How do pathologists help patient care?

Diagnostic tests

Diagnostic tests are those that are made on a sample from a patient, the result allocating the case to a diagnostic grouping; ideally the results will help to classify patients into two groups — presence or absence of a disease. An example would be the histopathology examination of a biopsy of a breast lesion leading to definite classification into a benign or malignant (i.e. cancer) category. Some quantitative measurements, such as haemoglobin (Hb) concentration or arterial blood oxygen tension, may be used in the clinician's diagnostic process but they do not by themselves assign a patient to a diagnostic category.

A diagnostic test may be based on:

• quantitative measurement , such as the level of beta-human chorionic gonadotrophin in blood for the diagnosis of trophoblastic disease

• subjective assessment , based on expert knowledge such as a histopathologist's assessment of a biopsy of the breast.

The ideal diagnostic test would produce complete separation between two diagnostic categories; usually, however, there is some overlap. This problem can be illustrated by taking as an example a screening test for colorectal carcinoma which makes measurements on a sample of faeces (such as blood contained in the faeces). An ideal diagnostic test would produce complete separation of patients with and without colorectal carcinoma ( Fig. 12.1 ). The majority of real diagnostic tests do not provide complete separation between diagnostic categories and there is overlap ( Fig. 12.2 ). The effectiveness of a diagnostic test can be expressed by several statistical parameters such as sensitivity (ability to detect a disease), specificity (ability to separate those with disease from those without), predictive value of a positive result and predictive value of a negative result. The statistical references at the end of this chapter provide more detailed information on these.

Quantitative measurements

Many tests in pathology do not categorise results into discrete groups but give a quantitative result which is interpreted in relation to expected ranges in the overall patient population. Examples of such tests include measurement of Hb and electrolyte concentrations, blood oxygen and carbon dioxide levels.

The measures of performance for such tests differ from diagnostic tests. In quantitative tests the accuracy of the measurement (how close the measured value is to the ‘true’ value determined by an absolute method) and the reproducibility of the measurement (what variation there is when measuring the same sample many times) are important parameters. These can be assessed by using reference samples with ‘known’ values and putting these through the measurement system at regular intervals. Most laboratories will have their own reference samples (internal quality assurance), and graphs of single measurement and running mean values will be used to ensure that the test is performing within expected limits and not showing ‘drift’ away from the central expected value ( Fig. 12.3 ). Many countries also have external quality assurance schemes where reference samples are sent to all participating laboratories to ensure acceptable analytical performance.

When a laboratory gives a quantitative result for a parameter that is under physiological control, a reference range is often given to facilitate interpretation of the result. If a parameter shows normal (Gaussian) distribution in the local population, the ‘normal’ range is often given as two standard deviations below the mean to two standard deviations above the mean. If a value lies outside this range then it lies outside 95% of the results for that population ( Fig. 12.4 ) and may be regarded as abnormal, but 2.5% of the healthy population will have values lying outside the range at either end. Thus all the details of the individual case must be considered, including other measurements, as a number of results at the top end of the ‘normal’ range could be more significant than a single result just above the ‘normal’ range.

Prognostic tests

In many tumours, assignment to a diagnostic category (e.g. adenoma or carcinoma) gives an indication of the prognosis for the individual patient, but within such groupings (e.g. colorectal carcinoma) there may be wide variation in the biological behaviour of the tumour. To plan appropriate treatment and to be able to give useful information and counselling to individual patients, many prognostic pathological tests have been developed.

In tumour pathology one of the most predictive prognostic tests is staging of the tumour (extent of spread), which is always assessed in the histopathological examination of specimens. One of the best examples of this is Dukes' staging of colorectal carcinoma ( Ch. 15 ). The histological type of tumour has important prognostic implications, particularly in some organs; subjects with papillary thyroid carcinoma have a life expectancy that is the same as for the rest of the general population without the tumour, whereas subjects with anaplastic thyroid carcinoma have a median survival of a few months. The grade of the tumour, an assessment of its degree of differentiation and proliferative activity, also has predictive value; well-differentiated tumours (closely resembling parent tissue) with few mitoses have a better prognosis.

In tumours that produce substances that enter the blood or urine (e.g. alpha-fetoprotein produced by testicular teratomas) measurement of their levels at the time of diagnosis may be predictive of prognosis and can be used in follow-up. As more becomes known of the molecular abnormalities of tumours, the possibilities for specific molecular tests that will have prognostic value increase, but the translation of an apparently significant research result into a routinely used prognostic test is not straightforward. An example of the use of genomics in cancer management is a molecular diagnostic test that helps to determine the individual risk of recurrence in breast cancer patients. It analyses the specific biology of a breast cancer tumour by examining the activity of 21 genes in the tissue. The results of the analysis are fed into a formula that gives a “Recurrence Score” which provides information about how likely this particular breast cancer is to recur within 10 years of diagnosis helping to stratify patients into patients with minimal likelihood of benefit from adjuvant chemotherapy and patients with substantial likelihood of benefit from chemotherapy.

