Agglutination of red blood cells from patients with diabetes mellitus by a polyclonal human antibody specific for D-glucose.

We have previously described an antibody which will agglutinate red blood cells which had been incubated in vitro in D-glucose. This antibody is specific for the ring form of glucose, beta-D-glucopyranose. The current report demonstrates that without prior in vitro incubation with glucose, red blood cells from 36 of 38 patients with diabetes mellitus, and 7 of 70 patients not diagnosed as diabetic were agglutinated by this antibody. Strength of agglutination of red blood cells from diabetic patients correlated with both glucose (r = 0.61; p less than 0.001) and hemoglobin A1c levels (r = 0.50; p less than 0.01) in simultaneously obtained samples. This reactivity could be reversed by incubating red blood cells from diabetics for several hours in saline. This report suggests that red blood cells from diabetic patients have membrane-bound glucose that can be detected immunologically. Reversibility of the reaction and rapidity of in vitro glycosylation suggests short-term binding of glucose. To our knowledge, this is the first report documenting immunological detection of in vivo short-term reversible binding of glucose to cellular membranes.

Assessing the clinical significance of anti-Cra and anti-M in a chronically transfused sickle cell patient.

An alloantibody to a high-incidence antigen, associated with multiple other alloantibodies, made it impossible to supply antigen-negative red blood cells (RBCs) for a chronically transfused sickle cell anemia patient. Anti-Cra,-E,K,-S, -Fya, -Fyb, as well as anti-M reactive at 37 degrees C and in the antiglobulin phase of testing, were identified in the patient's serum. An extensive search of rare donor files at the American Red Cross and at the American ASsociation of Blood Banks (AABB) failed to identify Cr(a-),M-,E-,K-,S-, Fy(a-b-) donors. Various studies were performed to predict the clinical significance of the anti-Cra and anti-M. Results of 51chromium survival studies showed 91.8 percent survival at 10 minutes and 87.2 percent survival at 60 minutes with Cr(a +),M-, K-,S-,Fy(a-b-) donors. Various studies were performed to predict the clinical significance of the anti-Cra and anti-M. Results of 51chromium survival studies showed 91.8 percent survival at 10 minutes and 87.2 percent survival at 60 minutes with Cr(a +),M-,E -,K-,S-,Fy(a-b-) red cells, suggesting that immediate destruction of transfused CrCa+) red cells would he unlikely. However, further analysis revealed diminished long-term survival of the donor's red cells with only 60.1 percent recovery at six days (T 1/2 = 12 days) and 10.8 percent at 14 days (T 1/2 = 4.5 days). A monocyte- monolayer assay (MMA) indicated that both the anti-Cra (5.9%) and anti-M (18%) would probably be clinically significant (normal value 0-3%). Mass screening continues at several blood centers for Cr(a-),M-, E-,K-,S-,Fy(a-b-) donors. However, if no suitable donors are found, the results of the 51chromium survival studies and the MMA support the decision to transfuse this patient with Cr(a+),M-,F(a- b-),S-,K- ,E- red cells, if necessary.

Agglutination of an EDTA blood sample caused by an EDTA-dependent panagglutinin.

A novel example of an EDTA-dependent panagglutinin is described. A blood sample drawn into EDTA for a routine hematologic workup demonstrated strong agglutination due to EDTA-dependent panagglutinins. Previous examples have been detected because of discrepant ABO results. This is the first report of an EDTA-dependent panagglutinin that caused agglutination in the EDTA sample collection tube and a false positive direct antiglobulin test.

Elution of anti-Cra. Superiority of the digitonin-acid elution method.

Six examples of IgG anti-Cra were absorbed onto group O, Cr(a+) red cells and subsequently eluted by five elution methods. Five of the anti-Cra antibodies were eluted only by the digitonin-acid method. The sixth example was recovered by all elution methods; however, the digitonin-acid and Lui freeze-thaw methods recovered more antibody activity than did the dichloromethane, ether, or 56 degrees C heat methods. This is the first report of multiple examples of antibodies with the same specificity demonstrated preferentially by a single elution technique.

Use of a modified procedure for treating small amounts of red blood cells with 2-aminoethylisothiouronium bromide.

