The type and antibody screen, revisited.

The type and antibody screen is a safe, economical substitute for "routine" two-unit crossmatch in those elective surgical procedures rarely necessitating blood transfusion. This report confirms a previous finding that the type and antibody screen is 99.99% effective in preventing the transfusion of incompatible blood. The type and antibody screen is even safer than the stated 99.99%, when the immediate spin, saline and albumin procedures (the initial portion of the authors' complete crossmatch) are performed prior to releasing blood in an emergency situation. The immediate spin procedures not only verify the ABO compatibility but also detect any high-titer antibodies directed against low-incidence antigens that might not have been present on the screening reagent erythrocytes. The use of the type and antibody screen is applicable only in those medical facilities that have moderate reserves of blood readily available for transfusion.     

Antibodies to low-incidence antigens and elimination of the antihuman globulin phase of the crossmatch-case report: anti-Wra

An antibody to a low-incidence antigen was identified in the serum of a nontransfused male patient. The antibody was subsequently identified as anti-Wra and was only detectable at the antihuman globulin (AHG) phase of the crossmatch. Instances of severe hemolytic transfusion reactions have been reported following the transfusion of red blood cells containing low-incidence antigens in patients with antibodies directed toward these antigens (e.g., anti- Wra, -Cob, -Jsa, etc.). Elimination of the AHG phase of the crossmatch can result in either risks or benefits. Since patients seen at this facility primarily have been multitransfused or are multiparous females, the AHG phase of the crossmatch has been maintained.     

Safety and cost-containment data that advocate abbreviated pretransfusion testing

Abbreviated pretransfusion testing, although permitted by American Association of Blood Banks Standards for unimmunized patients, is not widely practiced. Concerns remain about optimal antibody screening methods, antibodies missed by deleting the antiglobulin crossmatch, and cost-effectiveness. The authors prospectively tested 3,380 serum samples for blood type, antibody screen, and antiglobulin crossmatch. Antibody screens for 2,000 samples, performed with the use of a two-cell screen, were compared with 1,380 samples studied with a three-cell screen. Also, all 3,380 sera had major crossmatches performed carried through the antiglobulin phase. Two and three screening cells gave comparable results, with 5.45% of patients tested by two-cell and 5.22% by three-cell screens having a positive antibody screen. Of those with negative screens, 0.5% screened by two-cell screens and 0.8% by three-cell screens had a positive major crossmatch. Among these (negative antibody screen, positive crossmatch), only 0.03% (1 of 3.380) had a clinically significant alloantibody (anti-Kpa); 0.27% (9 of 3,380) had antiglobulin crossmatch positive with polyspecific antisera but negative with anti-IgG; and 0.12% (4 of 3,380) had positive crossmatch because of passive anti-A. By cost accounting of labor and reagents, 84 per unit would be saved using abbreviated versus complete pretransfusion testing. Blood banks now performing complete pretransfusion testing should reconsider abbreviated crossmatching for unimmunized patients as a safe, efficacious means of cost-containment.     

Safety in transfusion practice. Is it safe to eliminate the major crossmatch for selected patients?

If a patient has no clinically significant unexpected antibodies, a major crossmatch is not required prior to blood transfusion so long as a test method that demonstrates ABO incompatibility is done. In this study, the safety of using a noncrossmatch method for detecting ABO incompatibility was compared with the use of an immediate spin crossmatch (ISCX). This noncrossmatch method consisted of the duplicate ABO testing of blood recipients, the repeated ABO testing of donor blood, and a clerical check to assure that only ABO matched or compatible blood was selected for transfusion. During the one-year study, 7124 patient samples were tested in duplicate for ABO, 26,942 U of red blood cells received from blood collection facilities were retested for ABO, and 23,962 U of blood selected for transfusion based on the noncrossmatch method were tested by an ISCX. ABO test results were concordant for 7115 of 7124 patient samples and discordant for nine. Seven of the nine discordant patient test results were resolved prior to transfusion, and two were inadvertently overlooked. ABO test results were concordant for 26,922 of 26,942 donor units and discordant for 20. Seventeen of the 20 discordant donor test results were resolved prior to transfusion and three were inadvertently overlooked. Two ABO incompatibilities were missed by the noncrossmatch method but were detected by the ISCX. Unless clerical errors can be totally eliminated, it may be safer to retain the ISCX.     

