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:

The density of the suspension = 1.0 + Hb of suspension (g/l) × 0.097/340, assuming that packed red cells have a mean cell haemoglobin concentration (MCHC) of 340 g/l and a density of 1.097.

Inject the suspension intravenously without delay and note the time; at 10, 20 and 30 min later, collect 5–10 ml of the patient’s blood and add it to the appropriate amount of K₂ EDTA anticoagulant. This blood should preferably be drawn from a vein other than that used for the injection. However, it is often convenient to insert a self-retaining needle; in this case, care must be taken to ensure that the isotope is well-dispersed into the bloodstream when injected by flushing through with 10 ml of sterile saline. When the mixing time is likely to be prolonged, as in splenomegaly, cardiac failure or shock, another sample should be taken 60 min after the injection.

Measure the PCV of each sample. PCV should be obtained by microhaematocrit centrifugation for 5 min or for 10 min if the PCV is more than 0.50 and correcting for trapped plasma by deducting 2% from the measurement. A more accurate measurement of the PCV can be obtained by the International Council for Standardization in Haematology (ICSH) surrogate reference method (see p. 30).

Deliver 1 ml volumes into counting tubes and lyse with saponin; a convenient method is to add 2 drops of 2% saponin. Measure their radioactivity in a scintillation counter. Then dilute an aliquot of the original suspension that was not injected 1 in 500 in water (for use as a standard) and determine the radioactivity of a 1 ml volume. Then:

Technetium Method

^99mTc is available as sodium pertechnetate. This passes freely through the red cell membrane and will become attached to the cells only if it is present in a reduced form as it enters the cells when it binds firmly to β chains of haemoglobin. For this to occur, the red cells must be treated with a stannous (tin) compound by the following in vivo procedure.

Dissolve a vial of Stannous Reagent (stannous fluoride and sodium medronate [Amerscan, Amersham International]) in 6 ml of sterile saline and inject intravenously 0.03 ml/kg body weight.

After 15 min, collect 5 ml of blood into a sterile container to which has been added 200 iu of liquid heparin. Add 2 MBq of freshly generated ^99mTc in approximately 0.2 ml of saline or 100 MBq if measurement of splenic red cell pool and scanning are also required. Allow to stand at room temperature for 5 min. Centrifuge; wash twice in cold sterile saline and resuspend in a sufficient volume of cold sterile saline for an injection of 5–10 ml. Draw 5 ml into a syringe that is weighed before and after injection and carry out subsequent procedures as for the chromium method. Because of the short half-life of ^99mTc, radioactivity must be measured on the day of the test. Because 5–10% of the radioactivity is eluted from the red cells within an hour, the method is less suitable than the chromium and indium methods when delayed mixing is suspected (e.g. in splenomegaly).

Indium is available as ¹¹¹In chloride. The labelling procedure is simpler than with ^99mTc and, because there is less elution than with technetium during the first hour,¹⁰ it is particularly suitable for delayed sampling. For labelling blood cells, the indium is complexed with oxine¹¹ or tropolone.¹²

Calculating Total Blood Volume

The TBV can be calculated by multiplying the value for RCV by 1/(whole-body PCV) (see below). Plasma volume can be calculated by subtracting RCV from TBV.

If a sample has been taken at 60 min in cases in which delayed mixing is suspected and there is a significant difference between the measurements at 10–30 min and 60 min, then the 60 min measurement should be used for calculating the RCV.

Plasma Volume

¹²⁵l-Human Serum Albumin Method

Human serum albumin (HSA) labelled with ¹²⁵I or ¹³¹I is available commercially (from ISO Pharma, Norway; Mallinckrodt Medical (Nuclear Medicine Division), Northampton, UK). The albumin concentration should not be less than 20 g/l. The user must be reassured that only donors who are negative for human immunodeficiency virus (HIV) and hepatitis B and C have been used as the source of albumin. ¹²⁵I is readily distinguishable from ⁵¹Cr, ^99mTc and ¹¹¹In and this makes possible the simultaneous direct determination of RCV and plasma volume (see below). If further doses of the radioisotope are to be administered for repeat tests, it is advisable to block the thyroid by administering 30 mg of potassium iodide by mouth on the day before the test and daily for 2–3 weeks thereafter.

Withdraw approximately 20 ml of blood into a syringe containing a few drops of sterile heparin solution and transfer to a 30 ml sterile bottle with a screw cap. After centrifuging at 1200–1500 g for 5–10 min, transfer approximately 7 ml of plasma to a second sterile bottle and add 2.5 × 10³ Bq of the radionuclide-labelled HSA per kg body weight (approx. 0.2 MBq in total). Inject a measured amount (e.g. 5 ml) and retain the residue for preparation of a standard.

After 10, 20 and 30 min, withdraw blood samples from a vein other than that used for the original injection (or after flushing through with 10 ml of sterile 9 g/l NaCl [saline] if a butterfly needle has been used) and deliver into bottles containing EDTA or heparin.

Measure the PCV (see above), centrifuge the sample and separate the plasma. Prepare a standard by diluting part of the residue of the uninjected HSA 1 in 100 in saline.

Measure the radioactivity of the plasma samples in a scintillation counter and, by extrapolation on semilogarithmic graph paper, calculate the radioactivity of the plasma at zero time. If only a single sample is collected 10 min after the injection, the radioactivity at zero time may be approximated by multiplying by 1.015 to allow for early loss of the radioisotope from the circulation. Reliance on a single 10 min sample will lead to error if the mixing of the albumin in the plasma is delayed. After measuring the radioactivity of the standard, the plasma volume (ml) is calculated as follows:

Calculating Total Blood Volume

As has already been indicated, the TBV is frequently calculated from the RCV and PCV. Before this can be done, however, the observed PCV has to be corrected for the difference between the whole-body and venous PCV.

