Comparative Studies of the Function and Morphology of 111In-and 51Cr-Labelled Human Platelets

Comparative studies of the aggregability in vitro and ex vivo, and of the surface/volume ratio of ¹¹¹In- and ⁵¹Cr-labelled human platelets were carried out. The ADP-induced aggregation in vitro of ¹¹¹In-platelets was superior to that of ⁵¹Cr-platelets, as was that of ⁵¹Cr-platelets labelled in plasma as compared to ⁵¹Cr-platelets labelled in buffer. These differences seemed to be reversed in vivo, as identical collagen-induced aggregation responses were observed ex vivo when comparisons were made between ¹¹¹In- and ⁵¹Cr-platelets, and between labelled and unmanipulated platelets. Morphometric determination of the surface/volume ratios of the labelled platelets indicated a higher degree of platelet activation of ⁵¹Cr-platelets labelled in buffer as compared to those labelled in plasma. In this respect, no difference seemed to be present between ¹¹¹In- and ⁵¹Cr-platelets. The results of the ex vivo aggregation studies were unaffected by the time spent by the platelets in the circulation within 24 h post-injection, by platelet isolation yield, and by the medium used in ⁵¹Cr-labelling. Our results indicate that it will be possible to conduct comparative studies of simultaneously induced aggregation ex vivo of different platelet populations labelled with ¹¹¹In and ⁵¹Cr.     

The in vivo kinetics of 111In- and 51Cr-labelled platelets: a comparative study using both stored and fresh platelets

The in vivo kinetics of simultaneously injected 51Cr- and 111In-labelled platelets was investigated in 20 healthy male volunteers. The studies were carried out using both fresh platelets and platelets stored for 5 d at 22 degrees C; the disappearance of platelet-bound radioactivity was measured on whole blood samples as well as platelet pellets. Compared to 111In, the labelling efficiency was notably lower for 51Cr, and a higher amount of 51Cr was bound to contaminating red cells. As regards the fresh platelets, only small dissimilarities were observed in the in vivo kinetics obtained with the two labels. 51Cr yielded a slightly higher platelet recovery and longer T1/2 than 111In, when whole blood samples were used for calculations; no differences were seen when using platelet pellets. When stored platelets were studied, 51Cr gave significantly longer T1/2 and mean life-span (MLS) than did 111In. This difference was present for all mathematical models used for the calculation of MLS, and when whole blood samples as well as platelet pellets were employed. It was demonstrated that the estimation of MLS was also highly dependent upon techniques of blood sampling and curve fitting. Calculation, in which the initial data points were excluded, gave consistently longer MLS (P less than 0.0001), as compared to when all data points were included. Furthermore, when all survival studies were grouped together, calculations using platelet pellets gave a significantly (P less than 0.001) shorter platelet MLS than calculations using whole blood samples. It is concluded that both 51Cr and 111In are acceptable as radiolabels for both fresh and stored platelets. However, it appears that the viability of stored platelets may be influenced by the choice of label, and caution must be taken when these isotopes are used together in dual tracer experiments. Also, our results show that a meticulously standardized processing of blood samples and experimental data is required to enable interlaboratory comparisons of the results.     

Comparative Studies of the in Vivo Kinetics of Simultaneously Injected 111In- and 51Cr-Labelled Human Platelets

The kinetics of simultaneously injected ¹¹¹In- and ⁵¹Cr-labelled platelets have been assessed in 40 subjects, 13 of them thrombocytopenic. 4 platelet survival models were applied. The mean life-time (MLT) of ⁵¹Cr-platelets from non-thrombocytopenic individuals was found to be slightly, but significantly, longer than that of ¹¹¹In-platelets by applying linear and exponential models for data fitting. The in vivo recovery (IVR) of ¹¹¹In-platelets was significantly higher than that of ⁵¹Cr-platelets in this patient group when using all 4 models. In the group of thrombocytopenic patients no statistically significant differences in MLT or IVR were found between ¹¹¹In- and ⁵¹Cr-platelets. However, for each of the 11 ⁵¹Cr-labelled platelet suspensions with the shortest MLT, a longer MLT was observed in the corresponding ¹¹¹In-platelets, a finding probably related to antibody-induced elution of ⁵¹Cr-activity. The same mechanism might be responsible for an increasing ¹¹¹In-/⁵¹Cr-recovery ratio in the early post-injection period. The efficiency of platelet isolation from blood prior to labelling seemed to influence the IVR, inasmuch as the difference in IVR between ¹¹¹In- and ⁵¹Cr-platelets was eliminated in the group where the yield of ¹¹¹In-platelets surpassed that of the ⁵¹Cr-platelets by more than 15 %.     

