WO2016054033A1

WO2016054033A1 - Processes, systems, and devices for hemolysis detection via measurement of methemoglobin

Info

Publication number: WO2016054033A1
Application number: PCT/US2015/052929
Authority: WO
Inventors: David Marston CRISP; Paul William COTTON
Original assignee: Siemens Healthcare Diagnostics Inc.
Priority date: 2014-09-30
Filing date: 2015-09-29
Publication date: 2016-04-07

Abstract

There is provided a process 100 for determining hemolysis in a sample. The method comprises measuring 102 a first amount of methemoglobin in a sample and thereafter contacting 104 the sample with an oxidant such as potassium ferricyanide to oxidize free hemoglobin in the sample to a second amount of methemoglobin. The method further includes measuring 106 the second amount of methemoglobin in the blood sample following the addition of the oxidant.

Description

PROCESSES, SYSTEMS, AND DEVICES FOR HEMOLYSIS DETECTION VIA MEASUREMENT OF METHEMOGLOBIN

[0001] The subject application claims benefit under 35 USC § 1 19(e) of U.S. Provisional Application No. 62/057,421 , filed September 30, 2014. The entire contents of the above-referenced patent application are hereby expressly

incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of diagnostic testing, and more particularly to devices, systems, and processes for determining an extent of hemolysis in a blood sample.

BACKGROUND OF THE INVENTION

[0003] Point-of-care testing refers generally to medical testing at or near the site of patient care, such as in an emergency room. A desired outcome of such tests is often rapid and accurate lab results to determine a next course of action in the patient care. A number of such point of care tests involve analysis of a blood sample from the patient. The ideal blood sample is pure plasma separated from the source whole blood sample. However, even in such plasma samples, there are often residual broken blood cells as a result of hemolysis due to imperfections in obtaining the sample from the subject, pre-analytical blood sample handling, and the whole blood separation process. In certain cases, these hemolysed cells can interfere with the integrity of analytical test results.

[0004] For example, if hemolysis occurs, resulting free hemoglobin in the sample may cause interference in a number of tests, thereby leading to a signal reduction, reduced measurement accuracy and precision, or to false positive results at the other end of the spectrum. For one, it has been found that the potassium

concentration in a corresponding sample may increase significantly and cause a high risk of misdiagnosis in a diagnostic test for potassium levels. See Clinical Chemistry, December 2003, vol. 49, 12, 2105-2106.

[0005] To determine whether hemolysis has occurred, a number of tests have been developed to determine hemoglobin (Hb) levels in a blood sample. One common reagent used for determining Hb levels or hemolysis in a blood sample is referred to as Drabkin's Reagent. Drabkin's Reagent comprises a mixture that works by lysing red blood cells and quantitatively converting all Hb in a sample into one form, cyanomethaemoglobin, which is then be measured on a spectrometer using a single wavelength. As such, Drabkin's Reagent measures intracellular hemoglobin as well as free hemoglobin. For at least this reason, Drabkin's Reagent does not provide a realistic picture of the extent of free Hb present at a particular point in time in a sample, which is indicative of hemolysis.

SUMMARY

[0006] In accordance with one aspect of the present invention, there is provided a diagnostic method. The method comprises:

a) measuring a first amount of methemoglobin in a sample;

b) contacting the sample with an oxidant, wherein the oxidant is effective to oxidize free hemoglobin in the sample to a second amount of methemoglobin; and c) measuring the second amount of methemoglobin in the sample following the contacting.

[0007] In accordance with another aspect, there is provided a cartridge for a blood gas analyzer. The cartridge comprises a housing; at least one vessel disposed in the housing, wherein the vessel comprises an oxidant for oxidizing an amount of free hemoglobin in a blood sample to methemoglobin; and at least one sensor unit for detecting an amount of methemoglobin in a sample introduced thereto.