Whole-body and venous packed cell volume ratio

PCV measured on venous blood is not identical to the average PCV of all the blood in the body. This is mainly because the red cell:plasma ratio is less in small blood vessels (capillaries, arterioles and venules) than in large vessels. The ratio between the whole-body PCV and venous blood PCV is normally about 0.9,⁹ and it is thus necessary in the calculation of TBV from measurements of RCV to multiply the observed PCV by 0.9. Thus, TBV is given by the following:

However, the ratio varies in individuals, especially in splenomegaly and it is better to estimate RCV and plasma volume by separate measurements rather than to attempt to calculate one of these from an estimate of the other.

Simultaneous Measurement of Red Cell Volume and Plasma Volume

Collect blood and label the red cells by one of the methods described earlier. If ^99mTc is used, it is necessary first to inject stannous reagent (see p. 378). Then add ¹²⁵I HSA (see above) and mix it with the labelled red cell suspension. Inject an accurately measured amount and dilute the remainder 1 in 500 in water for use as a standard. Collect three blood samples at 10, 20 and 30 min, respectively, after the administration of the labelled blood and estimate the radioactivity of a measured volume of each sample and a similar volume of the standard.

When ^99mTc has been used in combination with ¹²⁵I, count on the same day; then leave for 2 days to allow the ^99mTc to decay and count again for ¹²⁵I activity. Because the radioactivity in the preparation from ¹²⁵I is much smaller than that from ^99mTc, the count from the red cells is not likely to be significantly affected by interference from ¹²⁵I in the initial count. However, if necessary, a correction can be made by subtracting the ¹²⁵I counts on day 2 (corrected for decay) from the original counts to obtain a measurement of the counts owing only to the ^99mTc.

When ⁵¹Cr has been used in combination with ¹²⁵I and a multichannel counter is available, measure the radioactivity owing to the ⁵¹Cr and ¹²⁵I at the appropriate settings for ⁵¹Cr and ¹²⁵I.

Calculate the radioactivity owing to the red cell label in the blood from the mean of the 10-, 20- and 30-min samples and obtain that owing to ¹²⁵I from the value extrapolated to zero time. Calculate RCV as described on p. 378.

Plasma volume is calculated from the formula:

Expression of Results of Blood Volume Estimations

RCV, plasma volume and TBV are usually expressed in ml/kg of body weight. Because fat is relatively avascular, low values are obtained in obese subjects and the relation between TBV and body weight varies according to body composition. Blood volume is more closely correlated with lean body mass (LBM).¹³ Earlier methods for determination of LBM were not practical as a routine procedure and discounting excess fat by using an estimate of so-called ‘ideal weight’ is arbitrary and tends to overcorrect for the avascularity of fat. The International Council for Standardization in Haematology (ICSH) developed two formulae, based on body surface area, which provide normal reference values in men and women, respectively.¹⁴ They are as follows.

Mean Normal Red Cell Mass (ml)

Men: [1486 × S] − 825; ±25% includes 98% limits

Women: [1.06 × age (years)] + [822 × S]; ± 25% includes 99% limits

Mean Normal Plasma Volume (ml)

Men: 1578 × S; ±25% includes 99% limits

Women: 1395 × S; ±25% includes 99% limits

where S = surface area (m²), W = weight (kg), H = height (cm).

However, the problem of establishing the LBM has been overcome to some extent because there are now instruments that are simple to use for estimating body composition by the different response of fat and other tissues to electrical impedance (body composition analyser, Holtain Ltd, Crosswell, Dyfed, Wales; body fat monitor, Tanita Corporation, IL, USA).^13,¹⁵

Thus, RCV can now be obtained by a direct measurement that discounts the effect of fat. The graph in Figure 17.2 shows the normalization of the RCV in ml/kg LBM.¹³ It is obtained as follows: on arithmetic graph paper with % fat on the horizontal (x) axis and RCV in ml/kg total body weight on the vertical (y) axis, plot the intercepts of the following:

Figure 17.2 Normalization of red cell volume in ml/kg lean body mass. This example shows a patient with 25% body fat; from the slope of the graph the normalized red cell volume for that person should be 28 ml/kg body weight. A measurement of more than 120% of this value (i.e. 33.6 ml) would indicate polycythaemia.

Fat 20% with RCV 29 ml; Fat 50% with RCV 19 ml

Join these two points and extend the line to the right and left.

When the % fat is known in any individual (male or female), draw a line vertically from this reading on the x axis to the slope and where this line intersects the slope draw a horizontal line to the y axis. The reading of this line on the y axis is the normalized RCV for that individual. When the measured RCV is >120% of this figure, it is equivalent to 43 ml/kg LBM and a diagnosis of polycythaemia can be made with confidence in men or women.

Range in Health

The TBV is 250–350 ml at birth. After infancy, the volume increases gradually until adult life when the RCV in men is 30 ± 5 ml (2SD)/kg and in women it is 25 ± 5 ml (2SD)/kg. Plasma volume (for men and women) is 40–50 ml/kg; TBV is 60–80 ml/kg.

