Laboratory haematology I – Blood and bone marrow

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Laboratory haematology I – Blood and bone marrow

Diagnosis of most blood disorders is possible from a combination of clinical history, clinical examination and relatively routine laboratory tests. Haematology laboratories are heavily dependent on complex electronic machinery. The ubiquitous full blood count (FBC) is the archetypal haematological investigation and is performed by specialised automated cell counters. However, despite the accessibility of modern technology, the more simple traditional techniques of blood and bone marrow film spreading, staining, and light microscopy remain essential parts of the haematologist’s repertoire.

The blood count

Many of the diseases discussed in this book are first suggested by an abnormality in the blood count (often referred to as the full blood count). The test is performed on a small specimen of anticoagulated venous blood; the normal anticoagulant is ethylene diamine tetra-acetic acid (EDTA). A typical report is illustrated in Figure 9.1. As can be seen, it contains a large amount of numerical information pertaining to the three cell lines in the peripheral blood: red cells (and haemoglobin), white cells (with a differential count of each specific cell type) and platelets.

When interpreting the report it is sensible to initially focus on the haemoglobin (Hb) concentration, total white cell count (WBC) and platelet count – most blood abnormalities of clinical significance are associated with a derangement of at least one of these values. Much of the remaining information details the nature of the red cells and their degree of haemoglobinisation, and the precise make-up of the white cell count. The former values are helpful in the diagnosis of anaemia, and the latter in the diagnosis of a variety of diseases of white cells (e.g. leukaemias) and reactions to systemic disease. To understand the role of the automated blood count in clinical practice, and particularly its limitations, it is helpful to understand how the numerical values are generated.

Automated haematology counters

The two essential functions of the automated blood cell counter are the measurement of Hb concentration in the blood and the counting and sizing of blood cells.

Most counters use a modification of the traditional cyanmethaemoglobin method to measure Hb concentration. In essence, blood is diluted in a solution where Hb is converted to cyanmethaemoglobin and then the Hb concentration derived from the light absorbance (optical density) of the resultant solution measured by a spectrophotometer. Automated machines have at least two channels for cell counting. In one, red cells and platelets may be counted and in the other red cells are lysed leaving white cells for analysis. Extra channels are often used for differential white cell and reticulocyte counting.

There are two basic methods for cell counting and sizing: electrical impedance and light scattering. The electrical impedance method relies on blood cells being very poor conductors of electricity. Thus, when the cells are passed in a stream through a narrow aperture across which an electrical current is maintained, the individual cells create an increase in electrical impedance of a size proportional to the cell volume. In the light scattering method the cells deflect a beam of light (often a laser beam) and a detector converts the scatter into pulses proportional to cell size. For sophisticated measurements such as the differential white cell count the two methods can be used together with the addition of other modalities reliant on biochemical reactions and light absorbance.

Sophisticated though this technology is, automated cell counters are ultimately no substitute for the trained human eye. Results outside the machine’s numerical normal range or the presence of unusual circulating cells (e.g. leukaemic cells) should be flagged as being abnormal. This alerts the operator who will return to the original blood sample to make a film.

The blood film

A blood film is simply made by smearing a drop of anticoagulated venous blood onto a glass slide with a glass spreader (Fig 9.2a). In larger laboratories film spreading can be automated. Following drying, the film is fixed with methanol and stained. Routine stains are based on Romanowsky’s method – commonly used variants are the May–Grünwald–Giemsa (MGG) stain and Wright’s stain. Constituent dyes include methylene blue, azure B and eosin. Once stained, the blood film should be systematically studied under the light microscope – the normal appearance of a film stained by the MGG method is illustrated in Figure 9.2b. Alternative stains are sometimes needed. Visualisation of reticulocytes requires the use of a dye such as methylene blue on live unfixed cells (‘supravital stain’). Malarial parasites are most easily seen following staining at a specific pH.

The first step in film examination is a decision as to whether the film is of adequate quality. Either poor staining techniques or prolonged storage of the specimen may make the film worthless. Any comment on the film appearance is usually appended to the blood count report. The nomenclature used in film reporting can appear obscure; some more commonly used morphological terms are listed in Table 9.1. Microscopic images of blood cells are now routinely photographed using digital cameras. These images may increasingly be used to create ‘virtual slides’ or employed with cell recognition systems for automated morphological screening.

Table 9.1

Some morphological terms used in blood film reports

Red cells  
Hypochromia Pale staining of cells
Polychromasia Grey-blue tint to cells (usually reticulocytes)
Anisocytosis Variation in cell size
Poikilocytosis Variation in cell shape
Macrocytosis/microcytosis Increase/decrease in cell size
Spherocyte Small spherical densely stained cell
Burr cell Crimpled cell membrane
Target cell Increased staining in middle of area of central pallor – suggests increased surface area
Basophilic stippling Small basophilic inclusions in cytoplasm (RNA)
Howell–Jolly bodies Nuclear remnants in cytoplasm
Schistocyte Fragmented cell
White cells  
Hypersegmented neutrophils Increased nuclear segmentation
Left-shifted neutrophils Reduced nuclear segmentation
Toxic granulation Increased neutrophil cytoplasmic granularity
Atypical lymphocytes Morphology variable; often seen in viral infections
Blasts Leukaemic cells
Platelets  
Clumping Sticking together; can cause artefactually low count

Note: Causes of these morphological abnormalities are discussed in the disease sections.

Where the film is significantly abnormal, examination of the bone marrow can give further diagnostic information.

Bone marrow examination

The clinical procedure for obtaining samples of bone marrow is described on page 106. From the favoured site, the posterior iliac crest, it is possible to obtain both a marrow aspirate sample and a marrow trephine biopsy.

Aspirate

The aspirate is simply sucked through the needle and spread onto a glass slide; the marrow particles are normally easily visible (Fig 9.3a). The marrow is fixed and stained as for a blood film and additionally stained by Perl’s method to demonstrate iron. Microscopy and reporting is systematic with reference to the overall cellularity, the appearance and number of each normal cell line, possible infiltration by malignant cells, and any other pathological features. The advantage of the aspirate specimen is that individual cells are well preserved and subtle morphological changes can be detected. The major disadvantage is that the normal architecture of the marrow is lost. In the investigation of haematological malignancy (e.g. leukaemia) marrow aspirate samples are often also used for immunophenotyping and cytogenetic and molecular genetic testing.

Trephine biopsy

The trephine biopsy (Fig 9.4) is sectioned and normally stained by haematoxylin and eosin (H&E) and Giemsa methods. Silver impregnation can be used to demonstrate marrow fibrosis and Perl’s stain to highlight iron. The trephine is less good than the aspirate for identifying morphological abnormalities of individual cells but it is better for detecting abnormalities of marrow architecture and infiltration by solid malignancy. The two types of bone marrow sample are thus complementary.

Laboratory haematology I – blood and bone marrow

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