C56-A Hemolysis, Icterus, and Lipemia/Turbidity Indices as Indicators of Interference in Clinical Laboratory Analysis; Approved Guideline

Transcription

1 C56-A Hemolysis, Icterus, and Lipemia/Turbidity Indices as Indicators of Interference in Clinical Laboratory Analysis; Approved Guideline Presented by: Merle B. Smith, MBA, MS, MT(ASCP) Siemens Healthcare Diagnostics Newark, Delaware, USA Authors Merle B. Smith, MBA, MS, MT(ASCP) Yung W. Chan, MT(ASCP) Alberto Dolci, MD Mark D. Kellogg, PhD Christopher R. McCudden, PhD, DABCC, NRCC Michelle McLean, MS, MT(ASCP) James J. Miller, PhD, DABCC, FACB Jack Zakowski, PhD, FACB 2

2 Acknowledgment Renze Bais, PhD Richard J. Baltaro, MD, PhD, FCAP Cynthia Foss Bowman, MD Jian Dai, PhD, FACB, FCACB Alberto Dolci, MD Raymond E. Karcher, PhD Jeffrey E. Vaks, PhD Jason A. Kellogg, PhD Christopher M. Lehman, MD Karen L. Warren, MS Dror Yahalom, PhD, MBA 3 Intended Use For manufacturers responsible for establishing the automated hemolysis, icterus, and lipemia/turbidity (HIL) detection systems in their clinical laboratory instruments For laboratory directors, managers, supervisors, and medical technologists responsible for establishing or evaluating HIL indices and making judgments about the acceptability of specimens and test results 4

3 Recommendations Establishing HIL indices to assess sample quality Estimating interference effects of hemoglobin, bilirubin, and turbidity Reporting (by manufacturers) hemolysis, icterus, and lipemia/turbidity in the reagent instructions for use (IFU) Establishing error flags for HIL interference Verifying HIL indices in the clinical laboratory Managing potential process outcomes of HIL measurements 5 HIL Indices Hemolysis, icterus, and lipemia/turbidity are common in serum and plasma samples. Each may interfere with certain laboratory tests by optical or chemical mechanisms. These interferents may exert their effects by more than one mechanism. The magnitude and direction of interference may vary with the concentration of the interferent, the number of interferents present, the concentration of the analyte, the specific methodology of the test, and the amount of sample in the final reaction mixture. 6

4 HIL Indices (cont d) HIL indices are (semi)quantitative markers for hemolysis, icterus, and lipemia/turbidity in patient samples. The accuracy and precision of individual H- or I- index estimates should not replace a standard assay for hemoglobin or bilirubin, respectively. 7 Sample Quality Hemolyzed (red) Icteric (yellow) Lipemic (turbid) mg/dl g/l mg/dl µmol/l mg/dl mmol/l * Hemoglobin Total Bilirubin Intralipid * Concentrations based on dilutions of 20% Intralipid (or the equivalent). 8

5 Spectra of Hemoglobin, Bilirubin, and Lipemia/Turbidity In Figure 1, the spectra of bilirubin, hemoglobin, and lipemia/turbidity overlap. However, above 625 nm, the absorbance is due to lipemia/turbidity alone. The spectrum of hemoglobin overlaps most of the spectrum of bilirubin, but absorbance due to hemoglobin without contribution from bilirubin can be measured in the range of 550 to 575 nm. Reprinted with permission from Synermed International, The Infrared Revoluation in Clinical Chemistry Testing. Copyright Syndermed International, Pioneers in Near Infrared Clinical Chemistry. 9 Mechanisms of Hemoglobin Interference Spectral interference: absorbance in the hemoglobin spectral range ( nm; nm; peak = 420) Chemical reactions of heme or its iron with reagent components Release of analytes found in high concentrations in erythrocytes, which will falsely elevate the values of these analytes (eg, K, Mg) Release of enzymes that participate in chemical reactions (eg, AK) 10