Kinetics and distribution in vivo of 111In-labelled autologous platelets in idiopathic thrombocytopenic purpura

The kinetics of autologous 111In-labelled platelets were studied in 26 patients with ITP. The platelet mean life time (MLT) was considerably shortened, the platelet in vivo recovery slightly lowered and the platelet turnover normal. Comparative studies of the kinetics of simultaneously injected 111In- and 51Cr-labelled platelets in 10 patients showed the MLT and turnover of 51Cr-platelets to be shorter and higher, respectively, than those of 111In-platelets, suggesting that 51Cr-labelling in ITP may underestimate platelet MLT and overestimate platelet turnover. Our results confirm that accelerated platelet destruction is an important pathogenetic factor in ITP, and that the platelet concentration may be influenced by increased splenic platelet pooling and by inability of the bone marrow to respond adequately to the low platelet count. Our scintigraphic studies showed that the spleen played an important role for platelet destruction in most patients, with the liver contributing in some patients.     

Concurrent label method with 111In and 51Cr allows accurate evaluation of platelet viability of stored platelet concentrates

Summary. The precision and reproducibility of ¹¹¹In and ⁵¹Cr platelet radiolabel agents for in vivo kinetic studies of stored platelet concentrates (PC) were investigated. The objective was to develop a precise method with concurrent labelling of two platelet populations using different isotopes, which would allow identification of small differences in in vivo platelet quality. Identical labelling procedures were used to investigate the effects of PC storage age, different methods of red cell (RBC) and white cell (WBC) contamination correction, and label elution correction on the results of ¹¹¹In and ⁵¹Cr kinetic studies. ¹¹¹In and ⁵¹Cr platelet survival curves from the same PC, even when uncorrected for elution and RBC contamination, exhibited excellent correlation, irrespective of the age of the concentrate and its viability. However, slightly higher, but statistically significant, post-infusion per cent recoveries with ⁵¹Cr labelled platelets were found. Two factors were identified as the cause for this difference. There was a higher affinity of contaminating RBC/WBC in PC for ⁵¹Cr than for ¹¹¹In. With determination of RBC/WBC activity by centrifugation/density separation, RBC/WBC fractions from the injectate were found to contain 12.6 ± 3·8%v 7·1 ± 3·6% of total ⁵¹Cr and ¹¹¹In activity, respectively, in 20 studies. In addition, there was a significantly higher ¹¹¹In activity in plasma immediately post-infusion than with ⁵¹Cr, 5·2 ± 1·3%v 2·8 ± 1·6%, respectively, suggesting more label elution or carryover. After correction for the activity of RBC/WBC and for elution or carryover, essentially identical ⁵¹Cr/¹¹¹In platelet survival curves were found. In 31 stored PC studies, the absolute average difference between ⁵¹Cr and ¹¹¹In per cent recoveries was only 4 ± 3% in a group of donors whose platelet recoveries ranged from 10% to 80%. Similarly, the average difference between ⁵¹Cr and ¹¹¹In survival was only 8±4 h within a range of survivals from 40 to 220 h. In conclusion, after correction for elution and contaminating RBC/WBC binding, these studies show that ⁵¹Cr and ¹¹¹In may be used interchangeably for labelling of stored PC, and that small differences between test and control platelets could be reliably detected using concurrent labelling with simultaneous infusion.     

Platelet size does not correlate with platelet age

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense- body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.     

Loss of 111Indium as indicator of platelet injury

We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.     

Labelling autologous platelets with 111In tropolonate for platelet kinetic studies: limitations imposed by thrombocytopenia

An in vitro method of radiolabelling platelets with 111In tropolonate in plasma has been devised enabling imaging and cell kinetic studies to be performed in patients with thrombocytopenia (TP) using autologous, rather than donor, platelets. Platelets from 10 TP patients, with platelet counts ranging from 4-91 x 10(9)/l, were labelled in 50% plasma with 111In tropolonate, containing the optimum tropolone concentration of 2 x 10(-4) mol/l, at platelet concentrations ranging from 0.08-4.5 x 10(9)/ml resulting in labelling efficiencies (LE) between 51 and 86%. In 4 patients, red cells contaminated the platelets but this was corrected for by measuring the proportion of 111In on the platelets prior to injection and in the post-injection blood samples. Platelet recovery (PR), mean platelet life-span (MPLS) and platelet production rate (PPR) were calculated and splenic and hepatic images were taken. The results clearly show that useful clinical data can be obtained by this method even in patients with severe TP.     

