Comparative electrophoretic analysis of human and porcine plasminogen activators in SDS-polyacrylamide gels containing plasminogen and casein.

Electrophoretic analysis of plasminogen activators from pig heart, human uterus, human plasma and human melanoma cells was performed in SDS-polyacrylamide gradient slab gels containing plasminogen and casein. Direct visualization of activator activity bands in polyacrylamide gels was achieved after removal of SDS, incubation in buffer, and staining with Coomassie brilliant blue. Tissue activator extracted from pig hearts displayed a molecular weight of 72000 and migrated similarly to activator secreted by human melanoma cells and to one activator component present in extracts of human uterus. Immunoadsorption experiments with melanoma cell activator antiserum indicated that these 72-kDa activators are all related immunologically. Human uterus also contained a second activator component with a molecular weight 55000, which migrated similarly to a higher molecular weight component of urokinase and cross-reacted with urokinase antiserum. We conclude that the 72-kDa uterine activator component represents a tissue activator and the 55-kDa component represents a urokinase-like activator. A euglobulin solution from venous occlusion plasma displayed multiple bands of plasmin activity in the Mr range 85000-96000. Two activator components were also present, one of Mr 72000 and another of Mr 62000. The 72-kDa euglobulin activator was adsorbed by MCA antiserum, and we conclude that this component represents vascular activator. The 62000 activator also had weak plasminogen-independent caseinolytic activity and was not affected by either melanoma cell activator or urokinase antisera. Conclusions concerning its identity cannot be made at this time.

Immunohistochemical identification of mast cells in paraffin- and epon-embedded tissues using platelet factor 41.

An immunoperoxidase method has been developed for staining heparin in the granules of mast cells. The method employs human platelet factor 4 (or anti-heparin) and a rabbit antiserum to this polypeptide. Platelet factor 4 binds to mast cell heparin and provides the basis for immunoperoxidase staining using the rabbit antiserum. Preliminary studies of mast cells in various tissues indicate that the stain is quite specific for the content of mast cell granules, presumably heparin and possibly other glycosaminoglycans.

A high prevalence of hypomagnesemia and hypermagnesemia in hospitalized patients.

The prevalence of hypomagnesemia and hypermagnesemia among hospitalized patients was studied by determining magnesium levels in 621 serum samples randomly selected from those submitted to the clinical chemistry laboratory for a biochemical test panel. The reference range for serum magnesium was established in this study as 1.2 to 1.9 mEq/L from measurements of serum magnesium on 341 healthy volunteers. Hypomagnesemia (less than 1.2 mEq/L) was present in 68 patients or 11.0%, and hypermagnesemia (greater than 1.9 mEq/L) occurred in 58 patients or 9.3%. The degree of association between hypomagnesemia and hypocalcemia was assessed by measuring serum magnesium on a separate group of 61 patients with hypocalcemia (corrected calcium less than 8.6 mg/dL). Hypomagnesemia was present in 23.3% of patients hypocalcemic in the absence of renal failure; this proportion was higher significantly than the 11.0% who were hypomagnesemic in the hospitalized patient group (P less than 0.025).

Heterogeneity of serum creatine kinase activity among racial and gender groups of the population.

To develop reference ranges for creatine kinase (CK) appropriate for the patient population served by this hospital, levels of serum CK were measured in 1,537 individuals in our employee population. There was substantial heterogeneity in mean, median, and range of CK levels among the several race/gender subgroups in the population studied. The race/gender subgroups could be placed into three broad groups: a high CK group, composed solely of black men; an intermediate CK group, consisting of nonblack men plus black women; and a low CK group, comprised of nonblack women. Mean CK level of the high CK group was twice that of the intermediate CK group, which, in turn, was twice that of the low CK group. Differences in mean CK values among the subgroups placed into either the intermediate CK group or the low CK group were not significant when tested with analysis of variance. Therefore, practical reference ranges for these groups are as follows: 52-520 U/L for the high CK group; 35-345 U/L for the intermediate CK group; and 25-145 U/L for the low CK group.

Immunohistochemical characterization of reactive and neoplastic mast cells.