[0008] In accordance with another aspect, there is provided a diagnostic method, wherein the diagnostic method comprises:

a) measuring a first amount of methemoglobin in a first sample;

b) contacting the first sample with an oxidant, wherein the oxidant is effective to oxidize free hemoglobin in the first sample to a second amount of methemoglobin;

c) measuring the second amount of methemoglobin in the first sample following the addition of the oxidant;

d) determining whether hemolysis has occurred in the first sample based upon a difference between the second measurement and the first measurement; e) measuring potassium levels in the first sample or a second sample from the same source as the first sample, wherein the measuring potassium levels produces potassium level results; and

f) upon determination of hemolysis in the first sample or the second sample, providing a notification along with the potassium level results that hemolysis has occurred.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 illustrates a method for determining a presence of hemolysis in a sample in accordance with an aspect of the present invention.

[0010] FIG. 2 illustrates a blood analyzer incorporating an oxidant for oxidizing free oxygenated to MetHb in accordance with an aspect of the present invention.

[0011] FIG. 3 illustrates a cartridge incorporating an oxidant in accordance with an aspect of the present invention.

[0012] FIG. 4 is a graph showing a corresponding increase in MetHb levels with increasing hemolysis.

[0013] FIGS. 5A, 5B, 6A, 6B, 7A, 7B, 8A, and 8B illustrate the effect of the addition of 20 μΙ_ΛηΙ_ of 0.05 M potassium ferricyanide to hemolysed whole blood samples.

DETAILED DESCRIPTION

[0014] In accordance with one aspect, there are provided devices, systems, and processes for determining a presence of hemolysis in a sample. Advantageously, devices, systems, and processes described herein determine whether hemolysis has occurred in a sample based upon extracellular levels of MetHb. Thus, the results are not influenced by intracellular levels of hemoglobin from hemolysed cells. Further, the extent of hemolysis can be quickly determined. In this way, a sample may be quickly flagged accordingly if the sample shows evidence of hemolysis. In one aspect, if a degree of hemolysis in the sample is above a predetermined threshold value, a notification of the same may be generated and proper diagnostic action may be taken accordingly. If the amount of hemolysis is less than the predetermined threshold level, further testing of the sample (or a second sample from the same source) within a blood gas analyzer or the like may be performed or results of previously run diagnostic tests may be interpreted with greater confidence.

[0015] As used herein, the term "about" refers to a value that is ± 10% of the stated value.

[0016] As used herein, the terms "sample" or "blood sample" includes a whole blood, serum, or plasma sample, or a sample derived therefrom.

[0017] As used herein, by the phrase "effective amount," it is meant an amount of material suitable for bringing about an intended result.

[0018] As used herein, the term "hemoglobin" refers to all naturally occurring forms of glycated and non-glycated hemoglobin and any derivatives thereof. The term may refer to any molecule comprised of at least two globin subunits or domains (dimeric) of hemoglobin. Hemoglobin can be free in solution or contained within in a cell, liposome or the like. In addition, hemoglobin may comprise one or more ligands therewith, e.g., oxygen, carbon monoxide or nitric oxide, or may be in the unliganded (deoxygenated) state.

[0019] As used herein, the term "subject" refers to any human or non-human mammal.

[0020] Now referring to FIG. 1 , there is shown a process 100 for determining an extent of hemolysis in a sample. The process 100 first includes step 102 of performing a first measurement for a first amount of methemoglobin (MetHb) in the sample. MetHb is a form of the oxygen-carrying metalloprotein hemoglobin, wherein the iron in the heme group of the hemoglobin molecule is in the Fe3+ (ferric) state, not the Fe2+ (ferrous) of normal hemoglobin. Unlike oxyhemoglobin, MetHb does not bind oxygen. The normal adult has from 1 -2% MetHb of the total hemoglobin content in their blood at a given time. The initial measurement in step 102 is performed to determine a baseline MetHb level for a subject as it is appreciated a subject might have an elevated amount of MetHb naturally and/or at a given point in time due to causes not associated with hemolysis.

