Robert S. Hillman

Rapid Platelet-Function Assay An Automated and Quantitative Cartridge-Based Method

BACKGROUND The platelet glycoprotein (GP) IIb/IIIa receptor is important in mediating platelet thrombus formation, and the GP IIb/IIIa antagonist abciximab (c7E3 Fab; ReoPro) is effective in preventing thrombotic ischemic cardiovascular complications of unstable angina and percutaneous coronary interventions. Small-molecule antagonists of GP IIb/IIIa based on the Arg-Gly-Asp (RGD) sequence show similar benefit, and some of these agents are orally active. However, there may be significant interindividual variation in response to such antagonists, especially with chronic oral therapy. It will be essential to balance the beneficial antithrombotic effect of these drugs with their potential for causing bleeding. In response to this need, we have developed a rapid platelet-function assay (RPFA), a point-of-care system that provides a quantitative measure of the competence of the GP IIb/IIIa receptor as reflected in the ability of platelets to agglutinate fibrinogen-coated beads. METHODS AND RESULTS Polystyrene beads were coated with fibrinogen and placed in a cartridge along with a lyophilized peptide that activates the thrombin receptor. Anticoagulated whole blood was added to the cartridge, and then a microprocessor-controlled operation mixed the reagents and detected agglutination between platelets and coated beads. Quantitative digital results were displayed within 3 minutes. Because there is no dilution of the blood, the assay can be used to measure platelet activity in samples that have been treated with GP IIb/IIIa antagonists with high dissociation rates. RPFA results of whole-blood samples treated with different GP IIb/IIIa antagonists correlated well with both conventional turbidimetric platelet aggregation (r2=0.95) and the percentage of free GP IIb/IIIa molecules in the sample (r2=0.96). The mean difference in measurements between RPFA and aggregometry was -4% (+/-4% SD), and the mean difference in measurements between RPFA and free GP IIb/IIIa receptors was -2% (+/-6% SD). CONCLUSIONS The RPFA provides rapid information on platelet function that mirrors turbidimetric platelet aggregation and reflects GP IIb/IIIa receptor blockade.

Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion

Platelets are known to become activated during storage, but it is unclear whether such activation affects recovery or survival after platelet concentrate (PC) transfusion. With the use of flow cytometry to determine the percentage of platelets expressing the alpha‐granule membrane protein 140 (GMP‐140), a known adhesive ligand appearing on the platelet surface after activation, several studies were conducted. These investigations evaluated 1) the occurrence of significant platelet activation over time in PCs (n = 46) stored under standard blood bank conditions; 2) the correlation between platelet activation and platelet recovery in normal subjects after PC storage (n = 12), as assessed by the recovery of Indium‐labeled platelets; and 3) the recovery of activated and unactivated platelets in thrombocytopenic cancer patients transfused with standard PCs (n = 11). It was determined 1) that an increasing duration of storage of PC was associated with increasing platelet activation as measured by the percentage of platelets expressing GMP‐140, progressing from a mean of 4 +/− 2 percent (SD) on the day of collection to a mean of 25 +/− 8 percent by 5 days of storage: 2) that, in normal subjects, posttransfusion recovery of autologous platelets stored for 2 to 4 days and then labeled with In111 was inversely correlated with the percentage of activated platelets in the transfused PC (r = ‐0.55, p = 0.05); and 3) that, when thrombocytopenic patients were transfused with standard PCs, the recovery of the activated platelets in the transfused PCs averaged only 38 +/− 15 percent of the number predicted by the absolute platelet increment. It can be concluded that significant platelet activation occurs with standard platelet storage over 5 days and that activated platelets that express GMP‐140 are preferentially cleared from the circulation after transfusion.

Effect of Alcohol on Serum Folate Level

Alcohol was given orally and intravenously to normal and chronic alcoholic volunteers to study its effect on folate metabolism. Oral alcohol, given to nine subjects on a low folate diet, caused a greater fall in serum Lactobacillus casei folate levels than that seen in eight subjects on a low folate diet alone. This alcohol-induced fall in serum folate level occurred largely during the 1st day of the protocol. Although brief infusions of intravenous ethanol had no effect on serum folate level, a 13 h infusion caused a striking fall in serum folate level between the 8th and 10th h. When ethanol was stopped, the serum folate level returned rapidly to normal. Two chronic alcoholic subjects with different basal levels of serum folate were studied for several weeks on a low folate diet plus alcohol. The serum folate level fell promptly in each subject, rose when alcohol was temporarily stopped, and fell when alcohol was resumed. Folate-deficient megaloblastic anemia developed in 3 wk in the subject with initially marginal serum folate levels, but failed to develop in almost 7 wk in the subject with normal folate stores, as reflected by initially high serum folate levels. Thus, the alcohol-induced fall in serum folate level was apparently not a result of depletion of folate stores. In vitro experiments ruled out an assay artifact as an explanation for the alcohol-induced fall in serum L. casei folate level. It seems likely that alcohol interferes with the delivery of n-5-methyltetrahydrofolic acid from storage areas.

