Ultrastructural morphometric observations on serial sectioned human blood platelet subpopulations.

The relationships between the volume of human platelets and their cytoplasmic organelles were studied by morphometric analysis. Platelets were separated into four density-dependent subpopulations on an arabino-galactan gradient. In vitro activation of platelets was effectively prevented by maintaining them at a constant ambient temperature of 37 degrees C. Serial sections were cut through platelets, morphometrically analyzed and the platelets reconstructed. The volumes of the individual platelets and their constituent granules, mitochondria and open canalicular systems (OCS) were calculated. Individual organelles were counted. The mean volumes of the platelets of the subpopulations decreased significantly as density decreased (p = 0.01). Also, as the density of platelets decreased, there was a decrease in the mean unit volume of their granules (p = 0.003). In contrast, independent of platelet volume or density, the OCS occupies about 10% of the platelet volume. These findings indicate that it is possible to prevent in vitro platelet activation by maintaining their environment at 37 degrees C. Our study confirms the direct relationships between platelet volume and density; and platelet density and granule content. There is no ready explanation for the constant relationship between platelet volume and that of the OCS.

The identification of human T and B lymphocytes by electron microscopy.

Lymphocytes of peripheral blood, bone marrow, spleen, vermiform appendix, tonsil and Mantoux skin reaction were examined by electron microscopy (EM) and classified as T, B and Null cells by E-rosette and immunoglobulin membrane-receptor characteristics. The low pH and ionic strength of the fixative solution for EM, and some other minor procedural modifications, made it possible to distinguish B and T lymphocytes morphologically. T-cells have electron-dense cytoplasm and euchromatin in the nucleus whereas B-cells constantly have electron-lucent cytoplasm and euchromatin in the nucleus. A proportion of lymphocytes were unclassifiable by their ultrastructural features. These unclassifiable cells may be Null cells as determined by the classical techniques. The specificity and simplicity of this EM technique for T and B lymphocytes is especially useful for studies of immunocompetent-cell topography and cell-to-cell interaction in lymphoid organs. It may also be utilized for diagnostic purposes in immunocytic dyscrasias.

Uranyl-labelled antibody (ULA) method and its application for Rh antibodies.

The existent labelling materials for studies of antigen--antibody interaction at ultrastructural level, namely ferritin and peroxidase, because of their large molecular size do not fulfill all requirements of excellent markers for electron microscopy (EM). Uranyl acetate has a molecule 354 times smaller than IgG and its uranium atom is electron-dense. These physical characteristics of uranyl acetate make it a labelling material par excellence as described in this article. Quantitative and qualitative studies of Rh antigen-antibody interactions are for the first time presented at the ultrastructural level, and the application of the uranyl-labelled antibody (ULA) method for weak antisera (dilutions 100 to 1000 time higher than the Coombs range of sensitivity) is demonstrated. The ULA method opens a new era for studies of antigens, antibodies and their interactions because it will demonstrate visibly details of the antigen-antibody interaction and is especially suitable for studies of weak antisera.

Evaluation of mathematic models to assess platelet kinetics.

