Sensitive Two-Stage Papain Technique without Cell Washing

A two-stage papain technique is described in which cell washing after papain treatment is replaced by the addition of a specific papain inhibitor. This technique permits optimal enzyme treatment of red cells while digestion of immunoglobulin following the addition of serum is avoided. The technique therefore combines the design and consequent sensitivity advantage of two-stage tests with the convenience of one-stage tests, rendering it suitable for use in compatibility testing.     

Differences in Substrate Specificities for Cysteine Proteinases Used in Blood Group Serology, and the Use of Bromelain in a Two-Phase Inhibitor Technique

Because substrate specificities differ between proteolytic enzymes and because knowledge of the optimal enzyme activity levels is necessary in order to standardize procedures used in antibody screening, a study was made of the best common assay method for the routinely employed enzymes bromelain, papain and ficin. Casein degradation was found better suited to this purpose than azoalbumin. With standardization achieved, a useful two-phase bromelain inhibitor technique was devised using bromelain at 20 casein units of activity. This method improved upon the one-stage bromelain technique in terms of sensitivity, freedom from false positive reactions and it compared well with the two-phase papain inhibitor technique.     

Eighteen Months'' Experience with a Manual Polybrene Crossmatch in a Large Hospital Transfusion Laboratory

For 18 months in this laboratory the manual polybrene technique (MP) has been used as the only crossmatching procedure preceded or accompanied by an antibody screen comprising two-stage papain and LISS antiglobulin techniques. There were 17,161 requests representing 43,006 blood units crossmatched and 20,841 units transfused. Non-specific reactivity with the MP required use of an antiglobulin crossmatch in approximately 0.2% of patient samples. Heparin in excess of 20 IU/ml reduced polybrene aggregation of red cells necessitating an antiglobulin crossmatch for 2 patients. Of 288 antibodies detected 20 reacted exclusively by MP compared with 18 by the papain procedure. The data supported the use of MP as an alternative to enzymes in antibody screening protocols. The polybrene technique was found to be a superior abbreviated crossmatch compared with the immediate spin technique and was applicable to all patients including those with known antibodies.     

Effect of papain on the interaction between human monocytes, erythrocytes, and IgG

The mechanism by which papain detaches IgG-sensitized erythrocytes from the monocyte surface has been explored in an in vitro assay for the monocyte IgG receptor using red cells quantitatively sensitized with IgG anti-Rh D immunoglobulin. Papain treatment of IgG-sensitized erythrocytes diminished the ability of these cells to bind to the monocyte surface; however, treatment of erythrocytes with papain prior to sensitization with IgG did not inhibit binding, and at papain concentrations is greater than or equal to 38 mug/ml binding was enhanced. IgG receptor activity was not diminished by prior treatment of monolayer cells with papain and was enhanced with high concentrations of papain. These studies suggest that papain detaches erythrocytes from the monocyte surface by virtue of its proteolytic effect on IgG and not by an effect of papain on the D antigen of red cells or the IgG receptor on monocytes.     

An Antibody Screening Test Based on the Antiglobulin Gel Technique, Pooled Test Cells, and Plasma

The recently introduced gel technique offers significant advances compared to traditional tube tests. The purpose of the present study was to design an antibody screening test based on the gel technique, pooled cells, and plasma and to evaluate this test by comparison with a conventional spin-tube indirect antiglobulin test (IAT) combined with a two-stage papain technique. Pilot studies were performed to establish optimal parameters during the design phase, and finally 5,446 consecutive samples were screened for irregular antibodies by the gel technique in parallel with routine techniques. Irregular erythrocyte antibodies were detected in 151 samples, and the gel technique proved equal or superior to the tube test in the detection of all antibodies except 'enzyme-only' anti-E and anti-Le^a. We conclude from this study that screening for unexpected antibodies using the gel IAT in our set-up, which includes: (1) omission of enzyme technique; (2) the use of stabilized (EDTA) plasma instead of serum as test material in order to facilitate automation; and (3) pooling 2 by 2 of 4 test cells (to make the gel technique price competitive), is a fast, reliable and sensitive procedure that maintains transfusion safety and compares favourably with our previous routine of saline IAT combined with a two-stage papain technique.     

