Effect of background region of interest and time-interval selection on glomerular filtration ratio estimation by percentage dose uptake of ^99mTc-DTPA in comparison with ⁵¹Cr-EDTA clearance in healthy cats

Abstract

Evaluation of glomerular function is a useful part of the diagnostic approach in animals suspected of having renal disease. Time-interval and background region of interest (bg ROI) selection are determining factors when calculating the glomerular filtration ratio (GFR) based on percentage uptake of ^99mtechnetium-labelled diethylene triamine penta-acetic acid (^99mTc-DTPA). Therefore, three different time intervals (60–120 s, 120–180 s, 60–180 s) and three different bg ROIs (C-shape, caudolateral, cranial + caudal) were investigated. In addition, global GFRs based on percentage dose uptake of ^99mTc-DTPA for the different time-intervals and bg ROIs were compared with the global GFR based on ⁵¹chromium-ethylene diaminic tetra-acetic acid (⁵¹Cr-EDTA) plasma clearance in nine healthy European domestic shorthair cats. Paired Student’s t-tests and linear regression analysis were used to analyse the data. Different time intervals seemed to cause significant variation (P <0.01) in absolute GFR values, regardless of the choice of bg ROI. Significant differences (P <0.01) between bg ROIs were only observed in the 120–180s time interval between the C-shape and cranial + caudal bg ROI, and between the caudolateral and cranial + caudal bg ROI. The caudolateral bg ROI in the 60–180 s time interval showed the highest correlation coefficient (r = 0.882) between ^99mTc-DTPA and ⁵¹Cr-EDTA, although a significant difference (P <0.05) was present between both techniques.

Introduction

The glomerular filtration rate (GFR) is considered as one of the most reliable indices of kidney function as it is related directly to the number of functioning nephrons.^{1 –5} Urinary clearance of inulin is accepted as the gold standard for GFR measurements.^2,5 Unfortunately, this method has several disadvantages.^{1,5 –9} Radionuclide-based determination of GFR, like ^99mtechnetium-labelled diethylene triamine penta-acetic acid (^99mTc-DTPA) and ⁵¹chromium-ethylene diaminic tetra-acetic acid (⁵¹Cr-EDTA), correlates well with inulin clearance and is proved to be a sensitive determinant of renal function in dogs, cats and hum-ans.^{1,6,8,10 –14} Imaging nuclear medicine techniques (ie, ^99mTc-DTPA) provide a quick and non-invasive means of estimating GFR, whereas non-imaging nuclear medicine techniques (ie, ⁵¹Cr-EDTA) rely on the measurement of the clearance of a substance from the blood.^2,6

Both ^99mTc-DTPA and ⁵¹Cr-EDTA have been used in cats.^{3,7,8,11,13,15–17} Both radiopharmaceuticals are considered as exclusive indicators of the filtration function because they are filtered completely by the glomerulus, are not secreted or reabsorbed by the renal tubules, are not metabolized or produced by the body and are not bound appreciably to plasma protein.^{2,10,11,18,19}

GFR with ^99mTc-DTPA by dynamic renal scintigraphy is calculated from ^99mTc-DTPA uptake in each kidney using a regression equation that was developed by plotting DTPA uptake data against plasma inulin clearance data. ⁸ This technique was first described by Gates in humans ¹⁰ and was later modified for use in dogs^1,6,20 and cats, ⁸ as the regression formula is considered to be species-specific. ²

As mentioned earlier, ^99mTc-DTPA is ideal for determining GFR. Nevertheless, there are two things, among other influences, that can potentially decrease the accuracy of nuclear imaging for GFR determination: the selection of inappropriate background regions for subtraction from the kidney counts and the selection of inappropriate time intervals. ⁶ Many background regions of interest (bg ROIs) have been used in previous studies.^2,6,10,20,21 Because over- or under-correction of background radioactivity may cause errors in the estimated GFR, the choice of the background ROI is an important step. ²¹ The time interval used for data collection is also a determining factor. When looking at a normal renogram, three phases can be identified: a circulation phase with a rapid initial rise (15–20 s), an uptake phase with peak renal activity reached 2.5–3.5 min after injection and an excretory phase.^18,22 Nevertheless, most studies have used the 60–180 s time interval for data collection, and discrepancy exists in choosing the optimal time interval.^10,20