Kell null red blood cell samples can be prepared using 2-aminoethylisothiouronium bromide (AET). This article describes a modification whereby only three drops of a 5% red blood cell suspension may be AET treated. This procedure has been used routinely in the authors' laboratory for more than a year. One patient's serum contained anti-Kpb plus anti-C and anti-D. Tests with panel cells pretreated with AET made it possible to identify underlying Rh antibodies without using a panel of genetic Kpb negative red blood cells. Of 24 red blood cell eluates from patient sample with warm autoantibodies, one had specificity within the Kell blood group system. This small volume AET-treatment method is a quick screen for the differentiation of a Kell-related specificity from a non-Kell specificity of both warm autoantibodies and alloantibodies to high-incidence antigens.

A human alloanti-N enhanced by acid media.

The serum from a 47-year-old man who had not been transfused previously was tested with reagent red cells selected from different commercial manufacturers to confirm the presence of an alloanti -N. Agglutination of homozygous N-positive red cells at 22 degrees C ranged from score 3 to 12. This variable reactivity was found to be due to pH, and the optimal reactivity was in test media below pH 7.

Autoantibodies mimicking anti-Jkb plus anti-Jk3 associated with autoimmune hemolytic anemia in a primipara who delivered an unaffected infant.

A serum sample from a nontransfused 25-year-old Caucasian primipara contained weak anti-Jkb plus anti-Jk3. The direct antiglobulin test on the patient's red cells was strongly positive. Anti-Jkb was recovered in a heat eluate. An ether eluate contained anti-Jk3. Her red cells types as Jk(a-b+)Jk:3. The anti-Jkb and anti-Jk3 reactivity was completely absorbed both by red cell samples lacking and red cells possessing the corresponding antigens, indicating these antibody specificities to be of the mimicking variety. At 41 weeks gestation she delivered a normal, healthy infant with no detectable serum or cell bound antibody.

Absorption and elution of anti-Hy from one of four Gy(a-) human red blood cell samples.

A red blood cell sample typed as Gy(a-) on direct testing did not absorb or elute anti-Gya, but unexpectedly absorbed and eluted anti-Hy. Three other Gy(a-) red blood cell samples from unrelated individuals failed to absorb and elute anti-Hy.

Absorption of warm autoantibodies using glutaraldehyde-treated human red cells.

Serum from patients with warm autoimmune hemolytic anemia often contains antibodies which react with all red cell samples and interfere with compatibility testing. We describe a modification of the triple absorption procedure to absorb this "non specific" antibody reactivity from the patient's serum without decreasing the reactivity of clinically significant alloantibodies. The alloantibodies can thus be identified and compatibility testing can be performed. Red cells from selected outdated donor bloods were treated with ficin and fixed with glutaraldehyde. Samples from 79 patients whose red cells had a positive direct antiglobulin test and whose serum contained warm autoantibodies were absorbed with these cells. The results were in agreement with those obtained by warm autoabsorption or the triple absorption procedures which were used routinely in our laboratory. It was concluded that glutaraldehyde treatment of red cells does not decrease the sensitivity of the triple absorption procedure and in addition the method is practical, economical, and simple to use for personnel trained in blood bank techniques. It takes less time than standard procedures because the red cells can be prepared and stored until needed. It is particularly useful for testing sera from patients who have a low hematocrit or who have received blood transfusions in the past three months, situations in which the warm autoabsorption method may not be effective.

Absorption of anti-Chido from serum using autologous red blood cells coated with homologous C4.

A method is described which absorbs anti-Chido from serum without reducing the strength of other alloantibodies. Autologous red blood cells are coated with homologous C4 by a low-ionic-strength method and then used for the absorption. This is a practical method for removing anti-Chido from serum so that underlying alloantibodies may be identified and compatibility testing performed.

Transient anti-Gya in an untransfused man. Serologic characteristics and cell survival study.

Anti-Gya was identified in the serum of a 71-year-old untransfused man of German descent. The anti-Gya was reactive in the antiglobulin test phase and was of the IgG1 subclass. The patient's red cells typed as Gy(a-). A survival study using 10 ml of ABO compatible Gy(a+) 51Cr labelled red blood cells indicated normal survival up to six days. The anti-Gya diminished in reaction strength, and three months after the patient's initial hospitalization the antibody was no longer demonstrable by standard manual techniques. We conclude that intravascular hemolysis would have been unlikely if this patient were transfused with Gy(a+) cells. However, due to the possibility of a delayed hemolytic transfusion reaction, Gy(a+) cells would have been given only if Gy(a-) cells were not available and the patient's red blood cell deficit were life-threatening.

Auto-anti-A1: another cause of ABO discrepancy.