A naturally occurring, warm-reactive macroglobulin specific for papain-treated human platelets: preliminary characterization

Sera from 28 of the 113 normal children and adults (25%) studied were found to contain an immunoglobulin capable of causing complement-dependent lysis of normal platelets treated with small quantities of papain. This factor reacts equally well at 4 degrees C and at 37 degrees C with a determinant induced on platelets from normal subjects by treatment with papain or bromelain, but not by trypsin, chymotrypsin, or neuraminidase. It does not bind to red cells treated with any of these enzymes. The site(s) for which the factor was specific could not be induced on platelets from six patients with type I Glanzmann's thrombasthenia (lacking glycoproteins IIb and IIIa), in contrast to platelets from each of 20 normal donors. Isolation and characterization of the factor has been difficult because of its intolerance to chemical and physical manipulation. In 11 of the 20 individuals studied, however, it was found to have the properties of an IgM immunoglobulin. The factor appears to be different from any previously described, naturally occurring human immunoglobulin. It has not yet been shown to be associated with any disease state, but in the presence of complement, it is capable of causing profound damage to platelets previously subjected to minimal proteolysis, and the possibility that it can provoke platelet destruction in some conditions deserves further study.     

Serological Studies of the H Activity of Oh Red Cells with Various Anti-H Reagents

Several examples of Oh cells have been investigated with various anti-H reagents. After enzyme treatment of the cells with either papain or neuraminidase, very high titres were obtained, particularly with some preparations of Ulex europeus. Fractionation of anti-H Ulex showed that the anti-H component inhibitable by L-fucose required papain treatment of the Oh cells for optimal activity whereas the component not inhibitable by L-fucose agglutinated preferentially Oh cells treated with neuraminidase. The anti-H lectin Cytisus sessifolius which is not inhibitable by L-fucose gave no reaction with papain-treated Oh cells but reacted well when the Oh cells were treated with neuraminidase. Normal O cells were almost equally well agglutinated by the lectins when treated by either of the two enzymes. That the various lectin components activated by enzymes had H specificity was shown by the fact that their activity was inhibited by purified H substance, partially inhibited to L-fucose and 2-fucosyllactose and not at all by purified Lea substance and non-secretor salivas. In spite of the high titres obtained with Oh cells by the enzyme technique, the amount of H present on the red cells, judged by inhibition tests, in comparison with normal group O cells was very small.     

The effect of naturally occurring Rh antibodies on the survival of serologically incompatible red cells

Investigations on six males with naturally occurring Rh antibodies are described. In two subjects in whom the antibody (one anti-E and one anti-D) could be detected only by a two-stage papain technique, the survival of incompatible red cells was normal. In the remaining four subjects, the antibodies (two anti-E and two anti-D) could be detected by the indirect antiglobulin test and, in these, incompatible red cells were destroyed at an accelerated rate; in two of the subjects, 75-99% of the cells were cleared within 24 h; in the other two, 50% of the cells were cleared within 24 h and the remaining cells were cleared far more slowly. All six antibodies were mainly or wholly IgG; a clear-cut immune response was observed in only one case.     

Quantitative evaluation of antibody-dependent lymphocyte-mediated lysis of human red cells

The ability of lymphocytes to lyse human red cells coated with anti-D antibody was assessed by measuring 51 Cr release from labeled red cells incubated with peripheral blood leukocyte suspensions from 12 normal donors. Mixed mononuclear cell suspensions (containing monocytes and lymphocytes) from all donors produced lysis of sensitized red cells. Treatment with carbonyl iron reduced monocyte concentration to less than 1.2% in all donors, as measured by morphologic criteria, esterase staining and ingestion of latex particles. Lysis of red cells following monocyte depletion was markedly reduced in 8 of the 12 donors. Despite depletion of monocytes, unchanged or increased lysis was noticed with the leukocytes of the remaining 4 donors. This lysis was due to lymphocytes, not to residual monocytes. If target red cells were treated with papain or trypsin prior to sensitization, marked lysis occurred with lymphocytes of all donors, including those which did not lyse unmodified red cells. Direct cytolysis of sensitized red cells during contact with small lymphocytes was recorded using microcinematography, which confirmed the role of lymphocytes in mediating lysis. Lymphocyte-mediated lysis of red cells increased with mounting levels of antibody sensitization regardless to prior treatment with papain. Papain increased antibody coating per red cell, yet lysis per molecule of antibody bound was also increased. Lysis was inhibited by IgG1 and IgG3 in the fluid phase but not by IgG2 or IgG4. At an equivalent level of antibody sensitization lysis was augmented by concurrent coating of the red cells with C3b, C3d and/or C4b, though these components could not produce lysis in the absence of antibody coating.     