The purpose of this study was to determine the best time interval (60–120 s, 120–180 s, 60–180 s) and bg ROI (C-shape, caudolateral, caudal + cranial) for estimating global GFR based on percentage kidney uptake of ^99mTc-DTPA. Therefore, we compared GFR based on the percentage dose uptake of ^99mTc-DTPA with a two-blood sample plasma clearance of ⁵¹Cr-EDTA, as this latest technique was validated recently in cats by one of the authors. ¹⁷

Materials and methods

Animals

Nine European domestic shorthair cats (seven female spayed and two male castrated) with a mean age of 56 months (range 24–67 months) and mean weight of 5.23 kg (range 3.9–6.1 kg) were included in this study. All cats were obtained from the population of laboratory animals of the University of Ghent. The study was conducted according to guidelines for animal care, with the consent of the Ethical Committee of the Faculty of Veterinary Medicine from University of Ghent, Belgium. All cats were considered healthy based on a general physical examination, complete blood count, blood urea nitrogen, serum creatinine concentrations, and routine urinalysis (urine specific gravity, urinary protein-to-creatinine ratio, urinary sediment and urinary dipstick).

A catheter was placed in the cephalic vein of one of the forelimbs and flushed with 1–2 ml 0.9% sodium chloride (NaCl). Each cat received a single mean bolus injection of 3.7 MBeq ⁵¹Cr-EDTA. After flushing the catheter again with 1–2 ml 0.9% NaCl, each cat received a mean bolus injection of 104.18 MBeq ^99mTc-DTPA.

^99mTc-DTPA imaging technique

Before ^99mTc-DTPA was injected, the syringe was positioned on the center of a gamma camera, fitted with a low energy parallel hole collimator. A pre-injection syringe count was made during 1 min and the result was expressed in counts per minute (cpm). The cats were anaesthetized with a bolus of 4–5 mg/kg propofol followed by a constant rate infusion of 0.3 mg/kg/min. The cats were positioned in dorsal recumbence with the gamma camera centred dorsal to the kidneys. The dynamic image acquisition, performed with a Toshiba GCA 7200 A/DI gamma camera, was initiated simultaneously with the intravenous injection. Frames were acquired at 6-sintervals over 6 mins and stored into a 128 × 128 digital matrix. After the scanning was completed, the empty syringe was positioned again on the center of the gamma camera and residual activity was determined from a 1-min post-injection scan. The total injected dose was determined as the difference between the pre- and post-injection syringe counts, and was expressed in cpm.

ROIs were drawn manually around both kidneys using the high count density-summed image of all frames, thereby avoiding overlap with the aorta, spleen, ureters or stomach. Furthermore, three different background ROIs were drawn manually: a C-shaped ROI around the entire kidney (Figure 1), a caudolateral ROI (Figure 2) and a combination of two rectangular ROIs (Figure 3), one at the cranial pole and one at the caudal pole of the kidney. To avoid influence of ROI placement on evaluation of the three different time-intervals (60–120 s, 120–180 s and 60–180 s), this set of ROIs was saved per cat so that GFR calculations for the three selected time-intervals was performed using the same ROIs.

Figure 1.

^99mTc-DTPA acquisition of a cat in dorsal position. A region of interest (ROI) is drawn around each kidney (1). A C-shaped ROI around the entire kidney is added for background correction (2)

Figure 2.

^99mTc-DTPA acquisition of a cat in dorsal position. A region of interest (ROI) is drawn around each kidney (1). A caudolateral ROI is added for background correction (2)

Figure 3.