Mast cells are connective tissue elements likened to unicellular endocrine organs because of the wide diversity of physiologic and pathologic events associated with the secretion of biologically active compounds. Using an immunoperoxidase method (PAP), we studied tissue from patients with benign and malignant systemic mastocytosis and with a variety of reactive conditions. The following immunoreactive antigens were identified in mast cells: a heparinlike compound or compounds (HLC), prostaglandin, serotonin, and fibronectin. HLC is constantly present, staining mast cells in a granular fashion from most lesions. Serotonin and prostaglandin stain in a diffuse cytoplasmic manner in occasional lesions. Fibronectin is found in a surface location in selected cases. We found no clear association between the immunoreactivity of one compound in mast cells and one clinical symptom, e.g., HCL with bleeding, prostaglandin, or serotonin with systemic vasomotor activity or fibronectin with increased tissue fibrosis. However, patients with localized and systemic disease had symptoms that might have been attributed to more than one compound. Only occasional patients with reactive conditions showed such symptoms. The presence of these compounds, either alone or in combination, did not separate benign from malignant conditions. Other cells within selected tissues also stained with the antibodies tested. Despite the lack of exclusivity, these antibodies are useful in identifying mast cells within tissue sections and may have a role in the study of mast cell constituents.

Role of prostaglandins in IUD-associated uterine bleeding--effect of a prostaglandin synthetase inhibitor (ibuprofen).

Prostaglandins have been shown to be increased in the endometrium of women and experimental animals wearing intrauterine devices (IUDs). As prostaglandins may cause increased vascularity and vascular permeability, and as certain prostaglandins (PgI2) inhibit platelet activity, the local generation of prostaglandins may contribute to endometrial bleeding. Thus, the effect of ibuprofen, a prostaglandin synthetase inhibitor, was tested by quantifying menstrual blood loss in 20 women wearing IUDs in a double-blind, 2-period crossover study. Ibuprofen produced a significant reduction in menstrual blood loss; the percentage reduction was greater in women using a Lippes Loop and who had heavier blood loss (39%) than in women using a copper device and who had lighter blood loss (25%). These findings support the contention that prostaglandin synthesis is important in the genesis of IUD-associated menorrhagia and that prostaglandin inhibitors may be useful in the therapy of this condition.

Menstrual blood quantitation in the rhesus monkey: an experimental tool for improving intrauterine contraceptive devices (IUDS).

PIP: 6 normally menstruating and 21 ovariectomized rhesus monkeys were used to measure the menstrual blood loss (MBL) during an estrous cycle. A reliable method of quantitating MBL should provide better evaluation of the bleeding problem associated with IUDs. The technic used in measuring the MBL and the volume of MBL are presented. The ovariectomized animals were given 25-mcg estradiol im daily for 22 consecutive days of each month and 1.5-mg progesterone daily during the second half of the injection schedule. These injections supported normal endometrial histology. Intravaginal pads were used for the collection of the menstrual blood. MBL was calculated by the Hallberg-Nillson method. Average MBL for all menstrual periods was 2.01 ml. This should provide a necessary parameter for evaluation of IUD-associated uterine hemorrhage.^ieng

Uterine bleeding in the rhesus monkey in response to various designs of intrauterine devices.

PIP: To study quantitative menstrual blood loss (MBL), 10 normally rhesus monkeys and 18 ovariectomized, hormonally supplemented monkeys (estradiol and progesterone) were inserted with IUDs: a silastic cylinder and 3 modifications the polyethylene Tatum T device. A 92% increase (p.01) in MBL was noted in the Y-T device monkeys (n=7) and a 70% increase (p.05) in the large oval T ones (n=5). MBL was unaffected by the small oval T, and by the cylindrical Silastic devices after the second postinsertion period. Intermenstrual bleeding was observed in 4 of 7 Y-T device animals but was uncorrelated with MBL. It was concluded that rhesus monkeys may serve as good research models for designing human IUDs.^ieng

A rapid and highly sensitive solid-phase radioassay for plasminogen activators.