Kinetics of the Normal Folate Enterohepatic Cycle

Detailed studies were undertaken to better define the role of the liver and the folate enterohepatic cycle in folate homeostasis. Three isotopes of folate were employed in a rat model to study several parameters: (a) intestinal transport; (b) variation in hepatic uptake after different routes of administration; (c) hepatic reduction, methylation, and polyglutamate formation; (d) biliary excretion; (e) transport of folate to tissue and its return to liver for re-entry into the enterohepatic cycle. Folate absorption was not affected by the type of folate administered, but subsequent liver accumulation was greater when PteGlu(1) was given rather than CH(3)H(4)PteGlu(1). After liver uptake, CH(3)H(4)PteGlu(1) is rapidly and quantitatively excreted into bile, whereas nonmethylated folates are either methylated and transported into bile or incorporated into a hepatic polyglutamate pool. Bile folate is then reabsorbed for distribution to both tissue and liver, completing the enterohepatic cycle. The importance of this cycle was demonstrated by long-term bile drainage and by transport studies with two isotopes of CH(3)H(4)PteGlu(1). With bile drainage, serum folate levels fell to 30-40% of normal within 6 h, a much more dramatic drop than that seen with folate-free diets alone. Studies with labeled CH(3)H(4)PteGlu(1) demonstrated that about one-third was taken up by tissue, demethylated, and returned to liver for remethylation and recirculation through the bile and gut. This establishes the enterohepatic cycle as a major factor in folate homeostasis and, for the first time, demonstrates a transport pathway between tissue and liver for nonmethylated folate.

Maturation of the Macroreticulocyte

With anaemia the erythroid marrow maturation pattern of the rat is altered so that macroreticulocytes containing increased amounts of haemoglobin, water, ribosomes and ribonucleic acid are released into circulation. This was shown to be accompanied by a reduction in the marrow iron transit time, a measurement of the maturation time of marrow reticulocytes, and possibly by the occurrence of skipped mitoses during maturation. Despite the premature release, increased volume and haemoglobin content of these macroreticulocytes, they matured normally without apparent splenic sequestration or significant cell death in the first 10 days. At the same time they underwent shrinkage by loss of both cell water and some haemoglobin. This appeared to be an intravascular phenomenon where macrocytes were remodelled, possibly by cell fragmentation, until a population with near normal indices was obtained.

The Relationship between the Supply of Cardiac Catheterization Laboratories, Cardiologists and the use of Invasive Cardiac Procedures in Northern New England

Objectives: Utilization rates of coronary angiography and cardiac revascularization have been found to vary between areas. This study addresses the relationship between resource supply and procedure rates. Methods: We compared the association of per capita catheterization laboratories, per capita cardiologists and multi-provider markets (where more than one hospital offers coronary angiography services) with the utilization rates for angiography and cardiac revascularization in northern New England, USA. Administrative data were used to capture invasive cardiac procedures. Small area analyses were used to create coronary angiography service areas. Linear regression methods were used to measure associations between the resource supply and utilization rates. Results: Variation in the use of invasive cardiac procedures was strongly associated with the population-based availability of catheterization facilities and multi-provider markets and unrelated to cardiologist supply or need (as reflected in the hospitalization rates for myocardial infarction). In the multivariate model, an increase of 1 catheterization laboratory per 100 000 population was associated with an increase in the angiography rate of 1.62 per 1000 population; those service areas with multi-provider markets were associated with an additional increase in the angiography rate of 1.27 per 1000 population (R 2= 0.84, P = 0.0006). There was a moderately strong relationship between the catheterization laboratories per capita and the revascularization rates (R 2 = 0.43, P = 0.029). Angiography rates were highly associated with cardiac revascularization rates: An increase in the angiography rate of 1 per 1000 population was associated with a 0.46 per 1000 increase in the cardiac revascularization rate (R 2 = 0.85, P = 0.0001). Conclusions: Our work suggests that current efforts to address variation in cardiac procedures through activities such as appropriateness criteria, guidelines and utilization review are misdirected and should be redirected towards capacity, in this case the supply of catheterization facilities.