[0021] The sample may be any sample that could exhibit an unacceptable amount of hemolysis therein. In an embodiment, the sample is one that could potentially provide elevated levels of potassium in an analytical test for potassium as a result of hemolysis. As noted above, if hemolysis occurs in a sample, a potassium value of the sample in question may be increased significantly, for example. When a subject's potassium levels are not actually as high as indicated by the potassium assay, a false positive result may in turn result in misdiagnosis and mistreatment of a disorder characterized by elevated potassium levels. For example, as a result of hemolysis, a subject might be misdiagnosed with having hyperkalemia or another disorder or condition characterized by elevated potassium levels over a

predetermined threshold value, e.g., Addison's disease or hemolytic anemia.

Further, the subject could be misdiagnosed as having elevated potassium levels as a side effect of taking medications, such as water pills (diuretics) or blood pressure drugs, and unnecessarily instructed to cease taking such medications to the subject's detriment. In addition, a false positive result could inadvertently lead to one unnecessarily being provided with agents to remove potassium from the intestines before potassium is absorbed or other unnecessary treatments. Thus,

advantageously, the processes described herein may be utilized in a screening process for an unacceptable level of hemolysis prior to analysis of the sample for potassium levels or another analyte, or may be utilized for confirming the integrity of diagnostic test results already run.

[0022] The MetHb content may be measured by any suitable technique or instrument known in the art. In one embodiment, the MetHb content in the sample in step 102 is measured by a spectroscopy method, such as by absorption or transmission spectroscopy. See e.g., Fishkin, JB, Appl Opt. 1995 Mar 1 ;34(7):1 143- 55, the entirety of which is hereby incorporated by reference herein. In an embodiment, the determining of the first amount of MetHB is done by absorption spectroscopy at one or more wavelengths such as 560 nm, 630 nm, or 635 nm.

[0023] Once the first amount of MetHb has been determined, the process 100 further comprises step 104 of contacting the sample with an amount of oxidant effective to oxidize free hemoglobin in the blood sample to a second amount of MetHb. The oxidant solution may be prepared according to known methods and may have an oxidant concentration of from 0.01 - 1 .5 M, for example. In an embodiment, the oxidant comprises potassium ferricyanide. Potassium ferricyanide oxidizes oxyhemoglobin (Hb02), for example, to methemoglobin (MetHb) according to the following formula (I):

(I) Hb0₂ (Fe²⁺) + Fe³⁺ (from K₃Fe(CN)₆) > MetHb (Fe³⁺) + 0₂ +

Fe²⁺ [0024] In a particular embodiment, the oxidant comprises a 0.05 M potassium ferricyanide solution. The oxidant may be added for a sufficient time and under suitable conditions effective to fully oxidize available free hemoglobin in the sample to a second amount of MetHb. The sample and oxidant may be mixed by any suitable structure or method known in the art. In an embodiment, the mixing is done by passive mixing.

[0025] Referring again to the process 100, the process comprises step 106 of measuring the second amount of MetHb in the blood sample following the contacting with the oxidant in step 104. Thus, after the oxidant has reacted with the free hemoglobin in the sample, a second amount of MetHb may be measured. In an embodiment, the duration of time between the first measurement and the second measurement is relatively short such as from 10 seconds to 2 minutes, and preferably less than one minute, such that potassium ferricyanide does not leach into cells. For consistency, the second measurement of MetHb may be done in the same manner as the first measurement such as via spectrometry at one or more suitable wavelengths for MetHb.

[0026] In an embodiment, the process 100 may further include a step of determining from a difference between the second amount and the first amount of MetHb whether hemolysis has occurred in the sample. A positive difference between the second measurement and the first measurement is indicative of hemolysis in the sample. In certain embodiments, for every 1 % increase in hemolysis, there will be at least a corresponding 0.5% increase in MetHb fraction levels. Thus, in one embodiment, if the difference between the second

measurement and the first measurement is greater than 0.5 % MetHb, the sample may be understood to have experienced a greater than a 1 % degree of hemolysis. A greater than 1 % level of hemolysis may be deemed unacceptable in certain instances. For example, a greater than 1 % degree of hemolysis will likely result in at least a 0.5 mmol increase in potassium levels, which may be deemed unacceptable. In an embodiment, the step of determining whether hemolysis has occurred in the sample thus comprises determining whether a difference between the second measurement and the first measurement exceeds a predetermined threshold value. The predetermined threshold value may be at least about 0.5 % MetHb. In further embodiments, the predetermined threshold value may be at least about 0.6 % MetHb, or at least about 0.7 % MetHb.