Automated Red Cell Antibody Analysis. A Parallel Study

Abstract. A parallel study of red cell antibodies was undertaken using two well-known AutoAnalyzer techniques: the bromelin-methylcellulose (BMC) method as described by Marsh, and the low-ionic strength-Polybrene (LISP) technique of Lalezari. In the present study, minor modifications were made to both methods, without changing the basic principle of antibody detection. The results obtained in this parallel study were compared to standard manual techniques. Screening of 13,135 sera gave 8.7% positive reactions. Of these, 22% were identified manually as being caused by red cell antibodies. Further antibodies could be identified by the machine [described elsewhere].
The sensitivity of the automated methods was generally higher than manual techniques, about 12 times for the BMC and 70 times for the LISP method. Anti-s,-Fy^a,-Jk^a,-Jk^b, and-K were less sensitively detected by the BMC than the most appropriate manual method in a test of 14 commercial typing sera. In no case was the LISP less sensitive than manual techniques. However, hemolyzing antibodies can be missed in rare instances. Anti-D was found at a lower sensitivity level of 20–50 ng/ml with the indirect antiglobulin technique, 5–15 ng/ml with the two-stage papain technique, 24 ng/ml with the BMC method and 0.2–0.4 ng/ml with the LISP method. Some weak anti-D's (5%) reacted more strongly in the BMC than LISP. When anti-D preparations used for Rh prophylaxis were quantitated with both methods, the results agreed well.     

Modulation of autoimmunity to beta-cell antigens by proteases

AIMS/HYPOTHESIS: Proteases are used in therapy for autoimmune diseases yet the mechanism of their action remains to be determined. We studied the immunological basis of protease therapy in the context of Type I (insulin-dependent) diabetes mellitus. METHODS: We studied the effects of proteases (trypsin, papain, chymotrypsin, bromelain) on immune reactivity of a series of autoreactive T-cell clones from prediabetic subjects and patients with a recent onset of Type I diabetes and specific to the autoantigens GAD65, IA-2 and insulin-secretory granule protein. RESULTS: Cell surface expression of adhesion, co-stimulatory and homing molecules on both antigen-presenting cells and T cells was changed after protease treatment. Cytokine analyses showed a selective inhibition of proinflammatory (Th-1) but not Th-2 cytokine production. Autoreactive T-cell proliferation was inhibited at pharmacological serum concentrations, whereas non-specific proliferation to phytohaemagglutinin was not affected at these concentrations. Preincubation experiments on T cells and antigen-presenting cells separately showed that this effect was mediated by APCs, but not T-cells. CONCLUSION/INTERPRETATION: Proteases have pleiotropic immunological effects supporting an immunomodulatory potential for the intervention of chronic inflammatory diseases and Th-1 mediated oedema formation.     

pH-dependent cytotoxicity of contaminants of phenol red for MCF-7 breast cancer cells

The pH indicator phenol red (phenolsulfonphthalein) is present in most tissue culture media. Contaminants of this indicator have shown substantial estrogenic activity for estrogen-dependent cells in culture, including the human breast cancer-derived MCF-7 cell line. In the course of other studies, we observed that brief (1- to 4-h) incubations of these cells at 37 C in serum-free medium (Hanks' or Earle's Balanced Salts Solution) could be toxic to MCF-7 cells when the pH was increased above 7.4, but only if phenol red (10 micrograms/ml) was present in the medium. Because damaged/killed cells detached from the substratum (greater than 98% of detached cells stained with trypan blue), we used DNA assay of the cells remaining after treatment and wash (98% of the remaining cells were dye excluding) to further assess cytotoxicity. The MCF-7 cells were more susceptible to the cytotoxicity at lower cell densities, so further characterization of phenol red cytotoxicity was performed at cell densities of 1-10 micrograms DNA/2-cm2 well, or approximately 40,000-400,000 cells/ml medium. In the pH range of 7.0-8.2, 50% cell death was observed in the presence of phenol red at pH as low as 7.6-7.7, with nearly 100% of the cells killed by pH 8.0. Little effect was seen in phenol red-free medium at any part of the tested pH range or in medium that contained phenol red at pH less than or equal to 7.4. In time-course studies of cytotoxicity at pH 8.0 (phenol red, 10 micrograms/ml), greater than 50% cell damage could be observed after less than 1 h, and little cell recovery was observed if the pH was restored to 7.4. For phenol red samples from two major commercial sources, the concentration for half-maximal cytotoxicity (TD50) in dose-responses after 4 h at pH 8.0 showed TD50 values of 2 and 6 micrograms/ml, while the estrogenic activities, as half-maximal stimulation of estrogen-dependent proliferation, were identical at 2 micrograms/ml. Both the cytotoxic and estrogenic activities could be removed from the phenol red by extraction with diethyl ether. A number of contaminants of the commercial phenol red were detected by reverse phase C18 HPLC. Cytotoxicity and estrogen bioassays of each of the HPLC fractions indicated that the pH-dependent cytotoxicity was separate from the estrogenic activity and confirmed that neither activity was associated with the phenol red itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and Mobility of the A, D and c Antigens on Human Red Cell Membranes: Studies with a Gold-labelled Antiglobulin Reagent

