The interaction of normal lymphocytes and cells from lymphoid cell lines: IV. HL-A typing of the cell line cells

Cells from thirty-three human lymphoid cell lines and sublines have been typed for HL-A antigens by a microcytotoxicity test. Similar patterns of HL-A antigens were found for the cell line cells and for the fresh lymphocytes of the donor of the line (eleven cases). However, certain typing sera gave positive reactions with the cell line cells which were not found with the fresh lymphocytes. No correlation was noted between the pattern of these `extra' reactions and the HL-A typing of the cells. These same typing sera often gave positive reactions with blood lymphocytes cultured for several days in conventional media. These positive reactions were quantitatively more pronounced and sometimes quantitatively different if the cells had been stimulated. A range of normal sera failed to react with cell line cells suggesting that the HL-A typing sera giving `extra' reactions are detecting antigens in some way related to a histocompatibility system. Absorption studies performed with two of the lines confirmed the HL-A typing by the direct cytotoxicity test. Two sera giving `extra' reactions were also tested in the absorption experiments. The results indicated that antibodies other than those of the HL-A specificity designated for these sera were responsible for the `extra' reactions. It is suggested that `extra' reactions indicate a change in the apparent antigenic expression of lymphoid cells reflecting altered membrane characteristics as they adapt to a culture environment.     

A one-step monoclonal antibody typing procedure that simplifies HLA class I and class II typing

Abstract: We have developed monoclonal antibodies to most HLA specificities, making it possible for us to devise a simple, rapid, one-step micro-cytotoxicity test. The test is performed by adding 1 μl of cells to 1 μl of antibody-complement mixture predotted on the microtest tray. The reactions are read following a 1-hour incubation period (30 minutes in some instances). The analysis of reactions seen on testing 105 class I antibodies and 50 class II antibodies is shown. A comparison of typing by the standard NIH method and the new one-step procedure showed a > 96% concordance in the 500 T cells and 200 B cells we examined. Class I and class II typing could be performed using B cells, thus obviating the need to isolate both T and B cells for HLA typing.     

Avoidance of Certain Systematic Pitfalls in the Detection of HLA–DR Antibodies Defining Fine Specificities

A selective screening program has been established to identify rapidly and effectively the fine specificity of HLA-DR antibodies in pregnancy anti-HLA sera. Following initial HLA-A, B, C screening sera with extra- or multispecific reactions were selected and specifically tested after platelet absorption on isolated B- and T-lymphocyte populations of the serum donor's husband. Identification of the HLA-DR type of the husband, as well as screening and typing on HLA, A, B and -C typed heterozygous panel B cells led to a more precise characterization of the major specificity of a detected anti-HLA-DR serum. Multispecific anti-DR sera were defined and rendered operationally monospecific by titration.
Some critical steps in a reliable assessment of HLA-DR typing reagents could be worked out. Weak HLA-A, B antibodies, B-cell auto- and Lewis antibodies may cause positive reactivity preferentially or even selectively on B lymphocytes. Of particular importance was the hidden presence of HLA-C specific antibodies, since they cannot be absorbed out by stored platelets. In addition they are not readily detectable through screening on typed panel cells. Because of the frequently very high linkage disequilibrium between HLA-B and HLA-C alleles it is difficult to select appropriately dissecting panel cells. The two points demonstrated above gain even more weight when isolated T and B cell populations are used for HLA-DR typing, because HLA-C antibodies preferentially kill B cells. In this fashion contaminating HLA-C antibodies are not only difficult to detect but can mimic the presence of HLA-DR antibodies.     

A new rapid and more sensitive microcytotoxicity test

A new rapid microcytotoxicity test based on the use of ethidium bromide is described. This substance diffuses rapidly through the membrane of damaged cells and gives an intense red fluorescence with the nuclear DNA. The results can be read in a regular fluorescence microscope 30 min after addition of the cell suspension to standard microtrays, and are easier to interpret than the currently used microcytotoxicity tests. With longer incubation times this method permits the use of dilute HLA typing sera. It is speculated that this would open the way to the preparation of absorbed HLA sera as reagents.     

Sensitivity of Various HL-A Typing Techniques

It has become apparent that the crossmatch procedures for the HL-A typing of organs, tissues and leukocytes for transplant and transfusion recipients are assuming a more prognostic role than conventional HL-A typing. Recent data has shown that regardless of the grade of the HL-A match, a positive crossmatch invariably indicates a troublesome clinical course for the patient. The antiglobulin lymphocytotoxicity technique is a relatively new HL-A crossmatch procedure. This technique was evaluated against the NIH standard lymphocytotoxicity test, with standardized HL-A typing sera. Pre- and post-transplant sera from several patients were compared using the two test systems in an attempt to show correlations with clinical courses. In addition, sera from patients who exhibited febrile reactions, which were not attributable to red cell incompatibilities were evaluated for crossmatch and conventional HL-A incompatibilities.     

