The main properties of the radioisotopes useful in diagnostic haematology are shown in Table 17.1. The units used to express radioactivity and the effects of radiation on the body are given in the previous edition. Anyone handling radioisotopes must be aware of the potential radiation hazard. It is also important to be aware of the potential biohazard of handling blood products and administering them to patients (see Chapter 24).

Table 17.1 Radioisotopes used for diagnostic investigations in haematology

Sources of radioisotopes

Radioisotopes that emit γ-rays are particularly useful because they have the advantage of emissions that penetrate tissues well, so they can be detected at the surface of the body when they have originated within organs. The radioisotope should have as short a half-life (T_½) as is compatible with the duration of the test. A radioisotope with a very short half-life can be administered in much higher amounts than those that are likely to remain active in the body for a considerably longer time.

The longer-lived radioisotopes that are used for haematological investigations are generally available from commercial suppliers. The usual way of obtaining certain short-lived radioisotopes is by means of a radioisotope generator, in which a moderately long-lived parent radioisotope decays to produce the required short-lived isotope. In this way ^99mTc (T_½ = 6 h) can be derived from ⁹⁹Mo (T_½ = 66 h).

Radiation protection

The quantity of radioactivity used in diagnostic work is usually small and good laboratory practice is all that is necessary for safe working. However, before using radioisotopes, workers should be familiar with the regulations concerning radiation protection for themselves, their fellow workers and patients.⁴

The effect of radiation on the body depends on the amount of energy deposited and is expressed in grays (Gy). The unit that describes the overall effect of radiation on the body, or the ‘effective dose,’ is measured in sieverts (Sv) or millisieverts (mSv). The annual whole-body dose limit for somebody working with radioisotopes is in the order of 20 mSv, whereas 1 mSv is the annual limit for the general public. To put this into perspective, 1 mSv is produced by normal background radiation in about 6 months and the radiation dose from a single chest X-ray is 0.02 mSv.⁵ No statutory limit of total annual radiation dose has been set for patients, but it is an important requirement that radioisotopes should be handled only in approved laboratories under the direction of a trained person who holds a certificate from the appropriate authority specifying the radioisotopes that the individual is authorized to use and the dose limits that must not be exceeded. In the UK, this authority is the Administration of Radioactive Substances Advisory Committee (ARSAC).⁵ Radioisotopes should not be given to pregnant women unless the investigation is considered imperative; if an investigation is necessary during lactation, breast-feeding should be discontinued until radioactivity is no longer detectable in the milk. When radioisotope investigations are necessary in children, the dose relative to that for an adult should be based on body weight (Table 17.2).

Table 17.2 Radioisotope doses for children as a decimal fraction of the adult dose

Weight (kg)	Fraction of adult dose
10	0.3
15	0.4
20	0.5
30	0.6
40	0.75
50	0.9
60	0.95
70	1.0

The laboratory (premises) using radioisotopes should be registered to store, handle and dispose of radioactive materials, and appropriate permits are obtained under the Environmental Permitting Regulations 2010.^5a

In general, the radioactive waste from radioisotopes used in haematological diagnostic procedures may be poured down a single designated laboratory sink. It should be washed down with a large quantity of running water. If the waste material exceeds the amount allowed for disposal in this way, it should be stored in a suitable place until its radioactivity has decayed sufficiently for it to be disposed of via the refuse system. All working and storage areas and disposal sinks should be clearly labelled with the internationally recognized trefoil symbol. Records should be kept of the amount of radioactive waste disposed down the drains and this should not exceed the permitted amount on the premises’ registration.

Decontamination of working surfaces, walls and floors can usually be achieved by washing with a detergent such as Decon 90 (Decon Laboratories, Ltd). Glassware can be decontaminated by soaking in Decon 90 and plastic laboratory ware can be decontaminated by washing in dilute (e.g. 1%) nitric acid.

Protective gloves must always be worn when handling radioisotopes; any activity that does get on the hands can usually be removed by washing with soap and water or, if that fails, with a detergent solution. For each laboratory in which isotopes are used, a radiation protection supervisor (RPS) should be nominated to supervise protection procedures and to ensure that a careful record is kept of all administered radioisotopes. This RPS should work in association with the departmental safety officer (see p. 579) and must ensure that all personnel working with radioactive materials wear dosimetry badges (available from an approved dosimetry service provider, e.g. Landauer, Oxford; ISO Pharma, Norway), which must be checked at regular intervals.

Apparatus for Measuring Radioactivity in Vitro

The radioisotopes used for most haematological tests are measured in a scintillation counter with thallium-activated sodium iodide crystals. These are available in various shapes and sizes. A ‘well-type’ crystal contains a cavity into which is inserted a small container or test tube holding up to 5 ml of fluid. Because the sample is almost surrounded by the crystal, counting is achieved with high efficiency. Because the geometric efficiency of a well-type counter depends on the position of the sample in relation to the crystal, it is important to use the same volume for each sample in a series. Another form of crystal detector is a solid circular cylinder, 2.5–10 cm in diameter. In this form, it is used for in vivo measurements and occasionally for the measurement of bulky samples (e.g. samples of faeces or 24-h urine specimens), thus avoiding the need to concentrate them to a smaller volume.

An alternative method for measuring bulky material is by using two opposed detectors in a single counting system. The sample is placed in a 450 ml waxed cardboard carton with a screw-top lid and is positioned between two counters placed above and below it with a plastic ring over the lower counter to ensure that the specimen in the carton is approximately equidistant from both crystals. The counting system is surrounded by lead and the responses of both crystals are counted together. If a single detector system is used, it is essential to homogenize the samples.