Anti Kell allo-antibody in a thalassaemic

We describe a case of incidental detection of anti Kell antibody in a child with transfusion dependent thalassaemia. Kell antibody detection may be missed by routine indirect antiglobulin test (IAT) crossmatch procedure because of low prevalence of Kell antigen in the general population. A false negative result can be avoided by using sensitive cross matching techniques and screening cells representing antigens in homozygous state, against all clinically significant antibodies. A transfusion alert card describing the nature of antibody and future transfusion policy should be given to such allo-immunized patients.     

Intravenous Rh immune globulin prevents alloimmunization in D- granulocyte recipients but obscures the detection of an allo-anti-K

Rh immune globulin (RhIG) has been used to prevent alloimmunization in D(-) recipients of apheresis platelet transfusions from D(+) donors that may contain up to 5 mL of D(+) red blood cells (RBCs). Granulocyte concentrates contain approximately 30 mL of RBCs and it has been necessary to give D(-) recipients granulocyte transfusions from D(+) donors. Intravenous RhIG has not yet been demonstrated to be effective in preventing D alloimmunization with granulocyte transfusions. Four D(-) recipients received multiple D(+) granulocyte transfusions from D(+) donors and multiple injections of intravenous RhIG at a standard dose of 600 microg for each D(+) transfusion. Two D(-) males with chronic granulomatous disease were given 32 and 13 daily granulocyte transfusions, 18 and 2 of which, respectively, were D(+). After the first dose of intravenous RhIG, both patients exhibited circulating anti-D that was undetectable 3 to 4 years later. Two patients with severe aplastic anemia were given 5 and 14 granulocyte transfusions, 4 and 7 of which, respectively, were D(+). Both patients died before the effectiveness of RhIG could be assessed. In one of these patients the indirect and direct antiglobulin tests became positive after the first dose of intravenous RhIG, which required that subsequent granulocyte transfusions from D(+) donors be crossmatched by immediate spin (IS) testing only. A delayed hemolytic reaction attributed to allo-anti-K occurred after granulocytes from a K(+) donor were given to this patient. These results suggest that intravenous RhIG can be used to prevent alloimmunization to D in D(-) patients receiving large quantities of RBCs from D(+) granulocyte transfusions. However, anti-D and other passive antibodies from RhIG prohibit the use of the antiglobulin crossmatch with antigen-positive granulocyte donor samples. It may be important to frequently collect new samples to screen for newly formed allo-antibodies when IS crossmatches are used in place of the antiglobulin crossmatch.     

Analysis of the routine use of polyethylene glycol (PEG) as an enhancement medium

This study compared the performance of polyethylene glycol (PEG) and low-ionic saline solutions (LISS) as enhancement media for routine use in a large transfusion service. A PEG additive solution (PEG plus LISS) was compared to a LISS additive (LISS plus polymers) and to an albumin-indirect antiglobulin test (A-IAT). Fifty serum samples containing clinically significant alloantibodies and fifty samples without alloantibodies were tested. Following an acute hemolytic transfusion reaction (HTR) involving an anti-K that was not detected with LISS but was retrospectively found to be reactive with PEG, an additional 151 samples received for antibody screening were prospectively evaluated in parallel using PEG and LISS. PEG detected all clinically significant antibodies in the 50 previously tested samples, with mean reactivity scores greater than LISS or A-IAT. In the prospective study, PEG detected 35 clinically significant antibodies and 10 clinically insignificant antibodies, while LISS detected only 15 clinically significant antibodies and 33 clinically insignificant antibodies. PEG appears to increase detection of significant antibodies while decreasing detection of insignificant antibodies. PEG was therefore substituted for LISS as an enhancement medium and has been in routine use for 12 months, with no reported acute or anamnestic HTRs in 6,353 transfusions.     

