How reliable are analytical results?

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Introduction

The accuracy of laboratory result can be affected by a wide range of factors. They are mainly those found in the pre-analytical phase of the testing process, as well as the analytical and the post analytical phase. Problems often arise before the specimen reaches the laboratory.

Pre-analytical errors can sometimes be misleading which can be caused by problems that occur prior to sample analysis. Controlling pre-analytical factors prior to testing is an important factor in order to get accurate results and is essential to patient safety. The quality of results provided by the laboratory is dependent on how the pre-analytical factors are controlled such as specimen collection, specimen handling, interfering substances and patient-related factors, as it has been estimated that 32–75% of errors occur in the pre-analytical phase. (Ajeneye 2007)

Guidelines for collecting samples and for evaluating submitted specimens are essential because those factors could affect patient care, and their identification and the use of continuously reviewing can help quality improvements in the laboratory services

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Pre-Analytical is the step when test is ordered and sample is collected.

Analytical Activities refers to the stage of what happens in the lab where the test is conducted

Post-Analytical Activities refers to what happens between the time the test is run and the results are reported to the physician. Before reporting to the physician the results are reviewed for any anomalies by a BMS.

The most common type of specimen used for analysis is blood. There are 2 basic reasons why analysis of blood is required, 1) diagnosis 2) monitor treatment. Laboratory test are not always reliable as errors can occur, these errors reduced by analytical quality controls.

In the following assignment the reliability of analytical results will be discussed with reference to pre-analytical, analytical and post analytical errors.

Pre-analytical phase

(Ajeneye 2007)

Specimen Collection Techniques

The major cause of haemolysis is the way in which the specimens are collected and handled. Therefore, proper attention needs to be given to reduce the number of hemolyzed specimens received in the laboratory and this can be achieved through training and education. The factors listed below are all known to cause haemolysis to a range of degree and should all be taken into consideration during collection. (Bush 2003)

  • Vein size and trauma- puncturing small fragile veins
  • Alcohol preparation- must be allowed to dry before the vein puncture.
  • needle size- using an appropriate sized needle
  • loose connections- making sure all connection components are tightened
  • under filled tubes-ensuring tubes are filled to correct volume
  • Syringe collections-improper syringe collections are known to cause haemolysis. (Bush 2003)

Prolonged use of tourniquet

Using the tourniquet for a pro-longed time can increase the localised pressure. Fluid leaks out and increases concentration of intracellular constituents. This effect increases with time, e.g. from 5 to 15 minutes therefore first sample will be different from the last sample drawn.

Speed of drawing blood

If blood is taken too quickly up the needle and into the syringe, bubbles can form which can cause haemolysis. The RBC will lyse and empty into extracellular fluid. This can lead to Increase in concentrations of analytes, e.g. electrolytes. These can release enzymes which metabolise other components therefore reducing the correct physiological level

Evacuation of blood into bottle

Haemoglobin interferes with spectrophotometric assays as it absorbs light at the visible and near UV wavelength

Contamination

Heparin or other anti-coags are commonly used to keep canulas patent, i.e. open.

Blood will clot in the tube unless anticoags are used.

Heparin can interfere with other substances. Eg, measurement of PP5

-Contamination & timing eg, immunosuppressive drug Cyclosporin

Drug used to prevent rejection in transplant patients. Who are on it for rest of their lives, they must be monitored as it is toxic.

Sample must be taken just before next dose (Hills 2007)

Specimen transportation and Specimen Handling Techniques

It is important to be aware of the way in which specimens are transported to the laboratory, as poor transportation can lead to the specimen being analyzed incorrectly. Delayed separation results in loss of ions and enzymes such as potassium (K+), magnesium (Mg2+), phosphate (PO4

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2–) and the enzymes lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate transaminase (AST); while coagulated samples require a fast analysis if not preserved. Also, ribonucleic acid (RNA) is rapidly lost without centrifugation. (Ajeneye 2007)

Temperature

A delay of transporting the sample up to an hour or so is ok.