As a rule, the TBV remains remarkably constant in an individual and rapid adjustments take place within a few hours after blood transfusion or intravenous infusion. In pregnancy, both the plasma volume and TBV increase. The plasma volume increases especially in the 1st trimester and the total volume increases later; by full term the plasma volume will have increased by about 40% and TBV will have increased by 32% or even more. The blood volume returns to normal within a week postpartum.¹⁶

Bed rest causes a reduction in plasma volume and muscular exercise and changes in posture cause transient fluctuations. In practice, the patient should always be allowed to rest in a recumbent position for 15 min before measuring the blood volume.

Splenic Red Cell Volume

The red cell content of the normal spleen (the red cell ‘pool’) is <5% of the total RCV (i.e. <100–120 ml in an adult). In splenomegaly, the pool is increased (e.g. by perhaps as much as 5–10 times in myelofibrosis, polycythaemia vera and hairy cell leukaemia and other lymphoproliferative disorders).¹⁷ An increase in the volume of the splenic red cell pool may itself be a cause of anaemia; measurement of the pool may be useful in investigating the anaemia in these conditions. It is also useful in determining the cause of erythrocytosis because the expanded pool in polycythaemia vera contrasts with that in secondary polycythaemia, which is normal.¹⁸

An approximate estimate of the splenic RCV can be obtained from the difference between the RCV calculated from the measurement of the blood sample that has been collected 2–3 min after the injection of labelled cells and that measured after mixing has been completed (i.e. after a delay of 20 min). The splenic RCV can be estimated more accurately by quantitative scanning, after injecting viable red cells labelled with ^99mTc.¹⁹ The blood volume is measured in the usual way using 100 MBq of ^99mTc. The splenic area is scanned 20 min after the injection or after 60 min when there is splenomegaly. To delineate the spleen more precisely, it may be necessary to carry out a second scan after an injection of heat-damaged labelled red cells (see p. 388). From the radioactivity in the spleen, relative to that in a standard, and knowledge of the total RCV, the proportion of the total RCV contained in the spleen can be calculated. This technique has also been used for demonstrating localized accumulation of blood in haemangiomas in the liver,²⁰ telangiectasia and other vascular abnormalities.²¹

Ferrokinetics

Whereas much can be learned about the rate and efficiency of erythropoiesis from the red cell count and reticulocyte counts, studies of iron metabolism and measurement of red cell lifespan with radioactive isotopes may provide useful additional information.

Radioactive iron (⁵⁹Fe) has a moderately short half-life, 45 days, and labels haemoglobin after injection. It also labels the plasma iron pool and this allows the measurement of iron clearance and calculation of plasma iron turnover. Its subsequent appearance in haemoglobin permits the assessment of the rate of haemoglobin synthesis and the completeness of the utilization of iron. Because it is a γ-ray emitter, radioactivity can be measured in vivo and the sites of distribution of the administered iron and the probable sites of erythropoiesis can thus be determined. (⁵⁹Fe is not available at present from the former supplier, Amersham plc, but it may be available from POLATOM, www.polatom.pl.)

Iron Distribution

Principle

Iron is transported to the bone marrow bound to transferrin. The transferrin–iron complex binds to transferrin receptors of the erythroblast membrane and the complex enters the cell by endocytosis; iron is then released into the cytosol, with transferrin and its receptor being recycled to the cell surface where transferrin is released into the plasma. Iron not bound to transferrin finds its way to the liver and to other organs rather than to the bone marrow, whereas colloidal particles of iron are rapidly removed by phagocytic cells.

The ferrokinetic studies with ⁵⁹Fe that provide information on erythropoiesis include the rate of clearance of the radioiron from the plasma and iron incorporation into circulating red cells (iron utilization). These are relatively simple procedures but they do not take account of the recirculation of iron that returns to the plasma from tissues, nor of iron turnover resulting from dyserythropoiesis or haemolysis. To take account of these factors requires much more complex and time-consuming procedures with multiple sampling over an extended period,²² but the simpler tests provide sufficiently reliable and useful measurements for clinical purposes.

In ferrokinetic studies, it is important to ensure that any iron administered is bound to transferrin. In most cases, plasma has an adequate amount of transferrin. However, the unsaturated iron-binding capacity (UIBC) or transferrin concentration of the patient’s plasma should be measured before the test is carried out and, if the UIBC is <1 mg/l (20 μmol/l) or the transferrin concentration is <0.6 g/l, normal donor plasma (HIV and hepatitis B and C negative) should be used instead of that of the patient for the subsequent labelling procedure.

Method

Under sterile conditions, obtain 5–10 ml of plasma from freshly collected heparinized blood. Add 0.4 MBq of ⁵⁹Fe ferric citrate (specific activity >0.2 MBq/µg). Incubate at room temperature for 15 min. Fill a syringe with all but 1 ml of the mixture. Weigh the syringe to the nearest 10 mg. Inject its content intravenously into the patient, starting a stopwatch at the midpoint of the injection. Reweigh the empty syringe and calculate the volume injected:

Dilute the residual portion of the dose (1 ml) 1 in 100 in water and use as a measure of the total amount of radioactivity and as a standard in subsequent measurements.

Plasma Iron Clearance

Take a sample at 3 min and four or five further samples over a period of 1–2 h, collecting them into heparin or EDTA. Retain a portion of one sample for measurement of plasma iron. Measure the radioactivity in unit volumes of plasma from the samples and plot the values obtained on log linear graph paper. A straight line will usually be obtained for the initial slope. The radioactivity at the moment of injection is inferred by extrapolation back to zero time and the time taken for the plasma radioactivity to decrease to half its initial value (T_½-plasma clearance) is read off the graph (Fig. 17.3).