Phage typing of Staphylococcus epidermidis.

Thirteen phages were isolated from lysogenic cultures of Staphylococcus epidermidis from a clinical laboratory and used to type 223 clinical isolates of this organism. The 18 phages isolated in The Netherlands were used to type these same cultures. No correlation was observed between phage type, biotype, or clinical source of isolation. At phage concentrations of 100 times the routine test dilution, 35.0% of the cultures were typable with out phages and 21.5% were typable with the phages from The Netherlands. When only cultures in biotype 1 were considered, 43.3 and 24.1% of 141 cultures were typable with our phages and those from The Netherlands, respectively. The lytic reactions obtained with our phages were generally stronger and easier to read and the lytic patterns were, almost invariably, shorter. The typability of untypable cultures was increased 12.0% by incubation at 45 C prior to phage typing and 20% by heat shock (55 C for 5 min) prior to typing. Phage typing 5 subcultures of 20 typable cultures on 5 successive days showed that the lytic patterns were reproducible. The present status of phage typing S. epidermidis and the work needed to obtain a set of typing phages for epidemiological studies of infections by this organism are discussed.     

Techniques used to define human MHC antigens: serology

The classical, routine test employed for definition of HLA antigens expressed in humans (tissue typing) is the complement-mediated cytotoxicity assay developed by Terasaki and McClelland in the early 1960s. In both healthy persons and patients, the assay target cells are usually lymphocytes obtained from peripheral blood, but when typing cadaver donors, splenic or lymph node lymphocytes can be used. HLA-A, B, Cw (class I) antigens are expressed on all nucleated cells while HLA-DR, DQ (class II) are restricted to B lymphocytes and immune activated cells. Tissue typing has been achieved using culture cells from amniocentesis and typing of cell lines is possible with small modifications to the standardised cytotoxicity assay. Usually, target cells are incubated under oil with typing antisera at 22 degrees C in a 60- or 72-well Terasaki tray. After 30 min rabbit serum is added as a source of complement. After a further 60 min incubation the test is stained. A positive reaction results in target cell death. There are local variations to this test. Automation of the assay is now commonplace, from reagent dispensing to automated reading of finished assay. The use of antibody-coated magnetisable microspheres has enabled separation of pure B lymphocyte samples for class II typing and has reduced incubation times through antigen modulation. It is possible to define antibodies to HLA antigens in the same assay using target cells with known HLA phenotypes.     

Solid phase radioimmunoassay for typing herpes simplex viral antibodies in human sera.

An indirect solid phase radioimmunoassay (RIA) was developed for typing antibody to herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2) in human sera. The test is based upon absorption of sera with uninfected, HSV-1-infected cells and testing for residual antibody. The high sensitivity of the RIA method for detecting HSV antibody permits examination of sera at high dilutions, and thus relatively small volumes of virus-infected cells are required for cross-absorption of antibodies. Results obtained in RIA typing of HSV antibodies showed good agreement with those obtained by microneutralization and inhibition of passive hemagglutination. The HSV antibody type(s) determined by RIA also showed good correlation with the virus type isolated from the individual, either from clinical specimens or sensory nerve ganglia. The technique was very sensitive for detection and typing of HSV antibdodies in cerebrospinal fluids. The RIA method was highly suitable for detecting two types of HSV antibody in the same serum specimen, and it was possible to show that a marked, type-specific antibody response to HSV-2 does occur in individuals with a primary HSV-2 infection who have experienced a prior infection with HSV-1.     

New human monoclonal antibody reagents for detecting C, c, E, e, K1, Jk(a), and Jk(b) red cell antigens

Using routine methods, human monoclonal Rh, Kell, and Kidd antibody reagents compared favorably with licensed human- source polyclonal antibody reagents when typing random donor blood specimens. In three different clinical trials, using standard methods and both types of reagents, we tested 2,866 samples by tube, 381 samples by slide, and, using only monoclonal antibodies (MAbs), 1,043 samples by microplate. No discrepant typings were found. More than 95 percent of all reactions using MAbs were the same as or stronger than those with polyclonal antibodies (PAbs). Use of MAbs instead of PAbs for routine typing will result in distinctly stronger reactions, shorter testing times, and decreased anti-human globulin reagent and equipment needs. Product quality will be more standardized, since uniform batches of antibody from immortalized hybrid cell lines can be made available in virtually unlimited supply. Cost-effective production should decrease or eliminate dependence on hyperimmunized human and animal donors from whom variably reactive reagents are currently obtained. This ease of use and more rapid results should improve patient care by facilitating availability of phenotype-appropriate red blood cells for special needs.     