[0027] In one aspect, the determination of the presence or absence of hemolysis may be critical for one or more analytical tests utilizing the same sample or a second sample from the same source (e.g., same source as the sample being screened for hemolysis). In certain embodiments, the one or more analytical tests may be performed on a blood gas analyzer as is known in art along with the hemolysis screen. The one or more additional analytical tests include but are not limited to measurements for pH, pC02, p02, Hb, Na, K, CI, iCa, glucose, lactate, bilirubin, CO- oximeter fractions (f02Hb, fC02Hb, fMetHb, fHHb), and pleural fluid pH.

[0028] If the difference between the second measurement and the first measurement is greater than a predetermined threshold value, a notification of hemolysis may be communicated in a suitable manner. In this way, the sample may be designated as one exhibiting evidence of hemolysis preceding, concurrent with, or following subsequent analytical test(s) one or more analytes or properties such as pH, pC02, p02, Hb, Na, K, CI, iCa, glucose, lactate, bilirubin, CO-oximeter fractions (f02Hb, fC02Hb, fMetHb, fHHb), and pleural fluid pH as described above. The notification may occur by any suitable method such as by an audio or visual alarm and/or by providing any suitable notification that hemolysis over the threshold value was found in a particular sample. Alternatively, the presence of hemolysis may be indicated on the output of the associated results - whether the associated results are printed or on a display. When the presence of hemolysis is associated with properly identified corresponding analytical test data, overtreatment or mistreatment of conditions involving elevated potassium results or the like as a result of hemolysis in the blood sample may be avoided or the presence of hemolysis may at least fully considered by the interpreter of test data.

[0029] The oxidant, e.g., potassium ferricyanide, may be provided in any suitable form for introduction to the sample. In an embodiment, the oxidant may be incorporated within a cartridge of a blood gas analyzer as is well known in the art. In this way, the process 100 may be carried out on a blood gas analyzer as is known in the art along with the analysis for one or more analytes. Exemplary blood gas analyzers are available from Siemens Healthcare and are currently sold under the trademarks: RAPIDLab^® 1200, RapidLab 348EX, RAPIDPoint^® 500, RAPIDLab 248/348, RAPIDPoint 400/405, and RAPIDPoint 340/350 Systems. In this way also, the determination of the extent of hemolysis in a given sample may take place in the same instrument as the testing of the same sample or a second sample from the same source for pH, pC02, p02, Hb, Na, K, CI, iCa, glucose, lactate, bilirubin, CO- oximeter fractions (f02Hb, fC02Hb, fMetHb, fHHb), and pleural fluid pH. The "second sample from the same source" may be a blood sample taken from the same subject at or near the same point in time (e.g., within an hour) and processed or stored (if at all) under the same conditions as the blood sample which is analyzed for hemolysis.

[0030] Thus, upon analysis of a sample for potassium levels, for example, the results of a hemolysis screen can be conveniently correlated with the analytical test results such as by outputting or printing out a notification along with the one or more analytical results that hemolysis has been detected or has likely occurred. If there is no indication of hemolysis, there may simply be no notification provided or a statement may be provided as to the absence of evidence of hemolysis along with analytical tests results.

[0031] By way of example only, FIG. 2 shows a schematic of an exemplary blood gas analyzer 200 which incorporates an oxidant for oxidizing free hemoglobin to MetHb. The blood gas analyzer 200 comprises a cassette magazine 202 for storing a plurality of disposable cassettes 204. The cassette magazine 202 is in operable communication with a control unit 206 which is programmed to instruct the cassette magazine 202 to release a cassette 204 one by one into communication with a fluid sample station 208 (which is also in communication with the control unit 206). After use, the used cassette 204 may be transported to a waste location (not shown) for disposal thereof. A rinse fluid may be introduced through the analyzer 200 as is necessary after sample analysis.