A rapid and highly sensitive solid-phase radioassay for the measurement of plasminogen activators is presented. The method employs a convenient and stable 125I-fibrinogen-latex bead product and can reproducibly detect 0.25 milli Ploug units/ml of urokinase. This represents a 100-fold increase in sensitivity over previously published radioisotopic solid-phase technique and a 120-fold increase over the sensitivity of the fibrin plate method. Since the assay can readily detect plasminogen activator levels in euglobulin solutions prepared from pre- and post-venous occlusion plasma, it may be useful for rapidly detecting and monitoring the fibrinolytic potential of patients predisposed to thromboembolic disease.

Ultrastructural hemostasis in response to vascular injury induced by intrauterine devices in human endometrium.

PIP: 8 micrographs are presented to help elucidate the mechanism of endometrial bleeding caused by IUDs. A previous study, which reported endometrial vessels with defects or gaps in the superficial portion of IUD-exposed endometrium, is expanded. Since the endothelial cells forming these gaps were in stages of degeneration to complete necrosis, perhaps IUD/endometrial interactions injured some of the superficial vessels which then degenerated, formed gaps, and allowed blood to escape. However, the same endothelial degeneration should initiate platelet adhesion, aggregation, and thrombosis. Yet the micrographs showed degenerated endothelial cells at the vessel gaps, gaps which represent disintegrated endothelial cells, and collagen which appeared to be directly exposed to luminal blood through the gaps. All of these hemostasis-initiating conditions resulted in a surprizing low level of activity. Rarity of platelet and/or fibrin thrombi plugging vessel gaps in IUD-exposed endometrium supported the feasibility of bleeding through small gaps. The interstitial hemorrhage is apparent by electron micrograph. When the concentration of red cells within the superficial endometrium became sufficiently high, apparently the erythrocytes either exuded through the spaces between surface epithelial cells or ruptured into the uterine caivty and resulted in clinical bleeding.^ieng

Selection of reference laboratory services.

This article is directed primarily to hospital pathology and clinical laboratories that require the testing services of other laboratories. However, many of the observations and principles presented should apply to other users of reference laboratory services, such as outpatient clinics, public health laboratories, occupational health laboratories or clinics, and even the individual doctor's office.

Cost-containment and the use of reference laboratories.

Hospital laboratories and hospital-independent reference laboratories will need to change in order to provide comprehensive, medically appropriate, and reasonably priced laboratory services in the cost-containment age we are entering. The change must be economically and technologically innovative and relevant to society's next generation of health care needs. Hospital laboratories and commercial laboratories may become weaker or stronger relative to one another, but our guess is that they will ultimately become more like one another or even may join forces to provide optimal patient care in the future. Until that time comes, hospital laboratories must decide whether to employ reference laboratory services more or less, enter a joint venture with a reference laboratory, or become a reference laboratory. Some of the items that could be considered in arriving at this decision are listed in Table 2. Some items favor hospital laboratories; some favor reference laboratories; some are a toss-up; and some suggest there are advantages in a team approach. For the present, we believe there are many arguments favoring a continuation and possibly even an expansion of hospital laboratory services, but this will likely be most feasible in financially sound and progressive hospitals having forward-looking administrators and imaginative but fiscally minded laboratory directors and managers. If decisions are made to send more tests to reference laboratories, each hospital or user laboratory must seek the best and most cost-effective services available. Various financial, technical, and medical considerations are described that should aid in the evaluation of where to have tests performed. We have provided suggestions on how agreements with reference laboratories can be established in either a formal (contractual) or an informal (verbal) way. Additionally, we have described methods for evaluating (or monitoring) the quality and quantity of services received from a reference laboratory. In general, for any significant agreement with a reputable reference laboratory, little more may be necessary for monitoring purposes than periodic financial and quality assurance audits and follow-up on any clinical complaints regarding test results. With a large contract, the user laboratory is advised to spot check results on submitted blind duplicates of patient samples (to test provider lab precision) and occasionally to split samples between the provider and one or more other reference laboratories (as a first look at possible inaccuracy).(ABSTRACT TRUNCATED AT 400 WORDS)

Morphologic studies on IUD-induced metrorrhagia. II. Surface changes of the endometrium and microscopic localization of bleeding sites.