The Effect of Diet and Alcohol on the Development of Folate Deficiency in the Rat

Studies of the rate of depletion of serum and tissue methylated and nonmethylated folates were carried out in rats maintained for long periods on either a folate deficient (sucrose‐water/sulphathiazole) diet or a deficient diet plus high alcohol intake. By means of implantation of a feeding gastrostomy tube, it was possible to sustain constant blood ethanol levels of between 50 and 300 mg/dl for 3–4 weeks with relatively normal calorie intake and without death of the animal. Using this animal model, which closely resembles severe alcoholism in man, a very rapid depression in serum 5‐methyl tetrahydrofolate was observed similar to that reported in alcoholic man. At the same time, release of folates from liver stores was unimpaired by alcohol ingestion. Liver folate store depletion rates were identical for alcoholic and folate starved animals. The explanation for the sudden alcohol suppression of serum folate levels must, therefore, be sought at a point in the internal metabolic sequences of folate other than the delivery of folate stores to plasma.

Variations in the hematologic and medical status of alcoholics.

Sixty-five skid-road and 47 middle- to upper-class alcoholics underwent hematologic and medical evaluation. Despite major differences in life style and nutrition between these two groups, there was no significant difference in extent of liver disease or anemia. Moreover, the extent of liver disease, anemia, leukopenia, and thrombocytopenia in this community study of functioning alcoholics was significantly less than in a prior hospital study of acutely ill alcoholics. Iron deficiency was the commonest cause of anemia, probably because of frequent donations of plasma and blood by the skid-road alcoholics. Though megaloblastic anemia was infrequent, subnormal serum folate levels were commonly found in the skid-road group. In the middle- to upper-class group serum folate levels were normal in the absence of high blood ethanol levels. Ethanol causes depression of serum L. casel folate level, thus complicating assessment of folate balance in alcoholics.

THE MISUSED RETICULOCYTE

When supravital dyes were first employed in the study of formed elements of the blood, a special population of cells was identified which contained a precipitable material now known to be ribonucleic acid. These reticulocytes were recognized as red cells bearing the hallmark of youth. In the hands of haematologists of the early 20th century their number became a powerful tool for the evaluation of red-cell production. The reticulocyte percentage was used to define the response of the anaemic patient to specific therapy, and haemolytic states were often identified on the basis of a high circulating reticulocyte count. In the past three decades further information has accumulated concerning this particular stage of red-cell development. It is now appreciated that in the normal subject there is a reticulocyte pool within the marrow equivalent in size to that of the circulating reticulocyte (Seip, 1953 ; Reiff, Nutter, Donohue and Finch, 1958). Under the influence of increased levels of erythropoietin stimulation, red-cell maturation is modified and a premature delivery of marrow reticulocytes into the blood occurs (Gordon, LoBue, Dornfest and Cooper, 1962; Hillman, 1969). These ‘shift’ reticulocytes require 1-3 days longer than the normal circulating reticulocyte to lose their reticulum (Ganzoni, Hillman and Finch, 1969; Hillman, 1969). Just as the level of erythropoietin stimulation increases proportionately to the degree of anaemia produced, so does the immaturity of the circulating reticulocytes (Adamson, 1968 ; Hillman, 1969; Hillman and Giblett, 1965). This immaturity is recognizable on the blood smear through the relative increase in size and basophilia of the ‘shift’ cells. It is apparent, therefore, that the crude reticulocyte percentage so extensively used to indicate erythropoietin activity is dependent not only on the number of reticulocytes in circulation but also on the number of adult red cells to which they are related and on the reticulocyte maturation time. In order to derive a more meaningful expression of effective marrow production, appropriate corrections must be applied both for variations in the circulating red-cell count and for changes in the maturation process. The former may be eliminated as a variable either by determining the number of reticulocytes per cubic millimetre of whole blood, or more conveniently by normalizing the PCV or red-cell count according to the following formula :

Folic Acid Metabolism in Normal, Folate Deficient and Alcoholic Man

Summary. Folate metabolism was studied in normal, folate‐deficient and alcoholic man by tracer measurements of plasma clearance, urinary excretion, tissue storage and release of folate using both [3H]pteroylglutamic acid (3H‐PteGlu) and 14C‐methyl‐H4PteGlu. Alcohol ingestion did not adversely affect tissue uptake of folates. Whether in normal or folate deficient subjects, the relative clearance rates of 3H‐PteGlu and 14C‐methyl‐H4PteGlu were maintained in the face of alcohol ingestion and there was no evidence of increased urinary loss of intact vitamin or labelled breakdown products. As measured by the flushing technique, the rate of storage or tissue binding of 3H‐PteGlu was not influenced by folate deficiency, folate store depletion or alcohol ingestion. However, alcohol may retard the release of methyl‐H4PteGlu from tissue stores to plasma. A significantly greater recovery of 14C‐methyl‐H4PteGlu with flush was observed in those normal subjects who ingested alcohol for 6 d. A partial block in the rate of release of tissue folate stores would be a possible mechanism behind the rapid depression in serum methyl‐H4PteGlu levels and early induction of megaloblastic erythropoiesis which has been observed following acute alcohol ingestion.