Limitations of the crossmatch for detection of incompatibility between A2B red blood cells and B patient sera

A recent evaluation of the immediate-spin crossmatch has revealed limitations when used for the detection of ABO incompatibilities in tests between group B patient sera and group A2B donor red blood cells. Presented with the option of deleting the anti-globulin crossmatch, the study reported here was conducted to determine whether the anti-globulin crossmatch would detect the ABO incompatibilities missed by the immediate spin. Results of 1,000 crossmatches between group B patient sera and group A2B red blood cells showed that the anti-globulin phase was capable of detecting a higher percentage of incompatibilities than the immediate-spin procedure, 79.4-86.8% versus 40-64.4%, respectively, depending on the enhancement used. Although anti-globulin testing did detect a substantially higher number of incompatibilities, are the increased cost of reagents and technologists' time used in performing the anti-globulin crossmatch justified for the detection of ABO incompatibilities? Due to limitations observed with the anti-globulin test in addition to budgetary and fiscal constraints, retention of the anti-globulin testing purely for ABO compatibility is not warranted.     

Reducing number of laboratories performing complement dependent cytotoxicity crossmatching: Reasons and conclusions

Complement dependent cytotoxicity crossmatch (CDC-XM) has been the original standard crossmatch test, whereas, flow cytometry crossmatch (FCXM) is an enhanced and highly sensitive crossmatch assay performed to detect donor specific anti-HLA antibodies (DSA). We analyzed American Society for Histocompatibility and Immunogenetics (ASHI) proficiency testing data (2011–2020) and examined the number of laboratories performing CDC-XM vs. FCXM, the overall efficiency of laboratories in reporting ≥80% consensus CDC-XM vs. FCXM result, and reasons for non-consensus results in the two assays. Of 600 crossmatches in each crossmatch category, the percentage of laboratories reporting T cell CDC-XMs reduced from 40% in 2011 to 13% in 2020, T cell anti-human globulin (AHG) CDC-XM reduced from 56% in 2011 to 21% in 2020, and B cell CDC-XM reduced from 51% in 2011 to 20% in 2020. The percentage of laboratories performing T cell and B cell FCXM remained at approximately 80% throughout. CDC-XM performed on par with FCXM in providing a consensus negative result using negative DSA serum, but under-performed in comparison to FCXM in providing a consensus positive result using positive DSA serum. In addition, only minority of CDC-XMs was reported positive in presence of complement fixing DSA. This study shows that non-consensus CDC-XM was always in presence of HLA IgG DSA and that laboratories may be struggling to interpret the low sensitive CDC-XM results, where highly sensitive solid phase multi-antigen or single antigen assay shows the presence of HLA IgG DSA in serum.     

Significance of histocompatibility tests

Pretransplant tests necessary for kidney transplantation are HLA typing, mixed lymphocyte culture response (MLR), and direct crossmatch. HLA typing and MLR are closely related to graft survival rates. The significance of HLA matching is generally known. In our analysis of 25 living related kidney grafts, graft survival rate of two haplo identical donor transplants was 4/4 (100%), while that of one haplo identical donor transplant was 18/21 (85.7%). Acute rejection rate in the MLR low response group (S.I. less than or equal to 5) was 2/7 (28.6%), while that in the high response group (S.I. greater than 5) was 11/19 (57.9%). HLA typing and MLR are useful in selecting the most suitable recipient. In order to reduce the risk of hyperacute or accelerated graft rejection, T warm direct crossmatch is performed. Anti-T warm antibodies are mainly produced by blood transfusion. In our study of 239 hemodialysis patients, there were 31 patients with positive T warm (13.0%), the positive rate became higher in proportion to increases in blood transfusion. Recently, there have been reports of kidney transplants successfully performed across T warm-positive crossmatches due to IgM antibodies. We also investigated the immunoglobulin class. Not only pretransplant crossmatch but also posttransplant crossmatch is necessary. In the case of accelerated, acute or chronic rejection, anti-donor HLA antibodies are produced in patients' peripheral blood. Thus, the test of posttransplant anti-donor antibodies is useful for the early detection of rejection, its diagnosis, and index of the prognosis. The sensitivity of flow cytometry crossmatches was compared to standard cytotoxicity crossmatch. Titration studies indicate a 32-64 fold range of greater sensitivity than the cytotoxicity test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening and Identification of Unexpected Red Cell Antibodies by Simultaneous LISS/Coombs and NaCl/Enzyme Gel Methods

