ClinIcal ComparIson of Cardiac Blood Pool Visualization

Dynamic imaging of the cardiac blood pool toanalyze regional myocardial motion and to calculatethe ejection fraction has become increasingly important in myocardial disease, both in the initialdiagnosis and in the serial evaluation of therapeutic

interventions (1—4). Fundamental to the procurement of high-quality blood-pool studies is the useof a suitable radiopharmaceutical that remains withinthe vascular space.

There are two major categories of tracers available for blood-pool imaging: labeled red blood cells(RBC) and labeled human serum albumin (HSA).Recently, Pavel and associates (5) have describedan in vivo procedure for RBC labeling with [aomTc]pertechnetate. The in vivo approach affords severaldistinct advantages over in vitro methods of labelingRBC (6,7) and potential advantages over the useof HSA. Preliminary data obtained from serial bloodsampling indicates that the in vivo approach provides good initial labeling efficiency, with the recovered activity remaining in the red-cell fraction forprolonged periods, allowing blood-pool images ofgood quality to be readily obtained (5,8,9).

We have undertaken a prospective study to evaluate imaging of the cardiac blood pool with technetium-99m red blood cells (Tc-RBC) labeled by the

in vivo approach, compared with two preparationsof Tc-99m human serum albumin (Tc-HSA).

MATERIALSANDMETHODS

Radiopharmaceutical preparation. Tc-HSA wasprepared using both a commercial stannous kit* anda modified inhouse electrolytic procedure (10).Manufacturer's instructions were followed for preparation from the commercial kit.

For the inhouse preparation, a tin wire anode anda stainless steel wire cathode are thoroughly cleanedand rinsed, and inserted through the diaphragm ofa sterile apyrogenic 10-mi shielded reaction vial.Added to this vial are: a) the desired activity of[99mTc]pertechnetate (Tc04) and 2.6 ml of physiologic saline; b) 0.2 ml of 25% HSAf; and c) 0.85ml of 1 N HC1. A constant current of 100 mA isthen applied to the electrolytic cell for 55 sec. During electrolysis, the reaction vial is agitated gently

Received Dec. 21, 1977; revision accepted Feb. 10, 1978.For reprints contact : James H. Thrall, University of Mich

igan Medical Ctr., Nuclear Medicine Sec., 1405 E. Ann St.,Ann Arbor, MI 48109.

C Present address: Manuel L. Brown, Div. of NuclearMedicine, Mayo Clinic, Rochester, MI 55901.

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with Technetium-99m Red Blood Cells Labeled in Vivo

and with Technetium-99m Human Serum Albumin

James H. Thrall, John E. Freitas, Dennis Swanson, W. Leslie Rogers, Jean M. Clare,Manuel1.Brown*,and BertramPitt

University of Michigan Medical Center, Ann Arbor, Michigan

Technetium-99m red blood cells (Tc-RBC) labeled by an in vivo technique were compared with two preparations of Tc-99m human serum albumm (HSA) for cardiac blood-pool imaging. Relative distribution of thetracers was analyzed on end-diastolic frames of gated blood-pool studies andon whole-body (head to mid-thigh) anterior pinhole images.

The Tc.RBC demonstrated greater relative percentage localization in thecardiac blood poo1, higher target-to-background ratios in the left ventricle,and less liver concentration. For cardiac blood-pool imaging, Tc-RBC labeledby the in vivo approach appears to be superior to the two Tc-HSA preparations studied.

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strips in a physiologic saline solution of HSA (5mg/ml) for 30 mm. The strips were rinsed for 5 secwith distilled water, allowed to dry, and stored at0—SOCuntil used. On presoaked ITLC-SG, usingethanol: ammonia :water (2 : 1:5 ) as the solvent,TcO4 and Tc-HSA migrate with an R@ of 1.0;whereas colloidal Tc-99m remains at the origin (Fig.1C, D). This chromatographysystemwas validated by separately analyzing the migration of TcO4and that of Tc-99m colloid produced by the dcctrolysis method in the absence of HSA.

Patients were injected intravenously with 20 mCiof Tc-HSA in an average volume of 3.3 ml. Scanning procedures commenced within 5 mm of theinjection.

In vivo Tc-RBC labeling was accomplished bythe method of Pavel et al. (1 ) . A commercial kitscontaining stannous pyrophosphate [15.4 mg (6.3mg Sn@+ )} is reconstituted with 5.0 ml of 0.9%NaCl. The height and weight of the patient are obtamed in order to estimate patient's blood volumefrom standard tables. The amount of stannous pyrophosphate to be injected is calculated by the formula:

Patient blood volume .5 400 1@ 2.5 ml volume injected

, m (blood volume standardman)

In no case is more than 2.5 ml (7.7 mg stannous pyrophosphate) administered. Injection is performed immediately following reconstitution.

