The monocyte monolayer assay, an in vitro method for prediction of in vivo survival of transfused incompatible red blood cells: a review.

It has long been a goal of transfusion medicine scientists to predict which patients will make clinically significant antibodies when transfused with donor red blood cells (RBCs). But this goal has yet to be achieved. Not all patients have an adverse response to an RBC transfusion by making an antibody to an RBC antigen, and for patients who do, in most cases, they form antibodies to common antigens for which provision of antigen-negative RBCs is not difficult. However, for patients who make antibodies to many antigens and for patients who make an antibody requiring rare blood that is negative for a high-prevalence antigen, knowing the clinical significance of that patient's antibody is important for effective and timely transfusion. This review of the literature provides information on the monocyte monolayer assays (MMAs) developed to predict the outcome of incompatible RBC transfusion. One of these assays has been used for almost 40 years in the United States to predict the outcome of RBC transfusion in patients with alloantibodies for whom provision of rare RBCs is very difficult. Because all transfusion medicine facilities and blood centers will not likely implement the MMA, it is important that the selection of the referral laboratory be carefully made. The MMA is a proven test in the prediction of incompatible transfusion outcomes in patients with IgG-only antibodies. It has been helpful in decision-making when rare blood components are not available or not available quickly, although decisions on blood transfusion must be made by the physician attending the patient and blood should not be withheld waiting for the MMA result in an urgent situation.

MAM: a "new" high-incidence antigen found on multiple cell lines.

BACKGROUND: Three women have been identified with an antibody to a "new" high-incidence antigen found on multiple cell lines. CASE REPORTS: The proposita, M.A.M., presented during her third pregnancy with an antibody reacting with all RBCs tested except her own. She delivered a thrombocytopenic infant with a 3+ DAT, but without symptoms of HDN. The second example, A.N., presented during her third pregnancy with an antibody reacting with all RBCs tested except her own and those of M.A.M. She delivered a slightly thrombocytopenic but severely anemic infant. The third example, F.K., a sister of A.N., has an antibody reacting with all RBCs tested except her own and those of M.A.M. and A.N. CONCLUSION: This "new" high-incidence antigen has been named MAM and assigned high-incidence antigen number 901016 by the International Society of Blood Transfusion. The corresponding antibody, anti-MAM, has been shown to cause HDN and has the potential to shorten RBC survival after the transfusion of incompatible RBC units, as determined by monocyte monolayer assay. Immunoblotting and flow cytometry show that this new antibody reacts with various WBC lines in addition to RBCs. This antibody also appears to react with platelets in some assays.

Naturally-occurring anti-Jka in infant twins.

Anti-Jka was detected by solid-phase red cell adherence (SPRCA) antibody detection and identification tests in the plasma of a 9-month-old female infant during a routine presurgical evaluation. The patient and her nonidentical twin sister, who also had anti-Jka in her plasma, were products of an uncomplicated in vitro fertilization, full-term pregnancy, and vaginal delivery. Neither twin had been transfused, recently infected, or treated with medication. Their mother had no prior pregnancies or transfusions. Red blood cells (RBCs) from the patient and her sister typed as Jk(a-b+) by direct hemagglutination, and this phenotype was confirmed by negative adsorption and elution studies. Both infants' plasma samples were strongly reactive with 20 examples of Jk(a+) RBCs and nonreactive with 20 examples of Jk(a-) RBCs by SPRCA assays. Anti-Jka was not detected in either twins' plasma by indirect antiglobulin tests by tube method in low-ionic- strength saline solution or polyethylene glycol, or with ficin- or papain-treated RBCs. Monocyte monolayer assays using Jk(a+) RBCs sensitized by either twins' serum were nonreactive (0%). RBCs from both parents typed as Jk(a+b+). Both parents' antibody detection test results by SPRCA assay were negative. The absence of a history of exposure to allogeneic RBCs or possible passive transfer of maternal or other alloantibody classifies these antibodies as naturally-occurring anti-Jka.

