HOW TO USE CLINICAL LABORATORY
Li Ping Department of Laboratory Medicine
West China Hospital and Medical School of Sichuan University
INTRODUCING CLINICAL LABORATORY
Clinical laboratory or medical laboratory is for the biological, microbiological, immunological, chemical, immunohaematological, haematological, biophysical, cytological, pathological, or other examination of materials derived from the human body for the purpose of providing information for the diagnosis, prevention, treatment of disease in, or the assessment of the health of, human beings These examinations also include procedures to determine, measure, or otherwise describe the presence or absence of various substances or microorganisms. Facilities which only collect or prepare specimens, or act as a mailing or distribution center, are not considered to be laboratories, although they may be part of a larger laboratory network or system. A laboratory may provide a consultant advisory service covering all aspects of laboratory investigation including the interpretation of results and advice on further appropriate investigation.
Pre-examination process：preanalytical phase steps starting in chronological order. From the clinician's request, including the examination requisition, preparation of the patient, collection of the primary sample, transportation to and within the laboratory and ending when the analytical examination procedure starts.
Postexamination process ：postanalytical phase all processes following the examination including systematic review, formatting and interpretation, authorization for release, reporting of results, transmission of the results, and storage of samples of the examinations.
Besides the medical history and diagnostic imaging, clinical laboratory makes an important and, not infrequently, essential contribution to the diagnosis of disease. The utility of laboratory is, however, not only limited to the diagnosis of diseases but it has also gained increasing value both in monitoring their course and treatment as well as in estimating their prognosis. Laboratory investigations are part of the evaluation of health and the assessment of risk factors for disease.
The development of new methods, the improvement of existing methods, high quality standards as well as increasing national and international standardization have resulted in an increase in the clinical sensitivity and specificity of clinical laboratory investigations.
The breadth of the spectrum of clinical laboratory tests and the increase in special and costly investigations pose the danger the attending physician uncritically uses a multitude of laboratory tests, the clinical pathologist or clinical chemist, whose work is far removed from the actual patient, is unable to tell whether each clinical laboratory request is based a clearly defined clinical concern and whether the preanalytical prerequisites for the collection of the specimen were met.
Clinicians should learn more about laboratory tests. The clinical laboratory is not simply a “black box” where one requests tests and always receives equivalent results. There are pitfalls related to specimen collection and handling, methodologies, and the skills of the individuals performing the tests. All laboratory tests are not created equal, and test differences can affect results, which in turn can affect medical decisions. Greater knowledge about laboratory tests not only will enable clinicians to use tests more effectively, but also will help them to better understand the nuances of office laboratory testing and home (self) testing. In some situations, it may occasionally be helpful for clinicians to actually know how to perform some tests themselves: (1) preparation and examination of a Gram-stained smear of the sputum for pneumonia and of the urine for urinary tract infection, (2) performance of a macroscopic (dipstick) urinalysis and (3) preparation and examination of a Wrightstained peripheral blood smear for anemia. For certain medical decisions, it is imperative to know not only which test or tests to request but also which method or methods to use and how the methods should be standardized and controlled. For example, one cannot use the National Institutes of Health (NIH) recommendations for desirable, borderline-high, or high levels for serum cholesterol unless one uses a method for measuring cholesterol that is equivalent to the NIH method. It is also important to know the diagnostic utility of tests, that is, how effective they are in confirming or excluding a diagnosis.
Clinicians should use the reference ranges of the laboratory that performs their tests. Common reference ranges are provided inside the front and back covers of the book, and special reference ranges are included in the test evaluation section of each problem. Reference ranges and other laboratory values are given in conventional units followed by international units in parentheses. Appropriate reference ranges should address important variables (analytic; biologic, genetic, ethnic; environmental; and life-style).