Twelve mathematic methods used to calculate the mean platelet survival time were compared by determining the "goodness of fit" of the models to the platelet survival curves of 15 reference subjects and 54 patients. Platelets were labeled with [111In]oxine. The linear (LN), exponential, weighted mean, multiple hit (MH), Dornhorst (DH), Meuleman (ML), alpha order (AO), and polynomial (PO) mathematic models were investigated. The goodness of fit for the exponential model was determined by the nonlinear least squares method (EP), and also by the linear least squares method on logarithmically transformed data (EX) as is recommended. The modified weighted mean (MWM) and the usual weighted mean method (WM) obtained with these exponential models were tested. The Dornhorst (DH10) and Meuleman (ML10) models, where the potential age-dependent platelet survival times were kept constant at 10 days, were also evaluated. The goodness of fit results, expressed as % s.d. indicated that the LN (5.2%), EX (5.0%), EP (4.4%), WM (3.7%), DH10 (3.7%), and ML10 (3.7%) models all fitted the data significantly worse than the MWM, MH, DH, ML, AO, and PO models (range 3.2-3.3%). The mean platelet survival time determined with the MH model differed significantly from the results with the DH, ML, and AO models. The results of mean platelet survival time calculated with different mathematic models cannot, therefore, be compared directly. The models that fitted the platelet survival curve well varied slightly in sensitivity to noise as is indicated by the coefficient of variation of the mean platelet survival time estimates for the reference subjects (range 7.9-12.0%). Fitting data to at least two mathematic models has definite advantages. Data on which the calculations are based are probably invalid if the following are true: (a) if the mean platelet survival time estimated with the alpha order model is shorter than that estimated with the EP, MWM, or MH models, or (b) the mean platelet survival time estimated with either the DH, ML, AO, or PO models, is longer than the LN, MWM, or MH estimate of the mean platelet survival time. We conclude that the mean platelet survival time can be reliably estimated by fitting the data to either the MWM method (if limited computing facilities are available) or the MH model. Confidence in the result will be increased if considered in conjunction with the finding obtained with one other model; in those cases where the platelet survival time is very short, the alpha order model is recommended.(ABSTRACT TRUNCATED AT 400 WORDS)

Reference values for red cell survival times.

The first purpose of this investigation was to investigate in 35 young normal male subjects the use of the Dornhorst function and the weighted-mean method to calculate reference values for mean red cell survival time with and without correction for elution of 51Cr. We compared survival times calculated with the Dornhorst and weighted-mean methods with survival time estimated with linear or exponential models. Two methods to correct for elution of 51Cr from red cells were investigated. For the first method, correction factors were generated using the Dornhorst function fitted to mean survival curves obtained from the normal subjects. In the second method, the new Dornhorst rate constant method, the survival time, corrected for elution of 51Cr, was directly calculated from the experimental survival curve without applying correction factors. Correction for elution using the Dornhorst rate constant method was not successful and resulted in nonphysiologic values. The 95% confidence range of red cell survival time for reference subjects without correction for 51Cr elution was 37-74 days for the weighted-mean method and 37 to 73 days for the Dornhorst method. The 95% confidence range for normal subjects when the survival curves were corrected for elution was 47-179 days for the Dornhorst method and 58-161 days for the weighted-mean method. The poor results obtained with the Dornhorst rate constant method and the large 95% confidence range were due to the rapid and large variation in elution rate of 51Cr from red cells.

Comparison of oxine and tropolone methods for labeling human platelets with indium-111.

The effect of the chelates oxine and tropolone, used to label platelets, on the kinetics of indium-111-(111In) labeled platelets was studied in twelve normal human subjects. Autologous platelets were labeled either in saline with 111In-oxine or in plasma with 111In-tropolone. Mean platelet lifespan was estimated by fitting the disappearance curve of platelets from the circulation to the multiple hit and other mathematical models. The in vivo distribution of platelets was quantitatively imaged with a scintillation camera. The in vivo recovery of 111In-oxine and 111In-tropolone did not differ, and the mean platelet lifespan was also similar (111In-oxine: 230 +/- 29 hr; 111In-tropolone: 226 +/- 13 hr). At equilibrium (90 min after reinjection of labeled platelets) and at the end of platelet lifespan, 111In-oxine and 111In-tropolone radioactivities in the spleen and liver were similar. These results demonstrate that the results of kinetics measured with 111In-oxine or 111In-tropolone do not differ significantly.

Kinetics, distribution, and sites of destruction of canine blood platelets with In-111 oxine.

In five normal dogs we have studied the survival, tissue distribution, and fate of autologous platelets labeled with indium- 111 oxine. The methods include blood sampling, computer-assisted scintigraphy, and whole-body profile scanning. Mean In- 111-platelet recovery in the circulation was 45 +/- 22.5 (s.d.) and survival 124.6 +/- 10.5 hr. Platelet survival curves fitted a linear function best. Initially platelets pooled rapidly in the spleen with a single exponential function, and at zero-time equilibrium (35 +/- 4)% of the injected In- 111 was located in this organ. Early hepatic uptake was also significant, and constituted (20 +/- 4)% of total-body radioactivity. As labeled platelets disappeared from the circulation, In- 111 activity in the spleen increased progressively and linearly to reach (59 +/- 9)% of the body activity at 120 hr. Hepatic radioactivity decreased with time but to a lesser extent than that of the heart. The results indicate that in the dog the major site of destruction of platelets is the spleen, with the liver playing a less important role.