[0032] The control unit 206 may be assembled by a number of computing components such as, for example, one or more processors (e.g., INTEL®, AMD®, SAMSUNG®) in communication with memory or other storage medium. The memory may be Random Access Memory (RAM), flashable or non-flashable Read Only Memory (ROM), hard disk drives, flash drives, or any other types of memory known to persons of ordinary skill in the art and having storing capabilities. The computing systems and devices may also utilize cloud computing technologies to facilitate several functions, e.g., storage capabilities, executing program instructions, etc. The computing systems and devices may further include one or more communication components such as, for example, one or more network interface cards (NIC) or circuitry having analogous functionality, one or more one way or multidirectional ports (e.g., bi-directional auxiliary port), in addition to other hardware and software necessary to implement wired communication with other devices.

Alternatively, the communication components may include wireless transmitters, a receiver (or an integrated transceiver) that may be coupled to broadcasting hardware of the sorts to implement wireless communication within the system. The control unit 206 may further comprise a display, a user interface, and hardware or software. The hardware and/or software, for example, one or more processors may be configured for carrying out any process steps as described herein, including but not limited to, the dispensing of sample and reagents, analysis and interpretation of data, and output of analytical test results, including those for hemolysis and potassium.

[0033] The fluid sample station 208 may comprise a pipette system as is known in the art for delivering a sample stored therein or delivered thereto to the cassette 204. Alternatively, the fluid sample station 208 may be operatively connected to a catheter for receiving a blood sample from a subject in real time. In either case, the cassette 204 receives a blood sample to be analyzed from the fluid sample station 208.

[0034] As shown in FIG. 3, each cassette 204 may comprise a housing 210 having a plurality of storage vessels 212 for housing any desired buffers, solutions, reagents, or other fluids to be delivered to one or more sample wells for the particular analytical test(s) being performed. It is appreciated that the oxidant as described herein, e.g., potassium ferricyanide, may be stored within any one of such vessels 212. A pipetting or metering system may be in place in the blood gas analyzer to direct the necessary fluids from the vessels 212 to one or more wells 214 which hold a sample. Further, each cassette 204 has one or more sensor units 216. Each sensor unit 216 may comprise an optical, electrochemical, or other sensor that can measure for an analyte or property of the sample in the wells 214. In an embodiment, the sensor unit 216 is configured to provide an absorbance or transmission spectrum for MetHb in the sample at a suitable wavelength for MetHb. The resulting data may be communicated from a communication interface on the cassette 204 to the control unit 206.

[0035] In certain embodiments, the control unit 206 is configured to provide a notification that a corresponding sample introduced to the blood gas analyzer is one that has shown evidence of hemolysis upon determination of the same. For example, the control unit 206 may be configured to provide a notification along with test results for potassium that a particular sample is one that is believed to have undergone an amount of hemolysis therein. In this way, treatment or diagnosis of the subject providing the sample can proceed at least with caution if hemolysis is indicated since hemolysis may potentially interfere with or cause misinterpretation of the analytical test results. In other embodiments, if hemolysis is indicated in a sample, associated test results may be simply discarded or not further considered.

[0036] Aspects of the present invention are demonstrated by the following examples, which are not intended to be limiting in any manner.

EXAMPLES

[0037] Example 1 : Determination concentration of potassium ferricyanide needed

[0038] 1 mole of K3Fe(CN)6 is needed to convert 1 mole of Hb (as monomer). At 15g/dl_ total hemoglobin (tHB), a 100 μΙ_ blood sample contains about 0.93 μιηοΙ of Hb. Assuming 100% hemolysis, just under 1 μιηοΙ of K3Fe(CN)6 would be needed for complete conversion. Assuming the mixing ratio is to be kept at 20 μΙ_ reagent / 100 μΙ_ of blood, the potassium ferricyanide concentration could be decreased to 0.05 M and still have a large excess for any sample likely to be encountered.

[0039] Example 2: Recovery of sensors after potassium ferricyanide-doped non-hemolysed blood sample

[0040] To show that ion selective electrodes were not adversely affected by repeated exposure to potassium ferricyanide, the following process was carried out:

1 . Preparel M potassium ferricyanide in deionized water.