Apparatus for Measuring Radioactivity in Vivo

Surface Counting

Surface counting depends on shielding the crystals by means of a lead collimator to exclude as far as possible the radiation from outside a well-defined area of the body. It is thus possible to measure the radioactivity in individual organs such as the spleen and liver.

Imaging

The most widely used method for imaging is by the scintillation camera (gamma camera). It consists of a lead shielding, a large thin sodium iodide detector, an array of photomultiplier tubes, a collimator with multiple parallel holes and a system for pulse height analysis and for storage and display of the data. By scanning down the body, an image of the distribution of the label is built up and recorded. It can also be used to measure the quantity of the isotope in various organs. By rotating the scintillation camera around the body, single-photon emission computed tomography (SPECT) can be performed to produce sectional images. Positron emission tomography (PET) has augmented scintillation scanning and uses radioisotopes that are positron emitters.^6,⁷

Measurement of Radioactivity with a Scintillation Counter

Standardization of Working Conditions

For each radioisotope, it is necessary to plot a spectrum of pulse height distribution and to identify a window corresponding to the energy at which the maximum number of pulses is emitted. Examples of spectra and selected settings are illustrated in Figure 17.1. The setting of the apparatus, once determined, should remain constant for many months.

Figure 17.1 Spectra of radioisotopes obtained on a scintillation spectrometer. (A) ⁵¹Cr, (B) ^99mTc, (C) ¹²⁵I and (D) ⁵⁹Fe. The radionuclides should be counted with the window set within the limits indicated by the vertical lines.

Counting Technique

Blood volume

The haemoglobin concentration (Hb), red cell count and packed cell volume or haematocrit (PCV/Hct) do not invariably reflect the total red cell volume (RCV). Whereas in most cases for practical purposes, there is adequate correlation between peripheral blood values and (total) RCV,⁸ there will be a discrepancy if the plasma volume is reduced or increased disproportionately. Fluctuation in plasma volume may result in haemodilution, giving rise to pseudoanaemia, or conversely, haemoconcentration, giving rise to pseudopolycythaemia.

An increase in plasma volume occurs in pregnancy, returning to normal soon after delivery. Increased plasma volume may also be found in patients with cirrhosis, nephritis and congestive cardiac failure and when there is marked splenomegaly. Reduced plasma volume occurs with oedema, with dehydration, following the administration of diuretic drugs, in smokers and sometimes as a persistent unexplained phenomenon. It also occurs during prolonged bed rest.

In contrast to the fluctuations in plasma volume, RCV does not fluctuate to any extent if erythropoiesis is in a steady state.

Measurement of blood volume should thus be considered whenever the Hct is persistently higher than normal; demonstration of an absolute increase in RCV is necessary to diagnose polycythaemia and to assess its severity. However, it should be noted that the discovery of a recurring JAK2 mutation (JAK2 V617F) in the great majority of patients with polycythaemia vera means that blood volume studies are now rarely needed for the confirmation of this diagnosis. The component parts of the TBV (i.e. red cell and plasma volume) can also be measured separately in the elucidation of obscure anaemias when the possibility of an increase in plasma volume cannot be excluded.

Measurement of Blood Volume

Principle

The principle is that of dilution analysis. A small volume of a readily identifiable radioisotope is injected intravenously, either bound to the red cells or to a plasma component, and its dilution is measured after time has been allowed for the injected material to become thoroughly mixed in the circulation but before significant quantities have left the circulation or become unbound. The most practical method now available is to use a small volume of the patient’s red cells labelled with radioactive chromium (⁵¹Cr), technetium (pertechnetate) (^99mTc) or indium (¹¹¹In). The labelled red cells are diluted in the whole blood of the patient and from their dilution the TBV can be calculated; the RCV, too, can be deduced from knowledge of the PCV. The plasma volume can be measured directly by injecting human albumin labelled with radioactive iodine (¹²⁵I) that is diluted in the plasma compartment.

In contrast to measurement of RCV, plasma volume measurements are only approximations because the labelled albumin undergoes continuous slow interchange between the plasma and extravascular fluids, even during the mixing period. For this reason, it is undesirable to attempt to calculate RCV from plasma volume on the basis of the observed PCV. However, because the RCV is generally more stable, calculation of TBV from RCV is usually more reliable, provided that the difference between whole-body and venous PCV is appreciated and allowed for (see p. 379). Measurement of red cell and plasma volumes separately by direct methods is to be preferred.

Red Cell Volume

Radioactive Chromium Method

For the radioactive chromium method,⁹ add approximately 10 ml of blood to 1.5 ml of sterile National Institutes of Health (NIH)-A acid–citrate–dextrose (ACD) solution (see p. 619) in a sterile bottle with a screw cap. Centrifuge at 1200–1500 g for 5 min. Discard the supernatant plasma and buffy coat and slowly, with continuous mixing, add to the cells 8 × 10³ Bq of Na₂⁵¹CrO₄ per kg of body weight. The sodium chromate should be in a volume of at least 0.2 ml, being diluted in 9 g/l NaCl (saline). Allow the blood to stand for 15 min at 37°C for labelling to take place. Wash the red cells twice in 4–5 volumes of sterile saline: for all procedures requiring sterile saline, this should be 9 g/l (0.9%) sodium chloride BP (non-pyrogenic); 12 g/l NaCl should be used when red cell osmotic fragility is greatly increased (e.g. in cases of hereditary spherocytosis).

Finally, resuspend the cells in a volume of sterile saline sufficient for an injection of about 5 ml and the preparation of a standard. Take up the appropriate volume into a syringe that is weighed before and after the injection. The volume injected is calculated from the following formula: