General Information

The main purpose of this Manual is to provide useful guidelines for the selection of pathology tests and to facilitate interpretation of results.

The Manual consists of several sections:

Clinical Problems

The clinical problems listing presents pathology tests in the context of the clinical problems which give rise to the need for diagnostic information. The tests listed are those likely to be useful, but consideration must always be given to the individual clinical situation. Where appropriate, the limitations of tests in providing definitive information are noted. Tests should never be ordered as a 'routine' or 'screen' an important principle is to request tests only when the results will contribute to diagnosis and/or management.

The clinical problems section is organised in two columns; a line is inserted at the end of each entry.

First column

In the first column, the main headings, or primary entries, are the 'clinical problems'.

Red dot points are provided for ease of reading only.

Subheadings indicate various categories, manifestations or complications.

Where the clinical problem has an alternative name, or acronym, the column one entry may provide a cross-reference or glossary link.

Second column

There may be a general comment or a definition of the problem, followed by a discussion or listing of appropriate tests.

Where comments or tests are appropriate only to the subsidiary entries, they appear across from those subsidiary entries.

If the nature of the specimen is not stated, the required specimen is blood, plasma or serum.

When the test requires other body fluids or tissue eg, urine, sputum, CSFCerebrospinal fluid or a biopsy, the nature of the required specimen is stated.

When different specimens or laboratories are involved in testing, the tests are separated by semi-colons.

If a test is not described in the test listing, the comments 'specialised laboratory' or 'consult pathologist' (for further information) are used.

If the test is described under a more general test description, a cross-reference to that test is provided.

If there is no entry in column two, no useful or additional pathology tests are available.

Pathology Tests

The pathology test listing includes most of the tests available to the clinician.

Tests which are performed infrequently or only in specialised laboratories are not described.

The entries are brief. Where further information is required consult the pathologist.

Test names reflect common usage.

Alternative names for tests are included as cross-references.

Other relevant test entries are provided as cross-references.

Each entry contains the following information:

• Specimen: Comments on the type and volume of the specimen, special requirements and precautions. The specimen volume specified is usually the ideal, rather than the minimum. If multiple tests are being performed on one specimen, the volumes are not additive. Paediatric samples can often be smaller than those generally specified.
• Method: A brief description of the methods used and/or the principles of the test.
• Reference interval: The 'normal range' for results is provided where this is possible. Many are dependent on analytical method, age, gender and other variables; these variables are described. A therapeutic interval is provided for drug assays.
• Application: The indications for performing the test and situations where useful information may be gained. For some tests, comment is made on situations where testing is inappropriate or an alternative test is of more value.
• Interpretation: A description of the significance of test results, factors which may influence the result, interpretation of results in specific clinical situations, comment on test sensitivity and specificity and predictive value, where possible.
• Reference: One or more books or journal articles which provide further information about the test and its clinical application.

General Information

This section includes:

• Specimen collection
• Validity and reliability
• Useful fact sheets for patients and requestors
• Interpretation guides
• Glossary

The Glossary from previous editions of the Manual has been included in the General Information section but has also been incorporated into the website. Hover your mouse over an abbreviation or acronym and the corresponding glossary entry will be shown in full.

Requests and collection

• The pathology laboratory provides and interprets analytical and morphological information to assist in the diagnosis of clinical problems and the monitoring of disease progress and treatment.
• The provision of a legible and appropriate clinical history on the request form, together with a properly collected specimen, allows the pathology laboratory to issue relevant and accurate results, and to assist the clinician in the interpretation of these results in the clinical context.
• The pathologist and/or laboratory staff should be consulted where uncertainty exists about the availability, appropriateness, or selection of tests, the nature of the specimen required, or interpretation of the results.

Anatomical pathology

• The correct handling of specimens submitted for morphological assessment is critical. Pathologists are often able to assist with appropriate collection and confirmation of adequacy of sample if contacted ahead of collection. When in doubt, consult the pathologist.
• Complete patient data and clinical information must be included on the request form.
• Gynaecological cytology can be fluid based or a conventional sample.
• For non-gynaecological or tissue cytology, aspirated fluid should be placed in cytology liquid transport media.
• If microbiological or immunological tests are required, send a separate sample.