Kinetics of In-111-platelets in the baboon: I. Isolation and labelling of a viable and representative platelet population.

A fully representative and viable platelet population was isolated from the blood of 15 baboons by a multiwash procedure, and labelled with In-111-oxine. The recovery of the total platelet population in the circulation was 85% +/- 9. Mean platelet life span was 146 hr +/- 13. Correcting for plasma radioactivity (always less than 3.5%) did not significantly affect the estimate of platelet life span (145 hr +/- 16) or recovery (85% +/- 12). Platelet survival estimates, repeated at different times, were reproducible. In 5 baboons, platelets were also harvested by a single step differential centrifugation. The mean life span of a representative platelet population was significantly longer than that of platelets harvested by a single step. Recovery values of the representative and non-representative population were similar. We conclude that it may be important to harvest and label a fully representative platelet population for kinetic studies. The proposed method is simple and reproducible, and may be applied in studies in humans.

Kinetics of In-111-platelets in the baboon: II. In vitro distribution and sites of sequestration.

The kinetics and sites of sequestration of a fully representative population of In-111-platelets were determined in 11 baboons. The in vivo method of quantification with computer assisted scintillation camera image analysis was validated by sacrificing 5 baboons and measuring and comparing the distribution of organ radioactivity. Recovery of platelets in the circulation was 87% +/- 7, and their mean survival time was 147 hr +/- 15. The mean splenic platelet pool was 16.0 +/- 1.9. At equilibrium 15.8% +/- 2.9 of the In-111-platelets were in the hepatic blood pool. Senescent platelets were destroyed in the reticulo-endothelial system. The major sites of sequestration were: liver (37.6% +/- 6.0), and the spleen (23.3% +/- 4.6). The bone marrow sequestrated 14.4% +/- 1.7 of the labelled platelets, and 15.5% +/- 4.0 were present in various other tissues. We conclude that the in vivo method of In-111-quantification is accurate. Senescent platelets are mainly sequestrated in the reticuloendothelial tissue, with the liver, spleen and the bone marrow important sites of sequestration.

Kinetics and fate of (111)Indium-oxine labelled blood platelets in asplenic subjects.

The survival, tissue distribution and fate of (111)Indium-oxine labelled autologous platelets was studied in four asplenic subjects with serial blood sampling, scintillation camera and computer-assisted imaging. Mean (111)In-platelet recovery in the circulation was 89 /+- 13% (/+- 1 SD). Platelet survival curves fitted a linear function best and was 238 /+0 41 h. The shape of the survival curves of normal and asplenic subjects differed: in the asplenic subjects the curve was linear whereas that of normal subjects was significantly more curvilinear if analyzed by least squares computer fitting to a gamma function. Early hepatic (111)In-activity was significant and transient and ascribed to the "collection injury". As labelled platelets disappeared from the circulation, (111)In-activity in the liver increased progressively and linearly to reach 42.5 /+- 14.1% of whole body activity at 240 h. Radioactivity also accumulated in the bone marrow, but could not be demonstrated in the vasculature of the lower limbs. These results would indicate that in asplenic subjects the majority sites of destruction of senescent platelets are the liver and bone marrow.

Kinetics and in vivo redistribution of (111)Indium-labelled human platelets after intravenous protamine sulphate.

The pathogenesis of thrombocytopenia induced by intravenous protamine sulphate was studied in six patients who underwent cardiopulmonary bypass surgery, and in three normal volunteers. Autologous platelets were labelled with (111)Indium-oxine. Platelet lifespan was determined. In vivo (111)In-platelet localization, organ redistribution and sites of destruction were quantitated with a scintillation camera and a computer-assisted imaging system. Protamine induced a transient thrombocytopenia, maximal 5-10 min after injection, and 30-40 min in duration. . The thrombocytopenia was accompanied by a transient accumulation of platelets in the liver. The splenic platelet pool remained unaltered and no platelets accumulated in the lungs. Platelet survival, measured in two volunteers, was slightly longer than normal and fitted a linear function best. There was a severe transient neutropenia during the period of thrombocytopenia. We conclude that protamine-induced thrombocytopenia is caused by hepatic accumulation of "activated" platelets or platelet aggregates, the process is reversible, and in the two normal volunteers studied, platelet survival was not affected.