2. Pool 2 tubes of non-hemolysed whole blood. 3. Measure non-hemolysed whole blood sample in triplicate on a RAPIDPoint 405 Blood Gas Analyzer for MetHb from Siemens Healthcare

Diagnostics, Inc.

4. Take 5 mL aliquot of non-hemolysed whole blood sample.

5. Add 20 μΙ_ΛηΙ_ of 0.1 M potassium ferricyanide to 5 mL of non- hemolysed whole blood.

6. Measure singleton sample of the doped sample on the Siemens RAPIDPoint 405.

7. Measure triplicate samples of non-doped, non-hemolysed samples to assess recovery of sensors back to levels observed in (3) above.

8. Freeze aliquots of whole blood samples for future use.

[0041] Example 3: Recovery of sensors after potassium ferricyanide-doped hemolysed whole blood

1 . Prepare 0.1 M potassium cyanide in 800 7.3 Cal Batch #286676 (mw 329.24 batch Sigma Aldrich # MKBH5539V)

2. Defrost whole blood aliquot from Example 2.

3. Utilize remaining stock whole blood sample and prepare 5 mL hemolysed aliquots having 0%, 0.4%, 0.6%, 0.8%, and 1 .0% hemolysis.

4. Take 1 % of each solution in (3) and add 20 μί/ιηί of 0.1 M potassium ferricyanide thereto.

5. Measure on a RAPIDPoint 500 (utilizing cart # 2315002126) from Siemens Healthcare Diagnostics, Inc.

[0042] Example 4: Linearity of elevated MetHb result with increasing hemolysis

[0043] Utilizing both potassium ferricyanide-doped and non-doped samples from Example 3, 0-1 .0% hemolysed non-doped sample were measured as singletons on 500 #30017. In addition, the 0-1 .0% hemolysed 0.1 M potassium ferricyanide doped samples were measured as singletons on a RAPIDPoint 500 Blood Gas Analyzer from Siemens Diagnostics Healthcare, Inc.

[0044] FIG. 4 shows the results in a graph. The graph shows both doped and non-doped MetHb results. As shown, there is a linear trend to the results even taking into account that baseline samples had all experienced mild hemolysis as indicated by the elevated potassium results.

[0045] Example 5: Increased MetHb results in increased hemolysis.

Increased hemolysis results in increased potassium

N= 6 10 mL vacutainer tubes of fresh whole blood were taken.

N = 1 vacutainer tube was placed in the freezer at nominally -18° C.

[0046] The remaining five tubes of whole blood were pooled and left gently rolling overnight.

[0047] After 24 hours, the vacutainer was removed from the freezer and the sample was placed on the roller, allowed to defrost, and equilibrate to room temperature.

[0048] Aliquots of the defrosted whole blood sample (now taken to be 100% hemolysed) were added to 3 mL volumes of the pooled whole blood sample to create a hemolysis range of 0-1 % hemolysis.

[0049] 1 mL aliquots of each of these samples within the hemolysis series were taken. To each aliquot, 20 μί of 0.05 M potassium ferricyanide solution was added.

[0050] The samples were then measured in order of increasing hemolysis with the aliquots not containing ferricyanide being measured before and after the sample containing ferricyanide.

[0051] Potassium and MetHb readings were taken for all samples and reviewed against increasing hemolysis levels.

[0052] FIGS. 5A-8B numerically and graphically depict test results obtained, on two separate occasions (FIGS. 5A-6B relating to a first test and FIGS. 7A and 8B relating to a second test), after the addition of 20uL/mL of 0.05M Potassium

Ferricyanide to hemolyzed whole blood samples. As shown in FIGS. 5A, 5B, 7A and 7B, MetHb fraction increases with increased hemolysis in the range of 0-1 % hemolysis by 0.7 % MetHb (FIGS. 5A and 5B) and 0.6 % MetHb (FIGS. 7A and 7B). As shown in FIGS. 6A, 6B, 8A and 8B, the % increase in hemolysis is confirmed by the increase in potassium, 1 .0 % hemolysis resulted in a 0.6 mmol/L (FIGS. 6A and 6B) and 0.5 mmol/L increase (FIGS. 8A and 8B) increase in potassium. The potassium sensor does not retain an offset due to the presence of potassium ferricyanide per pre- and post-potassium ferricyanide results. [0053] While various embodiments of the present invention have been shown and described herein, it will be obvious that such embodiments are provided by way of example only. Numerous variations, changes and substitutions may be made without departing from the invention herein. Accordingly, it is intended that the invention be limited only by the spirit and scope of the appended claims.