Although glucose levels will fall by 3% per hour at RT with no inhibitors

Must keep sample at correct temperature. Eg, Cryoglobulins must be kept at 37°C Temp too low – haemolysis. Temp too high – haemolysis. Therefore samples should not be kept above RT.

Sending away

Often samples have to be sent away to a reference or specialist lab. As stated before – temperature variations & delay. Either the serum or the plasma should be sent, so that it can be sent frozen.

(Hills 2007)

Sample processing

Samples must be centrifuged at a sufficient level so that all clotted material is removed, and standard operating procedures (SOPs) for sample processing must be followed. Proper centrifugation in the laboratory can prevent analytical problems.

(Ajeneye 2007)

Sample integrity

Specimens should be checked for correct labelling before analysis. If they are not labelled properly then they should not be analyzed and corrective action should be taken. The required volume, based on the hospital protocol, should be checked. Date and time of collection must be checked for suitability of the sample prior to processing. Also, checks should be undertaken for clot, haemolysis or haemodilution.

Results of a test can be affected by cylical variation, for example hormones can have an increase and decrease during the day or the night, and so analytes are affected by female sex hormones, e.g.: LH, FSH, oestrogen, which in turn can affect the calcium levels, cholesterol, and ADH. (Ajeneye 2007)

Physiological Factors that can affect pre-analytical errors

Urine analysis can be variable, as 1,25 dihydroxy-vitamin increases in the summer, urinary oxalate increases in the summer; therefore there would be variations if a test were undertaken in the summer compared to other seasons.

Exercise can affect levels of potassium, phosphate, creatine, serum, proteins can be altered, biliruben, and decrease in glucose levels.

Stress can cause decrease in gonadotrophins and sex hormones increase in prolactin. Increases ACTH, cortisol, prolactin, and cateocholamines, total cholesterol increases with stress, HDC decreases by 15%.

Diet- after food intake: increase in TG’s and glucose. Sodium, uric acid, iron, insulin and gastrin.

Posture: Standing when having blood taken can lead to fluid leakage due to increase in hydrostatic pressure. If fluid goes out leads to an increase in

concentration of constituents left inside. Lying down leads to decrease in concentration of cellular components

(Hills 2007)

Analytical phase

During the analytical phase

  • Quality control and quality assurance are important factors.
  • Maintenance and service plays a role- how the sample is maintained.
  • Finally analysis, test undertaken to meet standards.

The ideal analytical method is accurate, precise, sensitive and specific. It gives a correct result; it measures low concentrations of the analyte and does not interfere with other substances. In practice there is no test that is ideal, but the pathologist must ensure that the results are sufficiently reliable to clinical use. Analytical methods are subject to quality controls and quality assurance procedures at all time. However there will always be some errors to some extent, due to imprecision or analytical variation in a result. Analysis is repeated for assessment. (Marshall 2004)

Example

The following is a practical assignment which was carried out to measure out glucose. There are many methods used to measure glucose, below are some methods that were carried out:

  • Glucose oxidase method was used to measure glucose, also this practical was undertaken to examine the effects of vitamin C and galactose on glucose measurement

The principle of the glucose oxidase assay

(Lab handbook 2007)

Glucose is oxidized to gluconic acid and hydrogen peroxide by glucose oxidase. Hydrogen peroxide reacts with o-dianisidine in the presence of peroxidase to form a coloured product.

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Oxidized o-dianisidine reacts with sulphuric acid to form a more stable coloured product.

The intensity of the pink colour measured at 540nm is proportional to the original glucose concentration. (Lab handbook)

From the result of the practical, there were some variations in the amount of glucose concentration; this was due to one of the samples containing galactose as well as glucose, so there was some binding interference during the reaction.

Another sample did not contain any glucose but result indicated that glucose was present; this was due to the structure of galactose being similar to glucose.

Finally the effect of vitamin C was seen where the structure of vitamin C may have acted as an inhibitor causing the reaction to slow down.

Another method for measuring glucose was the hexokinase method, where glucose was measured again with the following reaction taking place.