^99mTc-DTPA acquisition of a cat in dorsal position. A region of interest (ROI) is drawn around each kidney (1). Two rectangular ROIs, one at the cranial pole and one at the caudal pole of the kidney, are added for background correction (2)

The net counts of ^99mTc-DTPA uptake in the kidneys (Net Kidney cts) were determined for each time interval and each background ROI by the formula:

$$\text{Net}\text{Kidney}\text{cts=kidney}\text{counts}-\left(\text{background}\text{counts}\frac{\text{kidney}\text{pixels}}{\text{background}\text{pixels}}\right)$$

For the 1-min time intervals (60–120 s and 120–180 s) the kidney counts were doubled when calculating the Net Kidney cts, as the regression equation used by Uribe et al ⁸ required the Net Kidney cts from a 2-min period.

The percentage dose uptake (ID) was determined by the formula:

$$\begin{array}{l}\%\text{dose}\text{uptake}\left(\%\text{ID}\right)\\ =\frac{\text{Net}\text{Kidney}\text{cts}}{\text{total}\text{number}\text{of}\text{injected}\text{counts}}\times 100\\ =\frac{\text{Net}\text{Kidney}\text{cts}}{(\text{preinjection}\text{count-postinjection}\text{count})}\times 100\end{array}$$

The global GFR (ml/min/kg) was calculated with a formula based on the regression correlation of the percentage dose uptake of ^99mTc-DTPA in each kidney with the GFR measured by plasma inulin clearance as determined by Uribe et al ⁸ .

$$\text{Global}\text{GFR}=0.284\left(\%\text{ID}\text{right}\text{kidney}+\%\text{ID}\text{left}\text{kidney}\right)-0.164$$

⁵¹Cr-EDTA clearance test

The ⁵¹Cr-EDTA plasma clearance test was performed as described previously by one of the authors. ¹⁷ Before and after injection of ⁵¹Cr-EDTA, the syringe was weighted on a high precision balance, allowing exact calculation of the injected dose. Two blood samples (1–2 ml) were taken, approximately 36 min (T1) and 240 min (T2) after administration of ⁵¹Cr-EDTA, from the jugular vein. The exact sampling time was registered and the blood samples were transferred to an EDTA-plasma tube. Plasma was then separated by centrifugation of the blood samples (1075 × g for 5 min). For each sample, 0.4 ml plasma was transferred into a counting vial. Each vial was counted in an automated well-counter for 3 min. The activity in the plasma samples was expressed in cpm. An empty vial was added for each cat and was used to correct the plasma samples for background activity. In addition, for each cat, a vial containing 2 ml of a standard dilution of 1/1000 of the stock solution used for injection was also counted to determine total injected counts (TIC) in relation to the plasma counts for the specific well-counter. The administered dose, expressed as TIC was calculated as follows:

$$\text{TIC}=\left(\text{WBI}-\text{WAI}\right)\times \text{DS}\times \text{SA}$$

where WBI is the weight of the syringe before injection (g), WAI is the weight of the syringe after injection (g), DS is the dilution of the standard solution and SA is the mean activity in the standard solution sample per ml (counts/ml).

Estimation of ⁵¹Cr-EDTA clearance, based on two blood samples (2BS), consisted of calculating the area under the curve (AUC) with a one-compartment model. The plasma activity (A) was assumed to follow a mono-exponential decrease (λ) over time (T). From the injected dose and 2BS, the GFR (expressed in ml/min) could be calculated as follows:

$$\text{AUC}={\displaystyle \underset{\text{T}=0}{\overset{\infty }{\int }}{\text{A}}_0{\text{e}}^{-\lambda \text{T}}}={\text{A}}_0/\lambda$$

The plasma concentration A₀ at T₀ and the mono-exponential decrease λ could be calculated from the plasma concentrations A₁ and A₂ (in counts/min × ml) at time T₁ and T_2:

$$\begin{array}{l}{\text{A}}_1={\text{A}}_0{\text{e}}^{-\lambda \text{T}1}\\ \lambda =\text{In}\left({\text{A}}_1/{\text{A}}_2\right)/\left({\text{T}}_2/{\text{T}}_1\right)\end{array}$$