Figure 17.3 Plasma iron clearance. ⁵⁹Fe activity in plasma at 10, 20, 30 and 60 min extrapolated to the vertical axis to obtain activity at zero time. The T_½ was 90 min.

Range of T_½-plasma clearance in health = 60–140 min.

The clearance rate is influenced by the intensity of erythropoiesis and also by the activity of the macrophages of the reticuloendothelial (RE) system, especially in the liver, spleen and bone marrow, where the iron is retained as storage iron. Also, to a lesser extent, circulating reticulocytes may take up some of the iron. A rapid clearance indicates hyperactivity of one or more of these mechanisms, as for instance in iron deficiency anaemia, haemorrhagic anaemia, haemolytic anaemia and polycythaemia vera. The clearance rate is decreased in aplastic anaemia. In leukaemia and in myelofibrosis, the results are variable, depending on the amount of erythropoietic marrow and the extent of extramedullary erythropoiesis; in myelofibrosis, however, rapid clearance is by far the more common finding. In dyserythropoiesis, the clearance may be normal or accelerated.

Iron Utilization

Collect blood samples daily or at least on alternate days, for a period of about 2 weeks after the administration of the ⁵⁹Fe. Measure the radioactivity per ml of whole blood and calculate the percentage utilization on each day from the formula:

where f is a PCV/Hct correction factor

When there is reason to suspect that the body:venous PCV ratio is not 0.9, measure the RCV by a direct method (see p. 378). Note, however, that because calculation of plasma volume from extrapolation of the ⁵⁹Fe disappearance curve is often unreliable, it should not be used as the basis for calculation of RCV.

Calculate the percentage utilization on each day from the formula:

Note: The radioactivity is adjusted for physical decay up to the day of measurement.

Plot the daily measured percentages against time on arithmetic graph paper. Record the maximum utilization (Fig. 17.4).

Figure 17.4 Iron utilization. Red-cell uptake of ⁵⁹Fe in (A) iron deficiency and polycythaemia, (B) a normal subject, (C) dyserythropoiesis and (D) severe aplastic anaemia.

The calculation gives a measure of effective erythropoiesis. In normal subjects, red cell radioactivity increases steadily from 24 h and reaches a maximum of 70–80% utilization on the 10th to 14th day.

A rapid plasma clearance is usually associated with early and relatively complete utilization and the converse also applies. The results are inconsistent in megaloblastic anaemia and in haemoglobinopathies, in which there is ineffective erythropoiesis; they also are inconsistent in myelofibrosis, depending on the extent of extramedullary erythropoiesis and whether the red cell lifespan is reduced. If there is rapid haemolysis, the utilization curve will be distorted by destruction of some of the labelled red cells; this may be recognized if frequent (daily) samples are measured. In aplastic anaemia, the utilization is usually 10–15%; in ineffective erythropoiesis, it is as a rule 30–50%.

The ferrokinetic patterns in various diseases are shown in Table 17.3 and Figure 17.4.

Table 17.3 Ferrokinetic patterns in various diseases

	Plasma clearance T_½	Red cell utilization^a
Normal	60–140 min	80%
Iron deficiency	Shortened	Increased (90%)
Aplastic anaemia	Prolonged	Decreased (10%)
Chronic infection	Slightly shortened	Normal
Dyserythropoiesis	Slightly shortened	Decreased (30%)
Myelofibrosis	Shortened	Decreased (50%)
Haemolytic anaemia	Shortened	Increased (85%)

a Average figures are shown, but there is a wide range, depending on the stage and severity of the disease.

Body Iron Distribution

An overall picture of ferrokinetics can be constructed from surface counting with a scintillation probe positioned over the liver, spleen, sacrum (for bone marrow) and heart (for blood pool) after an intravenous injection of ⁵⁹Fe. By counting over several days, it is possible to identify sites of erythropoiesis from early counts and sites of red cell destruction from later counts. This test is rarely performed nowadays, although it has some clinical value for determining the extent of extramedullary erythropoiesis in the spleen before splenectomy.

Where there are facilities for using cyclotron-produced ⁵²Fe and positron emission tomography (PET), high-resolution images of the intramedullary and extramedullary distribution of erythropoietic tissue can be obtained. This is especially helpful in the myeloproliferative neoplasms for diagnosing transition of polycythaemia vera to myelofibrosis and for differentiating essential thrombocythaemia from reactive thrombocytosis.²³ It is also useful in identifying residual skeletal erythropoiesis in aplastic anaemia.

Estimation of the lifespan of red cells in vivo

There is extensive literature on the survival of red cells in haemolytic anaemias using radioisotope labelling of red cells (see review by Bentley and Miller ²⁴). Although now undertaken less frequently than in the past, measurement of red cell survival can still provide important data in cases of anaemia in which increased haemolysis is suspected but not clearly demonstrated by other tests. In the usual procedure, a population of circulating red cells of all ages is labelled (‘random labelling’). By contrast, in ‘cohort labelling’ a radionuclide (e.g. ⁵⁹Fe) is incorporated into haemoglobin during its synthesis by erythroblasts and radioactivity is measured in red cells that appear in the circulation as a cohort of closely similar age. Red cell lifespan can be calculated from measurements of red cell iron turnover,²⁵ but the results have to be interpreted with caution because of the reutilization for haem synthesis of iron derived from red cells at the end of their lifespan. Random labelling is a much more practical method than cohort labelling.