The principle of the glucose hexokinase assay

(Lab handbook 2007)

Again there was some variation that had occurred, with the structure of galactose being similar to glucose. There was also some interference of vitamin C.

From the above examples and methods of measuring glucose we can see some forms of analytical errors that occurred during the analysis of the tests. These errors can lead to post-analytical errors, when interpreting the results. It can also be seen that during analysis the test can sometimes be subjected to interference of other substances.

The importance of Quality controls and quality assurance

QA comprises everything that the laboratory does to assure high quality service to its users. There are 2 kinds of processes, internal quality control (IQC) and external quality assessment (EQC). Each day laboratories analyze IQC materials with known concentrations of specific substances to ensure, in real time that they are producing reliable results. Laboratories have comprehensive QA programmers to ensure that every step of the process of producing results-requesting, sampling, analyzing, reporting- is monitored to ensure the correct tests are performed, that reliable results are produced, and that these are communicated to the appropriate doctor in timely manner. The QA programme defines requirements for staff proficiency equipment maintenance and monitoring, and other standards of operations. (Lab test online 2004)

  • The importance of Quality Assurance is the process whereby the quality of laboratory reports can be guaranteed
  • Comprises all the different measures taken to ensure the reliability of investigations
  • Seeks to minimize any variability in test results
  • Not limited to the technical procedures performed in the laboratory

If QA and QC are not maintained properly then a patient’s life is at risk, which is why Quality controls need to be accurate in order for samples to be analyzed correctly.

Most laboratories have introduced various automated machinery to assist with analysis of samples in order to minimize human error.

Most errors that occur during analytical stage are due to lack of precision, competence and accuracy in the following:

  • Conducting standard operating procedures
  • Performing routine maintenance of the automated machines
  • Preparing the analysers for use
  • Running the quality controls
  • Keeping record of maintenance
  • Keeping record of when the analyzer was last checked for efficiency of use
  • Pipetting techniques
  • Centrifugation time
  • Random errors - scatter of results about the true value
  • Variation in other techniques

If quality controls fail correct action needs to be taken, quality control material should be repeated. If it fails again, questions need to be asked: whether the reagent or buffers need changing, does the waste need to be disposed? If after all those checks are done and quality controls are still out of range then method should be suspended and switched to backup, and higher authority needs to be contacted.

The internal QC programmes are designed to detect the above errors and take corrective action in improving the service. (Lab test online 2004)

Post Analytical phase

Includes:

  • Systems training and knowledge
  • Reporting
  • Interpreting
  • Treatment
  • Clinical needs

Once analysis is completed and quality controls are met, a report is issued to interpret the result. Sometimes the results are subject to error, and have a potential to cause considerable harm. Errors can be minimized by following a strict protocol every step of the testing process. Errors can occur at various stages in the process:

During post-analytical, when a correct result is generated but it is incorrectly recorded in the patients’ record, this can be due to a transcription error.

When the result of a biochemical test is obtained, the following points need to be taken into consideration:

  • Is it normal?
  • Is it significantly different from the previous results?
  • Is it consistent with the clinical findings?

(Marshall et al 2004)

When reviewing the patients results the reference ranges need to be accurate and precise. Abnormal values need to be known. The normal range for an analyte can sometimes have limitations. It only identifies the most expected result that occurs most often in patients. It has been explained that 5% of people will by large, have a value for a given variable that is outside the reference interval. A second test is usually done, it follows that the more tests that are done on an individual then the greater the probability of that the result of one of them being abnormal.

If the result of a previous test is available then the clinician can then compare the results and decide whether any difference between them is significant. This all depends on the precision of the assay,

Sometimes problems arise from the point where the sample is put through so many unnecessary tests, in which during reviewing the BMS may avoid looking at that specific result assuming it is less significant.

If the result is consistent with the clinical findings, it is evidence in favour of the clinical diagnosis; if it not consistent then an explanation would be needed. There may have been a mistake in the process of sample collection, labelling, or analysis of the sample or in the reporting of the result. In practice, a request can be sought for another sample to repeat the test. This all would In turn affect the overall clinical diagnosis once the result is confirmed. (Marshall et al 2004)

(Ajeneye 2007)

Figure 1 shows the different stages of pre-analytical phase from the point of the test being ordered to when the result is reported which is the post analytical phase. Pre- analytical phase holds a very important phase in the whole process of relying on test results.