Surface changes were extensively studied by light and electron microscopy in human endometrium exposed to IUD's. A wide variety of alterations in the covering epithelium and its basal lamina (basement membrane) was observed. These ranged from essentially no alteration to a covering basement membrane completely denuded of its epithelium. Erosions or discontinuities of the surface basement membrane were uncommon, and when they occurred were most often associated with extrusion of fluid and cellular elements from the stroma into the uterine lumen. Metrorrhagia associated with IUDs probably results from two basic types of hemorrhage through the endometrial surface. Tissue adjacent to the IUD with interstitial hemorrhage bleed into the uterine cavity by (1) red cell transmigration through surface membranes (surface epithelium and its basal lamina), and (2) high interstitial pressure breaks in these same membranes.

Comparative quantitation of menstrual blood loss with the Lippes Loop, Dalkon Shield, and copper T intrauterine devices.

PIP: The magnitude of menstrual blood loss and the incidence of menorrhagia associated with 2 plastic intrauterine devices (IUDs), THE Lippes loop and standard-size Dalkon shield, were compared with a copper T device (TCu 300). In order to evaluate long-term as well as short-term effects, blood loss was measured at postinsertion levels of 6, 12, and 18 months in 72 women wearing the Lippes loop, in 73 wearing the Dalkon shield, and in 82 with TCu 300. Increases in the mean blood loss over controls (35.3 ml) for the Lippes loop at 6, 12, and 18 months were 112%, 60%, and 95%, respectively. Corresponding increases for the Dalkon shield were 71%, 112%, and 86%; those for the TCu 300 were 54%, 56%, and 41%. There was significantly less bleeding with the TCu 300 than with either of the other 2 devices (p.05). The Lippes loop and the Dalkon shield were associated with fluctuating blood loss values whereas blood loss with the TCu 300 was relatively stable at all study periods.^ieng

Quantitation of menstrual blood loss--further evaluation of the alkaline hematin method.

PIP: An evaluation of the photometric alkaline hematin procedure was carried out to determine its reliability and practicality in measuring menstrual blood loss (MBL). A reliable method for quantitating MBL was necessitated in order to measure the effect of IUD design on uterine bleeding. MBL was measured in 6 women during 2-4 consecutive cycles (total of 15 cycles). Test samples of blood, either as liquid or soaked into sanitary napkins, vaginal tampons, or cotton pads were incubated at room temperature in 5% (w/v) aqueous sodium hydroxide solution. After incubation, absorbance of the brown-colored alkaline hematin was measured at 550 nm in a Perkin-Elmer Model 124 dual beam spectrophotometer against a blank of either 5% sodium hydroxide or distilled water. The quantity of blood in each sample, represented by eluted, hematin-converted hemoglobin, was determined from a standard curve relating various known volumes of incubated blood to absorbance at 550 nm (A550). Standard volumes of blood were always incubated for the same length of time as the samples. When the samples were diluted with more sodium hydroxide solution than the standards, sample absorbances were multiplied by an appropriate dilution factor. The photometric alkaline hematin procedure indicated a sensitivity to less than .1 ml of blood, within-batch precision of 5% or less (2 C.V.), and an accuracy of measuring human menstrual blood from most sanitary devices generally within plus or minus 5%. The method appeared to be specific for menstrual blood and independent of other materials in genital fluids. Menstrual discharge could be stored for at least 1 month prior to determination without alteration of results. The procudre, once optimized for the authors' purposes, was found to be very practical. The results of the study were consistent with the available literature which indicated that maximum normal menstrual blood flow is between 60 and 80 ml, and the mean is near 30 ml. Variation of measured flow between consecutive menstrual cycles in some individuals (both normal and menorrhagic) revealed the necessity for repeated measurements to properly assess average blood flow in certain cases.^ieng

Morphologic studies on IUD-induced metrorrhagia. I. Endometrial changes and clinical correlations.