Claims

CLAIMS The invention claimed is:

1 . A diagnostic method comprising:

measuring a first amount of methemoglobin in a sample;

contacting the sample with an oxidant, wherein the oxidant is effective to oxidize free hemoglobin in the sample to a second amount of methemoglobin; and measuring the second amount of methemoglobin in the sample following the contacting.

2. The method of claim 1 , further comprising determining whether hemolysis has occurred in the sample based upon a difference between the second measurement and the first measurement.

3. The method of claim 1 , wherein the oxidant comprises potassium ferricyanide.

4. The method of claim 3, wherein the oxidant is provided in an amount of from 0.01 to 1 .5 M potassium ferricyanide.

5. The method in any of preceding claims, wherein the determining whether hemolysis has occurred comprises determining whether a difference between the second measurement and the first measurement exceeds a

predetermined threshold value.

6. The method of claim 5, wherein the predetermined threshold value is at least 0.5 % MetHb.

7. The method of claim 1 , further comprising performing one or more analytical tests on the sample or a second sample from the same source as the sample for a member selected from the group consisting of pH, pC0₂, p0₂, Hb, Na, K, CI, iCa, glucose, lactate, bilirubin, CO-oximeter fractions (f0₂Hb, fC0₂Hb, fMetHb, fHHb), and pleural fluid pH.

8. The method of claim 1 , wherein the performing one or more analytical tests comprises testing for potassium levels.

9. The method of claims 7 or 8, further comprising outputting results one or more analytical tests and providing a notification along with the output results that hemolysis has been determined to have occurred.

10. The method of claim 9, wherein the notification that hemolysis has been determined to have occurred in the sample is in the form of a printed notification, an audio notification, or a visual notification.

1 1 . The method of any of the preceding claims, wherein the process is performed on a blood gas analyzer.

12. A cartridge for a blood gas analyzer comprising:

a housing;

at least one vessel disposed in the housing, wherein the vessel comprises an oxidant for oxidizing an amount of free hemoglobin in a sample to methemoglobin; and

at least one sensor unit for detecting an amount of methemoglobin in a sample introduced thereto.

13. The cartridge of claim 12, wherein the oxidant comprises potassium ferricyanide.

14. A blood gas analyzer comprising the cartridge of claim 12.

15. A diagnostic method comprising:

measuring a first amount of methemoglobin in a first sample;

contacting the first sample with an oxidant, wherein the oxidant is effective to oxidize free hemoglobin and its derivatives in the first sample to a second amount of methemoglobin; measuring the second amount of methemoglobin in the first sample following the addition of the oxidant; and

determining whether hemolysis has occurred in the first sample based upon a difference between the second measurement and the first measurement;

measuring potassium levels in the first sample or a second sample from the same source as the first sample, wherein the measuring potassium levels produces potassium level results;

upon determination of hemolysis in the first sample or the second sample, providing a notification along with the potassium level results that hemolysis has occurred.

16. The method of claim 15, wherein the oxidant comprises potassium ferricyanide.

17. The method of claims 15 or 16, wherein the determining whether hemolysis has occurred comprises determining whether a difference between the second measurement and the first measurement exceeds a predetermined threshold value.

18. The method in any of claims 15 to 17, outputting results one or more analytical tests and providing a notification along with the output results that hemolysis has been determined to have occurred, wherein the notification that hemolysis has been determined to have occurred is in the form of a printed notification, an audio notification, or a visual notification.

19. The method of any of the preceding claims, wherein the process is performed on a blood gas analyzer.