Radioactive Chromium (⁵¹Cr) Method

Radioactive chromium (⁵¹Cr) is a γ-ray emitter with a half-life of 27.8 days. As a red cell label, it is used in the form of hexavalent sodium chromate. After passing through the surface membrane of the red cells, it is reduced to the trivalent form that binds to protein, preferentially to the β-globin chains of haemoglobin.²⁶ In this form, it is not reutilized or transferred to other cells in the circulation.

The main disadvantage of ⁵¹Cr is that it gradually elutes from red cells as they circulate; there may be, too, an increased loss over the first 1–3 days and uncertainty as to how much has been lost makes it impossible to measure red cell lifespan accurately. Chromium, whether radioactive or non-radioactive, is toxic to red cells, probably by its oxidizing actions; it inhibits glycolysis in red cells when present at a concentration of 10 μg/ml or more ²⁷ and blocks glutathione reductase activity at a concentration exceeding 5 μg/ml.²⁸ Blood should thus not be exposed to >2 μg of chromium per ml of packed red cells.

Na₂⁵¹CrO₄ is available commercially at a specific activity of about 15–20 GBq/mg Cr. For administration, the stock solution usually must be dissolved in 9 g/l NaCl (saline) (see below). ACD must not be used as a diluent because this reduces the chromate to the cationic chromic form.

Care must be taken to avoid lysis when the red cells are washed; it may be necessary, especially if the blood contains spherocytes, to use a slightly hypertonic solution (e.g. 12 g/l NaCl). This should certainly be used if an osmotic fragility test has demonstrated lysis in 9 g/l NaCl. In patients whose plasma contains high-titre, high-thermal-amplitude cold agglutinins, the blood must be collected in a warmed syringe and delivered into ACD solution previously warmed to 37°C; the labelling and washing in saline should be carried out in a ‘warm room’ at 37°C.

Method

The technique of labelling red cells is the same as for TBV measurement (see p. 378).²⁹ To ensure as little damage to red cells as possible, with subsequent minimal early loss and later elution, it is important to maintain the blood at an optimal pH. This can be achieved by adding 10 volumes of blood to 1.5 volumes of NIH-A ACD solution (see p. 619).

For a red cell survival study, 0.02 MBq per kg body weight (an average total dose of c2 MBq) is recommended. If this is to be combined with a spleen scan or pool measurement, a higher dose (4 MBq) should be used, bearing in mind that <2 μg of chromium should be added per ml of packed red cells.

After injection, allow the labelled cells to circulate in the recipient for 10 min (or for 60 min in patients with cardiac failure or splenomegaly, in whom mixing may be delayed). Then collect a sample of blood from a vein other than that used for the injection (or after washing the needle through with saline if a butterfly needle is used) and mix with EDTA as anticoagulant. The radioactivity in this sample provides a baseline for subsequent observations. Retain part of the labelled cell suspension that was not injected into the patient to serve as a standard. This enables the blood volume to be calculated if required.

Take further 4–5 ml blood samples from the patient 24 h later (day 1) and subsequently at intervals, the frequency of the samples depending on the rate of red cell destruction: in general, three specimens between day 2 and day 7 and then two specimens per week for the duration of the study. Measurements should be continued until at least half the radioactivity has disappeared from the circulation.

Measure the Hb or PCV in a part of each sample; then lyse the samples with saponin, mix well and deliver 1 ml into counting tubes, if possible in duplicate.

Measurement of Radioactivity

Estimate the percentage survival (of ⁵¹Cr) on any day (t) by comparing the radioactivity of the sample taken on that day with that of the day 0 sample (i.e. the sample withdrawn 10 [or 60] min after the injection of the labelled cells). Thus, ⁵¹Cr survival on day t (%) is given by the following:

No adjustment is necessary for the physical decay of the isotope, provided that the standard is counted within a few minutes of the day t sample.

Carry out the measurements in any high-quality scintillation counter, at least 2500 counts being recorded to achieve a precision within ±2%.

Processing of Radioactivity Measurements

Before the data can be analysed and interpreted, factors, other than physical decay, that are involved in the disappearance of radioactivity from the circulation have to be considered. There are two processes: ⁵¹Cr-labelled cells are lost from the circulation by lysis, phagocytosis or haemorrhage and, in addition, ⁵¹Cr is eluted from intact red cells that still circulate.

Elution

The rate of elution differs to a small extent from one individual to another. It is thought to vary to a greater extent between different diseases, especially when the red cell lifespan is considerably reduced. However, in such cases, elution and variation in the rate of elution become unimportant. The rate of elution is also influenced by technique, especially by the anticoagulant solution into which the blood is collected prior to labelling. With the NIH-A ACD solution, the rate of elution is about 1% per day.²⁹

Early Loss

Sometimes, in addition to the elution that occurs continuously and at a relatively low and constant rate, up to 10% of the ⁵¹Cr may be lost within the first 24 h. The cause of this major early loss is obscure and several components may be involved. If this major loss does not continue beyond the first 2 days, it is often looked on as an artefact, in the sense that it does not denote an increased rate of lysis in vivo, and it can be, and typically is, ignored by replotting the figures as described on p. 387. This procedure is acceptable, at least for clinical studies, but it does not take into account the possibility that a small proportion of red cells are present that lyse rapidly. It is common practice to calculate the T₅₀Cr (i.e. the time taken for the concentration of ⁵¹Cr in the blood to fall to 50% of its initial value) after correcting the data for physical decay but not for elution. T₅₀ is used rather than T_½ because the elimination of the label is not a constant exponential fraction of the original amount. The chief objection to the use of T₅₀Cr is that it may be misleading without additional information on the pattern of the survival curve. Moreover, the mean red cell lifespan cannot be directly derived from it. With the technique described earlier, the mean value of T₅₀ in normal subjects is 30 days, with a range of 25–33 days (Table 17.4).

