A high-sensitivity enzyme-linked immunosorbent assay for serum thyroglobulin.

A sensitive enzyme-linked immunosorbent assay (ELISA) for measuring serum thyroglobulin (Tg) is described. The assay has a functional sensitivity of 0.03 ng/mL and values obtained in sera from patients with treated differentiated thyroid cancer (DTC; n = 24, 17 of whom showed some evidence of recurrence) and from healthy blood donors (n = 48) were in agreement with those obtained by Tg immunoradiometric assay (IRMA) (functional sensitivity = 0.6 ng/ml) (r = 0.99 and 0.98 for the two groups, respectively). The Tg levels measured by ELISA in 47 of the healthy blood donor sera ranged from 2.3 to 139 ng/ml with 1 serum giving a value of 0.03 ng/mL. The mean +/- standard deviation (SD) Tg concentration for the healthy blood donors was 20.3+/-23 ng/mL. Studies with a recovery test suggest that Tg measurements by ELISA were not always reliable when Tg autoantibodies were present. Analysis of samples from 167 patients treated successfully for DTC (papillary carcinoma, 94; follicular carcinoma, 73) showed that 139 were negative for Tg autoantibodies and of these 106 (76%) had Tg levels measurable by ELISA (0.03 ng/mL or greater). In contrast, only 7 (5%) of these 139 sera had Tg levels measurable by IRMA (0.6 ng/mL or greater). It is possible that this ability to measure Tg simply and easily in most treated DTC patients will have significant advantages for patient care. In particular, the Tg level after initial ablative treatment will usually be measurable rather than undetectable. Furthermore, any increases in serum Tg levels which may herald relapse will be detectable earlier.

Stimulation of hematopoiesis by amifostine in patients with myelodysplastic syndrome.

The aminothiol, amifostine (Ethyol; U.S. Bioscience, West Conshohocken, PA), is a cytoprotective agent that ameliorates the toxicities of anticancer therapy. In vitro, amifostine promotes the formation and survival of primitive hematopoietic progenitors derived from myelodysplastic bone marrow (BM) specimens. To evaluate the hematological effects of amifostine, 18 patients with myelodysplastic syndrome (MDS) and one or more refractory cytopenias received treatment with amifostine in a Phase I/II study. Four cohorts received intravenous treatment with 100, 200, or 400 mg/m2 amifostine three times a week, or 740 mg/m2 weekly for three consecutive weeks followed by 2 weeks observation. Nonresponding patients received a second course of therapy at the next higher dose level depending upon drug tolerance. Bone marrow (BM) progenitor growth was assessed before treatment and after day 21. Diagnoses included refractory anemia (7), refractory anemia with ringed sideroblasts (5), refractory anemia with excess blasts (RAEB) (4), and RAEB-in transformation (RAEB-t) (2). Single- or multi-lineage hematologic responses occurred in 15 patients (83%) treated with the three-times-a-week dose schedule. Fourteen patients had a 50% or greater increase in absolute neutrophil count with amifostine treatment (range, 426 to 11,348/microL). Platelet count increased in 6 (43%) of 14 patients with thrombocytopenia (absolute increase, 16, 000 to 110,000/microL), and 5 of 15 red blood cell transfusion-dependent patients had a 50% of greater reduction in transfusion needs. Assayable hematopoietic progenitors increased in 13 of 15 evaluable patients; including CFU-GEMM (12), BFU-E (8), and CFU-GM (6). Amifostine doses less than or equal to 200 mg/m2 were well tolerated, whereas grade II nausea, vomiting, and fatigue was limiting at higher doses. Three patients with excess blasts before enrollment experienced an increase in BM blast percentage and two patients had evolution to acute leukemia that persisted after treatment withdrawal. We conclude that amifostine administered at doses

Plague. A clinical review of 27 cases.

We reviewed the medical records of 27 patients with plague seen at the Gallup (NM) Indian Medical Center between 1965 and 1989. Nineteen patients had bubonic plague and eight had septicemic plague. Three patients with septicemic plague and three with bubonic plague died. The patients presented with five different clinical pictures. Ten patients presented with classic signs of plague, five with the appearance of an upper respiratory tract infection, five with a nonspecific febrile syndrome, four with the appearance of a gastrointestinal or urinary tract infection, and three with the appearance of meningitis. Blood cultures were positive in 24 of 25 cases, and bubo aspirate cultures were positive in 10 of 13 cases. All six patients who died were under 30 years old, and all the deaths were related to a failure to treat initially with an antibiotic appropriate for plague. Plague is a treatable disease, but clinicians must have a high index of suspicion and give appropriate antibiotics at the earliest possible time to patients whose presentation suggests plague.