Flow cytometry related to red cells.

Researchers in Transfusion Medicine Science have benefited from the use of the flow cytofluorometer. The flow cytometer has distinct advantages over visual examination of antigen-antibody reactions. The flow cytometer measures fluorescence per cell, and through the use of anti-IgG tagged with a fluorochrome, cells with differing levels of cell-bound IgG can be quantitated. This has been used in the study of allo- and autoantibodies. Immunohematologists, with the wide range of red cell alloantibodies in many blood group systems, have a seemingly unending supply of materials to enable studies of red cells. This article describes the published reports involving flow cytometry related to red cells. Four areas are discussed: detection of red cell-bound IgG, detection of red cell immunogobulins other than IgG, detection and quantitation of red cell antigens, and detection and quantitation of red cell populations.

Monocyte monolayer assay as a predictor of severity of hemolytic disease of the fetus and newborn.

The monocyte monolayer assay (MMA), an in vitro model of in vivo antibody-mediated red blood cell destruction, was previously reported to predict the severity of hemolytic disease of the fetus and newborn accurately when only Rh antibodies and antigen-positive babies were studied. We studied 33 women whose serum contained antibodies with the potential to cause erythroblastosis fetalis; 7 of the 33 women had antibodies other than Rh. None of the babies of the ten women who had consistently negative test results required intrauterine or neonatal transfusions. False-positive MMA results were sometimes found when the fetus was antigen negative. Although the predictive value of a negative MMA was 100%, the efficiency of the MMA was no better than that of the antibody titer. Because of the lack of advantage of the MMA as well as the time and expense it requires, we cannot recommend the general clinical application of this test at this time.

Predicting hemolytic disease of the newborn: a comparison of the monocyte monolayer assay and the chemiluminescence test.

The ability of the monocyte monolayer assay (MMA) and the chemiluminescence test (CLT) to predict the clinical significance of alloantibodies associated with hemolytic disease of the newborn (HDN) was assessed by the use of 22 well-characterized antisera--predominantly anti-D--from alloimmunized pregnant women. Seventeen sera were obtained before delivery from women whose infants were antigen positive for the antibody specificities identified in the maternal serum. With testing of these 17 sera by MMA, 10 results were in agreement with the presence or absence of HDN, but there were 5 false-positive and 2 false-negative results. With the CLT, 16 results were in agreement with the presence or absence of HDN, and there was 1 false-negative result. Five sera were obtained from women whose infants were antigen negative for the antibody specificities identified in the maternal serum. The CLT and the MMA were both subject to false-positive results with these sera. These results suggest that the CLT may be more valuable than the MMA as a noninvasive test for predicting the clinical significance of alloantibodies in HDN.

Anti-i causing acute hemolysis following a negative immediate-spin crossmatch.

A unique case of acute hemolysis following transfusion of red cells (RBCs) that were found compatible by immediate-spin (IS) crossmatch technique is reported. Screening tests for unexpected antibodies, using low-ionic-strength saline (LISS), 10 minutes' incubation at 37 degrees C, and anti-IgG, were nonreactive; however, 1 transfused unit was found crossmatch incompatible by indirect antiglobulin technique (IAT). An anti-i (titer 512 at 4 degrees C) that was not an autoantibody was identified in the patient's serum. Unlike the incriminated donor RBCs, most I+ RBCs did not react by LISS-IAT. Variable reactivity was seen with ficin-treated I+ RBCs, and there was marked hemolysis of iadult and icord RBCs. In marked contrast, dominant Lu(a-b-) RBCs, with reduced expression of i, did not react by any test method; nor did autologous I+, Lu(b+) RBCs. The in vivo clinical significance of this anti-i was confirmed by monocyte monolayer assay and RBC survival studies. The patient's i antigen may have been altered, by either chemotherapy or disease, and lacked part of the i antigen-mosaic. Her antibody was directed at epitopes of i that were absent from her RBCs. Those i epitopes missing from her RBCs are also absent on dominant Lu(a-b-) RBCs. This anti-i represents a unique cause of an acute hemolytic transfusion reaction. It also represents a case of acute immune-mediated hemolysis following transfusion of IS crossmatch-compatible blood when screening tests for unexpected antibodies are nonreactive. Because of the rarity of such cases (less than 1/200,000 RBC units transfused), modifications to pretransfusion testing protocols are not proposed.