6 Mechanisms of Hemoglobin Interference (cont d) Dilutional (water release): release of erythrocyte intracellular fluid, which dilutes analytes Negative interference in haptoglobin assays by hemoglobin binding to haptoglobin Inhibition of reactions: released hemoglobin can directly inhibit chemical reactions, as in polymerase chain reaction based assays on viral and bacterial DNA and RNA 11 Hemolysis Index The H index value closely approximates the hemoglobin concentration in the patient sample. The potassium released by a variable number of erythrocytes is approximately proportional to the H index. However, the amount of potassium per erythrocyte varies due to acid-base and sodiumpotassium balance variation. The correction of potassium results based on the H index is not reliable and is not recommended. When the H index is elevated, a new patient sample free of hemolysis needs to be collected and retested. 12

7 Mechanisms of Bilirubin Interference Spectral interference: absorbance in the bilirubin spectral range ( nm) Chemical reaction with reagent components (eg, H 2 O 2 ) 13 Mechanisms of Lipemia/Turbidity Interference Light scattering due to lipemia/turbidity affects measurements based on light scattering principles, such as nephelometry and turbidimetry. The lipoproteins that are the primary cause of turbidity are the triglyceriderich VLDL and chylomicron fractions. Very low-density lipoprotein (VLDL) exists in three size classes: small (27 35 nm), intermediate (35 60 nm) and large ( nm). Chylomicrons ( nm) vary greatly in size distribution and number among individuals. The degree of scattering depends on the number, size, and refractive index of the suspended particles. Because patient serum or plasma samples are a mixture of various particle sizes, the sample appears lipemic/turbid because the light is scattered at all angles. 14

8 Mechanisms of Lipemia/Turbidity Interference (cont d) Volume exclusion effect due to their large size and hydrophobic nature. Absorption of hydrophobic analytes, reagents, or reaction products into the lipid interior due to their lipophilic nature. Regardless of the cause of turbidity, it results in light scattering. In the absence of a sample blank, light scattering from a sample decreases the spectral operating (linear) range and causes positive interference. Other possible causes of turbidity include erythrocyte debris, platelets, leukocytes, fibrin clots, or contaminating particulate matter. 15 Intralipid * Unlike hemoglobin and bilirubin, there is no simple chemical substance that can be used to mimic the physical/chemical interfering properties of lipemic/turbid samples. Reagent manufacturers use Intralipid (or the equivalent) to simulate lipemia/turbidity. Intralipid is a registered trademark of Fresenius Kabi AG, Bad Homburg, Germany. Intralipid is a synthetic, sterile nonpyrogenic fat emulsion for intravenous administration that can be added to serum or plasma to simulate lipemic samples. The particles in Intralipid (or the equivalent) range in size from 200 to 600 nm (mean 345 nm) and completely miss the range of values for large VLDL and the lower and upper ranges for chylomicrons. Samples with added Intralipid (or the equivalent) do not perfectly mimic lipemic samples. * (or the equivalent) 16

9 Additional Interferences Green biliverdin Brown methemalbumin Orange beta-carotenes Exogenous compounds such as dyes, drugs and contrast media Interfering substances that do not absorb in the visual spectrum (eg, heterophile antibodies) Paraproteins (eg, M-proteins [monoclonal immunoglobulins]). Sample is not visibly lipemic/turbid. Turbidity is created when the sample is mixed with the reagent in the cuvette. 17 Interference Testing for Hemolysis, Icterus, and Lipemia/Turbidity Recommendations for analyte and interferent concentrations for interference testing are based on CLSI document EP07: HIL testing should be performed at two different medical decision levels of the analyte. Clinically significant interference may be missed if testing is performed at one medical decision level. HIL testing should be performed at concentrations representing the various indices to be used. For example: An I index of 1 for bilirubin represents a bilirubin concentration of up to 2 mg/dl (bilirubin testing performed at 2 mg/dl). An index of 2 represents a bilirubin concentration of 2 to 5 mg/dl (bilirubin testing is performed at 5 mg/dl). 18