Information on clinical decision levels is also included. The term “decision level” refers to a threshold value above which or below which a particular management action is recommended. The use of decision levels recognizes the importance and additional information content in knowing not only whether a test result is high or low, but also how high or how low. For example, the reference range (normal range) for serum calcium is 8.4 to 10.2mg/dl (2.10 to 2.55mmol/L).The decision level above which hypercalcemic coma can occur is 13.5mg/dl (3.37mmol/L),and the decision level below which tetany can occur is 7.0mg/dl (1.75mmol/L).It would be inappropriate to assign the cause of a patient’s coma to hypercalcemia if the level was 11.5mg/dl (2.87mmol/L),since, even though 11.5mg/dl is above the upper limit of the reference range for serum calcium, it is below the decision level for hypercalcemic coma.Ⅶ
Information is provided on the sensitivity and specificity of test results in regard to the disease or disorder under consideration. Take thyroid function tests for the diagnosis of hyperthyroidism, for example. A serum thyroxine (T4),a triiodothyronine resin uptake (T3RU),and a calculated free thyroxine index (FT4I) are only about 95% sensitive for hyperthyoidism, and if the T4 and FT4I are normal in the presence of clinical findings suggestive of hyperthyroidism, a serum triiodothyronine (T3) or the new highly sensitive immunoradiometric assay for thyrotropin, the S-TSH, is indicated to confirm the diagnosis. On the other hand, a serum T4 or FT4I is even less sensitive to detect hypothyroidism, and if the T4 and FT4I are normal in the face of clinical features of hypothyroedism,a serum TSH or S-TSH is indicated to confirm the diagnosis.
If a test result does not make sense or is indeterminate, a useful tactic is to repeat it. For example, when testing for human immunodeficiency virus (HIV) infetion, if the enzyme-linked immunoassay (ELISA) is positive and the Western blot analysis is indeterminate, repeat the Western blot test monthly.
How to Monitor the Patient’s Disorder
Finally, if a diagnosis is confirmed and the patient is treated, which tests are useful to monitor the patient’s condition and how often these should be ordered. For example, in patients with acute hepatitis B who are antigen positive, it is appropriate to test for anti-hepatitis B antibody monthly, since development of the antibody will indicate when the patient is less infectious to others.
Sometimes, test results are available that are not really necessary for the diagnostic strategy but have been ordered as part of a profile or for some other reason. It is important to know whether or not these test results can occur in the condition under consideration and whether or not one has to hypothesize another disease process to explain them .If available, information on the pathophysiologic derangements responsible for these abnormal test results is also included here.
Instead of an alphabetical listing of tests and their reference range values, the abnormal test results are organized conceptually according to the way the results are usually grouped and considered. This same organization is used for common reference ranges located inside the front and back covers of this book..
It is hoped that a more scientific approach to medical decision making using laboratory tests will help assure the quality of patient care. This quality assurance through appropriate laboratory testing is not only good for patients but also is economical. Moreover, clinicians’ anxiety about medicolegal risk should be lessened.
INTRODUCING CLINICAL BIOCHEMISTRY
Clinical biochemistry, chemical pathology and clinical chemistry are all names for the subject, that branch of laboratory medicine in which chemical and biochemical methods are applied to the study of disease. While in theory this embraces all non-morphological studies, in practice it usually, though not exclusively, confined to studies on blood, urine and other body fluids because of the relative ease in obtaining such specimens. Clinical biochemical tests comprise over one-third of all hospital laboratory investigations.
THE USE OF BIOCHEMICAL TESTS
Biochemical investigations are involved, to varying degrees, in every branch of clinical medicine. The results of biochemical tests may be of use to the clinician in diagnosis and in the monitoring of treatment. Biochemical tests may also be of value in screening for disease or in assessing the prognosis once a diagnosis has been made. The biochemistry laboratory is often involved in research into the biochemical basis of disease and in clinical trials of new drugs.
CORE BIOCHEMISTRY
Biochemical facilities are provided in every hospital, although not necessarily to the same extent. Most biochemistry laboratories provide the “core analyses”, commonly requested tests which are of value in many patients, on a frequent basis. The clinician will often request specific groupings of tests, and clinical biochemistry assumes a cryptic language of its own as request forms arrive at laboratory reception for “U and Es” (urea and electrolytes), “LFTs” (liver function tests) or “blood gases”.
SPECIALIZED TESTS
There are a variety of specialties within clinical biochemistry. Not every laboratory is equipped to carry out all possible biochemistry requests. Large departments may act as reference centers where less commonly asked for tests are performed. For some tests which are needed in the diagnosis so rare diseases, there may be just one or two laboratories in the country offering the service.