Anti-A1 was found in the serum of a patient of blood group A1 who had never received a blood transfusion. The patient's serum caused agglutination of his own red blood cells. The anti-A1 could be totally absorbed by red blood cells from the patient and from other A1 individuals. The anti-A1 was inhibitable by soluble group A-specific substance and was denatured by 2-mercaptoethanol. The A and H serum transferases were normal. The presence of auto-anti-A1 in the serum of an A1 individual is yet another cause of ABO discrepancy.

Characteristics of an antibody causing agglutination of M-positive non-enzymatically glycosylated human red cells.

An alloagglutinin was identified in the serum of an M-negative diabetic patient. The agglutinin reacted with all commercial M-positive red cell samples. Routine crossmatches showed no incompatibility. This anti-M would only agglutinate M-positive red cell samples that had been incubated in 2% glucose for a minimum of 2 h at 37 degrees C, 2 days at 22 degrees C, or 1 week at 4 degrees C. Reactive red cell samples, when washed and incubated in saline, gradually became non-reactive. This antibody reacted optimally in low ionic strength solution at 16 degrees C for 20 min where MM red cells were agglutinated to a titer of 256, score 85; and MN red cells were agglutinated to a titer of 128, score 66. The antibody was denatured by 2-mercaptoethanol and was inhibited by a crude M tryptic isolate and by 2% glucose, but not by other sugars prepared at a 2% concentration.

Failure of normal red blood cells to become Tn-activated when transfused into a patient with Tn-polyagglutination.

A Group O patient with Tn-polyagglutinable RBCs was transfused with two units of Group O packed RBCs. Patient and transfused RBCs were separated by differential agglutination in blood samples drawn over a period of two months after transfusion. The transfused RBCs did not become polyagglutinable.

An evaluation of Re/coagulan for blood center tests.

Re/coagulan was compared to the standard calcium chloride (CaCl2) method for effectiveness of recalcification of plasma and whole blood. The time required for clot formation was determined. The effect of Re/coagulan on the specificity of hepatitis tests, direct antiglobulin tests, indirect antiglobulin tests, and agglutination tests were investigated. Hepatitis testing was performed on untreated serum and Re/coagulan-treated ethylenediamine tetraacetic acid (EDTA) plasmas by radioimmunoassay, reversed passive latex agglutination, and reversed passive hemagglutination methods. Direct antiglobulin tests were performed on untreated, CaCl2 treated, and Re/coagulan treated EDTA, acid-citrate-dextrose (ACD), and citrate-phosphate-dextrose (CPD) samples of whole blood. Red cell antibody specificity and titer were determined before and after clot formation by both the CaCl2 and Re/coagulan methods. Examples of agglutinating and sensitizing antibodies were tested. Antibodies directed against antigens in the Rh, Kell, MNS, Lewis, P, Kidd, Duffy, and Lutheran blood groups systems were investigated. Re/coaglulan didnot have a detrimental effect on the tests performed and compared favorably to the standard recalcification method.

Positive direct antiglobulin test on thawed deglycerolized units of erythrocytes: prediction and prevention.

Occasional units of red blood cells develop a positive direct antiglobulin test (DAT) when frozen and deglycerolyzed by the Meryman method. The presence of cold autoagglutinins with a high titer at 4 degrees C or autoreactivity at room temperature were generally predictive of which units would develop a positive DAT, but neither was reliable in all cases. An indicator system is presented in which a DAT is performed on a segment that has been glycerolized and deglycerolized in a manner similar to the Meryman method for freezing a donor unit. This system is extremely reliable in predicting which units will convert from DAT-negative to DAT-positive during processing. The positive DAT of deglycerolized red blood cells was due to C4 and weak C3d binding. This complement binding can be prevented by addition of ethylenediaminetetraacetic acid (EDTA) or removal of plasma before the addition of glycerol.

The effect of delayed refrigeration on red blood cells, platelet concentrates and cryoprecipitable AHF.

The effect of delaying blood processing for six hours while maintaining it at ambient temperature was investigated. Blood drawn from volunteers on two occasions was processed immediately (I) or after a six-hour delay (D). The effects of the delay on the efficacy and safety of red blood cells, platelet concentrates and cryoprecipitable AHF were studied. There was a more rapid decrease in 2,3-DPG in the delayed group during 21 days of refrigerated storage. ATP levels declined at similar rates. 24-hour survival of 51CR-labeled autologous cells was slightly better (p = .05) in the (I) group but excellent for both. Total platelets, per cent recovery and pH at 72 hours were identical for both groups. All cultures were sterile. There was no difference in total AHF recovered or per cent recovery between the two groups. To increase the availability of blood components, a six-hour processing delay seems warranted.