Approximately 30 mm after the injection of stannous pyrophosphate, patients receive 20 mCi TcO4i.v. Scanning procedures then start within 5 mm.

Imaging methods. For clinical studies, multiplegated blood-pool images of the heart are obtained.A format is used that provides 28 frames per cardiaccycle, and imaging is continued long enough to collect 300,000 counts per frame. A low-energyparallel-hole collimator is used on a scintillationcamera with conventional field of view, interfacedto a computer. All patients are studied in both theanterior and left anterior oblique projections. Thelatter view is used to calculate the ejection fractionusing an automated edge-detection algorithm to define regions of interest over the left ventricle (12).The images from both projections are reviewed ina dynamic-display format to analyze regional wallmotion.

Fifty consecutive studies obtained with Tc-HSA(inhouse preparation) and 50 with Tc-RBC werereviewed to assess their relative quality in the dynamic-display format and to identify any factorsadversely affecting the image. To determine relativequantitative tracer distribution in the field of viewin clinical studies, the following calculations were

FIG. 1. SampleTc-HSAchromatography.Tc04(A)andTc-HSA(B) are readily separated by conventional ITLC-SG system. In (C)and (D), ITLC-SG strips were presoaked in albumin, allowing separation of Tc-HSA from Tc-colloid.

using a circular motion. The reaction mixture is thenallowed to stand at room temperature for 30 mm.Following removal of the electrodes, 0.6 ml of1 N sodium hydroxide and 0.8 ml of 8.4% NaHCO3solution are added, buffering the preparation to afinal pH of about 7.4. The product is subsequentlyassayed and passed through a 0.22-it bacterial filter.The final volume is about 5 ml.

Quality-control procedures are routinely performed on all technetium radiopharmaceuticals.Sterility and pyrogen testing are done on pooledsamples radiometrically@ and limulus lysate testing,respectively. All preparations are tested, before administration, for correct pH using narrow-range pHindicator paper. Radionuclidic and radiochemicalpurity of the generator eluate is ascertained beforethe preparation of any Tc-99m radiopharmaceutical.

Both the inhouse and the commercial-kit Tc-HSAwere analyzed before injection for the presence ofunreacted, unreduced TcO4 and colloidal forms ofTc-99m. Instant thin-layer chromatography, usingsilica gel (ITLC-SG) as the support medium and85 % methanol as the solvent, resulted in the separation of Tc04 (R@ = 1) from Tc-HSA and colloidal Tc-99m (both R@= 0) (Fig. IA, B). Becauseof the albumin loading effect (1 1 ) the presence ofcolloid was determined after presoaking ITLC-SG

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A B

THRALL, FREITAS, SWANSON, ROGERS, CLARE, BROWN, AND PITT

liminary studies this field of view was shown toinclude over 95 % of administered activity. Sincethe major areas of interest are in the center of thefield, no field correction was made for the pinhole.The images were run for 5 mm at the end of routineclinical studies (about 45 mm after injection) andstored on the computer system for analysis. Regions of interest over the central blood-pool activity(heart and great vessels) , the liver, and the bladderwere defined with a light pen. Gross counts fromeach region were determined and expressed as apercentage of the total image counts to normalizebetween subjects.

To estimate maximum regional tracer concentrations, the maximum count in a single image elementin the central blood-pool region, and another in theliver region, were determined for each of the pinholeimages, and ratios between these maxima were calculated.

The Wilcoxan rank sum test (13) and Student'st@testwere used respectively for statistical analysisof the pinhole and clinical study data.

RESULTS

Chromatographic analysis of Tc-HSA preparedby electrolysis or from the commercial stannous HSAkit revealed total labeling efficiencies greater than91 % in all cases. With the electrolytic method, labeling efficiency averaged 96.21 % ± 1.89 s.d.(range 94. 1—99.5) . Colloidal impurities in this preparation averaged 2.13% ± 1.73 s.d. (range 0.2—5.2) . No colloidal impurities were detected in thecommercial stannous-HSA kit, and the total labeling efficiency averaged 94.5% ± 2.78 s.d. (range91—98.6). These results are consistent with previousreports describing in vitro analysis of Tc-HSA preparations (10,11,14—16).