Statistical analysis

Paired sample t-tests were performed to evaluate if global GFR based on percentage dose uptake of ^99mTc-DTPA differs according to the background ROI and time interval that had been used. Paired Student’s t-tests were also used to evaluate if significant differences existed between the GFR values based on percentage dose uptake of ^99mTc-DTPA and GFR values based on ⁵¹Cr-EDTA clearance. Linear regression analyses were performed to determine the correlation coefficients (r value) between the GFR obtained with ^99mTc-DTPA and GFR obtained by ⁵¹Cr-EDTA clearance for the different background ROIs and the different time intervals. Additionally, Bland–Altman plots were generated by plotting the differences between GFR estimated with ^99mTc-DTPA and ⁵¹Cr-EDTA on the y-axis versus the average of the estimated GFRs with ^99mTc-DTPA and ⁵¹Cr-EDTA on the x-axis.

Results

GFR values calculated with one bg ROI, whether this was the C-shape, caudolateral or cranial + caudal bg ROI, but using different time intervals, significantly differed from each other (P <0.01). The 120–180 s time interval, showed a significant difference between the C-shape and cranial + caudal bg ROI (P <0.0001), and between the caudolateral and cranial + caudal bg ROI (P = 0.006). No significant difference was observed between the C-shape and caudolateral bg ROI (P = 0.591) for this time interval. For the 60–120 s and 60–180 s time interval, however, no significant difference (P >0.01) was observed between the different bg ROIs, although lower P-values were noted between the cranial + caudal bg ROI and caudolateral bg ROI (P = 0.026 and P = 0.034, respectively), and between the cranial + caudal bg ROI and C-shape bg ROI (P = 0.028 and P = 0.025, respectively) than between the C-shape and caudolateral bg ROI (P = 0.655 and P = 0.765, respectively).

The mean values and standard deviations of the GFR calculated with ^99mTc-DTPA for the different bg ROIs in the different time intervals and for the GFR calculated with ⁵¹Cr-EDTA (single injection, 2BS) are noted in Table 1.

Table 1.

Mean value and SD of glomerular filtration rate (GFR) calculated with ^99mTc-DTPA for the different background regions of interest at the different time intervals, and mean value and SD of GFR calculated with ⁵¹Cr-EDTA (single injection, two blood samples) in nine healthy European domestic shorthair cats

		Mean (ml/min/kg)	SD
99mTc-DTPA	60–120 s
	C-shape	1.95	0.39
	Caudolateral	1.97	0.40
	Cranial + caudal	2.08	0.37
	120–180 s
	C-shape	2.33	0.45
	Caudolateral	2.36	0.50
	Cranial + caudal	2.54	0.44
	60–180 s
	C-shape	2.13	0.41
	Caudolateral	2.15	0.48
	Cranial + caudal	2.26	0.46
51Cr-EDTA	Two blood sample	2.42	0.29

The P-values of paired Student’s t-test and correlation coefficients (r value) between the global GFR calculated with ⁵¹Cr-EDTA (single injection, 2BS) and the global GFR calculated with ^99mTc-DTPA based on the formula of Uribe et al ⁸ , for the different bg ROIs at the different time intervals, are summarized in Table 2.

Table 2.

P-values of paired Student’s t-tests and correlation coefficients (r) between the global glomerular filtration rate (GFR) calculated with ^99mTc-DTPA, based on the formula of Uribe et al, ⁸ and the global GFR calculated with ⁵¹Cr-EDTA (single injection, two blood samples) for the different background regions of interest (ROIs) at the different time intervals

Time interval and background ROI	P	r
60–120 s
C-shape	0.0007	0.743
Caudolateral	0.0004	0.827
Cranial + caudal	0.0022	0.788
120–180 s
C-shape	0.406	0.764
Caudolateral	0.588	0.797
Cranial + caudal	0.267	0.750
60–180 s
C-shape	0.012	0.759
Caudolateral	0.014	0.882
Cranial + caudal	0.113	0.850

A good positive correlation coefficient (r >0.74) was present, for all three bg ROIs in all three time intervals, between ^99mTc-DTPA and ⁵¹Cr-EDTA. The caudolateral bg ROI yielded the highest correlation coefficients in all three time intervals, but only the 120–180 s time interval did not show a significant difference (P <0.05) between ^99mTc-DTPA and ⁵¹Cr-EDTA. This could be visualized by the Bland–Altman plots (Figure 4). The caudolateral bg ROI in the 120–180 s time interval, however, had a remarkably lower correlation coefficient (r = 0.797) compared with the other time intervals (r = 0.882 and 0.827 for the 60–180 s and 60–120 s time interval, respectively). The best correlation was found using the caudolateral bg ROI in the 60–180 s time interval.