10 Recommendations The maximum concentration of interferent tested should correspond to the concentration designated at the highest index. For example, hemoglobin is tested up to 1000 mg/dl, thus, the highest H index should include up to 1000 mg/dl of hemoglobin. A manufacturer may perform a: Screening (paired-difference) test followed by a dose-response test if interference is observed during the screening test Dose-response test (skipping the screening test) to assess interference and establish alert indices 19 Materials Used for Determination of Indices Manufacturers should provide information on the type, source, supplier, preparation of the hemolysate, bilirubin (unconjugated, conjugated, or icteric patient samples), and lipemic materials (fat emulsions). CLSI document EP07 may be used in the preparation of hemolysate and unconjugated bilirubin. Lipemic/turbid samples may be prepared by dilutions of the 20% Intralipid (or the equivalent) in serum pools. 20

11 Interferent Test Concentrations The range of hemoglobin concentrations tested should go up to at least 1000 mg/dl, bilirubin to at least 30 mg/dl, and lipemia/turbidity to at least 1000 mg/dl. Index values increase as the interferent concentration increases. 21 Sample Volumes for HIL Testing The volume of sample consumed for index measurements should be provided by the manufacturer. Many samples are available in smaller volumes such as neonatal, geriatric, and critical care. The sample volume required for these precious samples should be minimized to the lowest volume that will yield an accurate measurement. 22

12 Sample Dilution and Diluent Sample dilution and diluent (if applicable) should be specified if and how samples are diluted and what diluent is used (deionized/distilled water, saline [0.9% NaCl], special buffer, etc.). Dilutions and diluents are important considerations to avoid precipitation of paraproteins. 23 Read Wavelengths There is an overlap in the spectra of hemoglobin, bilirubin, and lipemia/turbidity. It is important that appropriate wavelengths and correction methods are used to provide the most accurate concentration estimates. There is currently no defined best method for measuring the serum indices, but manufacturers should describe how they estimate the presence of each interferent and provide information on cross-interferences of hemoglobin, bilirubin, and lipemia/turbidity indices. Detection methods should account for the absorbance spectrum overlap of hemoglobin, bilirubin, and lipemia/turbidity. 24

13 Number of HIL Indices (Bins) The manufacturer should provide the number of indices or "bins" and the concentration ranges associated with each bin. For example, an H index of 1 has a hemoglobin concentration of 10 mg/dl; an H index of 2 has a hemoglobin concentration between 10 and 25 mg/dl. This may be provided graphically, in table form, or as text. The number of indices for each interferent is decided by the manufacturer. It is recommended that a minimum of five "bins" be used (this equates to the standard visual estimation). 25 Number of HIL Indices (Bins) (cont d) There are currently no data to suggest that any particular number of "bins" is of greater utility. Assay imprecision should be considered when determining the number of " bins." Care must be taken in assigning HIL and alert indices to properly flag patient results that are affected by hemolysis, icterus, lipemia/turbidity and avoid false interference flags. 26

14 Read Time Manufacturers should indicate how long it takes to measure each index. The impact of HIL testing on system throughput should be minimized. 27 Calculation Algorithms The presence and degree of hemolysis, icterus, and lipemia/turbidity are detected by measuring the absorbance or reflectance on at least two different wavelengths over the measured spectrum and preferably on the absorbance peaks of the specific substance. Correction factors are required to correct for spectrum overlap for hemoglobin, bilirubin, and lipemia/turbidity. 28

15 Existing Automated HIL Detection Systems on Various Chemistry Analyzers Interferent Material Used HIL System Hemolysis (hemoglobin) Icterus (bilirubin) Lipemia/Turbidity/ Intralipid 1 Erythrocyte Hemolysate Unconjugated 20% Intralipid 2 Erythrocyte Hemolysate Unconjugated 20% Intralipid 3 Erythrocyte Hemolysate Unconjugated 20% Intralipid 4 Erythrocyte Hemolysate and Patient Samples Unconjugated, Conjugated, and Patient Samples 20% Intralipid and Patient Samples 5 Erythrocyte Hemolysate Unconjugated 20% Intralipid 6 Erythrocyte Hemolysate Unconjugated, Patient Samples 7 Erythrocyte Hemolysate Unconjugated, Conjugated 20% Intralipid 20% Intralipid Intralipid (or the equivalent) concentrations were based on dilutions of 20% Intralipid (or the equivalent). 29 Existing Automated HIL Detection Systems on Various Chemistry Analyzers (cont d) Maximum Concentration Tested [mg/dl] HIL System Hemolysis (hemoglobin) Icterus (bilirubin) Lipemia/Turbidity/ Intralipid N/A

16 Existing Automated HIL Detection Systems on Various Chemistry Analyzers (cont d) Sample Volume [ml] Hemolysis Icterus Lipemia/Turbidity HIL System * 35 * 35 * 5 2.0/ / / *The analysis does not consume the sample. 31 Existing Automated HIL Detection Systems on Various Chemistry Analyzers (cont d) Diluent Used/Diluent Volume Used to Determine Hemolysis, Icterus, and Lipemia/Turbidity for Each Sample HIL System Diluent Used Diluent Volume [µl] 1 Water Saline (Reagent 1) Saline (AST/ALT 200 Reagent) 4 Undiluted Undiluted 5 Saline Tris Buffer ph Saline (0.9% NaCl) 150 Abbreviation: AST/ALT, aspartate aminotransferase/alanine aminotransferase. 32

17 Existing Automated HIL Detection Systems on Various Chemistry Analyzers (cont d) Read Wavelength(s) [nm] Hemolysis Icterus (bilirubin) Lipemia/Turbidity/Intralipid HIL System (hemoglobin) 1 405/ / / / / /604; 628/ /524; 572/604; 500/524; 572/604; 572/ / /480; 600/ /570; 600/ / , 340, 470, 600, , 410, 470, 600, , 410, 470, 600, / / / Existing Automated HIL Detection Systems on Various Chemistry Analyzers (cont d) # of HIL Indices/Levels Reported Hemolysis Icterus Lipemia/Turbidity HIL System Concentration reported in Concentration reported in Concentration reported in units units units Concentration reported in units Concentration reported in units 1 60 Concentration reported in units

18 Example of HIL Testing: Hemoglobin Abbreviation: RBC, red blood cell. 35 Alert Index (Threshold Value) An alert index indicates the lowest tested concentration of hemoglobin, bilirubin, and lipemia/turbidity (Intralipid [or the equivalent]) at which the analyte concentration would be falsely increased or decreased. An interference flag is generated if the HIL index of the sample is equal to or exceeds the alert index. At a specific alert index of hemolysis, icterus, and lipemia/turbidity, the interference effect and magnitude of the interference at a specific analyte level is specified in the manufacturer s product labeling. 36

19 Alert Index (Threshold Value) (cont d) When establishing alert indices the manufacturer should consider the following: The magnitude of the bias observed relative to the clinical acceptability criteria. The frequency of false or unnecessary flagging. This may occur if the observed interference is due to the interferent concentration at the upper limit of the concentration range within the HIL index. For example, interference is at 500 mg/dl of hemoglobin; bin of 4 ( mg/dl). No data between mg/dl; using 4 as the alert index, will flag samples containing hemoglobin between 250 and 249 mg/dl causing unnecessary flagging. Customer may consider using 5 ( mg/dl) as the alert index. 37 Gray Zone HIL indices represent estimates of the degree of hemolysis, icterus, and lipemia/turbidity; therefore, the manufacturer may consider defining a gray zone. Gray Zone Indicating Maximum Interferent Concentration With No Known Interference A gray zone may also be established to indicate an HIL index containing a concentration above the highest tested interferent concentration for which no interference data are available. For example: The results of the hemoglobin interference testing for glucose at 100 mg/dl indicated no observed interference up to 1000 mg/dl of hemoglobin. Based on the manufacturer s hemoglobin indices, an H index of 8 indicates a hemoglobin concentration > 1000 mg/dl. An H index of 8 is assigned a gray zone. 38

20 Gray Zone (cont d) The following results are obtained from the dose response (hemoglobin) testing for interference in a glucose assay. The interferograph is as follows: Hemoglobin Glucose mg/dl mg/dl Bias % N/A The allowable bias used for this assay is 10%. Bias outside of ± 10% is considered an interference. The observed percent bias due to hemoglobin for the glucose assay is acceptable. 39 Gray Zone (cont d) The hemoglobin indices are evaluated. Gray Zone H Index Hemoglobin (mg/dl) 1 H < H < H < H < H < H < H H >