Although diagnostically useful images were ob

A

B

.@j

C

FIG. 2. (A) End-diastolicframein left anteriorobliqueprojection. (B) Computer-generated outline of left ventricle (bright dots)is used to define area of interest. (C) End-systolic frame with background area indicated by highlighted area.

made in the first 20 consecutive cases with Tc-HSA,and in the first 20 with Tc-RBC: a) net left-ventricular counts at end-diastole as a percentage of thetotal image count; b) average net counts per imageelement in the left ventricle at end-diastole; c) average background counts per image element; and d)the target-to-background ratio for the left ventricle( = average net left-ventricular counts per channeldivided by average background counts per channel).All calculations were made using regions defined byan automated edge-detection algorithm (Fig. 2) onthe end-diastolic frames. Background in this cornputer program is taken from a crescentic area lateralto the left ventricle at end-systole (Fig. 2C).

To determine the relative tracer distribution inthe central blood pool, liver, and bladder with theTc-RBC and the two HSA preparations, ten consecutive patients were imaged in the anterior projection with a pinhole aperture. The field of view ineach case was from head to mid-thigh. The average

subject-to-collimator distance was 120 cm. In pre

FIG. 3. End-diastolicframe from Tc-HSAstudy.In (A), computer display of heart is suboptimal because of scaling to highestcount portion of liver image. In (B), cardiac blood pool is bettervisualized after foreground suppression with scaling of display tohighest count in heart. Display intensity was not changed.

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AverageAveragenet[Netleftbackgroundleft-ventricularventricularcounts

percountspercounts]X 100image elementimage elementTarget-to

total image(64 X 64(64 X64backgroundCaseNo. Total counts countsarray)array)

A. Tc-HSA

CLINICAL SCIENCESINVESTIGATIVE NUCLEAR MEDICINE

TABLE 1. Tc-HSA AND Tc-RBCDISTRIBUTION AT END-DIASTOLE

1300,0006.07367.922300,0008.08459.703300,0007.343541.264300,0006.556571.025300,00010.47265.906300,00010.441972.227300,0008.87362.858300,0009.368691.019300,0007.08732.3710300,0009.46556.8611300,0004.647481.0212300,0005.16451.8013300,0007.89533.3414300,00010.27168.9615300,0003.87146.6516300,0002.08349.5917300,0005.16059.9818300,0005.57473.9919300,0009.959771.3120300,0005.16558.89Mean

±s.d.Range7.1

±2.412.0—10.467.6

± 14.143—9559.0

± 14.832-@97.93

± .39.372.221300,00011.440541.342300,00011.0491022.093300,0008.375951.274300,0006.28776.875300,00010.842831.986300,0007.636782.177300,0007.150831.668300,0005.09067.759300,00010.49366.7110300,0008.77667.8911300,0008.560881.4712300,00010.25952.8813300,0006.3120102.8614300,0008.4821181.4415300,00010.0841331.5916300,0008.19583.8717300,00014.8821401.7218300,00016.31051301.2519300,00013.3961501.5620300,0006.88467.79Mean

± s.d.Range9.4

±2.805.0—15.375.2

±23.336—12091.7

± 29.052—1501.31

± .47.71—2.17

B.Tc-RBC

tamed with both types of agent, a striking differencewas observed in the qualitative appearance of theTc-RBC images compared with Tc-HSA images.With Tc-RBC, the liver had consistently less activity, relative to the cardiac blood pool, than was notedwith either Tc-HSA preparation. When liver activitywas significantly greater than cardiac blood-pool activity in the Tc-HSA images, the computer's displaywas adversely affected as a result of scaling to the

highest channel count in the image; this requiredforeground suppression for optimum viewing of thecardiovascular structures (Fig. 3).

In the left anterior oblique images, the net enddiastolic activity in the left ventricle, as a percentageof total image counts, averaged 9.4 ±2.80 s.d. withTc-RBC and 7.1 ± 2.41 s.d. with Tc-HSA (p <0.01, Table 1). The left-ventricular target-to-background ratios averaged 1.3 1 ±0.47 for the Tc-RBC

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Maximumcountsperchannel—

centralCentralblood

poolblood-poolCentralMaximumBladdercountLiver

countsblood-poolcountspercountstotalimagetotal imagecountschanneltotalimageCase

No. countscountsliver counts— livercounts

Tc-99m HSA (NEN)

THRALL, FREITAS, SWANSON, ROGERS, CLARE, BROWN, AND PITT

TABLE 2. ACTIVITY DISTRIBUTION BY PINHOLE IMAGING

Tc-99m HSA(electrolytic)Tc-99m

RBCTc-99m

HSA (NEN)Mean ± s.d.Range.13

± .04.08—.23.19

± .07.12—.3533

±.22.35—1.13.94

± .10.77—1.08.04

±.03.01—.09Tc-99m

HSA (electrolytic)Mean ± s.d.Range.15

± .03.10—.20.18

±.06.12—.31.87

±.30.47—1.461.13

±.25.79—1.55.03

±.02.01—.07Tc-99m

RBCMean ±s.d.Range.24

±.06.15-.36.12

± .03.06—.162.04

± .741.31—3.142.38

± .781.44—4.27.03

± .02.01—.07

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and averaged 0.93 ±0.39 s.d. for studies with TcHSA (p < 0.01, Table 1).