Can the reading for serologic reactivity following 37 degrees C incubation be omitted?

The need to detect antibodies that agglutinate and/or hemolyze red cells (RBCs) directly at 37 degrees C, but do not react in subsequently performed indirect antiglobulin tests (IATs), is of concern relative to the streamlining and automation of antibody detection methods. To determine incidence and significance of such reactions, data from 87,480 tests, which used low-ionic-strength saline, 10-minute incubation at 37 degrees C, and anti-IgG, were analyzed for unexpected antibodies. There were 3590 positive tests, of which 475 showed reactions at 37 degrees C but not in subsequently performed IATs (37 + IAT-). Of these, 196 reactions were due to autoantibodies or other factors usually considered insignificant with respect to the survival of transfused incompatible RBCs, 176 were due to alloantibodies of questionable clinical significance (M, Lea, P1, etc.), and 103 were associated with alloantibodies of potential clinical significance (63 E, 27 K, 5 Jka, 4 D, 3 cE, and 1 C). This latter reaction was seen in 72 patients, with two 37 + IAT-antibodies occurring in each of 3 patients. Of the 75 potentially significant 37 + IAT-antibodies, 57 were seen in patients recently exposed to homologous RBCs, 13 in patients with a history of transfusion and/or pregnancy, and 5 in patients with no known exposure to homologous RBCs. IAT reactivity was observed in subsequent samples with 27 of these antibodies. The predictive value of a 37 + IAT-test was 21.7 percent for a potentially significant antibody. The incidence was 0.12 percent of all tests for unexpected antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between in vivo hemolysis and the amount of red cell-bound IgG measured by flow cytometry.

A flow cytometry method was used to compare the amount of red cell (RBC)-bound IgG in 73 patients with and without immune hemolytic anemia (IHA). The positive results in 10 of the direct antiglobulin tests (DATs) were idiopathic, and those in 25 were due to methyldopa therapy; 38 of the 73 DAT-positive patients were babies born to women with IgG alloantibodies of potential clinical significance. Normal blood donors with (n = 30) and without (n = 121) positive DATs were also tested. RBCs that had been strongly sensitized (4+ indirect antiglobulin test) in vitro with different quantities of IgG anti-D, but that had similar antiglobulin test (AGT) titration scores, could easily be differentiated by flow cytometry. The mean percent fluorescence of RBCs, incubated with fluorescein-labeled anti-IgG, from neonatal patients with IHA was higher than that of RBCs from those without IHA, but there was no statistical difference in the other groups. There was considerable overlap in the respective ranges of percent fluorescence of RBCs from patients with and without IHA in all groups. It was not possible to define a clear quantitative threshold differentiating patients with IHA from those without. Although flow cytometry was more precise and reproducible than standard serology (e.g., AGT titration scores), correlations of the amount of RBC-bound IgG and in vivo hemolysis were similar.

Monocyte monolayer assay: an efficient noninvasive technique for predicting the severity of hemolytic disease of the newborn.

The authors performed monocyte monolayer assays (MMAs) with the use of normal donor monocytes, and homologous reagent red blood cells, sensitized in vitro, with antibodies in maternal sera. The sera were from 16 pregnant women with Rh antibodies, drawn at the time of amniotic fluid analysis. They compared the predictive value (PV) of the MMAs and the delta OD 450 of amniotic fluid in forecasting the need for transfusing the infant. The PV of the delta OD 450 result in Liley zone mid II-III was 100%, but in zones 0 to low II it was only 60% (13 samples from ten women were in these zones but four babies required transfusion). In contrast, the PV of a positive (greater than 20% reactivity) MMA was 91% (one false positive result) and the PV of a negative MMA was 100%. Thus, the MMA was more efficient than amniotic fluid analysis at predicting hemolytic disease of the newborn severe enough to require transfusion. The noninvasive MMA could be used as a screening test, reducing the number of amniocenteses or ultrasound evaluations performed on Rh-sensitized women.