Morphologic studies on human hysterectomy specimens indicate the IUD elicits a vascular reaction which is most pronounced in the endometrium adjacent to the device. This reaction includes increased vascularity, congestion and increased permeability, and degeneration with defect formation. In addition, there is poor hemostatic responsiveness to vascular permeability and damage. The reaction leads to interstitial hemorrhage which undoubtedly causes metrorrhagia. A likely cause for initial vascular damage is mechanical stress transmitted by the IUD through the endometrium to its vascular network. Vascular reaction and poor hemostatic responsiveness may be perpetuated during each cycle by the products released from endothelial cell degeneration and necrosis. Bleeding is one of the most frequent complications leading to discontinuation of an otherwise effective form of long-term contraception and family spacing. Therefore, its solution could be of crucial importance to world-wide population control. Our findings suggest that better understanding of the mechanism of IUD-induced metrorrhagia should result from closer study of the endometrium adjacent to that which is compressed by the IUD.

Menstrual blood loss and body iron stores in Brazilian women.

Menstrual blood loss (MBL) studies are relevant for developing world women as this could be an important cause of anemia. Whenever a contraceptive method is to be used by such women, consideration should be given to the method which least affects the volume of MBL. In 309 women considered as clinically healthy, MBL, serum ferritin, serum iron and hemoglobin levels were measured: a mean MBL of 23 ml was found. Age, weight, height and previous oral contraceptive use did not affect MBL. Higher parity women may have higher MBL levels but their hematologic indices are not altered. While body iron stores (as judged by serum ferritin levels) are depleted in women who bleed more than 60 ml per cycle, clinical anemia may not be present until their blood loss exceeds 80 ml per menstruation. Brazilian women who lose more than 60 ml of menstrual blood associated with multiple pregnancies without adequate iron supplementation may have a depletion of their body iron stores.

Changes of plasminogen activator in human uterine tissue induced by intrauterine contraceptive devices.

Potassium thiocyanate-extractable uterine plasminogen activator activity was determined to be highest in the endometrium surrounding intrauterine devices (IUDs). Such activity was significantly higher than that encountered in control endometrium or in the endometrium remote to IUDs. As in control cases, extracted endometrial activity fluctuated during the intermenstrual ovarian cycle. It was highest in the pre- or periovulation part of the cycle, and it rose again prior to menstruation. These peaks of activity seem to correspond to times in the cycle when metrorrhagia and abnormal menstruation are usually encountered. Possible implications of the myometrial and endometrial patterns of plasminogen activator in control and IUD-exposed uterine tissue are discussed.

PIP: This research study was designed to determine the relative activator concentration in uterine tissue depressed by, adjacent to, and remote from the IUD and to compare these concentrations to control uterine tissues. Uterine tissue used in the study was obtained following hysterectomy in 22 women wearing IUDs. All analyzed tissues were acquired from uteri containing either Lippes loop D or large Saf-T-Coils. These IUDs had been in place for a minimum of 1 year and a maximum of 8 years. Hysterectomies were performed primarily for indications related to symptomatic pelvic relaxation secondary to multiparity. No patient was taking hormonal medication or contraceptives for 12 months prior to hysterectomy. No cases included specimens with significant uterine pathology other than that related to the IUD. Ovarian cycle phase was determined for all specimens analyzed by histologic criteria on adjacent slices of the excised samples. Cases included the following phases: menstrual; early proliferative; mid to late proliferative; early to mid secretory; and late secretory. The myometrium contained considerably more extractable activator than the endometrium in terms of activity per milligram of tissue protein. Variation of endometrial plasminogen activator (PA) showed the same pattern as did previously analyzed control specimens at different stages of the intermenstrual ovarian cycle. Values reached the highest levels in mid to late proliferative endometrium with a fall during the early to mid secretroy phase to the lowest levels, followed by a rise in late secretory endometrium. Myometrium did not parallel endometrium as closely as reported for control cases. In IUD cases, the highest myometrial levels were encountered in the late follicular phase and a fall occurred in the secretory phase, as was the case with endometrium, but no myometrial rise occurred prior to menses. Comparison of mean activator results of depressed, adjacent, and remote endometrial sites from IUD cases and mean endometrial activator levels from control cases revealed the highest values in IUD depressed endometrial tissues. The next highest were in IUD adjacent endometrium. Significantly lower results occurred in IUD remote endometrial samples. These comparisons were not considered to be biased by interphase differences in activator since equal proportions of all 4 menstrual phases existed among the 3 IUD sampling sites and the total control material studied. In sum, uterine PA activity plays an important role in IUD associated menorrhagia, and the results indicate that it is also important in IUD associated metrorrhagia. The causes of cyclic physiologic fluctuation and of IUD stimulated increases in uterine PA activity are poorly understood at this time.