Table 17.4 Normal range for ⁵¹Cr survival curves with correction for elution

Day	% ⁵¹Cr (corrected for decay; not corrected for elution)	Elution correction factors^a
1	93–98	1.03
2	89–97	1.05
3	86–95	1.06
4	83–93	1.07
5	80–92	1.08
6	78–90	1.10
7	77–88	1.11
8	76–86	1.12
9	74–84	1.13
10	72–83	1.14
11	70–81	1.16
12	68–79	1.17
13	67–78	1.18
14	65–77	1.19
15	64–75	1.20
16	62–74	1.22
17	59–73	1.23
18	58–71	1.25
19	57–69	1.26
20	56–67	1.27
21	55–66	1.29
22	53–65	1.31
23	52–63	1.32
24	51–60	1.34
25	50–59	1.36
30	44–52	1.47
35	39–47	1.53
40	34–42	1.60

a To correct for elution, multiply the % ⁵¹Cr by the elution factor for the particular day.

Correction for Elution

When haemolysis is marked, elution is of minor importance and can be ignored. When haemolysis is not greatly increased, it is essential to correct for elution. This can be done by multiplying the measured survival by the factors given in Table 17.4.

Survival Curves

Normal red cell survival (corrected for elution) will be in the range shown in Figure 17.5. When survival is reduced, a survival curve should be drawn and from this the mean red cell lifespan can be derived.

Figure 17.5 ⁵¹Cr red cell survival. The hatched area shows the normal range.

Plot the % radioactivity figures or count rates per ml of whole blood (corrected for physical decay and for elution) on arithmetic and semilogarithmic graph paper and attempt to fit straight lines passing through the data points.

1. If a straight line can be fitted to the arithmetic plot, the mean red cell lifespan is given by the point in time at which the line or its extension cuts the abscissa (Fig. 17.6).

2. As a rule, however, a straight line is better fitted to the semilogarithmic plot; the mean red cell lifespan can be read as the exponential e⁻¹ (that is, the time when 37% of the cells are still surviving [Fig. 17.7]) or calculated by multiplying the half-time of the fitted line by the reciprocal of the natural log of 2 (0.693) (i.e. multiplying by 1.44).

Figure 17.6 ⁵¹Cr red cell survival curve. Patient with hereditary spherocytosis. The results give a straight line when plotted on arithmetic graph paper. The mean cell lifespan is indicated by the point at which its extension cuts the abscissa (20 days).

Figure 17.7 ⁵¹Cr red cell survival curve. Patient with autoimmune haemolytic anaemia. The results have been plotted on semilogarithmic graph paper and the mean cell lifespan was read as the time when 37% of the cells were still surviving (9–10 days). The T₅₀Cr was 6–7 days. MCL, mean cell lifespan.

A computer programmed curve-fitting procedure is more precise but is not likely to improve overall accuracy of the results for clinical purposes.

Interpretation of Survival Curves

In the autoimmune haemolytic anaemias, the slope of elimination is usually markedly curvilinear when the data are plotted on arithmetic graph paper. Red cell destruction is typically random and the curve of elimination is thus exponential and the data give a straight line when plotted on semilogarithmic graph paper.

In some cases of haemolytic anaemia (possibly only when there are intracorpuscular defects), the survival curve appears to consist of two components, an initial steep slope followed by a much less steeply falling slope. This suggests the presence of cells of widely varying lifespan. This type of ‘double population’ curve is seen in paroxysmal nocturnal haemoglobinuria, in sickle cell anaemia, in some cases of hereditary enzyme-deficiency haemolytic anaemia and when the labelled cells consist of a mixture of transfused normal cells and short-lived patient’s cells. The mean cell lifespan of the entire cell population can be deduced by plotting the points on semilogarithmic graph paper, as described earlier. The proportion of cells belonging to the longer-lived population can be estimated by plotting the data on arithmetic graph paper and extrapolating the less steep slope back to the ordinate; the lifespan of this population can be estimated by extending the same slope to the abscissa (Fig. 17.8). The lifespan of the short-lived cells can be deduced from the formula:

Figure 17.8 ⁵¹Cr red cell survival curve showing a ‘double population’. By plotting the data on semilogarithmic graph paper as described in Figure 17.7, the mean cell lifespan (MCL) of the entire cell population was deduced as 5 days. When plotted on arithmetic graph paper, by extrapolation of the less steep slope to the ordinate it was deduced that approximately 30% of the red cells belonged to one population, and by extrapolation of the same slope to the abscissa the MCL of this population was deduced as 35 days. The lifespan of the remaining 70% of cells was calculated to be 3.6 days (see formula). The T₅₀Cr was 3–4 days.

where S = short-lived population, L = longer-lived population, T = entire cell population and MCL = mean cell lifespan.

Correction for Early Loss

The simplest method is to ignore the early loss by taking as 100% the radioactivity still present at the end of 24–48 h. Alternatively, the following method can be used; it has the advantage that the slope of the survival curve is not altered. Plot the data on arithmetic graph paper, extrapolate the line of the slope beyond the initial steep part back to the ordinate and take the point of intersection as 100%; then calibrate the ordinate scale accordingly.