If the pre-analytical phase goes wrong then there will be analytical and post analytical errors and the result will be incorrect.

Error prevention/ error management

It is important to identify ways to reduce pre-analytical error. An increasing body of evidence demonstrates that error rate has improved significantly over time and is affected by education of healthcare workers, patient education and patient identification systems.

Reliable specimen quality can only be seen if potential phlebotomists receive the proper training and acquire the necessary skills. Also, the use of computerised ordering has improved test request and reduced order-related errors. (Ajeneye 2007)

Pre-analytical error is the most common cause of laboratory error and can occur at any stage of the test cycle. Therefore is very important that error prevention techniques are implemented at all stages of the analytical pathway, and also create a culture in which errors are reported.

In terms of analytical quality, clinicians depend on the laboratory for the detection and correction of errors; therefore, the following points should be addressed:

  • defining laboratory errors and their causes, and setting up plans for corrective strategy
  • creating a standard for laboratory error detection, and accurately defining, reporting and error risk
  • measuring error reduction and demonstrating, via process analysis, a reduction in risk
  • Creating a culture in which the existence of error is acknowledged, and regularly updating information given for sample collection and transportation protocols for non-laboratory personnel’s.
  • Carrying out protocols for procedures that are followed by all members of staff involved in requesting tests, taking blood, transporting and processing blood samples.
  • Being aware of ‘odd’ or ‘unusual’ results and taking corrective action

(Ajeneye 2007)

The doctors should evaluate all the relevant findings from test data plus and information from other sources, before settling down on diagnosis and developing a treatment plan. Given the amount of variability that a laboratory test can provide, no diagnosis should ever be made based on a single laboratory test. The clinician should always ask “do the test results fit with everything else” which includes the patient’s clinical status, personal and family history. Medical test are not fully reliable information, and can present some inaccuracy.

Conclusion

Careful evaluation and consideration of test findings can increase the reliability of a diagnosis and reduce the chance of medical errors. If the test doesn’t fit well with the clinical details, additional investigation may be required, a retest maybe appropriate to confirm the clinical findings.

Although laboratory testing should meet high standards of proficiency and reliability, however some test can sometimes be falsely negative or falsely positive. Many of the few errors that occur in some laboratories are usually detected by quality control measures, including data handling software or personal analysis of reports by laboratory staff. Many results are quite obvious when they go wrong, but more subtle ones go undetected and unfortunately the errors that do occur can never be eradicated.

However, analytical results are improving, as modern technology is advancing, where more and more automated systems are being used for precision and accuracy, which means more accurate results being obtained and less human error.

References and Bibliography:

  • Ajeneye. F. (2007). Pre-analytical quality assurance. Biomedical scientist. (pg 86-87)
  • Bonini P, Plebani M, Ceriotti F, Rubboli F. (2002). Errors in laboratory medicine. Clin. Chem.; 48:691-8.
  • Bush. V. (2003). The haemolysed specimen: cause, effects and reduction. Lab notes. Vol 13, no 1.
  • Bush V, Green S. Managing Pre-analytical Variability in Chemistry. ASCP Fall 2001 Teleconference Series; Program No: 9080, November 14, 2001
  • Kalra. J. (2004). Medical errors: impact on clinical laboratories and other critical areas Department. Science Direct (37) 1052– 1062
  • Hills. A. (2007). Specimen collection and sample journey. Clinical Biochemistry (lecture notes)
  • Laboratory handbook. (2007/2008). Clinical Biochemistry. Middlesex University. Department of Natural sciences.
  • Lab test online. (2004). How reliable is laboratory testing [online] available from:http://www.labtestsonline.org.uk/understanding/features/reliability-3.html
  • Marshall. W. J, Bangert. S. K. (2004). Clinical Chemistry. Fifth edition. Mosby