S ummary . Studies are presented in which a gold-labelled anti-IgG reagent has been used to map the distribution of A, D and c antigen sites on the human red cell membrane. Red cells were combined with IgG antibodies; ghosts were produced which were then labelled with gold-anti-IgG. D antigen sites were found to be single entities dispersed on the red cell membrane of lysed but otherwise untreated Rh positive cells. Following papain treatment prior to lysis, a clustered distribution was observed. Clustering of D sites was also observed on non-enzyme-treated red cells of the -D- phenotype. Further studies indicated that D and c antigen sites are closely associated on ccDEE red cells. The distribution of A antigen sites was found to be clustered in proportion to the amount of IgG anti-A combined with the cells. Reasons are given for considering such distribution as indirect evidence of mobility of A sites. There was an association between agglutinability of red cells by IgG antibodies and the extent of the clustering of the antigen sites.     

Increased Active Rosette Formation in Patients with Gastrointestinal Cancer after Enzyme Treatment of Lymphocytes In Vitro*

Summary: Increased active rosette formation in patients with gastrointestinal cancer after enzyme treatment of lymphocytes in vitro. B. A. J. Walters, J. C. Rutherford and J. R. Wall, Aust. N.Z. J. Med., 1978, 8, pp. 610–614. Total rosette forming cells (RFC) and active rosette forming cells (ARFC) were estimated in 33 patients presenting with gastrointestinal symptoms suggestive of cancer. When the 33 patients were grouped according to whether they had cancer or not, there was a distinct difference in the percentage of ARFC (P < 0001 Mann-Whitney Test); patients with cancer having much lower values (mean 11 6 ±4–5) than patients without cancer (mean 27 2 ± 3–6). Total RFC were generally lower in the cancer group (mean 54-8 ± 8-3) than the non-cancer group (mean 71-9 ± 4–5) a/though the range was greater. After treatment of the lymphocytes with papain, the percentage of ARFC increased. In the non-cancer group the levels reached a mean of 74-8 ± 1–8, indicating that the T lymphocyte population (mean 71-9 ± 4–5) was converted into cells all having high affinity receptors for sheep red blood cells (SRBC). In the cancer group, after papain treatment of the lymphocytes, the percentage of ARFC increased to levels greater than that obtained for total RFC, suggesting a contribution from the null cell population. Serum taken from these patients was shown to produce a decrease in the percentage ARFC obtained from normal, young individuals. Further, this decrease could be partially reversed by papain digestion indicating that serum from cancer patients may contain a factor that masks T     

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.     

Evaluation of Toluidine Blue for Measuring Erythrocyte Membrane Loss during in Vivo Ageing

A simple method using a cationic dye, toluidine blue (TB), to quantify changes of red cell membrane area has been developed and tested for its validity. After incubating a glucose-depleted red cell suspension with a fixed quantity of TB at 37 degrees C for 10 min, the remaining TB was measured spectrophotometrically at 640 nm. Using this technique, we were able to show differences in TB uptake by populations of young and old red cells. The exact mechanism for TB uptake by the red cells is not clear. Treatment of the cells with bromelin, papain and trypsin reduced the uptake of TB, but neuraminidase and ficin had little or no effect. No inhibition of TB removal by red cells was observed using heparin, D-glucose, glucuronic acid, or N-acetylglucosamine.     