Figure 4.

Bland–Altman plots of glomerular filtration rate (GFR) estimated with ^99mTc-DTPA with the caudolateral background region of interest for the different time intervals compared with the two blood samples ⁵¹Cr-EDTA. (a) The horizontal axis represents the mean of the GFR estimated with ^99mTc-DTPA (60–120 s) and ⁵¹Cr-EDTA; the vertical axis represent the difference between the GFR estimated with ^99mTc-DTPA (60–120 s) and ⁵¹Cr-EDTA. (b) The horizontal axis represents the mean of the GFR estimated with ^99mTc-DTPA (120–180 s) and ⁵¹Cr-EDTA; the vertical axis represents the difference between the GFR estimated with ^99mTc-DTPA (120–180 s) and ⁵¹Cr-EDTA. (c) The horizontal axis represents the mean of the GFR estimated with ^99mTc-DTPA (60–180 s) and ⁵¹Cr-EDTA; the vertical axis represent the difference between the GFR estimated with ^99mTc-DTPA (60–180 s) and ⁵¹Cr-EDTA. The mean difference is given by the straight middle line. The area between ±1.96 SD is given by the dashed outer lines

In general, the ^99mTc-DTPA technique had a tendency to underestimate the GFR when compared with the results of ⁵¹Cr-EDTA, with exception of the cranial + caudal bg ROI in the 120–180 s time interval. In the 60–180 s time interval ^99mTc-DTPA underestimated the GFR by approximately 12%.

Discussion

^99mTc-DTPA is an important radiopharmaceutical for GFR determination in clinical practice because of its labelling with ^99mTc rendering it the most suitable agent to use for conventional gamma camera imaging, the ease of preparation for either imaging or external counting of plasma samples, and the possibility of calculating the individual kidney GFR.^2,18

Correction for background radioactivity is an important step when determining GFR, with dynamic renal scintigraphy, based on the percentage uptake of ^99mTc-DTPA. Over- or under-correction of background radioactivity may under- or overestimate the GFR. Background radioactivity can be separated into extra- and intrarenal components. As intrarenal background radioactivity is included in the kidney ROI, it cannot be separated from the kidney. By using the regression equation of Uribe et al, ⁸ which was developed by plotting DTPA uptake data against inulin clearance data, the net counts are corrected using the GFR of inulin clearance data whereby the error induced by intrarenal background radioactivity is eliminated. Therefore, the external background radioactivity can be considered as a reasonable estimate of the total background radioactivity. ²⁰ For ^99mTc-DTPA, with its low extraction ratio (20%) and high background activity, the choice of location of the background ROIs is very important. Many regions around the kidneys can be chosen as background. Previous studies used backgrounds ROIs as small areas at the cranial and caudal poles of the kidneys,^2,6,18 at only the caudal pole of the kidney^10,18,21 and around the entire kidney.^10,20

In our study, significant differences between bg ROIs were only observed in one time interval (120–180 s) between the C-shape and cranial + caudal bg ROIs, and between the caudolateral and cranial + caudal bg ROIs. Why only a significant difference was seen in this time interval and not in the two others (60–120 s and 60–180 s) could not be explained. Although in those latest’s time intervals (60–120 s and 60–180 s) lower P-values were also noted between the C-shape and cranial + caudal bg ROIs, and between the caudolateral and cranial + caudal bg ROIs.