Another example is the detection of expression of the transmembrane receptor tyrosine kinase KIT (CD117), which is the product of the c-kit protooncogene in stromal tumours of the gastrointestinal tract. This can be detected by immunohistochemistry (IHC) ( Fig. 12.5 ), which, if positive, predicts that the patient's tumour will respond to treatment with a specific tyrosine kinase inhibitor, imatinib mesylate.

Clinical chemistry

Methods in clinical chemistry detect and measure subcellular substances in the blood or in bodily fluids and tissues. Molecules measured include electrolytes (sodium, potassium), lipids (cholesterol, triglycerides), large molecules (urea), proteins (including enzymes, hormones, antibodies) and exogenous molecules (e.g. carbon monoxide, therapeutic drugs). Since many of the tests in clinical chemistry are quantitative, the laboratory reports quote reference ranges. For many tests, the ranges appropriate for the age and sex of the patient and the critical values indicating the need for prompt clinical intervention (e.g. arterial partial pressure of oxygen or pO ₂ ) may be quoted.

As with all pathological tests, the clinician with direct responsibility for the patient must decide whether a particular test is an appropriate investigation and what sample is most appropriate for that test. These considerations are especially important in clinical chemistry where large automated machines can measure a wide range of substances on a single sample and, if not used selectively, may generate nonessential data which may be confusing or difficult to interpret and lead to unnecessary further investigations.

The type of sample and the circumstances in which it is taken are also important. It is outside the scope of this chapter to give specific recommendations for individual tests but examples of inappropriate samples would be blood taken for glucose analysis shortly after a large carbohydrate-rich meal, blood taken for electrolyte analysis from a vein in an arm receiving an intravenous infusion, and blood taken for a digoxin level immediately after a dose of the drug.

The interpretation of results also requires knowledge about the substances being assayed, and the advice of a specialist clinical chemist is often useful. An example of this is the use of cardiac enzymes measured to determine whether a myocardial infarct has occurred. The enzymes lactate dehydrogenase, aspartate transaminase and creatine kinase normally reside intracellularly in muscle cells; if muscle is damaged, they gain entry to the blood, and elevated levels may be detected. The interpretation of results requires knowledge about the time course of the enzyme release and the possible sources of these enzymes because different isoenzymes are present in cardiac muscle and in skeletal muscle. Fig. 12.6 shows typical curves of the enzymes in blood after a myocardial infarct; it can also be seen from this graph that total creatine kinase and aspartate transaminase reach their peaks earlier than lactate dehydrogenase. The interpretation of this pattern will require knowledge of enzyme properties and an estimate of when the ischaemic myocardial event is likely to have occurred in the patient. If the assay measures the total amount of enzymes, any damage to skeletal muscle would produce elevations. Thus if a patient had been found lying collapsed on the floor, measurement of the isoenzymes would be required to ascertain whether an ischaemic myocardial event had precipitated the collapse. Similar interpretative considerations apply to all tests in clinical chemistry.

Molecular genetics

Molecular genetics involving cytogenetics, with analysis of chromosomal abnormalities and molecular pathology looking at specific genetic changes to characterise diseases, is a dynamic discipline that has fundamentally changed the concepts of disease aetiology, disease classification and promising new therapy. In many situations, clinicians need to be as familiar with genetic testing as they are for other diagnostic methods used as essential adjuncts to diagnosis, for example, cystic fibrosis. Laboratory techniques may look at the number and form of chromosomes, the karyotype, or at more specific areas of DNA.

The karyotype can be examined using a sample of peripheral blood. Under special conditions, dividing T lymphocytes are arrested in metaphase after which the chromosomes will be visible with appropriate staining. The resulting alternating light and dark bands (G-banding) are viewed by light microscopy; the patterns of banding allow identification of each chromosome and visualisation of missing or additional material. A G-band usually represents several million to 10 million base pairs of DNA, a stretch long enough to contain hundreds of genes. Therefore cytogenetic method has a limited resolution; in practice, a karyotype is a good screening procedure, particularly to detect known cytogenetically visible chromosomal rearrangements (deletions, inversions, duplications, translocations). One of the classic chromosomal abnormalities associated with a malignancy is the Philadelphia chromosome in chronic myeloid leukaemia. This abnormality is a reciprocal translocation between chromosomes 9 and 22, resulting in the translocation of the abl oncogene to a breakpoint cluster region, which results in a chimeric gene producing a novel protein with properties playing a key role in the neoplastic transformation ( Ch. 23 ).