Platelet turnover and kinetics in immune thrombocytopenic purpura: results with autologous 111In-labeled platelets and homologous 51Cr- labeled platelets differ

Mean platelet survival and turnover were simultaneously determined with autologous 111In-labeled platelets (111In-AP) and homologous 51Cr- labeled platelets (51Cr-HP) in ten patients with chronic immune thrombocytopenic purpura (ITP). In vivo redistribution of the 111In-AP was quantitated with a scintillation camera and computer-assisted image analysis. The patients were divided into two groups: those with splenic platelet sequestration (spleen-liver 111In activity ratio greater than 1.4), and those with diffuse sequestration in the reticuloendothelial system. The latter patients had more severe ITP reflected by pronounced thrombocytopenia, decreased platelet turnover, and prominent early hepatic platelet sequestration. Mean platelet life span estimated with 51Cr-HP was consistently shorter than that of 111In-AP. Platelet turnover determined with 51Cr-HP was thus over-estimated. The difference in results with the two isotope labels was apparently due to greater in vivo elution of 51Cr. Although the limitations of the techniques should be taken into account, these findings indicate that platelet turnover is not always normal or increased in ITP, but is low in severe disease. We suggest that this may be ascribed to damage to megakaryocytes by antiplatelet antibody. The physical characteristics in 111In clearly make this radionuclide superior to 51Cr for the study of platelet kinetics in ITP.     

Five leucocyte labelling techniques: a comparative in-vitro study

During the past years, several authors have used labelled leucocytes to detect inflammatory foci. However, before routine use in man. It is necessary to control the viability of labelled cells. Five leucocyte labelling techniques (111In-oxine, 111In-oxine without extraction, 99mTc oxine, pyrophosphate 99mTc, 51Cr) were compared using the same separation methods, conservation medium, viability assays and migration studies. Electron microscopic studies allowed the assessment of cellular damage induced by the labelling techniques as well as the calculation of the percentage of cells disrupted during preparation. Results obtained in vitro using 111In-oxine were not satisfactory and in fact appeared contradictory to those published by the authors using this technique in vivo. Even the best method, pyrophosphate 99mTc labelling, was not completely atoxic, but the functional behaviour of the leucocytes did not seem affected. While in vitro studies offer much information concerning labelled cells, they cannot predict the in vivo behaviour of these cells.     

The role of nuclear medicine in infection and inflammation

Investigators have used various techniques and radionuclides such as 51Cr and 32P-diisofluorophosphate to label blood cells and to study cell survival. Early studies also used these radionuclides to label human leucocytes for cell survival by in-vitro counting. But external imaging could not be done with these agents. Starting with the use of the gamma-emitting radionuclide (111)In-oxine for in-vitro labelling of phagocytic leucocytes, external imaging became possible. This method was the basis of visualisation of cell distribution within the body. Because an abscess consists primarily of leucocytes, leucocytes labelled with (111)In localise within the abscess and are detectable by imaging. Nowadays other radiopharmaceuticals with other underlying uptake mechanisms are also used to detect inflammatory or infectious foci in patients. Nuclear medicine can be most useful in patients with fever of unknown origin, where a focus has to be defined, or in patients where a lesion is known by clinical symptoms or by a radiological imaging and the differentiation between infection and other pathologies has to be made.     

A simple,fluorescent method to internally label platelets suitable for physiological measurements

Current methods for studying platelet survival in vivo are limited by the use of radioisotopes, with their inherent safety and regulatory concerns, systemic drug administrations that produce biochemical modifications of platelet functions, or external labeling techniques, which may produce artifacts due to surface modifications. For these reasons, we sought to develop a simple, nonisotopic method for labeling platelets internally, thereby producing platelets more likely to have in vivo properties equivalent to native cells. Murine platelets in protein-free buffer were fluorescently labeled internally by incubation with 2.5 μM 5-chloromethyl fluorescein diacetate (CMFDA), and without washing, were injected into mice for platelet survival studies. CMFDA-labeled platelets were unactivated, as shown by minimal P-selectin expression. When tested in vitro for function by aggregometry, the response of CMFDA-labeled platelets to collagen and thrombin was identical to that of unlabeled platelets. Flow cytometric analysis demonstrated that CMFDA platelets were an intensely stained, unimodal population that was completely separated from unlabeled platelets. The mean half-life of labeled platelets in the murine circulation was 37.5 ± 4.5 hr (±1 SD), and the mean survival time was 3.1–3.3 days (n = 24), similar to results reported using ⁵¹Cr and ¹¹¹In. No evidence of in vivo transfer of dye from labeled platelets to unlabeled cells was observed. CMFDA produces a population of platelets that are nonradioactively, internally labeled with a highly fluorescent, stable product. The labeled platelets function equivalently to native platelets, as demonstrated by immunocytometry and aggregometry, and importantly, in vivo, by normal platelet survival. Am. J. Hematol. 56:17–25, 1997. © 1997 Wiley-Liss, Inc.     