Blood collection

Staff responsible for specimen collection will not collect from:

• Arms or hands receiving intravenous (IVIntravenous) fluids as IVIntravenous fluids can dilute the sample.
• Arms with working fistulas or grafts as venepuncture may cause damage to the fistula or graft, introduce infection or cause haemorrhaging.
• Badly bruised or thrombosed veins as venepuncture can cause a blood clot to dislodge causing an embolism.
• Recent catheter sites as this may cause haemorrhaging at the site.
• Arms or hands on the side of a mastectomy as the tourniquet pressure can cause lymphoedema.
• Inside of wrists as there are many nerve endings which may be damaged or cause pain. There is also a greater chance of hitting an artery or tendon when performing venepuncture in this area.
• Legs as complications may arise in diabetic patients.

Blood collection using evacuated tube system

1. Prepare venepuncture equipment and select tubes and position equipment next to patient.
2. Perform hand hygiene (moment 1 – before touching the patient).
3. Ensure patient is identified, request form and test conditions have been checked and the patient is positioned appropriately.
4. Apply tourniquet approximately 12 cm above the venepuncture site.
5. Palpate for vein position and direction.
6. Perform hand hygiene and don gloves (combined moment: moment 4 – after touching the patient and moment 2 – before a procedure).
7. Using a sterile alcohol swab, in one downward motion swab the area and allow to air dry.
8. Remove needle cover and using non-dominant hand, anchor vein to prevent movement. Ensure bevel is uppermost and keeping the needle at a 15° to 30° angle to the skin, insert the needle over and in-line with the vein. Ensure the needle remains steady by pressing the hand holding the needle holder firmly against the skin.
9. Using the other hand push the collection tube onto the needle using a ‘hypodermic’ action and using the wings as support.
10. Tubes must be collected in the correct order
11. Once the tube is ‘fully drawn’, remove from the needle using the holder wings as support.
12. Invert tubes at least six times to ensure mixing of contents of tube with blood.
13. Repeat steps 10-13 until all tubes have been collected.
14. Release tourniquet and place clean dressing over site. In one smooth and safe motion remove the needle. Apply pressure to the dressing once the needle has left the skin.
15. Immediately dispose of needle and needle holder directly into a sharps container.
16. Label each tube as labelling procedure describes
17. Date, time, sign and the request form, ensuring you have noted the number and types of tubes collected.
18. Show the patient all samples and request form and ask patient to identify that the information on all labels is correct.
19. Place all samples and request form into biohazard bag or into specimen rack.
20. Remove gloves and perform hand hygiene (combined moment: moment 3 – after a procedure or biohazard exposure risk and moment 1 – before touching a patient).
21. Check patient's venepuncture site for any sign of bleeding.  Apply clean swab and tape in place once bleeding has stopped.
22. Explain to the patient to monitor the site for a further 15 minutes and provide after care advice.
23. Perform hand hygiene (moment 4 – after touching a patient).
24. Transport specimens to testing laboratory.

NB: If at any time during the collection procedure you require additional equipment, please ensure you remove your gloves, perform hand hygiene and don a fresh set of gloves to adhere to infection control policies.

Blood collection using needle and syringe

1. Prepare venepuncture equipment and select tubes and position equipment next to patient.
2. Perform hand hygiene (moment 1 – before touching the patient).
3. Ensure patient is identified, request form and test conditions have been checked and the patient is positioned appropriately.
4. Apply tourniquet approximately 12 cm above the venepuncture site.
5. Palpate for vein position and direction.
6. Perform hand hygiene and don gloves (combined moment: moment 4 – after touching the patient and moment 2 – before a procedure).
7. Using a sterile alcohol swab, in one downward motion swab the area and allow to air dry. Meanwhile, attach needle to disposable syringe.
8. Remove needle cover and using non-dominant hand, anchor vein to prevent movement. Ensure bevel is uppermost and keeping the needle at a 15° to 30° angle to the skin, insert the needle over and in-line with the vein. Ensure the needle remains steady by pressing the hand holding the needle holder firmly against the skin.
9. Using the other gradually withdraw the plunger until the required amount of blood is obtained.
10. Release tourniquet and place clean dressing over site. In one smooth and safe motion remove the needle. Apply pressure to the dressing once the needle has left the skin.
11. Immediately dispose of needle directly into a sharps container.
12. Using a transfer device, and following the recommended order of draw, decant blood into evacuated tubes.
13. Invert tubes at least six times to ensure mixing of contents of tube with blood.
14. Label each tube as labelling procedure describes
15. Date, time, sign and the request form, ensuring you have noted the number and types of tubes collected.
16. Show the patient all samples and request form and ask patient to identify that the information on all labels is correct.
17. Place all samples and request form into biohazard bag or into specimen rack.
18. Remove gloves and perform hand hygiene (combined moment: moment 3 – after a procedure or biohazard exposure risk and moment 1 – before touching a patient).
19. Check patients’ venepuncture site for any sign of bleeding.  Apply clean swab and tape in place once bleeding has stopped.
20. Explain to the patient to monitor the site for a further 15 minutes and provide after care advice.
21. Perform hand hygiene (moment 4 – after touching a patient).
22. Transport specimens to testing laboratory.

