could be used for normalisation of the samples for their Hb content we run the same samples 100 times. Average values were then inserted in tables 4 and 5 in order to determine which wavelengths satisfied the criteria (less than 5% fluctuation from the mean ratio to the optimal wavelength at 414nm). Table 4 Hb NORMALIZATION PROCEDURE SCANNING THE 460-380 n RANGE

Reference Value OD at 414 nm (n=300)

WAVELENGTHS TESTED (nm) - RATIOS (ODx /

OD414 nm) SAMPLE DESCRIPTION dilution 460 RATIO 450 RATIO 440 RATIO Whole blood (March 98)^" 1 : 1 dil 0,352 0,2196 0,336 0,21 0,423 0,264 Whole blood (March 98) 1 :2.5 0,142 0,2082 0,118 0,173 0,103 0,151 dil

Sigma Normal control none 0,112 0,2086 0,083 0,155 0,059 0,11 Whole blood (Nov.97) 1 : 1 dil 0,191 0,174 0,188 0,171 0,217 0,198 Whole blood (Nov.97) 1 : 1 dil 0,154 0,1549 0,151 0,152 0,403 0,405 0,223

0,104

Peak observed at 415,5 nm in all cases

Acceptance criteria : % variance less then 5% for n=100 samples at least

430 RATIO 420 RATIO 417 RATIO 405 RATIO 390 RATIO 380 RATIO

0,82 0,512 1,48 0,9264 1,567 0,9775 1,2 6,749 0,69 0,431 0,529 0,33

2 8 5 1

0,29 0,434 0,62 0,9135 0,676 0,9912 0,50 0,74 0,25 0,374 0,194 0,284

6 3 5 5 5

0,22 0,417 0,47 0,892 0,527 0,9814 0,40 0,762 0,21 0,393 0,163 0,303

4 1 9 9 1 5

0,51 0,464 0,98 0,9007 1,065 0,9699 0,84 0,77 0,43 0,394 0,319 0,290 5 9 6 3 5 0,5

I

8 6 ' ^" . 7 4 8 6 0 038 0,029 0,015 0,02 0,02 0,020

9 5 ⁴ 1 1 * 2 4 7 \ ~ 8,33 3,2 1,53 3,01 6,25 7,03

CONCLUSION : RANGE OF ACCEPTABLE ODs (420 - 400 nm) Reference values:

414 1,603 0,682 1,17 0,537 1,098 0,994

Table 5 - Results of OD 405nm / 417nm

whole/417 whole/405 W405/417 spot/417 spot/405 S405/417 whole/417 whole

1131 0937 08284704 0384 0249 06484375 1131 0937

1216 0972 07993421 0625 0437 06992 1216 0972

12 0963 08025 0455 0294 064615385 12 0963

1137 0926 08144239 0482 0343 0,71161826 1137 0926

084 0799 09511905 0539 0355 065862709 084 0799

1366 1073 07855051 0568 0402 0 70774648 1366 1073

1273 1013 07957581 0453 0302 066666667 1273 1013

1223 0977 07988553 0557 0386 06929982 1223 0977

1276 1036 08119122 0504 0341 06765873 1276 1036

1411 1145 08114812 0.731 0.528 072229822 1411 1145

118 0959 08127119 053 034 064150943 118 0959

1215 1035 08518519 0635 0435 068503937 1215 1035

0973 0839 08622816 0478 0319 066736402 0973 0839

1246 1088 08731942 0537 038 070763501 1246 1088

1054 09 08538899 0465 0327 070322581 1054 09

1206 0999 0,8283582 0551 0389 070598911 1206 0999

1077 0928 08616527 0528 0375 071022727 1077 0928

1088 0934 08584559 047 0332 070638?98 1088 0934

1309 1038 07929717 0487 035 071868583 1309 1038

1.351 1.115 08253146 0609 0429 07044335 1.351 1.115

1109 0906 08169522 0.107 0.076 071028037 1109 0906 0.837 0.735 0.8781362 0.131 0.093 0.70992366 0.837 0.735