Serological Identification of la Antigens: Report of a British Region la Workshop

In preparation for the 7th International Histocompatibility Workshop 13 laboratories in the British Region participated in a local workshop. One hundred and twenty-three sera which had been previously shown to have activity on either normal B cells, CLL cells or B cell lymphoid lines in the absence of HLA-A, B or C activity were exchanged between the laboratories. These sera were tested on a total of 212 B cells, 101 CLL cells, 76 T cells and 76 lymphoid cell lines. The data was collected and analyzed in Oxford. The analysis showed that six groups of sera could be distinguished. When these groups were compared with the D locus typing of some of the lymphoid lines which were derived from individuals used as MLC typing cells, they were seen to have significant associations with D locus antigens. The serological groups defined were therefore given numbers corresponding to the D locus numbers they associate with, i.e. UK1 is associated with DW1 and so on for UK2, 3, 4, 5 and 7. Comparison of typing techniques showed that long incubation both with antiserum and then with complement, 1 hour + 2 hours gave the best and most reproducible reactions on normal B cells. Residual anti-HLA-A, B or C activity in some of the sera even after platelet absorption showed the importance of adequate checking on T cells after absorption.     

Review: monoclonal reagents and detection of unusual or rare phenotypes or antibodies

Monoclonal antibodies have been used in the formulation of commercially available blood grouping reagents since the early 1990s. It became apparent early on that introducing them into routine use along with, or instead of, human- or animal-derived reagents could and did lead to discrepant reactions. These discrepancies most often came to light when confirming a blood type obtained previously with human- or animal-source reagents or when using two or more sources of a reagent from the same or another manufacturer to perform blood typing or antibody detection or identification testing. A number of factors contribute to differences in reactivity of reagents that are of the same specificity but are from more than one source. One factor is the use of different clones of the same specificity to manufacture blood bank reagents. Another is the effect of the various diluents used by different manufacturers to formulate reagents that contain the same clone(s). In addition, RBCs having unusual or rare phenotypes can cause discrepant reactions when performing phenotyping. Discrepant reactions can also occur because of patient or donor antibodies that react in an unusual manner when antiglobulin tests are performed with monoclonal antihuman globulin (AHG) versus rabbit AHG reagent. It is important to know the identity of the unusual or rare phenotypes and antibodies and to be able to recognize the different types of reactions that will be observed when using more than one reagent of the same specificity. Most importantly, one must be able to interpret reactions correctly and establish the true blood type of the RBCs or specificity of the antibodies. This review will describe situations in which the use of monoclonal reagents from more than one source or manufacturer, or comparison with results of human- and animal-source reagents, resulted in discrepancies with unusual or rare phenotypes or antibodies. Many of the samples described in this review were sent to the reference laboratory at Gamma Biologicals, Inc., in Houston, Texas, which later became ImmucorGamma with sites in Norcross, Georgia, and Houston, Texas.     

HLA-DR and HLA-A, B, C typing of human fetal tissue

In anticipation of clinical trials of fetal pancreas transplantation we have investigated the feasibility of performing HLA-DR and HLA-A, B, C typing on fetal lymphoid cells other than PBL. Using the standard NIH microcytotoxicity test modified for HLA-DR typing it was possible to demonstrate HLA-DR antigens on subpopulations of bone marrow cells and splenocytes but not on thymocytes or hepatocytes. In contrast, HLA-A, B, C antigens could be detected on all four tissues. Excellent HLA-DR typing, confirmed by maternal typing, was obtained for 19 fetuses (14 to 23 weeks old) using bone marrow cells isolated by two-fold purification on discontinuous Percoll buoyant density gradients. Similar purification of splenocytes resulted in weak reactions with anti-DR sera; however, adherent splenocytes recovered from nylon wool columns proved to be primarily DR-bearing and also provided excellent DR typing. As a corollary to these results, non-adhering splenocytes depleted of DR-bearing cells were ideal for HLA-A, B, C typing since spurious reactions due to DR antigens were greatly diminished, whereas strong specific reactions were obtained with anti-HLA-A, B, C sera. Despite weaker reactions with HLA-A, B, C antisera obtained for thymocytes, reliable HLA-A, B, C typing could be obtained when results from thymocytes were evaluated together with typing from bone marrow cells or splenocytes. The possible benefits of fetal HLA typing for fetal pancreas transplantation are discussed.     