Blood Volume Changes

There is no need to correct the measurements of radioactivity per ml of whole blood for alterations in PCV provided that the total blood volume remains constant throughout the study. However, if it is suspected that the TBV may be changing (e.g. in patients suffering from haemorrhage or being transfused), serial determinations of TBV should be carried out and the observed radioactivity should be multiplied by the observed TBV and divided by the initial TBV. In practice, if a patient receives a blood transfusion during a survival study, it can, as a general rule, be assumed that the TBV will have returned to its pretransfusion level within 24–48 h.

Correction of Survival Data for Blood Loss

When there is a relatively constant loss of blood during a red cell survival study, the true mean red cell lifespan can be obtained by the following equation:

where Ta = apparent time of MCL (days), RCV = red cell volume (ml) and L = mean rate of loss of red cells (ml/day).

Normal Red Cell Lifespan

The mean red cell lifespan in health is usually taken as 120 days.

Determination of Sites of Red Cell Destruction Using ⁵¹Cr

Because ⁵¹Cr is a γ-ray emitter, the sites of destruction of red cells, with special reference to the spleen and liver, can be determined by in vivo surface counting using a shielded scintillation counter placed, respectively, over the heart, spleen and liver. This procedure is laborious, but occasionally it may provide clinically useful information on the role of the spleen in various types of haemolytic anaemia, especially by predicting response to splenectomy.³⁰

Compatibility test

The behaviour of labelled donor cells in a recipient will provide important information on the compatibility or otherwise of the donor blood:

1. When serological tests suggest that all normal donors are incompatible

2. When in the presence of an alloantibody no non-reacting donor can be found

3. When the recipient has had an unexplained haemolytic transfusion reaction.

Method

Remove 1–2 ml of blood from the tubing attached to donor bag using a sterile technique. Label 0.5 ml of the red cells with 0.8 MBq of ⁵¹Cr, 2 MBq of ¹¹¹In or 2 MBq of ^99mTc in the standard way (see p. 378) and administer to the recipient. Collect 5–10 ml of blood into EDTA or heparin at 3, 10 and 60 min after the injection from a vein other than that used for the injection. Prepare 1 ml samples in counting vials. Centrifuge the remainder of the specimens and pipette 1 ml of the plasma into counting vials. Measure the radioactivity in the usual way. Calculate the activity in the blood and plasma samples as a percentage of the 3 min blood sample.²⁹

Interpretation

With compatible blood, the radioactivity in the 60 min sample is, on average, 99% of that of the 3 min sample, but it may vary between 94% and 104%. If the blood radioactivity at 60 min is not less than 70% and the plasma activity is not more than 3%, the donor cells may be transfused with minimal hazard.²⁹

Visualization of the spleen by scintillation scanning

Anatomical features of organs, including the spleen, are usually studied in radiology or nuclear medicine departments by means of magnetic resonance imaging (MRI), computed tomography imaging (CT scans) or ultrasound imaging. Imaging of radioisotope-labelled red cells provides an alternative functional method. If red blood cells labelled with ^99mTc are heat damaged, they will be selectively removed by the spleen. ^99mTc-labelled colloid is also removed from the circulation by the spleen, but this is not as specific because it is also taken up by reticuloendothelial cells in the liver and elsewhere. The rate of uptake of the isotope by the spleen is a measure of its function (see below). Imaging by scintillation scanning is usually started about 1 h after the injection of the damaged cells, but it can be performed up to 3–4 h later. Accumulation of radioactivity within the spleen after administration of heat-damaged labelled cells thus provides a means of demonstrating its size and position, whether it is absent or has reduced function and the presence of splenunculi. Satisfactory scans can also be obtained with ⁵¹Cr or ¹¹¹In.

Method

With ^99mTc as the label, carry out pre-tinning in vivo by an injection of a stannous compound as described on p. 378. Then collect 5–10 ml of blood into a sterile bottle containing 100 iu of heparin. Wash twice in sterile 9 g/l NaCl (saline), centrifuging at 1200–1500 g for 5–10 min. Transfer 2 ml of the packed red cells to a 30 ml glass bottle with a screw cap; heat the bottle in a waterbath at a constant temperature of 49.5–50°C for exactly 20 min with occasional gentle mixing. Wash the cells in saline until the supernatant is free from haemoglobin and discard the final supernatant. Label with 40 MBq of ^99mTc by the method described on p. 378. After it has stood for 5 min, wash twice in saline. Resuspend in about 10 ml of saline and inject as soon as possible. After about 1 h carry out a gamma camera scan.³¹

Spleen Function

Information on splenic activity may be obtained by measuring the rate of clearance of heat-damaged labelled red cells from the circulation. A blood sample is taken exactly 3 min after the midpoint of the injection and further samples are collected at 5-min intervals for 30 min, at 45 min and finally at 60 min. The radioactivity in each sample is measured and expressed as a percentage of the radioactivity in the 3-min sample. The results are plotted on semilogarithmic graph paper, the 3-min sample being taken as 100% radioactivity. For consistent results, a carefully standardized technique is necessary to ensure that the red cells are damaged to the same extent.

The disappearance curve is, as a rule, exponential (Fig. 17.9). The initial slope reflects the splenic blood flow; the rate of blood flow is calculated as the reciprocal of the time taken for the radioactivity to fall to half the 3-min value (i.e. ), where 0.693 is the natural log of 2.