Analysis of enzyme-treated red blood cell surface and haemagglutination using a theory of soft-particle electrophoresis

Background and Objectives  Enzymatic treatment of red blood cells is thought to reduce the cell zeta (ζ) potential, effectively decreasing the distance between cells to less than the length of an immunoglobulin G antibody binding site, and resulting in agglutination of cells. However, the ζ potential given by Smoluchowski's formula is based on theories of the electrophoresis of hard colloidal particles. A theory has recently been developed for the electrophoresis of colloidal particles covered with polyelectrolytes, which we call 'soft particles'.
Materials and Methods  The electrophoretic mobility of red blood cell treated with papain and neuraminidase was measured as the electrolyte concentration of the medium using phosphate buffer. The results were analysed via the formula for 'soft particles'. This mobility formula involved two parameters, the fixed charge density ( ZN ) and parameter 1/λ characterizing the 'softness' of the cell surface layer.
Results  The best-fit curves of 0·1 units neuraminidase-treated red blood cells indicated that ZN decreased by 76% and 1/λ decreased by 8% compared to intact red blood cells. In contrast, in 5 units of papain-treated red blood cells ZN decreased by 45% and 1/λ decreased by 33% compared to intact red blood cells.
Conclusion  The present study shows that the change in ZN for neuraminidase-treated cells was very large, but the cells did not become agglutinable. Papain-treated cells had changes in both ZN and 1/λ, and the cells became agglutinable. 1/λ is one of the important factors for agglutination.     

Tophus-derived monosodium urate monohydrate crystals are biologically much more active than synthetic counterpart

Summary Monosodium urate monohydrate (MSUM) crystals [1] derived from a tophus surgically removed from patients suffering from gout and MSUM [2] prepared from a supersaturated solution of sodium urate were studied and compared with respect to their ability to: (1) stimulate chemiluminescence (CL) production by human polymorphonuclear (PMN) cells, (2) induce hemolysis of the human red blood cells and (3) induce inflammation when injected in the rat paw and knee joint. Human MSUM crystals were considerably more active in stimulating CL production by PMN cells and in inducing synovial inflammation. Both serum and papain pretreatment of human MSUM crystals caused inhibition of their enhancing effect on CL production by PMN cells. Papain pretreatment only reduced their phlogogenic activity. Uncoated and, to a much lesser extent, serum-coated human MSUM crystals induced secretion by mononuclear cells (MNC) of the factor(s) that considerably enhanced CL production by PMN cells. Both tophus-derived and synthetic crystals appeared to be weak hemolytic agents. Serum pretreatment of synthetic MSUM crystals reduced their hemolytic activity. These results suggest that surface coating, destroyed by papain treatment, was probably responsible for cell activation induced by human MSUM crystals.     

A Prospective Study of the Incidence of Red Cell Allo-Immunisation following Transfusion

To establish the incidence and timing of red cell allo-immunisation following transfusion, pretransfusion and serial post-transfusion samples were screened for allo-antibodies in a total of 452 patients who had undergone elective surgery. Antibody screening was performed by 2-stage papain, manual polybrene and indirect antiglobulin techniques (IAT). Red cell allo-antibodies were found in 42 patients and 38 of these (8.4% overall) demonstrated antibodies only after transfusion; 76% of them had Rh specificity. This rate of red cell allo-immunisation is higher than what would be expected if transfused patients were tested only once post-transfusion, as has been the case in several previous studies. For the type of patients studied, this finding may not be of clinical relevance at present because most patients undergoing elective surgery do not require further transfusion in their lifetime. However, this is changing with the longer life expectancy of the population and the increased probability of repeat surgery. Twenty-two (58%) of the antibodies were initially detected by the 2-stage papain and/or polybrene techniques, when the IAT was negative, although later 19 became positive by IAT; this added sensitivity of techniques other than the IAT, to detect early allo-immunisation may be relevant in pretransfusion testing to prevent haemolytic transfusion reactions in patients requiring repeated transfusions.     

Effect of Beta-Propiolactone on Blood Group Antibody Detection

A panel of 50 blood group antibodies covering a range of blood group antigens has been tested in the presence of 0.25% beta-propiolactone as a possible means of reducing infectivity of high-risk HTLV III/HBsAg samples. 11/50 (22%) antibodies could not be detected by the indirect antiglobulin test, and 6/40 (15%) were undetectable by the two-stage papain technique.