21 Gray Zone (cont d) Gray Zone Indicating Potential Interference For each analyte, a gray zone represents the HIL index containing the highest tested interferent concentration that is below or equal to the alert index. This gray zone lies between the highest concentration with acceptable bias and the lowest concentration with known unacceptable bias. For example: The results of the bilirubin interference testing for glucose at 100 mg/dl indicated interference starting at 15 mg/dl of bilirubin. Based on the following bilirubin indices, an I index of 5 is assigned as an alert index while an I index of 4 is assigned as a gray zone. 41 Bilirubin Interference on Glucose Assay Bilirubin Dose Response Testing Interferograph Bilirubin Glucose mg/dl mg/dl Bias % N/A The allowable bias used for this assay is 10%. Bias outside of ± 10% is considered an interference. The bias becomes higher at increasing bilirubin levels. 42

22 Bilirubin Icterus Index Assigned 43 Multiple Interferences More than one HIL interferent may be present simultaneously in a patient sample. The presence of one HIL interferent may adversely affect the measurement of another HIL interferent. Due to the overlapping nature of the absorbance spectra between lipemia/turbidity and the other two interferents, many automated systems have calculation algorithms to correct the raw signals at particular wavelengths to obtain the pure signal and report the index for each individual interferent. 44

23 Wavelength Corrections The H absorbance is read at 405 and 700 nm with correction at 452 nm to prevent high bilirubin samples from generating falsely elevated H index. The I absorbance is read at 452 and 700 nm with correction at 577 nm to minimize falsely high I index due to hemoglobin. The L absorbance is derived from a blanked 700 nm measurement. 45 Verification and Quality Control of HIL Indices in the Clinical Laboratory The manufacturer can verify the sensitivity and specificity of the established HIL indices. The evaluation can consist of assessing the sensitivity of I index to concentrations of hemoglobin and lipids; of H index to concentrations of bilirubin and lipids; and of L index to concentrations of bilirubin and hemoglobin. 46

24 Verifying Specificity of HIL Indices Prepare a sample with low I and L indices, and a medium H index. Aliquot 1 Add a small volume of saline. Aliquot 2 Add bilirubin concentrate equal to the volume of spiking saline. Aliquot 3 Add lipids concentrate equal to the volume of spiking saline. Measure H index of the above aliquots. 47 Verifying Specificity of HIL Indices (cont d) Similarly, effects of H and I indices on lipemia/turbidity, and of H and L indices on icterus, are evaluated using samples with medium levels of lipemia/turbidity and icterus, respectively. Because of the nature of these indices, the results should be considered qualitative or as estimates of the degree of hemolysis, icterus, and lipemia/turbidity. 48

25 Verifying Specificity of HIL Indices (cont d) If the readings of the H index do not change on samples with added bilirubin and lipemia/turbidity, it can be concluded that the H index is not affected by changes in (ie, cross-interferences from) I and L indices for the analytical method being evaluated. If there is a change in the hemolysis index, further investigation should be conducted to make sure there is no interaction between hemoglobin, bilirubin, or lipemia/turbidity, respectively. The number of indices and the range of hemoglobin concentrations within the H index should be considered. 49 Reporting HIL Interference Claims by Manufacturers in Product Labeling Manufacturers provide information on H, I, and L interferences in the reagent package insert or IFU. The information provided includes the concentration of analyte; the concentration of hemoglobin, bilirubin, Intralipid (or the equivalent) tested, respectively; and the bias observed. Interference claims may be listed in one or more of the following sections of the IFU as appropriate: 1) Specimen Collection; 2) Limitations (Interfering Substances); 3) Specificity (HIL Table, Noninterfering Substances). 50