We evaluated the clinical usefulness of simultaneous LISS/Coombs and NaCl/Enzyme testing using the gel method for screening and identification of unexpected antibodies in 15,014 samples. When unexpected antibodies were detected by either screening test, those antibodies were identified using both the LISS/Coombs and the NaCl/Enzyme gel test. The positive screening rates of the LISS/Coombs, NaCl/Enzyme, and combined tests (excluding 25 autoantibody cases) were 0.48%, 1.29%, and 1.39%, respectively. Among the 57 samples positive by both screening methods, the antibodies in 19.3% could be identified only by the NaCl/Enzyme method. Among the 137 samples positive only by NaCl/Enzyme screening, 74.5% showed positive results in antibody identification only by the NaCl/Enzyme test, although 7.3% were also positive in the LISS/Coombs test. The NaCl/Enzyme method thus showed about threefold higher detection rates than the LISS/Coombs method, especially in screening for Rh antibodies, and higher exact identification rates and discriminatory power for identifying mixed antibodies. Addition of the NaCl/Enzyme method to routine laboratory procedures may detect and identify considerable numbers of significant antibodies that might be missed if only the LISS/Coombs method is used.     

Severe hemolytic reaction due to anti-JK3.

A 35-year-old gravida 3, para 3 Filipino woman with a negative antibody screen, no prior history of transfusion, and no hemolytic disease of the newborn in her children suffered a massive postpartum hemorrhage requiring transfusion. A severe hemolytic transfusion reaction occurred 5 days after delivery. Subsequently, a panagglutinin on a routine antibody identification panel was identified as anti-Jk3. The patient's red blood cell phenotype was Jk(a-b-) and all of her children were Jk(a-b+), yet the antibody that formed reacted with equal strength against all Jk(a)- or Jk(b)-positive cells. The rare Jk(a-b-) phenotype is more common in Polynesians. Anti-Jk3, like other Kidd system antibodies, is difficult to detect because in vivo production may be absent between provocative episodes and because these antibodies often show weak in vitro reactions. The increasing numbers of Pacific Islanders in the United States could result in more frequent encounters with this rare phenotype. Increased awareness of ethnic variability in blood phenotypes and of the capricious nature of Kidd antibodies can help pathologists and technologists deal more effectively with these cases.     

Possible insensitivity of the polybrene antibody screen to detect anti-Jka

An acute hemolytic transfusion reaction (AHTR) occurred in a 28-yr-old gravida immediately after transfusion with leukocyte-reduced red cells. The patient gave no history of prior transfusion. Initial serologic testing by the polybrene method was negative for both antibody screening and cross-matching. Further testing by the indirect anti-globulin test (IAT) demonstrated the presence of anti-Jka antibodies. These observations suggest a limitation in polybrene testing for Jka antibodies associated with hemolytic transfusions. Caution is advised when the polybrene test is used as the sole determinant for anti-Jka.     