The reversible binding of vinblastine to platelets: implications for therapy

The ability of platelets to adsorb vinblastine has been used to treat patients with immune thrombocytopenia. It is hypothesized that the drug- platelet complex is coated with antibody, taken up by macrophages which are then destroyed by the drug. We gave 16 courses of vinblastine- platelets to six patients with immune thrombocytopenia. Only one patient responded, and therefore we examined possible reasons for the lack of benefit. Using 3H-vinblastine, the kinetics of vinblastine binding to platelets was studied in vitro. The binding of vinblastine to both human and rabbit platelets was identical with maximal binding occurring within 10 min at 600 microgram/ml vinblastine. Similarly, the plasma half-life of vinblastine in rabbits was close to that reported for man, and therefore, in vivo binding of vinblastine to platelets in rabbits was considered a suitable model for man. Homologous donor rabbit platelets were labeled with 51Cr alone, 51Cr plus vinblastine, or 3H-vinblastine and infused into recipient rabbits. Vinblastine had no effect on 51Cr survival, but all measureable vinblastine had left the platelets within 2 hr of the infusion. These observations suggest that delivery of the vinblastine to the macrophages depends on the platelets being phagtocytized before the drug leaves the platelets. This would be likely to occur only in those patients with severe immune thrombocytopenia. Further investigations into this treatment should be directed at methods to maintain the drug within the platelet.     

Biotinylated platelets: a new approach to the measurement of platelet life span

Summary The in vivo life span of dog platelets was determined by derivatizing whole blood with N-hydroxysuccinimido biotin, reinfusing the biotinylated blood and subsequently monitoring the survival of the biotinylated platelets with flow cytometry. We found that the biotinylated platelets had a mean life span of 6.0 ± 1.1 d as determined by curve-fitting the platelet disappearance data to gamma functions. These data are in good agreement with literature values of platelet life span for canine platelets labelled with either ¹¹¹Indium-oxine or ⁵¹Chromium. Biotinylated platelets were analysed after reinfusion and found to aggregate normally in response to the agonists adenosine diphosphate and phorbol myristate acetate. These experiments demonstrate that biotinylated platelets survive normally in vivo and that this labelling method can be used for determining platelet life spans.     

Function ex vivo of 111 In-Labelled Human Platelets Simultaneous Aggregation of Labelled and Unlabelled Platelets Induced by Collagen

The function of ¹¹¹In-labelled platelets has been assessed by collagen-induced aggregation of platelets in samples of whole blood. The blood samples were drawn after injection of autologous ¹¹¹In-labelled platelets in 19 subjects undergoing platelet kinetic studies. It was thus possible to measure the aggregability of labelled and unmanipulated platelets simultaneously. ¹¹¹In-labelled platelets aggregated to the same extent as unmanipulated platelets when tested from 10 min to 24 h after injection of the labelled platelets. The results confirm the assumption that minimal damage is inflicted on the platelets during the isolation and labelling procedures, and support the concept that platelets manipulated in vitro may recover in vivo within a few minutes after reinjection.     

A comparative study in volunteers of apheresis and buffy coat derived platelets

It is important that, at the time of transfusion, platelets prepared by different techniques are effective in vivo and meet acceptable criteria. A paired study was undertaken in 8 volunteer donors to compare apheresis platelets collected on the COBE Spectra with platelets derived from the buffy coat of whole blood donations after 5 days storage. In vivo recovery, survival and biodistribution were determined following indium-111 labelling of the platelets and autologous infusion into volunteers together with the in vitro evaluation of platelet function and biochemistry. The in vivo and in vitro characteristics of both types of platelet concentrate were not significantly different. Both products were equally viable after 5 d storage and both were of an acceptable quality as judged by currently used platelet products. Mean platelet survival (multiple hit) was 5.4 d for the apheresis platelets and 6.9 d for the buffy coat derived platelets and maximum recovery in circulation was 28.1% and 34.8%, respectively. There was a significantly higher platelet yield, as expected, from apheresis and a 1.5 log lower level of white cell contamination. This would be advantageous for patients in need of prolonged or recurrent transfusions as the number of donor exposures and the risk of alloimmunisation or viral transmission would be reduced.     