0.929 0.806 0.8675996 0.177 0.131 0.74011299 0.929 0.806

0.514 0.405 0.78793774 0.384 0.249

0.111 0.077 0.69369369 0.625 0.437

0.179 0.122 0.68156425 0.455 0.294

0.119 0.083 0.69747899 0.482 0.343 0.539 0.355

0.961871.158565 0.834035 0.307407 0.441704 0.6963636 average 0.568 0.402

0.1032170.155838 0.038382 0.126856 0.179513 0.030744 st.dev. 0.453 0.302

0.557 0.386 dil.blood/405 diI.blood/417 405/417 0.504 0.341

0.428 0.545 0.7853211 0.731 0.528

0.481 0.587 0.8194208 0.53 0.34

0.4545 0.566 0.802371 average 0.635 0.435

0.037477 0.029698 0.024112 st.dev. 0.478 0.319

0.537 0.38

0.465 0.327

0.551 0.389

0.528 0.375

0.47 0.332

0.487 0.35

0.609 0.429

0.107 0.076

0.131 0.093

0.177 0.131

0.514 0.405

0.111 0.077

0.179 0.122

0.119 0.08

It will be apparent to those skilled in the art that there are many variants of the method described herein before. For example, in some cases when a colour reagent is added at the end of the assay obtaining the necessary measurements at the wavelength needed are prohibited or the reaction produces a final product which also absorbs at 405 nm. In these cases it is necessary to instead of measuring at λ2 after the kinetic (ODCλ2 or ODSλ2 and Bsa or Bst) to measure before the addition of the chemicals, colour reagents or even before running the kinetic measurements.

Assays employing precipitation steps.

In other cases such as the normal enzymatic reactions employed in regular newborn screening assays, the first step includes precipitation of all peptides and proteins by using a 3% trichloroacetic buffer or other suitable media. In those assays the Hb Normalization is performed before the precipitation step. Thus, the Dried Blood Spot or whole blood sample is eluted with water or other suitable lysis buffer (like hemolytic buffers containing e.g. saponine or hypotonic solutions e.g. phosphate buffers 0.001 mM) or other known means. In whole blood samples, the determination of the blood content can be performed immediately. Any wavelength in the range 420-400nm or 535-555nm or 565-580nm will work in the same aspect. For the precipitation step of the assay, a more concentrated "elution" or precipitation buffer is required to compensate for the presence of the red blood cell lysis buffer used for Hb Normalization. In dried blood spot samples, since the disk blocks the light path, part of the eluant is transferred to another microplate or other suitable container and read at a suitable wavelength. Any wavelength in the range 420-400nm or 535-555nm or 565-580nm will work in the same aspect. The results are directly compared to the ones of the standard which is included in the assay. Since the standards are always rated at 55% hematocrite, a direct comparison is possible. In fact, the formula which will give a hematocrite estimate is the following: ((ODSλ2/ODCλ2))*55 = Hematocrite sample (%). Moreover, values indicating a very high Hb content in the samples (Hematocrite values over 60 %) should warn the person performing the assay that probably there have been more than one blood drops dried on the same spot. Those samples should be examined separately after completion of the assays and results obtained for them should be treated accordingly. High values for those samples should be interpreted as being the result of more sample and not a higher concentration of the analyte in question, which will lead to a considerable reduction of the number of "false positive" samples. Similarly, Hb readings, which correspond to a hematocrite value less than 30% (or less than 60% of the blood content of the standard, control or any tested sample of a healthy donor included in the test) should warn the person performing the test about a partially covered disk or the presence of a concurrent disease. Following the Hb Normalization procedure, the assay is then carried in the remaining eluant as usually. For the precipitation step a more concentrated "elution" or precipitation buffer is required to compensate for the presence of the red blood cell lysis buffer used for Hb Normalization. In general, the formula which normalizes the samples against the control, standard or known sample is the following:

ODSaλ2 x Bst = Bsa

ODStλ2 wherein ODsa is the optical density for the sample measured once at a particular wavelength (λ2), and ODst is the optical density of the standard standard or control or sample with known blood content measured once at that particular wavelength (λ2), λ2 is the wavelength at which a reading of Haemoglobin contained can be taken, Bst is the blood content of the standard, control or any examined sample expressed as % Hematocrite or any other unit which expresses blood content as stated by the manufacturer of the standard or control for the assay or the person who performed the blood analysis, Bsa is the blood content expressed as % Hematocrite or any other unit expressing blood content the standard, control or examined sample is expressed at.

Normalizing for more than one assays in one step.

If more than one assays are to be run, then an appropriate number of dried blood spot disks (number of tests + one) or volume of whole blood ((volume for one test * (number of tests + one)) should be taken. For example, if the test asks for one dried blood spot disk and we intent to perform 3 tests normalized for their Hb content (e.g. Phenylalanine [ PKU ], total galactose [ GAL ] and leucine [ MSUD ] ), then we should take 4 dried blood spot disks. Similarly, if the test requires 5 microliters of whole blood and we intent to perform perform 3 tests normalized for their Hb content, then we should take 20 microliters of blood. One fourth of the quantity will be used for the Hb Normalization while the rest will be used for the assays as previously described.

The advantages of the method of the present invention are as follows:

1) It provides a QUANTITATIVE measurement of enzyme activity due to the Kinetic Readings. Quantitative results are needed to differentiate between intermediate and deficient specimens. It is very difficult to do this "by eye" and is also subjective.