26 Reporting HIL Interference Claims by Manufacturers in Product Labeling (cont d) The interference claim may state the concentration of hemoglobin, bilirubin, Intralipid (or the equivalent), respectively, above which it causes a bias. This represents the lowest tested interferent concentration causing interference. This information is used to establish the alert index for the assay and to alert the user of the potential inaccuracy of the generated analyte result. The interference claim may state the concentration of hemoglobin, bilirubin, and Intralipid (or the equivalent), below which there is no interference. This represents the highest interferent concentration that does not cause interference. 51 Reagent Package Insert/Instructions for Use C56 recommends reporting HIL information in an HIL table format under the Limitations (Interfering Substances) or Specificity sections. Provides information if a particular assay is or is not subject to interference by HIL and includes: Concentration of analyte tested Concentration of hemoglobin, bilirubin, Intralipid (or the equivalent) tested Bias and percent bias (positive or negative) The smallest concentration of hemoglobin, bilirubin, and turbidity that caused an interference and that triggers the alert index to flag an analyte result must be reported in the IFU. 52

27 Instructions for Use: Reporting HIL Interference for Glucose The glucose method was evaluated for interference according to CLSI document EP07. Bias is the difference in the results between the control sample (without the interferent) and the test sample (with the interferent) expressed as a percentage. Bias exceeding 10% is considered interference. Table 1 Substance Tested Substance Concentration Glucose (mg/dl) Bias (%) Hemoglobin (hemolysate) 1000 mg/dl < 10 < 10 Unconjugated bilirubin 60 mg/dl < 10 <10 Lipemia/turbidity (Intralipid [or the equivalent]) 1000 mg/dl <10 <10 53 Instructions for Use: Claims and Alert Indices Assigned Manufacturers that use automated HIL detection systems recommend alert indices based on interference data. HIL IFU claims should align with the HIL indices assigned. Using Table 1, a patient sample with an I index of 5 (15 20 mg/dl of bilirubin) and above, and an L index of 6 ( mg/dl of Intralipid [or the equivalent]) and above, will have the glucose results flagged with bilirubin interference and lipemia/turbidity, respectively. There is no alert index assigned by the manufacturer for hemoglobin because no interference was detected. 54

28 Instructions for Use: HIL Claims The manufacturer may elect to report: Interferent concentration that shows no interference Additional concentrations of the interferent to demonstrate the actual concentration at which interference is first detected, and the highest interferent concentration tested In this example, interference is observed starting at 250 mg/dl of hemoglobin. The highest hemoglobin concentration tested is 500 mg/dl. Substance Tested Substance Concentration (mg/dl) Glucose (mg/dl) Bias (%) Hemolysis < 10 (hemoglobin) 250 < Instructions for Use: Reporting Example 1 When there is no interference: The highest concentration of hemoglobin, bilirubin, and Intralipid (or the equivalent) tested should be reported in the IFU. For example: hemoglobin at 1000 mg/dl; bilirubin at 60 mg/dl; Intralipid (or the equivalent) at 1000 mg/dl. Substance Tested Substance Concentration Glucose (mg/dl) Bias (%) Hemoglobin (hemolysate) 1000 mg/dl < 10 < 10 Unconjugated bilirubin 60 mg/dl < 10 <10 Lipemia/turbidity (Intralipid [or the equivalent]) 1000 mg/dl <10 <10 The alert indices to be assigned for this assay will be: H = 8; I = 8; L = 8 (corresponding to H > 1000 mg/dl, I > 60 mg/dl, and L > 1000 mg/dl). 56

29 Instructions for Use: Reporting Example 2 57 Instructions for Use: Reporting Example 2 (cont d) Manufacturers that use automated HIL detection systems recommend alert indices based on interference data. Icterus and lipemia indices of 5 and 6, respectively, will be assigned as alert indices for this assay because they are the bins that contain 15 mg/dl of bilirubin and 600 mg/dl of lipemia/turbidity (Intralipid [or the equivalent]). 58

30 Instructions for Use: Reporting Example 3 59 Instructions for Use: Reporting Example 4 60

31 Potential Process Outcomes of HIL Measurements/Estimates 61 Laboratory Guidelines It is recommended that the laboratory establish its own guidelines for addressing patient samples that have HIL error flags, such as when reporting interference error flags without results, rejecting the patient results, redrawing the specimens with considerations given to neonatal and geriatric populations, and notifying the health care provider (physician), which includes documenting the dialogue conducted with the physician. Laboratories must follow the regional, national, and local regulatory agencies for current applicable requirements. 62