Quantitation of fetal-maternal hemorrhage by flow cytometry. A simple and accurate method.

A simple and objective assay was developed for the detection and quantitation of fetal-maternal hemorrhage with the use of flow cytometry. In vitro prepared control mixtures of 10%, 2%, 1%, 0.5%, 0.25%, 0.125%, and 0.06% D+ RBCs in D- RBCs were tested (8-11) different times by flow cytometry and gave mean % D+ results of 11.10%, 1.90%, 0.92%, 0.45%, 0.24%, 0.11%, and 0.05%. The coefficient of variation of preparing and testing these mixtures ranged from 11.0 to 15.9% for the 10-0.125% mixtures. Thus, flow cytometry was accurate, reproducible, and sensitive. Flow cytometry was compared with Du tests, rosette tests, and acid elution. The Du test was highly variable because it was not sensitive enough to detect a significant bleed (approximately 0.6%) in some cases and too sensitive (necessitating quantitation of an insignificant bleed) in others. The rosette test was too sensitive. Acid elution and flow cytometry results did not always agree; acid elution results were approximately twice as high as flow cytometry. The authors believe flow cytometric detection of D+ red blood cells to be more accurate than the detection of fetal hemoglobin by acid elution techniques, which is known to have poor reproducibility. Postpartum samples from 56 D- women who delivered D+ babies were tested. Fifty-two had fetal bleeds less than 0.3% by acid elution and flow cytometry; all had negative Du test results, but there were two false positive results with the use of the rosette technique. Four had significant bleeds (greater than or equal to 0.6%); in all four cases the flow cytometry results were lower than the acid elution results. The authors were able to quantitate a bleed of fetal RBCs, which were D+ only by the Du test, in a D- mother with the use of flow cytometry, and D+ RBCs in a mother whose RBCs were of the rare DVI mosaic phenotype. This would not have been possible with the use of the standard Du or rosette techniques.

Clinical significance of anti-Yt(b). Report of a case using a 51chromium red cell survival study.

Several published reports have documented the variable survival of Yt(a+) red cells (RBC) in patients with anti-Yt(a) as measured by 51Chromium (Cr)-labeled RBC survival studies. Similar studies with anti-Yt(b) have not been reported. A 51Cr-labeled RBC survival study was performed using Yt(b+) RBCs and a monocyte monolayer assay in a young hemodialysis patient who required chronic transfusion therapy and who had developed anti-Yt(b). The survival of the transfused RBCs was 100 and 93 percent at 1 and 24 hours, respectively, with a half life of 21 days at termination of the study (normal, 28 to 32 days). These results showed no evidence of rapid destruction of the Yt(b+) RBCs, indicating that this patient could be transfused safely with blood from Yt(b+) donors. Long-term survival of the 51Cr-labeled Yt(b+) RBCs was shortened moderately, however, a finding that correlated with a slightly abnormal monocyte monolayer assay test.

Erythromycin-induced immune hemolytic anemia.

A 3-year-old female receiving Pediazole (erythromycin ethylsuccinate and sulfisoxazole) for tonsillitis and otitis media developed severe hemolytic anemia. No serum drug-dependent antibodies could be demonstrated with an in vitro 'immune-complex' method using Pediazole, pure erythromycin ethylsuccinate or pure sulfisoxazole. However, a method using red cells coated with erythromycin base showed in vitro lysis of the erythromycin-coated red cells. This is only the second case of immune hemolytic anemia associated with erythromycin and the first where in vitro drug-dependent hemolysis was demonstrable.