2) There is no interference from other enzymes due to the use of a Pre- incubation. Other enzymes can interfere with the reaction as has been described by Solem et. al. These enzymes react with the coenzyme to form the same end-product as with the enzyme to be measured. The solution to this is the use of a pre-incubation during which the interfering enzyme(s) are active until completion of their reaction. After this the enzyme to be measured solely uses the coenzyme to produce the measured end-product, which is called the linear phase. The invention makes use of this linear phase for the kinetic readings. 3) The reaction can take place at a wide range of temperatures 22-37C. Using a wide range of temperatures allows the laboratory to choose a method best fit for the routine workflow. Working at room temperature means there is no need for temperature controlled incubators.

4) It provides a truly QUANTITATIVE result by assessment of the haemoglobin content of the sample and calculation of the results in Units per gram Haemoglobin. This is an absolute necessity if one is to measure enzyme activity. Enzymes are often located inside the red blood cells and bound to haemoglobin. The amount of enzyme activity measured in a sample is directly proportional to the amount of haemoglobin. This must be measured in the same eluate used for the reaction. Elution of dried bloodspots can be incomplete and is always subject to variation. So far it has not been possible to asses the Hb content of an eluate.

5) The invention has particular utility for analytes of interest to be found in dried blood stains (DBS) but is surprisingly effective also for whole blood, including red blood cells in solution e.g. saline. The assay is particularly useful in the quantitative measurement of any analyte in such samples and enables assessment of the haemogolobin content of these samples and provides results in % hematocrite or any other unit the standards are calibrated by with reference to their haemoglobin content.

6) This invention identifies samples which have a higher than normal blood content and therefore reduces the number of "false positive" results in screening assays and identifies any sample mishandling and / or sample collection errors. The invention is of particular interest for neonatal screening assays.

7) This invention identifies samples which have a less than normal quantity of hemoglobin and should therefore be treated accordingly during screening. This could be the result of sample mishandling and / or sample collection errors or the result of a disease.

Claims

1. A qualitative or quantitative assay for an analyte of interest in blood, comprising (a) the preparation of a sample for analysis;

(b) a kinetic or not measurement (photometric, fluorimetric, mass measurement, chromatographic, enzymatic or other suitable method) measured in suitable units or physicochemical parameters; said assay being characterised by

(c) a photometric measurement of the sample at any wavelength in the range of 400-420 nm or 535-555 nm or 565-580 nm;

(d) conducting steps equivalent to (b) and (c) above upon a control; and

(e) calculating the blood content (haemoglobin) of the sample by solving the following equation

Odsaλ2 x Bst = Bsa

Odstλ2

wherein ODsa is the optical density for the sample measured once at a particular wavelength (λ2), and ODst is the optical density of the standard, control, or sample with known blood content measured once at that particular wavelength (λ2), λ2 is the wavelength at which a reading of Haemoglobin contained can be taken, Bst being the blood content of the standard, control or sample with known blood content expressed as % Hematocrite or any other unit which expresses the blood content which is reported by the manufacturer of the standard or control or the blood content of the known sample had been expressed at, Bsa being the blood content of the samples expressed as % Hematocrite or with the same units used to express the blood content of the standard, control or measured sample to which comparisons are made against.

2. A quantitative assay for an analyte of interest in blood samples, comprising

(a) the preparation of a sample for analysis;

(b) a kinetic photometric measurement of the sample, being periodic measurements recorded under controlled temperature conditions at a selected wavelength approximately 330-360 nm; said assay being characterised by (c) a photometric measurement of the sample at a wavelength in the range of 400- 420 nm or 535-555 nm or 565-580 nm;

(d) repetition of steps (b) and (c) described above upon a control while step c) can be performed immediately after the elution (or lysis) of the red blood cells of the samples; and

(e) calculating the results of the analysis by solving the following

δODsλl/min/δODcλl/min x Control Value = Sample Value (Activity in U/g Hb)

ODsλ2/ODcλ2 wherein δODs is the change in optical density (per minute) for the sample, λl is the wavelength at which optical density is measured under kinetic mode, δODc is the change in optical density (per minute) of a control measured under kinetic mode conditions at a particular wavelength (λl), ODs is the optical density for the sample measured once at a particular wavelength (λ2), and ODc is the optical density of the control measured once at that particular wavelength (λ2), λ2 is the wavelength at which a reading of Haemoglobin contained can be taken, the control value being determined by the change in optical density (per minute) of the control measured under kinetic mode at λl divided by the optical density of the control at λ2 i.e. δODcλl/min/ODcλ2.