32 HIL Reporting of HIL Indices/Flags Laboratories employ middleware or laboratory information systems for autoverification. Rules or algorithms can be used to alert laboratory personnel of error messages, including HIL flags. If the HIL index is below the alert index designated for the assay, there is no HIL interference and the assay result is reported. If the HIL index is equal to or greater than the alert index, the result is flagged with either the appropriate H, I, L comment indicating potential interference. The laboratory is responsible for rejecting, reporting, and handling of samples and test results that have been flagged with interference errors. 63 Example of Reporting HIL Indices 64

33 Verification and Quality Control of HIL Indices in the Clinical Laboratory Though HIL indices may not be subject to the requirements specified by regulatory and/or accrediting agencies for analysis of clinical specimens, laboratories should consider verification and Quality Control (QC) of expected performance of these parameters before (verification) and after (QC) implementation into laboratory practice. Because HIL parameters are produced using spectrophotometric measurements, they are subject to drift and failure like any standard spectrophotometric assay. Failure to maintain consistent measurements over time may lead to changes in the effective criteria for acceptance or rejection of specimens received by the laboratory. In addition, variability between analyzers may result in inconsistent acceptance/rejection criteria. To date, no HIL standards (calibrators) are available for use. 65 Verification and Quality Control of HIL Indices in the Clinical Laboratory (cont d) Visual examination of patient specimens for comparison with HIL indices is not recommended, because it has been shown to be inaccurate and variable due to its qualitative and subjective nature. For verifying the HIL indices, laboratories may consider the following options: The laboratory can challenge the HIL flagging system using hemolysates, and samples with added bilirubin and Intralipid (or the equivalent), respectively (see CLSI document EP07), that span the range of the H and I values that would generate a flag when exceeded. The laboratory may be able to use samples that are provided by a health care organization and are designated for verifying HIL interference effects using doseresponse methodology. The laboratory can measure the hemoglobin (eg, cyanmethemoglobin methodology) and bilirubin (total and direct) concentrations of patient specimens that have been evaluated by the instrument HIL system. 66

34 Verification and Quality Control of HIL Indices and Alert Indices in the Clinical Laboratory Each laboratory should consult with its manufacturers to identify the appropriate control materials for monitoring the performance of HIL parameters over time. For monitoring the H parameter, one option is to prepare a hemolysate for use as QC material for a period of time as validated by the laboratory. For monitoring the I parameter, one option is a total bilirubin control that is used daily in the laboratory for the bilirubin assay. For monitoring the L parameter, one option is to make up lipemia/turbidity controls by spiking Intralipid (or the equivalent) into serum or plasma at the desired concentration, aliquotting, and freezing for storage. Because HIL determinations are spectrophotometric measurements, monitoring and basic troubleshooting should include general spectrophotometric instruments/device investigations. 67 Verification and Quality Control of HIL Indices in the Clinical Laboratory The laboratory may be able to use samples that are provided by a health care organization and are designated for verifying HIL interference effects using dose-response methodology. These samples contain varying concentrations of interferents (hemoglobin, bilirubin) that can be analytically measured using the clinical instrumentation in the laboratory. The measured interferents (hemoglobin, bilirubin) may then be checked against the range of concentrations assigned for H and I indices provided by the manufacturers. These samples can also be measured for various analytes of interest. 68

35 Verification and Quality Control of HIL Indices in the Clinical Laboratory (cont d) The accuracy of interference flagging based on the assigned alert indices and interference claims made by the manufacturer can be evaluated. Each laboratory should consult with its manufacturers to identify the appropriate control materials for monitoring the performance of HIL parameters over time. 69 Conclusions C56-A Hemolysis, Icterus, and Lipemia/Turbidity Indices as Indicators of Interference in Clinical Laboratory Analysis; Approved Guideline focuses on the intended usefulness and challenging issues of HIL indices as estimates of interference that may impact the validity and clinical utility of reportable patient results. This document enhances the continuous education of health care personnel by explaining the mechanisms of HIL interference, which in some cases include the noncorrelation of visual and (semi)quantitative HIL indices, the strengths and limitations of HIL measurements, and the verification of HIL indices in the clinical laboratory. 70

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