Kinetics and distribution of platelets in man

⁵¹Cr sodium-chromate, though having been widely used in the last two decades for labeling platelets, suffers from several serious drawbacks, ie, low labeling efficiency, long physical half-life, and low gamma photon yields. ¹¹¹In-oxine and ¹¹¹In-tropolone overcome these shortcomings and have the potential of precise determination of platelet kinetics as well as visualization and in vivo quantification of the temporal and spatial distribution of platelets in man. Computer analysis of platelet kinetics reveals that the multiple-hit model fits the survival curve better than the linear or the exponential model. The multiple-hit model provides not only the mean platelet survival time but also information on the initial recovery and the shape of the survival curve. The application of these techniques in normal and disease states should greatly enhance our understanding of the physiology and pathophysiology of platelets.     

Platelet survival studies in man with diisopropylphosphorofluoridate (DF32P). Some observations in patients with hypocoagulability, 'hypercoagulability' and platelet disorders

Diisopropylphosphorofluoridate (DF³²P) acts as a cell label by combining irreversibly with esterases in the cell membrane. It was first used in man for tagging red cells (Cohen and Warringa, 1954) and later for platelets (Leeksma and Cohen, 1956) and leucocytes (Athens, Mauer, Ashenbrucker, Cartwright and Wintrobe, 1959). Since DF³²P allows platelets to be labelled directly in vivo it has obvious advantages over the ⁵¹Cr method (Aas and Gardner, 1958) which necessitates removal of platelets from the body for labelling and subsequent re-injection. The precise form of the normal platelet survival curve in man and the relative importance of intrinsic factors such as cell age, and extrinsic factors such as variations in the concentration of plasma coagulation factors, remains to be established. As part of an investigation into some of these factors, DF³²P has been used in a small comparative study of platelet survival in some hypocoagulable and some ‘hypercoagulable’ states. During the course of this study the opportunity arose to apply the method to patients with qualitative and quantitative platelet abnormalities. These studies are presented and discussed in this paper.     

A Comparative Study in Volunteers of Apheresis and Buffy Coat Derived Platelets

It is important that, at the time of transfusion, platelets prepared by different techniques are effective in vivo and meet acceptable criteria. A paired study was undertaken in 8 volunteer donors to compare apheresis platelets collected on the COBE Spectra with platelets derived from the buffy coat of whole blood donations after 5 days storage. In vivo recovery, survival and biodistribution were determined following indium-111 labelling of the platelets and autologous infusion into volunteers together with the in vitro evaluation of platelet function and biochemistry.

The in vivo and in vitro characteristics of both types of platelet concentrate were not significantly different. Both products were equally viable after 5 d storage and both were of an acceptable quality as judged by currently used platelet products. Mean platelet survival (multiple hit) was 5.4 d for the apheresis platelets and 6.9 d for the buffy coat derived platelets and maximum recovery in circulation was 28.1% and 34.8%, respectively. There was a significantly higher platelet yield, as expected, from apheresis and a 1.5 log lower level of white cell contamination. This would be advantageous for patients in need of prolonged or recurrent transfusions as the number of donor exposures and the risk of alloimmunisation or viral transmission would be reduced.     

Kinetics and imaging of indium-11-labeled autologous platelets in experimental myocardial infarction

The kinetics of accumulation and the external imaging patterns of indium-111-labeled platelets infused in a dog model of left anterior descending coronary artery occlusion with reperfusion were studied. The effects of infarct age and regional residual myocardial blood flow upon platelet accumulation were quantified, and the capacity of indium-111 platelets to image the experimental infarction was evaluated qualitatively. The endocardial accumulation of indium-111 platelets occurred primarily in infarct zones with residual blood flow less than 0.6 times normal and was maximal (24.98 +/- 2.76 times normal) in the lowest blood flow zone (less than 0.1 times normal). Indium-111 platelet accumulation in the epicardium occurred in the regions with blood flow less than 0.6 times normal and was maximal (17.83 +/- 1.20 times normal) in the lowest blood flow zone (less than 0.1 times normal). The maximal endocardial and epicardial platelet accumulation occurred 24 hours after reperfusion and was significantly decreased at 48 hours. In vivo cardiac images revealed discrete areas of increased myocardial radioactivity uptake in the anterior wall of dogs 24 hours after reperfusion. All images 48 hours after reperfusion were negative. Thus, in the experimental setting, indium-111 platelets allow quantification of platelet accumulation after myocardial infarction at a tissue level and provide a noninvasive means of in vivo imaging of reperfused infarcted myocardium.