The karyotyping of chromosomes is a relatively coarse method of detecting genetic abnormalities. The techniques of in situ hybridisation (ISH) have helped to narrow the gap between cytogenetics and molecular genetics, where the resolution is at the level of individual genes. ISH is a powerful technique for localising specific nucleic acid targets within fixed tissues, which allows obtaining information about gene expression while analysing the histology of the sample. The technique uses DNA probes (single-stranded sequences of DNA) that bind only to those complementary DNA and messenger RNA (mRNA) target sequences in the cells. The DNA probes are labelled with radioisotopes, biotin or digoxigenin, to visualise the site of hybridisation using a colorimetric (CISH) or fluorescent (FISH) agent. The DNA in the tissue section is made into a single-stranded form, under special conditions to allow the probe to bind. This technique is useful for detecting infectious agents in tissue sections, such as cytomegalovirus (CMV) or Epstein–Barr virus. It can also be used to detect production (rather than simply storage) of proteins by detection of the mRNA for the specific protein. A common use of FISH is to detect amplification of the HER2 gene in breast cancers, which indicates that trastuzumab will be effective treatment against that tumour ( Fig. 12.7 ). Whole genome sequencing is now relatively affordable and could be used to detect genetic abnormalities in cancers but does pose problems in the management of huge volumes of data and the interpretation of genetic variants of uncertain significance.

Cytopathology

Cytopathology specimens are widely used for the diagnosis of malignancy ( Fig. 12.8 ). Since cells are dissociated from their surrounding tissue, some key malignant features used in histopathological diagnosis, such as invasion, are not available for assessment. The main features used in cytopathological diagnosis are:

• variation in size of nuclei (nuclear pleomorphism)

• changes in chromatin quality (nuclear hyperchromatism)

• the ratio of nuclear area to cytoplasmic area (by subjective assessment).

Cells may be collected for cytological examination from epithelium shed or scraped from a body surface (exfoliative cytology) or by aspirating cells through a fine-bore needle into a syringe (aspiration cytology). The cells are either smeared on glass slides at the time the sample is taken or by centrifugation methods in the laboratory. The slides are stained — the most frequently used method is the Papanicolaou (Pap) technique — and examined by light microscopy. Many cytopathological specimens are taken to assess dysplasia or malignancy in tissues but infective pathologies may also be diagnosed; for example, Pneumocystis jiroveci pneumonia in immunosuppressed patients may be detected by cytological examination of alveolar washings.

Haematology

Laboratory haematology encompasses all blood tests, and provides both quantitative and qualitative characteristics of analysed blood; the pathology of these is described in Chapter 23 . The work of haematologists is usually divided into three areas:

• diagnosis of haematological disorders

• management of haematological disorders

• blood transfusion.

The diagnosis of haematological disorders is based on clinical history and examination, measurement of parameters in the blood, and microscopic examination of blood films, bone marrow aspirates and trephine samples. Automated machines measure multiple parameters in samples of blood; the most common are:

• Hb concentration

• red cell count

• haematocrit

• red cell indices (MCV, mean corpuscular volume; MCHC, mean corpuscular Hb concentration)

• white cell count, differential count (WCC, DIFF)

• platelet count and thrombocyte indices (MPV, mean platelet volume)

• coagulation screening

• fibrinogen concentration.

Analytical machines can produce a plethora of data and the same problems of interpretation may occur as described in the earlier section on clinical chemistry.

Examination of a blood smear gives information about the number and shape of blood cells as an integral part of a haemogram. It allows quantitation of the different types of leukocytes (DIFF), estimation of the platelet count, and detection of morphologic abnormalities which may reflect physiological processes or diseases. Blood film can reveal abnormalities of red blood cell shape and size (e.g. anisocytosis, poikilocytosis, macrocytosis; see Ch. 23 ) and abnormal white blood cells such as blast cells in leukaemia. Some features, such as rouleaux formation by red blood cells, may suggest abnormalities in the noncellular components of blood (in this case, possible overproduction of antibodies or immunoglobulin).

An essential tool for the diagnosis and follow-up of haematological neoplasms is flow cytometry, used for immunophenotyping. It is based on the identification and counting of single cells labelled with monoclonal antibodies to specific surface or intracellular antigens associated with cell lineage (T lymphocytes/B lymphocytes), cell function (presence of receptors, cytokines) and degree of maturation (i.e. pre-B cells/mature B cells).