Tuberculosis of the pancreas: a case report.

A 73-year-old female died of adult respiratory distress syndrome (ARDS) and multiple organ system failure (MOSF) after a routine cholecystectomy for acute cholecystitis. An autopsy revealed disseminated tuberculosis with a tuberculous abscess of the pancreas.

Determination of plasminogen in human serum or plasma by a fibrometric method.

A simple, fast, reproducible, and reliable fibrometer method for determination of plasma or serum plasminogen was developed. Results obtained with this method were compared to those obtained with a Coseinolytic method and a Chromogenic Substrate S-2251 method. For all three procedures, serum or plasma samples were acidified before the activation process. Conversion of plasminogen to plasmin during a controlled incubation time at 37 degrees C was accomplished with urokinase, and/or streptokinase. Precision which was determined on multiple aliquoted serum samples showed that the within day coefficient of variations were 4.35 percent and 4.82 percent, and an overall coefficient of variation of 7.68 percent. Satisfactory correlation was also demonstrated on 26 patients' plasmas. A correlation of 0.90 and 0.88 was found between the Caseinolytic assay and the Chromogenic assay, and the clotting time assay. A fibrometric normal range of 68 to 150 percent activity was obtained on 24 normal serum samples.

High erythrocyte distribution values and possibilities of hemoglobinopathies.

Patients with hemoglobinopathies were observed to have high erythrocyte distribution widths. In a representative study of 800 patients, 31% of patients with high erythrocyte distribution widths were observed to have hemoglobinopathies by hemoglobin electrophoresis. The potential of erythrocyte distribution width as a means of incidental detection of hemoglobinopathies is discussed.

Erythrocyte sedimentation, viscosity, and plasma proteins in disease detection.

In order to assess the relationships of erythrocyte sedimentation rates (ESR) and various acute phase reactants in diagnosing and following the remissions and exacerbations of various chronic diseases, multiple tests were performed on patients for whom an ESR was ordered. The Wintrobe and Westergren ESR, plasma and serum viscosity, hemoglobin, blood cell indices, fibrinogen, serum protein electrophoresis, triglycerides, and complement (C-3) were performed. A discussion of the relative merits of correcting the Wintrobe ESR by the Wintrobe anemia correction chart of the Hynes and Whitby method is presented. Although many significant correlations exist, only the Wintrobe and Westergren ESRs and plasma viscosity approached the same number of abnormal results. If the results of the Wintrobe and Westergren are combined 67 abnormal results are observed. The plasma viscosity exhibited a 10.4 percent false positive rate compared to the combined ESR's and a 6.0 percent false negative rate. The plasma viscosity was not only highly significantly correlated with the ESR's (P < < 0.001), but also with all acute phase reactants measured as well as total protein, albumin, and gamma-globulin. The plasma viscosity appears to give the best overall summation of the many non-specific changes observed in chronic disease.

Histochemistry of bone marrow aspirations.

The accuracy of diagnosis of hematological diseases from bone marrow aspirates and peripheral blood smears is considerably improved by use of histochemical stains. As hematopoietic cells differentiate and mature from the pluripotent stem cell and are committed to a particular cell line, the committed cell lines produce different substances, particularly enzymes, which can be identified by various histochemical stains. Histochemical stains are not diagnostic, but are aids in diagnosis, because various cell lines may produce similar substances and because the stains lack absolute specificity. A discussion of the use of histochemical stains for the diagnosis of leukemias is presented. Complete procedures for Prussian blue stain for iron, peroxidase, Sudan black B, naphthol AS-D chloroacetate esterase, nonspecific esterase, periodic acid Schiff (PAS), acid phosphatase, toluidine blue and alkaline phosphatase stains are given in the appendix.

Kinetic determination of granulocyte alkaline phosphatase by the GEMSAEC.