Influence of platelet membrane sialic acid and platelet-associated IgG on ageing and sequestration of blood platelets in baboons.

Platelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/10(8) platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p < 0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p < 0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/10(8) platelets was removed (p < 0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and in vivo distribution of in-111-labelled platelets and platelet function in familial hypercholesterolaemia.

The kinetics, in vivo distribution and sites of sequestration of autologous In-111-labelled platelets and other platelet function parameters were studied in ten patients with type IIa or IIb familial hypercholesterolaemia and thrombotic complications of atherosclerosis. The in vitro platelet aggregation response to ADP (P = 0.50) and collagen (P = 0.46); binding of fibrinogen to platelets (P = 0.61); and plasma beta-thromboglobulin levels (P = 0.42) of the patients and normal reference subjects did not differ significantly. The in vivo distribution of In-111-labelled platelets at equilibrium was within normal limits, and at the end of platelet life-span the sequestration pattern of labelled platelets in the reticuloendothelial system was also normal (spleen P = 0.31; liver P = 0.54). There was minimal evidence of in vivo platelet activation: only mean platelet lifespan (MPLS), 195 +/- 57 hours (difference between mean MPLS of patients and controls was 25 hours, with a 95% confidence interval from 23 to 31 hours; P = 0.02); mean platelet platelet turnover, 2298 +/- 824 platelets/microliter/hour (P = 0.005); plasma platelet factor 4 (P = 0.02); and the mean circulating platelet aggregate ratio, 0.8 +/- 0.1 (P = 0.02); differed significantly from normal. These results suggest that abnormalities of platelet function and kinetics observed in type II hyperlipoproteinaemia cannot be ascribed wholly to the hyperlipidaemia, but may be induced by the associated atherosclerosis.

Preparation of a viable population of indium-111-labelled human blood platelets.

Factors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22 degrees C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37 degrees C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.

Kinetics and sites of sequestration of indium 111-labeled human platelets during cardiopulmonary bypass.

A new approach for the study of the kinetics and quantification of the in vivo and ex vivo sites of sequestration of platelets during cardiopulmonary bypass (CPB) is described. Autologous platelets of four patients were labeled with 111In-oxine and reinfused on the day prior to CPB for coronary artery bypass grafting. Changes in blood 111In-labeled platelet radioactivity and blood platelet counts were monitored during the operation. In vivo 111In-labeled platelet redistribution was quantified with a scintillation camera and a computer-assisted imaging system before and after CPB. Sequestration of 111In-labeled platelets in the bubble oxygenator was measured. 111In-labeled platelet activity in the blood decreased by 46% +/- 5% within 5 minutes of CPB, but this decrease was mostly due to hemodilution; the true loss of platelets from the circulation was 13% +/- 4%. Intraoperatively, whole body 111In activity decreased by oxygenator 10.8% +/- 1.3% of administered platelets were sequestered, especially in the innermost active layers of the defoaming mesh of the bubble oxygenator. Mean survival time of circulating platelets was 58 +/- 8 hours and fitted an exponential function best. The bleeding time increased to 40 minutes during operation and returned to normal within 24 hours. During operation 111In-labeled platelets accumulated somewhat in the liver (10.7%) but not in the spleen, thorax, or head. In the 48 hours after operation, platelets were sequestered mainly in the liver. The scintillation camera with computer-assisted imaging allows in vivo quantitative studies of platelet kinetics of a type which has not been possible with previous techniques.

Platelet aggregation in chronic idiopathic thrombocytopenic purpura.