The pinhole images showed 24% of the total activity concentrated in the central blood pool forTc-RBC, compared with an average of only 14% forTc-HSA (Table 2) . At the same time, fractionalliver activity was 50% greater for Tc-HSA. Thesedifferences in activity were highly significant for bothregions (p < 0.01 ) . The observation of increasedblood-pool activity and reduced liver activity forTc-RBC is in agreement with the clinical image data

but direct numerical comparison cannot be madedue to differences in the fields of view. No differenceswere observed in the bladder regions.

The quantitative differences in relative distribution are readily appreciated by inspection of thepinhole images. Figures 4, 5, and 6 illustrate representative cases obtained with each of the three preparations, with corresponding isometric displays. Inthe albumin images (Figs. 5, 6), the large secondpeak seen in the isometric displays is due to the highliver activity.

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: FIG. 4. Whole-bodypinhole images(A) and isometricdisplays(B)from selectedcaseswith Tc-RBC.Note single large peakin isometric displays for cardiac bloodpool. (Numbersrefer to casenumbersfrom

:Tc@@2).

FIG.5. Whole-bodypinholeimageswith inhouse Tc-HSA preparation. Not.second large peak in isometric display,due to liver activity.

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THRALL, FREITAS, SWANSON, ROGERS, CLARE, BROWN, AND PITT

FIG.6. Whole-bodypinholeimageswith stannous-HSA kit. Note again doublepeaking in isometric display, due to liveractivity.

ing provides a useful additional method of analyzingtracers by allowing direct comparison of their actualin vivo distribution in the clinical setting.

The differences in in vivo localization of Tc-RBCand Tc-HSA have technical implications and directly affect the quality of cardiac blood-pool imaging. The greater count rate in the heart's blood poolis an advantage of Tc-RBC over Tc-HSA : it reducesimaging time for equivalent statistical accuracy indelineating the left ventricle. It must be stressed thatthis may not be appreciated if one considers thetotal count rate in the detector's field of view. Ifonly the photons arising in the area of diagnosticinterest (i.e., left ventricle) are considered, however,the data indicate that the useful count rate with Tc

RBC is greater per mCi of administered activity.The higher target-to-background ratio achieved withTc-RBC facilitates the application of automatededge-detection algorithms, which have become usefulin calculating the ejection fraction.

A less obvious but important consideration is theeffect of “out-of-view―activity on the energy spectrum and on the total events that the gamma camerais required to process. Any increased liver activitywith Tc-HSA increases the number of internallyscattered Compton photons in the image, with concomitant loss of contrast. Also, camera deadtimelosses are increased relative to the useful count ratebecause of the greater integral count rate.

DISCUSSION

Several investigators have suggested that Tc-RBCmight be superior to Tc-HSA for cardiac blood-poolimaging because of the in vivo stability of Tc-RBCand the observation that albumin leaks from thevascular space with time (5,6,16,17). Such leakagereduces effective tracer activity and the target-tobackground ratio. Although we did not documentthe leakage problem directly by obtaining serialblood samples, the lower heart-to-background ratiosachieved with Tc-HSA, as compared with Tc-RBC,are an indicator of this phenomenon.

We attempted to explain the marked liver uptakeOf the Tc-HSA preparations by chromatographicanalysis to detect Tc-colloids or other reduced-Tccontaminants that would be actively taken up by theliver. Both Tc-HSA preparations, however, demonstrated good in vitro labeling efficiencies. The smallamount of Tc-colloid detected chromatographicallyis not sufficient to explain the marked liver activitywith Tc-HSA, and confirms the observations ofRhodes that conventional in vitro procedures arenot always good predictors of in vivo behavior (16).