NB: If at any time during the collection procedure you require additional equipment, please ensure you remove your gloves, perform hand hygiene and don a fresh set of gloves to adhere to infection control policies.

Variables affecting laboratory test results

Preanalytical variables include physiological factors, patient preparation, specimen collection and transport.

Analytical variables include the precision and accuracy of the test method and factors which may interfere with a particular assay eg, lipaemia, in vitro haemolysis, and medications.

Post-analytical variables include data entry and calculations by laboratory staff, result validation, interpretation of the result, data transfer and the method used to report the results (electronic, paper or telephone).

Diagnostic testing

Pathology tests guide clinical decision making and the clinician should have some understanding of the factors which influence the reliability of a diagnostic test for such decisions to be valid.

The clinician has an important part to play in the avoidance, or control, of many of the preanalytical variables.

The clinician also needs to have an understanding of the sensitivity and specificity of tests and of their predictive value.

Profile testing, or 'screening', is an expensive process which, even with tests of high sensitivity and specificity, has a limited positive predictive value (PPV), if the prevalence is low. Highly accurate test results may be entirely meaningless or misleading when used in this fashion.

False positive results lead to unnecessary and expensive follow-up testing and patient anxiety.

False negative results place the patient at risk.

Diagnostic tests must supplement rather than be used as a substitute for clinical skills.

Careful clinical assessment followed by discretionary testing is cost-effective, efficient and leads to improved patient outcomes.

Diagnostic accuracy studies

TP, true positive; FP, false positive; FN, false negative; TN, true negative.

Diagnostic sensitivity

Diagnostic sensitivity (‘positivity in disease’) of a test refers to the probability of obtaining a positive result for a subject with a given disease.

Diagnostic sensitivity = TP / (TP + FN)

High sensitivity corresponds to a high negative predictive value (NPV) and is the ideal property of a ‘rule out’ test.

Diagnostic specificity

Diagnostic specificity (‘negativity in health’) of a test is the probability of obtaining a negative result in a subject without a given disease.

Diagnostic specificity = TN / (TN + FP)

High specificity corresponds to a high PPV and is the ideal property of a ‘rule in’ test.

Receiver-operating characteristic curve

A receiver-operating characteristic (ROC) curve compares sensitivity versus specificity across a range of values to enable the best cut-off for clinical purpose to be assigned.

Predictive value

A major factor in improving the PPV of a test is to limit the use of the test to those patients who are likely to have the disease in question.

PPV is the proportion of positive results that are true positives, which represents the diagnostic value of a positive result for the test.

PPV = TP / (TP + FP)

NPV is the proportion of negative results that are true negatives.

NPV = TN / (TN + FN)

As the prevalence increases, PPV increases and NPV decreases.

Diagnostic efficiency

Diagnostic efficiency of the test refers to the probability of all tested subjects being correctly classified with or without a given disease.

Diagnostic efficiency = (TP + TN) / (TP + FP + TN + FN)

The diagnostic efficiency of a test can be markedly increased by using it in a discretionary fashion in high risk groups or in patients with clinical features suggesting the disease in question.

This improved diagnostic efficiency does not significantly reduce its NPV.

Predictive values and diagnostic efficiency are significantly influenced by FP and FN rates and by the prevalence of the disease in the population being tested.

Prevalence

Prevalence = (TP + FN) / (TP + FP + TN + FN)

Thus, for a test with a diagnostic sensitivity of 95% and a diagnostic specificity of 95%, with a population prevalence of the disease of 1%, the PPV is only 16.1%.

Likelihood ratio

Ultimately, the value of a diagnostic test will depend upon its ability to alter the pre-test probability of a disease into a post-test probability that will influence a clinical management decision, which can be achieved by the application of likelihood ratios (LR), but note that probability needs to be converted to odds.

LR+ = sensitivity / (1 – specificity)

LR– = (1 – sensitivity) / specificity)

A LR+ >10 and a LR– <0.1 are considered to exert highly significant changes in probability, such as to alter clinical management.

Reference

Florkowski CM. Sensitivity, specificity, receiver-operating-characteristic (ROC) curves and likelihood ratios: communicating the performance of diagnostic tests. Clin Biochem Rev 2008; 29: S83-7.