A method for washing platelets by albumin density gradient separation has been modified to prepare platelet rich plasma of thrombocytopenic patients for platelet aggregation studies. The concentration procedure, consisting of centrifuging platelets into a specific gravity gradient between plasma and 40-45% aqueous solution of bovine albumin, does not affect platelet aggregation adversely. Platelet aggregation in eight patients with chronic idiopathic thrombocytopenic purpura was determined by this method. On the basis of the results the patients could clearly be divided into two groups: four patients with normal aggregation and four with a qualitative platelet defect. In contrast to the other patients, the group with an in vitro platelet functional defect all had more prolonged bleeding times and the presence of a serum antiplatelet antibody.

Kinetics, distribution, and sites of destruction of indium-111 oxine labelled red cells in haemolytic anaemia.

The survival of red cells labelled with indium-111 oxine in the circulation was determined. In vivo distribution at equilibrium and sites of deposition at the T50In--that is, the half life of labelled red cells--were quantitated with a scintillation camera and computer assisted image analysis. Although the rate of elution. Of 111In from the red cells was higher than that of chromium-51-disodium chromate, estimates of T50In and T50Cr corresponded reasonably well and were shortened in haemolytic anaemia. In normal subjects red cells were sequestered mainly in the liver and spleen. In five patients with different types of haemolytic anaemia two distinct patterns of red cell sequestration could be recognised: mainly splenic sequestration, and destruction of red cells in the liver, spleen, and the bone marrow. These patterns were expected for the particular disease studied.

Unsuitability of evacuated tubes for monitoring heparin therapy by activated partial thromboplastin time.

Activated partial thromboplastin times (APTT) for monitoring heparin therapy for venous thromboembolism tended to be inappropriately short if blood was collected in commercially available evacuated glass tubes. Five types of evacuated tubes marketed under the trade names Vacutainer and Venoject were examined. The APTT of heparinized blood collected in these tubes correlated poorly (r = 0.04 to 4 = 0.25) with that of blood samples from the same patients collected in plastic tubes. Most of the evacuated tube APTT were shorter than that of blood collected in plastic or siliconised glass tubes, but the results were unpredictable and varied from tube to tube and from batch to batch. This effect on heparin is apparently due to an unidentified substances which is eluted from the rubber stoppers of the tubes. Heparin control according to the APTT blood collected in these evacuated tubes is hazardous.

Kinetics and fate of indium 111 oxine-labeled platelets in patients with aortic aneurysms.

The survival and sites of sequestration of indium 111 oxine-labeled autologous platelets were studied quantitatively in six patients with aortic aneurysms. The in vivo distribution was quantitated daily with a scintillation camera and a computer-assisted imaging system. Data of platelet survival curves were fitted to a gamma function model. Mean platelet survival was shortened and the disappearance curves were exponential in all but two patients who had normal platelet survival. Platelet radioactivity in the aneurysm was 5.1 +/- 3% of whole-body radioactivity at the end of platelet survival. Platelet were sequestered in the spleen, liver, and bone marrow. Accumulation of platelets, presumably due to microembolization, was prominent in the lower limbs. This indicates that although platelets were deposited in the aneurysm, many are damaged and are eventually sequestered in the reticuloendothelial system.

Fanconi's anemia: a cytogenetic study on lymphocyte and bone marrow cultures utilizing 1,2:3,4-diepoxybutane.

Fanconi's anemia (FA) is an autosomal recessive genetic trait characterized by congenital abnormalities, pancytopenia with a late onset, and increased chromosome instability. A great deal of heterogeneity exists in the disease, making an early correct diagnosis very difficult. Previously chromosome instability was used as a diagnostic tool but was found to be unreliable. Auerbach et al. have described the use of a difunctional alkylating agent, 1,2:3,4-diepoxybutane (DEB), in lymphocyte, fibroblast, and amniotic fluid cultures for the accurate diagnosis of homozygotes and heterozygotes for the FA gene. We report here the findings on lymphocyte and bone marrow cultures from 18 FA homozygotes and 17 family members. Statistical analysis of the results with DEB at different concentrations showed a significant increase in induced chromosome breakage rates for homozygotes and heterozygotes when compared to those for a control group. The bone marrow cultures gave similar results.