3. A qualitative or quantitative assay for an analyte of interest in blood, comprising

(a) the preparation of a sample for analysis;

(b) a photometric measurement being a measurement taken (under controlled or not temperature conditions) at a selected wavelength; said assay being characterised by

(c) a photometric measurement of the sample at a wavelength in the range of 400- 420 nm or 535-555 nm or 565-580 nm;

(d) conducting steps equivalent to (b) and (c) above upon a control; and

(e) calculating the blood content (haemoglobin) by solving the following equation

Odsaλ2 x Bst = Bsa

Odstλ2

4. A quantitative assay according to Claim 1 or 2 characterised in that the sample preparation is by a pre-incubation of a sample within the temperature range of about 20°C to about 40°C.

5. A quantitative assay according to Claim 1,2,3 or 4 characterised in that the sample preparation is by a pre-incubation of a sample within the temperature range of about 20-26°C.

6. A quantitative assay according to Claim 1,2,3, or 4 characterised in that the sample preparation is by a pre-incubation of a sample within the temperature range of about 20-30°C.

7. A quantitative assay according to Claim 1,2,3 or 4 characterised in that the sample preparation is by a pre-incubation of a sample at a temperature of about 37°C.

8. A quantitative assay according to Claim 5 characterised in that the sample preparation is by a pre-incubation of a sample for about 10-60 minutes.

9. A quantitative assay according to Claim 7 characterised in that the sample preparation is by a pre-incubation of a sample for about 5-10 minutes.

10. A quantitative assay according to Claim 7 characterised in that the sample preparation is by a pre-incubation of a sample for about 0-5 minutes.

11. A quantitative assay according to any one of the preceding claims characterised in that the invention has particular utility for analytes of interest to be found in dried blood stains (DBS) but also for whole blood, including red blood cells in solution e.g. normal saline and enables assessment of the haemoglobin content of these samples and provides results in terms of units per grams haemoglobin.

12. A quantitative assay accordingly to any one of the preceding claims characterised in that the assay is particularly useful in quantitative measurement of enzyme activity in dried blood stains (DBS), whole blood and red blood cells in solution such as saline and enables assessment of the haemoglobin content of these samples and provides results in terms of Units per gram haemoglobin.

13. A quantitative assay accordingly to any one of the preceding claims characterised in that the assay is particularly useful in quantitative measurement of enzyme activity in dried blood stains (DBS), whole blood and red blood cells in solution such as saline and enables assessment of the haemoglobin content of these samples and provides results in terms of any chemical or physical measurement units per gram haemoglobin.

14. A quantitative assay according to any one of the preceding claims characterised in that the invention has particular utility for analytes of interest to be found in dried blood stains (DBS) but also for whole blood, including red blood cells in solution e.g. saline and enables assessment of the haemoglobin content of these samples and provides results in terms of Units per % hematocrite.

15. A quantitative assay according to any one of the preceding claims characterised in that the invention has particular utility for analytes of interest to be found in dried blood stains (DBS) but also for whole blood, including red blood cells in solution e.g. saline and enables assessment of the haemoglobin content of these samples and provides results in terms of any chemical or physical measurement units per % hematocrite.

16. A quantitative assay according to any one of the preceding claims characterised in that the invention has particular utility for analytes of interest to be found in dried blood stains (DBS) but also for whole blood, including red blood cells in solution e.g. saline and enables assessment of the haemoglobin content of these samples and provides results in terms of units per number of red blood cells per volume (erythrocytes).

17. A quantitative assay according to any one of the preceding claims characterised in that the invention has particular utility for analytes of interest to be found in dried blood stains (DBS) but also for whole blood, including red blood cells in solution e.g. saline and enables assessment of the haemoglobin content of these samples and provides results in terms of any chemical or physical measurement units per number of red blood cells per volume (erythrocytes).

18. A quantitative assay according to any one of the preceding claims characterised in that the invention has particular utility for analytes of interest to be found in dried blood stains (DBS) but also for whole blood, including red blood cells in solution e.g. saline and enables assessment of the haemoglobin content of these samples and provides results in terms of any chemical or physical measurement units per any unit which is used to express blood content of a sample.

19. A quantitative assay according to any of the preceding claims characterised in that the sample preparation may be such that the sample is obtained from a dried blood stain on a physical support or carrier such as filter paper which can be hole punched to obtain a suitable sample "spot", this spot may be then incubated with haemolysing solution, and an aliquot of that may subsequently be incubated with substrate, coenzyme and oxidising agent.

20. A quantitative assay according to any one of the preceding claims characterised in that elution buffer and dilution buffer may be present in the sample when presented for analysis.

21. A quantitative assay according to any of the preceding claims characterised in that the sample preparation may be such that the sample is obtained from a dried blood stain on a physical support or carrier such as filter paper which can be hole punched to obtain a suitable sample "spot", this spot may be then incubated with haemolysing solution, and an aliquot of that may subsequently be incubated with substrate, oxidizing agent, coenzyme and intermediate electron receptors.