Typing of polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients

The study assesses the reproducibility, typability and discriminatory power of several typing methods for Pseudomonas aeruginosa isolated from cystic fibrosis patients. 178 polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients were serotyped using polyclonal sera and monoclonal antibodies, phage typed, pyocin typed and reverse phage typed. 31 of these polyagglutinable isolates, six monoagglutinable isolates and three nontypable isolates were also typed by means of hybridization using a DNA probe. In a comparison of the methods used, on polyagglutinable isolates only, typability was 0% with polyclonal sera, 90% with monoclonal sera, 94% with phage typing, 85% with pyocin typing, 36% with reverse phage typing and 100% with DNA-prope typing. Using monoclonal antibodies, the reproducibility was 75%, while that of phage typing was 88%, pyocin typing 53% and reverse phage typing 62%. Typing with the DNA probe was not repeated. using polyclonal sera, repeated typing showed that 94% of the isolates were polyagglutinable. Using phage typing, 40% of the isolates belonged to phage type 31, while 60% were distributed amongst 32 phage types. Using monoclonal antibodies, 71% of the isolates belonged to 0-group 3, and these isolates showed 16 different phage types. Subdivision of the phage types was further achieved by both pyocin typing and reverse phage typing. The DNA probe typing made it possible in some cases to discriminate between isolates which were otherwise found identical with the conventional typing methods, while in other cases typing with the DNA probe recorded as identical isolates which conventional methods had typed as being different. These differences may be due to a high mutation rate caused by the selection pressure of antibiotics, and by the host immune response. According to our results, investigations of reproducibility and typability of old and new typing methods are essential when they are used in clinical situations. The low reproducibility of some of the typing methods in the present study affects the reliability of epidemiological investigations in cystic fibrosis patients. Usage of only one method may not be sufficient in cases of polyagglutinable strains from cystic fibrosis patients.     

A New Alloantigenic System on Human Lymphocytes

Certain otherwise well-defined HL—A typing sera contain extra antibodies which do not appear to be closely related to known HL-A specificities and which react in the cytotoxic test with chronic lymphatic leukemia lymphocytes but not with normal lymphocytes. Despite nonreactivity in the cytotoxic test, absorption studies show that the antigenic factors are well represented on normal lymphocytes. Based on cross-absorption experiments using both normal and leukemic cells, followed by back-testing with the cytotoxic test against a leukemic cell panel, evidence is presented suggesting the existence of a complex, probably multiple allelic system on human lymphoid cells which appears distinct from the HL-A system, and is definable by serologic techniques.     

The major histocompatibility complex of outbred chickens: II, Analysis of the typing response in mixed lymphocyte culture stimulated by homozygous typing cells

MLR phenotypes of outbred and inbred chickens typing B13 of the major histocompatibility complex (MHC) of the chicken were compared. F1 hybrids of outbred and inbred B13 positive chickens were analysed in mixed lymphocyte culture (MLC). The intermediate strength responses of cells from B13 heterozygous outbred chickens stimulated by inbred B13 homozygous chicken cells were not due to minor variations of B encoded lymphocyte activating determinants (Lads). Nor were Lads encoded by genes unlinked to the B complex responsible for these reactions. In contrast, F1 anti-parental type reactions were observed, and these alone are probably responsible for the intermediate strength reactions so often seen in typing of heterozygous outbreds with homozygous typing cells.     

Typing of herpes simplex virus by an enzyme-linked immunosorbent assay with monoclonal antibodies.

We developed a method that uses monoclonal antibodies for typing herpes simplex virus type 1 and type 2 strains. Clinical isolates from GMK cells were seeded directly into a monolayer of GMK cells. After incubation overnight, monoclonal antibodies were added to the infected monolayer, and antibody binding was indicated by a peroxidase enzyme-linked immunosorbent assay. Using prototype strains and previously typed patient strains, we verified the specificity of this technique. This method is now used routinely for typing herpes simplex strains in our laboratory. We have also used this technique for specific staining of type 1 plaques in a mixture of type 1 and type 2 plaques. With this method it is possible to find a single type 1 plaque among several hundred type 2 plaques on a single petri dish. Infectious virus can also be recovered from stained, unfixed type 1 plaques.     