By comparing ^99mTc-DTPA with ⁵¹Cr-EDTA, the caudolateral bg ROI revealed the highest correlation coefficients at all time intervals. This corresponds to the result of Gates ¹⁰ in human patients, where caudolateral bg ROI was compared with the C-shape bg ROI in comparison with a 24 h creatinine clearance. Compared with the caudolateral bg ROI, the C-shape bg ROI had a tendency to overestimate the extrarenal background radioactivity, whereas the combination of two rectangular bg ROIs, one at the cranial and one at the caudal pole of the kidney, had a tendency to underestimate the background radioactivity. Therefore, even though differences between bg ROIs were not all significant, consistency in GFR scan processing is advisable, especially in follow-up examinations of the same patient.

Different time intervals seem to cause significant variation in absolute GFR values, regardless of the choice of the bg ROI. The GFR values in the 60–180 s time interval are the average of the values in the two other time intervals, with the GFR values in the 60–120 s time interval being significantly lower than the GFR values in the 120–180 s time interval. When looking at a normal renogram, three phases can be identified. The first phase is the circulation phase where a rapid initial rise within 15–20 s of injection of the radiopharmaceutical is followed by a down slope, which reaches an inflection point after at about 20–40 s. The second phase is the uptake phase as the kidney accumulates the radiopharmaceutical in the nephrons through glomerular filtration, represented by a gradual increase in activity. Peak renal activity is reached 2.5–3.5 min after injection. This phase expresses the functional capacity of the renal parenchyma and is therefore the most suitable for GFR determination by gamma camera. The third phase or excretory phase is represented by a falling slope as kidney activity decreases as the radiopharmaceutical passes out of the collecting system into the lower urinary tract.^18,22 In conclusion the 120–180 s time interval seems to be the most accurate time period for determining GFR, and GFRs obtained in time intervals prior to this are expected to be lower, which was confirmed by our data.

In general, the ^99mTc-DTPA imaging technique had a tendency to underestimate the GFR when compared with ⁵¹Cr-EDTA clearance. Imaging studies are based on the first few minutes of data, whereas plasma clearance studies acquire data over longer time intervals and are therefore presumed to be more accurate. ² In dogs, Barthez et al ¹ compared GFRs determined by ^99mTc-DTPA plasma clearance and image-based quantitative renal scintigraphy to plasma and urinary inulin clearance. They concluded that both percentage uptake and plasma clearance of ^99mTc-DTPA give reasonable estimates of filtration function, but that ^99mTc-DTPA plasma clearance is a more accurate method.

Further validation of the ^99mTc-DTPA imaging technique is needed as our study showed some limitations. Only a low number (n = 9) of healthy cats was used and comparison was not tested over a representative range of GFR values, including cats with chronic kidney disease and hyperthyroidism. Ideally, for validation of an alternative method comparison to a gold standard method is indicated. The 2BS ⁵¹Cr-EDTA has proven its applicability as a GFR marker in cats, ¹⁷ but is not yet a well validated method and still requires testing in a different population of cats and against other methods of determining GFR. Although multi-sample methods give a better estimation of the actual GFR, they are also cumbersome to perform and cause stress and discomfort to the animals. During the scan the cats were anaesthetised with propofol. This should not have had an influence on the results of our study as propofol has no direct effect on the renal function.^23,24 Another limitation was that, as Kampa et al ²⁵ pointed out in their study, each laboratory using the gamma camera method for calculating GFR should make its own regression equation relating percentage uptake to GFR by plasma clearance because this relationship is affected by, for instance, camera sensitivity, imaging technique, time interval selection, kidney and background ROI drawing. In our study we used the regression equation of Uribe et al, ⁸ who used a caudolateral background and the 60–180 s time interval. Further tests plotting percentage ^99mTc-DTPA uptake against inulin clearance data for the different background ROIs and time intervals are indicated to determine the optimal regression equation.

Conclusions

Scintigraphic imaging with ^99mTc-DTPA is a sensitive technique to estimate the kidney function in cats, but different time intervals and different background ROIs can cause variation in absolute GFR values. Therefore, consistency in GFR scan processing is needed, especially in follow-up examinations of the same patient. In our study, the best estimate of the GFR using the percentage dose uptake of ^99mTc-DTPA was obtained in the 60–180 s time interval using data that were background corrected with the caudolateral bg ROI.