Quantitative menstrual and intermenstrual blood loss in women using Lippes Loop and Copper T intrauterine devices.

PIP: 127 women were studied during 3 consecutive menstrual cycles preceding, and 6 non-consecutive menstrual cycles during the 1st year following insertion of either Lippes Loop C or Copper T. Both (MBL) menstrual blood loss and (IMBL) intermenstrual blood loss were quantified during these cycles. When postinsertion MBL was averaged and compared to mean preinsertion MBL, the Lippes Loop and Copper T devices increased the volume of menstrual bleeding by 99 and 42% respectively. In spite of this, mean hemoglobin levels did not change significantly during the period of study. Quantifiable MBL was experienced primarily during the 1st cycle postinsertion. The incidence was 90% in women inserted with the Lippes Loop C and 48% in those women inserted with the Copper T during this cycle. The volume of IMBL was extremely variable among the women studied (0.7 - 398 ml). In several cases the volume nearly equalled or even exceeded the MBL of the 1st cycle. Incidence of IMBL fell to 6.5% and 5.0% in the 2nd postinsertion cycle for women with loops and copper devices, respectively. Thereafter the incidence was neglibible. This marked decrease in incidence was not due to closures for bleeding. Less than 10% of the total blood loss experienced during the 1st year postinsertion was the result of IMBL and following the 1st postinsertion cycle, it contributed less than 2% of the total blood loss. The conclusion is that IMBL, while contributory to IUD discontinuation rates, does not contribute significantly to total blood loss and thus to iron loss following IUD insertion.^ieng

Effect of prior pregnancy and combined oral contraceptives on baseline menstrual blood loss and bleeding response to intrauterine devices.

No effect of pregnancy was found on baseline menstrual blood loss (MBL) in women within one year of parturition or abortion. The increased bleeding response of women to intrauterine devices (IUDs) was found to be independent of pregnancy status during the year preceding IUD sertion. Women pregnant within a year of insertion had no different MBL than those pregnant more than a year prior to insertion. MBL quantified in subjects within three months of discontinuing combined oral contraceptives (OCs) was significantly lower than in prior non-OC users. Furthermore, MBL was significantly reduced in the former group during the first three menses following IUD insertion. At the sixth and twelfth menses post-insertion, MBL was still lower in prior OC users, but the difference between users and non-users was less and no longer statistically significant.

PIP: The effects of pregnancy and oral contraceptive (OC) use within 1 year of IUD insertion were studied on both preinsertion and postinsertion menstrual blood loss. Of 88 starting subjects, 36 (41%) had not used OCs for the previous 15 months or longer, and had had a pregnancy termination more than (n=21) or less than (n=15) l year of study (nonusers). 52 patients had used OCs (combined-type) for 3 months-13 years before study; they discontinued OCs from 38-88 days before IUD insertion (users). No effect of pregnancy was found on baseline menstrual blood loss in women within 1 year of parturition or abortion. Increased bleeding was found in all women after IUD insertion was independent of pregnancy status; women pregnant within 1 year of insertion had no different menstrual blood loss than those pregnant more than 1 year of prior. The OC user group had significantly lower menstrual blood losstthan prior non-users. Menstrual blood loss was significantly reduced in the OC user group during the 1st 3 menstrual flows after IUD insertion. Though still lower at the 6th and 12th months postinsertion, the difference was no longer statistically significant for either.