A rare case of clinically significant naturally occurring anti-Cw reported in a healthy male donor

Anti-C^w antibody is an immunoglobulin against the red cell antigen C^w which is a low frequency red cell antigen that belongs to the Rh antigen system. It is a clinically significant antibody and may cause haemolysis on exposure to antigen positive red cells. Due to its low frequency, it is not included in routine antibody screening panels. A 32 years healthy male donor with no history of transfusion donated whole blood at the department of Transfusion Medicine & Blood Centre of our institute. As a part of routine pre-transfusion testing, the donor’s samples were subjected to automated blood grouping and screening for unexpected red cell antibodies using 3 cells panel on solid-phase red-cell adherence (SPRCA) (Galileo Neo, Immucor, Norcross, USA). The antibody screening came out to be positive with a reaction in cell I of the antibody screening panel. Further the antibody was identified as anti C^w in using 16 cells panel, select cells and phenotyping. In the present case, the anti-C^w antibody was found to be reactive at 37 °C and AHG phase which could lead to haemolytic transfusion reaction. The fact that the male donor had no history of transfusion or transplant led us to the conclusion that it was a naturally occurring, but a clinically significant antibody. This case highlights the importance of performing an antibody screening for healthy donors as well and urges transfusion services to procure screening cells which incorporate C^w positive cells.     

Laboratory investigations following an unexpectedly positive crossmatch result in a patient awaiting renal transplantation

In the preparation of patients for renal transplantation tests of human leucocyte antigen (HLA) sensitisation are performed to detect "unacceptable" HLA antigens that, if present on donor cells, would be expected to result in a positive crossmatch. Individuals bearing such specificities may then be excluded from consideration as donors. Unexpected positive crossmatch results are sometimes obtained when a serum specificity has not been detected on screening. Failure to identify a donor relevant HLA antibody in a recipient at the time of crossmatch may result in hyperacute rejection of the graft. This report describes laboratory investigations performed after a positive crossmatch result in a live donor situation. The pattern of crossmatch results indicated that reactivity resulted from HLA class I antibody. Previously performed serum screening using a standard complement dependent cytotoxicity technique had failed to identify donor relevant antibody specificities in the recipient. Retrospective flow cytometric screening of the same serum samples identified an HLA-A24 specificity of donor relevance. The lower sensitivity of methods used for routine serum screening compared with those used for crossmatching accounts for the findings in this case. The laboratory has amended its serum screening protocol to include flow cytometric analysis.     

A reappraisal of pre-transfusion testing procedures in a hospital blood bank

In recent years, experience with screening protocols for the detection of red cell antibodies has led to a reappraisal of pre-transfusion testing procedures. In the U.S.A. in particular, it has become accepted practice to perform only an antiglobulin crossmatch when an antibody screen has been negative. There is now debate in that country regarding the necessity of retaining even the antiglobulin crossmatch. In this laboratory, as is usual in Australia, room temperature, enzyme and antiglobulin tests are used for both antibody screening and crossmatching. A review was therefore conducted of the results of screening 58,227 samples from approximately 40,000 patients, involving 126,771 crossmatched blood units over a 53 mth period. Eight hundred and seventy-two red cell antibodies were detected in 718 patients. Forty-four of these antibodies were detected only in the crossmatch, and of these 23 were of potential clinical significance. Fourteen of the 23 were detected by the antiglobulin test, 8 by an enzyme test and only 1 by the saline method. Of 107 antibodies detected only in the room temperature phase, none were of clinical significance. The results indicate that exclusion of room temperature tests from all pre-transfusion testing, and deletion of enzyme tests from the crossmatch will not compromise patient safety.     

Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab

Rituximab (RIT), a murine/human chimeric monoclonal antibody directed against human CD20 is under investigation for its role in transplantation. RIT causes B-cell crossmatches to appear positive. Pronase, a proteolytic enzyme that targets F(c) receptors removes CD20 from B cells. After CD20 is removed, RIT should not bind, making it possible to detect class I or class II antibodies on treated B cells. In this study, we incubated RIT with normal human serum (NHS, negative control) or pooled sera from highly sensitized (>50% panel reactive antibody, HLA+) subjects awaiting renal transplantation (positive control) and then performed B-cell flow cytometric crossmatches using untreated or pronase treated B cells as targets. We observed that untreated B cells incubated with RIT-spiked NHS displayed a significant increase in surface fluorescence compared with NHS without RIT, similar to the fluorescence that occurs with a positive crossmatch. In contrast, when CD20 was cleaved from the B cells with pronase, B cells displayed a negative crossmatch with the RIT-spiked NHS. In addition, there was no change in the crossmatches of pooled high panel reactive antibody (PRA) sera after pronase treatment. RIT could be used without worry about losing the ability to perform transplant immunologic monitoring.     