Granulocyte alkaline phosphatase (ALP) activity is measured kinetically using the GEMSAEC centrifugal analyzer and p-nitrophenylphosphate as substrate. Measurements of enzyme activity are made at pH 9.8 utilizing 1.0 M diethanolamine as a buffer containing 0.5 mM magnesium chloride. Granulocytes are separated from heparinized blood using dextran sedimentation, followed with ficoll-hypaque separation. The separated cells are suspended in saline, and the red cells are lysed. Manual counting of stained smears of the cells isolated showed that 95 percent of these cells were granulocytes. After lysing the red cells, the granulocyte suspension is rinsed in 12.5 percent heat inactivated serum in saline and separated by centrifugation. After 200 microliters of the diluent solution (12.5 percent heat inactivated serum in saline) is added to the cells, they are sonicated for seven sec at 0 degrees in an ice bath. The extracts are then assayed kinetically for ALP activity. Granulocyte ALP activity varies from day to day in normal individuals. The daily variation of ALP activity observed correlates closely with and is confirmed by the Sigma Histochemical Procedure based on Kaplow' Scoring Method. The coefficient of variation of the ALP method within assay performed on two consecutive days on a pooled sample is 0.75 percent and 1.38 percent, respectively, and between assays 4.82 percent.

In vitro and in vivo assessment of the viability of cryopreserved postmortem murine bone marrow cells.

The viability of cryopreserved mouse fresh and postmortem bone marrow cells was studied using methylcellulose clonal cell culture assay and the murine spleen colony technique. This study clearly indicates the presence of pluripotent hemopoietic stem cells in cryopreserved murine postmortem bone marrow cells. The viability of granulocytic precursors in both fresh and postmortem bone marrow cells was mildly to moderately decreased by the freezing process, while the proliferative capacity of erythrocytic stem cells is significantly decreased by cryopreservation. The results strongly suggest that frozen, fresh and cadaveric bone marrow bank for bone marrow transplantation may be of value.

Pluripotent hemopoietic stem cells in murine postmortem bone marrow.

Although 12-hours postmortem murine bone marrow cells exhibit extensive degeneration, these cells when infused into irradiated mice produce erythrocytic, granulocytic, megakarocytic and mixed colonies in their spleen. These observations clearly demonstrate the presence of pluripotent hemopoietic stem cells and their proliferative capability in 12-hours postmortem, murine bone marrow. These observations suggest that cadaveric bone marrow transplantation may be possible in patients with hematologic disorders.

Proliferative function of cadaveric bone marrow cells.

The proliferative function of human and murine cadaveric bone marrow was studied using methylcellulose clonal cell culture assays and the murine spleen colony technique. The study revealed persistence of hemopoietic functions for as long as 19 postmortem hours in cadaveric marrows of some patients. Studies of murine cadaveric marrows corresponded with those of human marrows. These results strongly suggest that human pluripotent hemopoietic stem cells survive in cadaveric marrows.

Correlation of platelet aggregation, plasma factor activity, and megathrombocytes in diabetic subjects with an without vascular disease.

Second-phase platelet aggregation induced by adenosine diphosphate (ADP) and epinephrine was measured in fasting platelet-rich plasma in normals, "prediabetics," and diabetics with or without vascular disease. "Plasma factor" potentiation of ADP-induced second-phase platelet aggregation was also estimated, as were megathrombocyte numbers in the same patient groups. There was an increased sensitivity of second-phase platelet aggregation noted with both aggregating agents in all diabetic groups except for the prediabetics. This activity was paralleled by an increase in plasma factor activity. In vivo evidence of an increased turnover of platelets in frank diabetics was suggested by increased numbers of megathrombocytes. These studies demonstrate that platelets from diabetics are sensitive to aggregating agents and that this sensitivity may be related to plasma factor(s) present in diabetics. In vivo platelet aggregation may be present in diabetics. Longitudinal studies will be necessary to establish the relationship of these findings to the genesis of diabetic vascular disease.

Increased platelet aggregation in early diabetus mellitus.

In view of the tendency toward vascular disease in diabetes mellitus, we studied platelet aggregation in 15 normal, 7 prediabetic, 12 latent, and 20 frankly diabetic subjects. Platelets from latent and frank diabetics showed increased platelet aggregation 4 minutes after adding adenosine 5'-diphosphate (60% verus 29% at 1.0 mu-M), epinephrine (46% versus 14% at 0.25 mu-M), and collagen (72% versus 17% at 0.25 mu-g/ml). Three prediabetics had increased platelet aggregation. Platelet sensitivity to aggregating agents was most marked in frank diabetics, intermediate in latent diabetics, and least in prediabetics. Second-phase platelet aggregation was reversed with acetylsalicylic acid, intravenous tolbutamide, and oral glucose administration. We conclude that platelet aggregation may be increased early in diabetes mellitus and may be involved in the genesis of diabetic microangiopathy. Prospective studies on the effect of therapeutic agents such as acetylsalicylic acid on the natural course of diabetic vascular disease are indicated.