The marked Tc-HSA liver activity may be due toin vivo loss of the Tc-99m label with subsequenthydrolysis to colloid, but it is more likely the resultof HSA denaturation at the acidic pH used in thelabeling procedures. Although the exact mechanismis still not known, it is obvious that whole-body imag

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Another direct advantage of the in vivo approachto Tc-RBC labeling is that it allows use of smallvolume, high-concentration bolus injections that facilitate “firstpass―studies performed in conjunctionwith equilibrium imaging. In the RBC labeling technique, the injected volume of radioactivity is dependent only on the specific concentration of the

generator eluate. In the case of the HSA radiopharmaceuticals, the volume of injection is dependent onboth the eluate concentration and the volume requirements of the involved preparation. The commercial stannous-HSA kit used in the study requiresa minimum volume of 2 ml for reconstitution, andthe electrolytic procedure results in a final volumeof 5 ml.

Two disadvantages of the in vivo Tc-RBC procedure are the inability to confirm whether the initial“cold―pyrophosphate injection has been extravasated and the requirement for two separate injections30mm apart.

In a small series reported by Stokely et al. (18),in vivo Tc-RBC labeling was insufficient for clinicalblood-pool imaging in two of 22 patients when99mTcpertechnetate was administered 24—48hr afterTc-99m stannous pyrophosphate. In the cases included in the current study and in over 200 subsequent cases with in vivo Tc-RBC, however, we havenot encountered a failure to achieve clinically usefullabeling; nor did Pavel et al. note such a labeling

problem in their first 75 studies (5). The 24—48hrdelay and the difference in the form of pyrophosphate administered may account for Stokeley's resuIts (18).

The results of this study suggest that Tc-RBC Iabeled by the in vivo technique is a superior cardiacblood-pool imaging agent compared with the twoTc-HSApreparationsstudied.Tc-RBCdemonstratesgreater cardiac blood-pool activity levels and superior target-to-background ratios, and should become the agent of choice, among the radiotracersexamined, for cardiac blood-pool studies.

FOOTNOTES

* Cardiolite, New England Nuclear Corp., Boston, Mass.

t Hyland,CostaMesa,Calif.@ Technescan PYP, Mallinckrodt.

IIBACTEC,JohnstonLaboratories,Inc.,Cockeysville,Md.

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2. PITT B, STRAUSS HW: Myocardial perfusion imaging

and gated cardiac blood pool scanning: Clinical application. Am I Cardiol 38: 739—746,1976

3. PIERSONRN, ALAM S, KEMP HG, et al: Radiocardiography in clinical cardiography. Seniin Nuci Med VII: 85—100, 1977

4. STRAUSSHW: Cardiovascular nuclear medicine: Anew look at an old problem. Radiology 121 : 257—268,1976

5. PAVEL DG, ZIMMER AM, PATrERSONVN: In vivolabeling of red blood cells with ‘@“Tc:A new approach toblood pool visualization. J Nucl Med 18: 305—308,1977

6. SMITH TD, RICHARDS P: A simple kit for the preparation of “mTc-labeledred blood cells. I Nucl Med 17: 216—132, 1976

7. Rvo UY, MOHAMMALZADEHAA, SIDDIQUIA, Ct al:Evaluation of labeling procedures and in vivo stability of‘@mTc-redblood cells. I Nuci Med 17: 133—136,1976

8. COLOMBETTILG, SIDDIQUIA: Efficiency of in vivolabeling of red blood cells with “Tc. Nuclear Medizin 15:211—213,1976

9. HAMILTON RG, ALDERSON P0: A comparative evaluation of techniques for rapid and efficient in vivo labelingof red blood cells with [@‘mTc]pertechnetate. I Nuci Med18: 1010—1013,1977

10. DWORKINHi, GuncowSKl RF: Rapid closed-systemproduction of ‘@mTc-albuminusing electrolysis. I Nuci Med12: 562—565,1971

11. LIN MS, KRUSE SL, GOODWIN DA, et al: Albuminloading effect: A pitfall in saline paper analysis of “@‘Tcalbumin. I Nuci Med 15: 1018—1020,1974

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13. HOLLANDER M, WOLFE DA : Nonparametric statistical

methods. New York, John Wiley and Sons, 1973, pp 68,282

14. MCLEAN JR. WELSH WI: Measurement of unbound@mTcin ‘@mTc-labeledhuman serum albumin. Letter to theEditor.INuclMed 17:758—759,1976

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16. RHODES BA: Considerations in radiolabelling of albumin. Semin Nuci Med 4: 281—293,1974

17. BLAHDWH: Nuclear Medicine. New York, McGrawHill, 1965, pp 598—610

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1978;19:796-803.J Nucl Med.   James H. Thrall, John E. Freitas, Dennis Swanson, W. Leslie Rogers, Jean M. Clare, Manuel L. Brown and Bertram Pitt  Blood Cells Labeled in Vivo and with Technetium-99m Human Serum AlbuminClinical Comparison of Cardiac Blood Pool Visualization with Technetium-99m Red

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