THE EMERGENCY LAB
All clinical biochemistry laboratories provide facilities for urgent tests. Only a small number of test types are available from the “emergency laboratory”. These are processed rapidly and reports phoned to the requesting clinician or ward. An urgent test is designated as one on which the clinician is likely to take immediate action. An “on call” service may be provided to ensure that such requests can be done outside the normal working hours of the laboratory. Table 1 shows some of the tests available on an emergency basis.
IN THE OPERATING SUITE OR CLINIC
In some large hospital, the facilities to perform biochemistry analyses are sited close to where they are needed, for example monitoring of patients undergoing major surgery, such as transplantation, or providing blood glucose results at the diabetic clinic. Increasingly, biochemical tests are being performed away from the laboratory .
AUTOMATION AND COMPUTERIZATION
Most laboratories are now computerized, and the use of bar-coding of specimens and automated methods of analysis allows a high degree of productivity and improves the quality of service. Links to computer terminals on wards and GP surgeries allow direct access to results by the requesting clinician.
TEST REPERTOIRE
There are over 400 different tests which may be carried out in clinical biochemistry laboratories. They vary from the very simple, such as the measurement of sodium, to the highly complex, such as DNA analysis, screening for drugs or differentiation of lipoprotein variants. Many high volume tests are done on large automated machines. Infrequently performed tests may be conveniently carried out by using commercially. Increasing workload and budgetary constraints mean that all laboratories continually reassess what is the most cost-effective way of providing the best service.
Dynamic tests require several specimens, timed in relation to a biochemical stimulus, such as a glucose load in the glucose tolerance test for the diagnosis of diabetes mellitus. Some tests provide a clear cut answer to a question; others are only a part of the diagnostic jigsaw.
LABORATORY PERSONNEL
The clinical biochemistry laboratory provides a consultative service. The laboratory has on its staff both medical and scientific personnel who are familiar with the clinical significance and the analytical performance of the test procedures, and they will readily give advice on the interpretation of the results. Do not be hesitant to take advantage of this, especially where a case is not straight forward.
The clinical biochemistry laboratory plays only a part in the overall assessment and management of the patient. For some patients, biochemical analyses may have little or no part in their diagnosis or the management of their illness. For others, many tests may be needed before a diagnosis is made, and repeated analyses required to monitor treatment over a long period.
Every biochemistry analysis should provide the answer to a question which the clinician has posed about the patient. Obtaining the correct answers can often seem to be fraught with difficulty.
SPECIMEN COLLECTION
In order to carry out biochemical analyses, it is necessary that the laboratory be provided with both the correct specimen for the requested test, and also information which will ensure that the right test is carried out and the result returned to the requesting clinician with the minimum of delay. As much information as possible should be included on the request form to help both laboratory staff and the clinician in the interpretation of results. Patient identification must be correct, and the request form should include some indication of the suspected pathology. The requested analyses should be clearly indicated. Request forms differ in design. Clinical biochemistry forms in Europe are conventionally colored green.
Blood specimens
If blood is collected into a plain tube and allowed to clot, after centrifugation a serum specimen is obtained. For many biochemical analyses this will be the specimen recommended. In other cases, especially when the analyte in question is unstable and speed is necessary to obtain a specimen which can be frozen quickly, the blood is collected into a tube containing an anticoagulant such as heparin. When centrifuged, the supernatant is called plasma which is almost identical to the cell-free fraction of blood but contains the anticoagulant as well.
Urine specimens
Urine specimen containers may include a preservative to inhibit bacterial growth, or acid to stabilize certain metabolites. They need to be large enough to hold a full 24 h collection. Random urine samples are collected into small “universal” containers.
Other specimen types
For some tests, specific body fluids or tissue may be required. There will be specific protocols for the handling and transport of these samples to the laboratory. Consult the local lab for advice.
All specimens from patients with dangerous infections should be labeled with a yellow “dangerous specimen” sticker. A similar label should be attached to the request form. Of most concern to the laboratory staff are hepatitis B and HIV, but all specimens should always be treated both be clinicians and biochemists as potentially hazardous.