Factors responsible for successful HLA-DR typing of mononuclear cells from cord blood

Of 115 cord blood samples tested, both HLA-DR antigens were identified in 79% of fresh samples, 66% of older samples and 55% of freshly frozen samples. Because of weak reactions, one or two questionable antigens were present in 21% of fresh samples as compared to 34% of older samples and 45% of freshly frozen samples. Older samples showed lower cell viability, more extra reactions, and a high frequency of weak reactions. Comparison of cord blood DR antigens with parental DR antigens revealed that antigens were missed or inappropriately assigned in 5 known cases. This was more frequent with older blood samples. The critical factors in successful DR typing of cord blood samples were 1) dilution of cord blood samples with medium, 2) processing of the samples as soon as possible, and 3) overnight incubation to remove adherent cells.     

Outer-membrane protein and lipopolysaccharide serotyping of Neisseria meningitidis by inhibition of a solid-phase radioimmunoassay.

A new procedure involving inhibition of a solid-phase radioimmunoassay was developed for specific determination of the outer-membrane protein and the lipopolysaccharide (LPS) serotypes of meningococci. Antigen was allowed to bind to the wells of a polyvinyl microtiter plate and then reacted with a limiting amount of homologous antibody which had been preincubated with buffer or a standard concentration of inhibiting antigen. The amount of antibody bound per well was quantitated by incubation with excess 125I-labeled goat anti-rabbit immunoglobulin. Typing sera for detecting eight LPS antigens and 18 protein antigens were made in rabbits by use of both the group C and group B bactericidal serotyping strains. Reactions between unabsorbed sera and purified LPS were inhibited in the LPS typing system, whereas reactions between absorbed sera and outer-membrane complex were inhibited in the protein typing system. Outer-membrane complex was used as the inhibiting antigen in both cases. Approximately 97% of the 80 group B and C strains tested were LPS typable, and 80% were protein typable. Of 51 group A strains tested, however, only 22% were LPS typable and 14% were protein typable. Several nonreciprocal correlations between the occurrence of particular LPS and protein serotype antigens on the same strain were observed, but in general the protein and LPS serotype antigens appeared to occur independently.     

The utility of class I HLA antibodies screening in a Tunisian obstetric department in order to select good HLA typing reagents

In order to reduce the cost of the serologic class I HLA typing, we have established a screening program for HLA antibodies among obstetric patients. Class I HLA antibodies were identified by standard microlymphocytotoxicity test using a panel of 100 lymphocytes. In the study period, carried out between January 2000 and June 2002, 817 women were tested, most of them (62%) during the last trimester of pregnancy and in the other cases after delivery. These patients, aged between 18 and 45 years, were in the majority (88%) multiparous. From the total number of 817 maternal serum samples screened, 194 specimens (23,74%) tested positive for HLA antibodies. Thirty eight HLA specificities were characterised in 110 maternal sera of which 62 contained monospecific HLA antibodies. HLA-A2 and HLA-B51(5) were the most common specificities characterised in this study. HLA alloimmunization was present since the first pregnancy. There was no statistically significant linear correlation between HLA alloimmunization and the number of pregnancies. Fifty maternal serum samples (6,11%) could be used as HLA typing reagents. Of these 50 sera, 36 had monospecific HLA antibodies. These useful antisera covered a total of 30 specificities: 8 HLA-A and 22 HLA-B. The cost of self - screening for useful antisera as estimated at dollars 62/mL, the personnel was not considered. However, the lowest cost of commercial HLA typing sera is approximately dollars 400/mL. Our study showed the utility and the net economic advantage to use maternal sera as reagents in our new HLA laboratory with limited budget.     

Herpes simplex virus type-selective enzyme-linked immunosorbent assay with Helix pomatia lectin-purified antigens

Helix pomatia lectin-purified antigens with specific reactivity to herpes simplex virus type 1 (HSV-1) and HSV-2 antibodies in human sera were used in an enzyme-linked immunosorbent assay. The type specificity of the antigens was assessed by double immunodiffusion precipitation in gel against rabbit HSV-1 and HSV-2 hyperimmune sera, and by enzyme-linked immunosorbent assay with human reference sera containing antibodies to either type of HSV. Fifty-two sera from patients with documented infection with either HSV-1 or HSV-2 were assayed for HSV type-specific immunoglobulin G antibodies. The reactivity of the sera against lectin-purified antigens correlated completely with the results of virus typing. We conclude that HSV type-specific immunoglobulin G antibodies can easily be measured by enzyme-linked immunosorbent assay with the use of Helix pomatia lectin-purified HSV-1 and HSV-2 antigens.