Variations in pretransfusion practices

A variety of pretransfusion tests have been developed to improve the safety and effectiveness of transfusion. Recently, a number of traditional tests have been shown to offer limited clinical benefit and have been eliminated in many facilities. A survey of pretransfusion test practices was distributed to 116 hospital transfusion services. Routine test practices and facility size were analyzed. Ninety-one responses were received. Many smaller laboratories include tests such as anti-A,B, an autocontrol, and DAT, and immediate spin and 37 degrees Celsius microscopic readings. Nine percent never perform an Rh control with anti-D typing on patient samples. Various antibody screening and crossmatch methods are utilized. Individual laboratory test practices should be periodically assessed to ensure that they comply with standards, represent the recognized best practice, and are cost-effective. The survey responses indicate that many laboratories perform tests that are not necessary or cost-effective. These facilities should review their processes to determine which tests contribute to transfusion safety. Smaller facilities may be reluctant to change or lack the expertise necessary for this decision making and often continue to perform tests that have been eliminated in larger facilities. Consultation with larger hospital transfusion services may provide guidance for this change.     

Use of a solid phase red blood cell adherence method for pretransfusion platelet compatibility testing

A solid phase red blood cell adherence method has been used for platelet antibody detection and crossmatching for refractory platelet recipients. Patient sera were first screened for HLA or platelet-specific antibodies, then crossmatched with potential apheresis platelet donors. The overall correlation of platelet crossmatch results with transfusion outcome was 97% in patients with no evidence of nonimmune platelet destruction. The solid phase red blood cell adherence method provided a feasible and effective alternative to HLA matching as a means of donor selection for refractory platelet recipients. The speed and simplicity of this method may allow most hospital laboratories to perform platelet antibody screening before routine platelet transfusions.     

Absence of hemolytic disease of fetus and newborn despite maternal high-titer IgG anti-Ku

Anti-Ku seen in K(o) (Kell-null) individuals has previously been shown to cause severe hemolytic transfusion reactions. Maternal anti-Ku can cause none or moderate to severe hemolytic disease of the fetus and newborn (HDFN). In two of four previously described HDFN cases, intrauterine transfusions were required because of severe anemia. We report a case in which maternal anti-Ku did not cause HDFN. Standard serologic methods were used for RBC antibody screening and identification, adsorption and elution of RBC antibodies, and antigen typing. A gravida 3, para 3 (G3P3) woman was first evaluated in 2006 and was found to have an IgG RBC antibody that reacted against all panel RBCs in the anti-human globulin phase. A panel of RBCs treated with DTT did not react with the antibody. The antibody failed to react with one example of K(o) RBCs. The patient’s RBCs typed negative for the following Kell blood group antigens: KEL1, KEL2, KEL3, KEL4, KEL6, KEL7, KEL11, KEL13, and KEL18. These results established the presence of anti-Ku in maternal serum. The newborn was group A, D+ and required phototherapy for hyperbilirubinemia, but did not require transfusion. The woman was seen again in January 2010 during the third trimester (G4P3). At this time, anti-Ku titer was 256. She delivered a healthy group O, D+ baby boy at 37 weeks' gestation. Cord RBCs were 4+ for IgG by DAT. An eluate reacted with all RBCs tested, but did not react when tested against a panel of DTT-treated RBCs. K(o) phenotype is rare to begin with, and the maternal anti-Ku formation may require more than one pregnancy. Therefore, cases that can be evaluated for anti-Ku–related HDFN are rare. Our case contributes to serologic and clinical aspects of such rare cases.