SAMPLING ERRORS
There are a number of potential errors which may contribute to the success or failure of the laboratory to provide the correct answers to the clinician’s questions. Some of these problems arise when the clinician first obtains specimens from the patient.
l Blood sampling technique. Difficulty in obtaining a blood specimen may lead to haemolysis with consequent release of potassium and other red cell constituents. Results for these will be falsely elevated.
l Prolonged stasis during venepuncture. Plasma water diffuses into the interstitial space and the serum or plasma obtained will be concentrated. Proteins and protein-bound components of plasma such as calcium or thyroxine will be falsely elevated.
l Insufficient specimen. Each biochemical analysis requires a certain volume of specimen to enable the test to be carried out. It may prove to be impossible for the laboratory to measure everything requested on a small volume specimen.
l Errors in timing. The biggest error in the measurement of any analyte in a 24-hour urine specimen is collecting an accurately timed volume of urine.
l Incorrect specimen container. For many analyses the blood must be collected into a container with anticoagulant and preservative. For example, samples for glucose should be collected into a special container containing fluoride which inhibits glycolysis; otherwise the time taken to deliver the sample to the laboratory can affect the result. If a sample is collected into the wrong container, it should never be decanted into another type of tube. For example, blood which has been exposed even briefly to EDTA (an anticoagulant used in sample containers for lipids) will have a markedly reduced calcium concentration, approaching zero.
l Inappropriate sampling site. Blood samples should not be taken “downstream” from an intravenous drip. It is not unheard of for the laboratory to receive a blood glucose request on a specimen taken from the same arm into which a dextrose drip is connected. Usually the results are biochemical incredible but it is just possible that they may be acted upon, with disastrous consequences for the patient.
l Incorrect specimen storage. A blood sample stored overnight before being sent to the laboratory will show falsely high potassium, phosphate and red cell enzymes such as laceate dehydrogenase, because of leakage into the extra cellular fluid from the cells.
HOW OFTEN TO INVESTIGATE
Many biochemical tests are repeated at intervals. How often depends on how quickly significant changes are liable to occur, and there is little point in requesting tests if a numerical change will not have an influence on treatment.
URGENT REQUESTS
The main reason for asking for an analysis to be performed on an urgent basis is that immediate treatment depends on the result.
ANALYSING THE SPECIMEN
Once the form and specimen arrive at the laboratory reception, they are matched with a unique identifying number or bar code. The average lab receives many thousands of requests and samples each day and it is important that all are clearly identified and never mixed up. All analytical procedures are quality controlled and the laboratory strives for reliability.
Once the results are available they are collated and a report is issued. Cumulative reports allow the clinician to see at a glance how the most recent result(s) compare with those tests performed previously, providing an aid to the monitoring of treatment.
UNNECESSARY TESTING
There can be no definite rules about the appropriateness, or otherwise, of laboratory testing because of the huge variety of clinical circumstances which may arise. Clinician should always bear in mind that in requesting a biochemical test they should be asking a question of the laboratory. If not, both the clinician and the laboratory may be performing unnecessary work, with little benefit to the patient.
THE LABORATORY REPORT
It can take considerable effort, and expense, to produce what may seem to be just numbers on pieces of paper. Understanding what these numbers mean is of crucial importance if the correct diagnosis is to be made, or if the patient’s treatment is to be changed.
HOW BIOCHEMICAL RESULTS ARE EXPRESSED
Most biochemical analyses are quantitative, although simple qualitative or semiquantitative tests such as those for the presence of glucose in urine are commonly encountered in methods used for biochemistry testing away from the laboratory. Many tests measure the amount of the analyte in a small volume of the sample, whether that is blood, plasma, serum, urine or some other fluid or tissue. The test results are commonly expressed in molar units. A mole of any compound always contains 6*1023 molecules. Describing how much of an analyte is present in “moles” indicates how many molecules of the substance are present. Molar units can be converted to mass units; one mole is the molecular weight of the substance in grams.
Results are reported as concentrations, usually in terms of the number of moles in one liter (mol/l). The concentration of any analyte in a body compartment is a ratio: the amount of the substance dissolved in a known volume. Changes in concentration can occur for two reasons:
l The amount of the analyte can increase or decrease.
l The volume of fluid in which the analyte is dissolved can similarly change.