Figure 17.9 Curve of disappearance of heat-damaged red cells from the circulation. The curve shows the sequence of blood flow, transient pooling, sequestration and irreversible trapping of cells.

When the spleen is functioning normally, the T_½ is 5–15 min and fractional splenic blood flow is 0.05–0.14 ml/min (i.e. 5–14% of the circulating blood per min). The clearance rate is considerably prolonged in some patients with thrombocythaemia and in other conditions associated with splenic atrophy such as sickle cell anaemia, coeliac disease and dermatitis herpetiformis.¹⁷ It thus provides some indication of spleen function. However, the disappearance curve is a complex of at least two components. The first (mentioned earlier) reflects the splenic blood flow and the second component mainly measures cell trapping, the consequence of both transient sequestration and phagocytosis with irreversible extraction of the cells from circulation.^32,³³ Measurement of phagocytosis alone is obtained more reliably with immunoglobulin G (IgG) (anti-D)-coated red cells.³⁴

Leucocyte Imaging

The main diagnostic value of ¹¹¹In-labelled granulocyte scintigraphy is to localize specific sites of infection and abscesses and, in investigation of patients with fever of unknown origin, to rule out an infectious cause for the fever.³⁵ For this it is necessary to prepare a granulocyte concentrate separated from other leucocytes (see p. 65). This is then labelled with ¹¹¹In in a procedure similar to that for labelling platelets (see p. 389)³⁶ and administered. The sites of granulocyte accumulation are shown by gamma camera scan.

Miscellaneous Imaging

In addition to the radioisotopes discussed earlier, there are other radioisotopes that can be used to provide information in haematological disorders. For example, ¹⁸F-FDG (fluorine-18 fluorodeoxyglucose), a tracer of glucose metabolism, with position emission tomography (PET) can be useful in the assessment of tumour metabolism.^37,³⁸ The information generated can assist in clinical staging of patients with malignancies, including lymphoma. Malignant tissue shows enhanced uptake of this tracer and this information can be used to monitor progress of patients receiving chemotherapy.

Measurement of blood loss from the gastrointestinal tract

The ⁵¹Cr method of red cell labelling can be used to quantitate blood lost into the gastrointestinal tract because ⁵¹Cr is neither excreted nor more than minimally reabsorbed. Accordingly when the blood contains ⁵¹Cr-labelled red cells, faecal radioactivity is at a very low level unless bleeding has taken place somewhere within the gastrointestinal tract. Measurement of the faecal radioactivity then gives a reliable indication of the extent of the blood loss.

Method

Label the patient’s own blood with approximately 4 MBq of ⁵¹Cr, as described on see p. 378. On each day of the test, collect the faeces in plastic or waxed cardboard cartons. Prepare a standard by adding a measured volume (3–5 ml) of the patient’s blood, collected on each day, to approximately 100 ml of water in a similar carton. Compare the radioactivity of the faecal samples and the corresponding daily standard in a large-volume counting system (see p. 375). Then:

Blood loss from any other source (e.g. surgical operation or menstruation) can be measured in a similar way by counting swabs, dressings and so on placed in a carton. It is not, however, possible to measure blood or haemoglobin loss in the urine (haematuria or haemoglobinuria) by this method because free ⁵¹Cr is normally excreted in the urine.

An imaging procedure has also been described in which blood is labelled with ^99mTc and a large-field scintillation scan is performed after 60–90 min and, if necessary, again at intervals for 24 h.⁴⁰

Measurement of platelet lifespan

Principle

The procedure for measuring platelet lifespan is broadly similar to that for red cell survival (see p. 383). A method using ¹¹¹In-labelled platelets was recommended by ICSH.⁴¹ A modification of this method especially for use with low platelet counts⁴² is described in the following.

Method

Collect 51 ml of blood into 9 ml of NIH-A ACD (see p. 619); a proportionately lower amount is required if the platelet count is normal or high. Distribute the blood equally into three 30 ml polystyrene tubes, each containing 2 ml of 60 g/l hydroxyethyl starch (Hespan, Bristol Myers Squibb). Mix and immediately centrifuge at 150 g for 10 min. Transfer the supernatant platelet-rich plasma into clean centrifuge tubes and add ACD, 1 volume to 10 volumes of the platelet-rich plasma. If necessary, centrifuge again at 150 g for 5 min to remove residual red cells.⁴²

Centrifuge the platelet-rich plasma at 640 g for 10 min to obtain platelet pellets. Carefully remove the supernatant plasma but do not discard. Add 1 ml of this platelet-poor plasma to the platelet pellets, gently tap the tubes to resuspend and pool the contents.

Prepare a solution of tropolone, 4.4 mmol/l (0.54 mg/ml) in HEPES-saline buffer, pH 7.6 (see p. 622). Mix 0.1 ml with 8 MBq (250 μCi) of ¹¹¹InCl in <50 μl of 40 mmol/l HCl. Add the platelet suspension with gentle mixing and leave at room temperature for 5 min. Then add 5 ml of platelet-poor plasma. Centrifuge at 640 g for 10 min. Remove the supernatant and resuspend the platelet pellet in 5 ml of platelet-poor plasma. Take up the platelet suspension into a 10 ml plastic syringe.

Add 0.5 ml of the platelet suspension to 100 ml of water in a volumetric flask as a standard. Weigh the syringe, inject the platelets into the patient through a butterfly needle and reweigh.