Enzymes are not usually expressed in moles but as enzyme activity in “units”. Enzyme assays are carried out in such a way that the activity measured is directly proportional to the amount of enzyme present. Some hormone measurements are expressed by comparison to standard reference preparations, also as “units”. Large molecules such as proteins are reported as grams or milligrams. Blood gas results (PCO2 or PO2) are expressed in kilopascals (kPa), the units in which partial pressures are measured.
Biochemical measurements vary for two reasons. There is analytical variation, and also biological variation.
Laboratory analytical performance
A number of terms describe biochemical results. These include:
l precision and accuracy
l sensitivity and specificity
l quality assurance
l reference ranges
Precision and accuracy
Precision is the reproducibility of an analytical method. Accuracy defines how close the measured value is to the actual value. A good analogy is that of the shooting target. The scatter of results which might be obtained by someone with little skill, compared with that of someone with good precision where the results are closely grouped together. Even when the results are all close, they may not hit the centre of the target. Accuracy is therefore poor, as if the “sights” are off. It is the objective in every biochemical method to have good precision and accuracy.
Sensitivity and specificity
Sensitivity of an assay is a measure of how little of the analyte the method can detect. As new methods are developed they may offer improved detection limits which may help in the discrimination between normal results and those in patients with the suspected disease. Specificity of an assay relates to how good the assay is at discriminating between the requested analyte and potentially interfering substances.
Quality assurance
Every laboratory takes great pains to ensure that the methods in use continue to produce reliable results. Laboratory staff monitor performance of assays using quality control samples to give reassurance that the method is performing satisfactorily with the patients’ specimens. These are internal quality controls which are analyzed every day or every time an assay is run. The expected values are known and the actual results obtained are compared with previous values to monitor performance. In external quality assurance schemes, identical samples are distributed to laboratories; results are then compared. In this way, the laboratory’s own internal standards are themselves assessed.
Reference ranges
Analytical variation is generally less than that from biological variables. Biochemical test results are usually compared to a reference range considered to represent the normal healthy state. Most reference ranges are chosen arbitrarily in include 95% of the values found in healthy volunteers, and hence, by definition, 5% of the population will have a result out with the reference ranges. In practice there are no rigid limits demarcating the diseased population from the healthy; however, the further a result is from the limits of the range, the more likely it is to represent pathology. In some situations it is useful to define ‘action limits’, where appropriate intervention should be made in response to a biochemical result.
There is often a degree of overlap between the disease state and the ‘normal value’. A patient with an abnormal is a false positive. A patient who has the disease but has a ‘normal’ result is a false negative.
Biological factors affecting the interpretation of results
The discrimination between normal and abnormal results is affected by various physiological factors which must be considered when interpreting any given result. These include:
l Sex of the patient. Reference ranges for some analytes are different for men and women.
l Age of the patient. There may be different reference ranges for neonates, children, adults and the elderly.
l Effect of diet. The sample may be inappropriate if taken when the patient is fasting or after a meal.
l Time when sample was taken. These may be variations during the day and night.
l Stress and anxiety. These may affect the analyte of interest.
l Posture of the patient. Redistribution of fluid may affect the result.
l Effects of exercise. Strenuous exercise can release enzymes from tissues.
l Medical history. Infection and/or tissue injury can affect biochemical values independently of the disease process being investigated.
l Pregnancy. This alters some reference ranges.
l Menstrual cycle. Hormone measurements will vary through the menstrual cycle.
l Drug history. Drugs may have specific effects on the plasma concentration of some analytes.
Other factors
When the numbers have been printed on the report form, they still have to be interpreted in the light of a host of variables. Analytical and biological variations have already been considered. Other factors relate to the patient. The clinician can refer to the patient or to the clinical notes, whereas the biochemist has only the information on the request form to consult. The cumulation of biochemistry results is often helpful in patient management.
The clinician may well ask the following questions on receiving a biochemistry report:
l Does the result fit in with what I expected on the basis of the clinical examination and history of the patient?
l If the result is not what I expected, can I explain the discrepancy?
l How can the result change my diagnosis or the way I am managing the patient?
l What should I do next?
What is done in response to a biochemistry report rests with the clinical judgment of the doctor. There is a maxim that doctors should always “treat the patient, rather than the laboratory report”.
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