| Abstract|| |
Background: The antiglobulin test is used to determine red blood cells (RBCs) having surface-bound immunoglobulin G (IgG) and/or complement (C3b, C3d, C4b, and C4d) free in serum or attached to antigens on RBCs. In such circumstances, the quality of the anti-human globulin (AHG) which is used in routine cross-match/indirect agglutination test plays a vital role in blood transfusion medicine. For potency testing of polyspecific AHG, it is recommended by the Food and Drug Administration and the Centre for Biologics Evaluation and Research to use fresh O +ve RBCs within 1 hr of collection for sensitization with anti-C3d.
Aim and Objective: Freshly collected red cells were cryopreserved and stored at a temperature of −70°C for 30 days. These cells were then sensitized with C3d and IgG after deglycerolization. Their viability was checked by potency testing using polyspecific AHG-containing anti-C3d and anti-IgG.
Materials and Methods: Anonymous left over fresh whole blood samples were collected from the Indian Red Cross Society, New Delhi, with CPDA as an anticoagulant, and the samples were treated within 1 h of collection. ABO and Rh (D) phenotyping was performed by test tube method. RBCs and plasma from the same donor were used throughout the study.
Results: In this comparative study, fresh RBCs (O +ve) maintained their viability after cryopreservation and were also found to be suitable for sensitization with C3d and IgG. The potency of polyclonal AHG did not differ significantly with fresh RBCs and cryopreserved RBCs for 30 days.
Conclusion: This result suggests that the sensitization of fresh RBCs with IgG and C3d is not affected by using cryopreserved cells for 30 days.
Keywords: Anti-C3d and anti-immunoglobulin G, cryopreservation, polyspecific anti-human globulin, red blood cells, sensitization
|How to cite this article:|
Ahuja K, Satapathy RK, Gulati G, Singh S. A comparative study showing the potency of anti-C3d and anti-immunoglobulin G in polyspecific anti-human globulin using fresh and cryopreserved red blood cells. Asian J Transfus Sci 2017;11:28-32
|How to cite this URL:|
Ahuja K, Satapathy RK, Gulati G, Singh S. A comparative study showing the potency of anti-C3d and anti-immunoglobulin G in polyspecific anti-human globulin using fresh and cryopreserved red blood cells. Asian J Transfus Sci [serial online] 2017 [cited 2017 Mar 25];11:28-32. Available from: http://www.ajts.org/text.asp?2017/11/1/28/200769
With Landsteiner's discovery in 1901 of the ABO blood group system, the first step toward a rational approach to blood transfusion became possible. Nearly a half century later, the next advance occurred: The introduction of serologic techniques permitting the detection of “incomplete” antibodies, which are nonagglutinating immunoglobulin G (IgG) antibodies that sensitized (coat) red blood cells (RBCs). Anti-human globulin (AHG) techniques were introduced in 1945 by Coombs et al. The direct antiglobulin test (DAT) was first reported in 1908, but found more widespread notoriety after being described in 1945 by Coombs et al.IgG antibodies are termed as nonagglutinating because their monomer structure is too small to agglutinate sensitized RBCs directly. The addition of AHG-containing anti-IgG to RBCs sensitized with IgG antibodies allows for hemagglutination of these sensitized cells. Some blood group antibodies have the ability to bind complement to the RBC membrane. Antiglobulin test detects IgG/or complement-sensitized RBCs. AHG is used to detect in vivo sensitization of RBCs, in two stage-technique which is referred to as the indirect agglutination test  and by a one-stage procedure which is referred to as direct agglutination test (DAT).
Garratty and Petz  reported on the significance of red cell-bound complement components. Using in-house antisera, they confirmed the need for anti-C3d activity in AHG for use in the direct test in 1976. Engelfriet et al. observed that given sufficient incubation time, cell-bound C3b could be degraded to C3d in vitro. According to Plunkett et al., 1980, cells coated with complement components (C3b-C4b and C4b cells) were prepared by various methods and stored at low temperatures by using a glycerol method.
In blood transfusion, cryopreservation has been particularly useful for long-term storage of RBCs with rare phenotypes.,, Glycerol is widely used as a cryoprotectant in case of RBC preservation due to its less toxicity, less penetrating power into RBCs membrane, requires few reagents for thawing of RBCs, and protects cells from freezing injury. Human RBCs could be cryopreserved, thawed, washed free of cryoprecipitate, and transfused with normal in vivo survival of 85–90%. Glycerol is either used as 20% or 40% w/v concentration, and these methods are termed as low glycerol concentration or high glycerol concentration (HGC). In HGC freezing method, RBCs to be frozen with higher glycerol concentrations could reduce the cell loss and subsequently make cryopreserved RBCs more applicable for routine clinical usage., A deep ultra-freezer is used for this which provides storage at −70°C. According to Valeri et al., red cells frozen with 40% w/v glycerol can be stored at −80°C for up to 37 years with acceptable in vitro results.
Polyspecific AHG reagent conforms to the regulations of the Food and Drug Administration (FDA) concerning biologics and its testing is performed in accordance with FDA-recommended methods. Before the AHG is available for purchase, manufacturers must subject their reagents to an evaluation procedure i.e. quaity control of AHG against IgG and complement coated RBCs to ensure a suitable reactivity.
As per the Centre for Biologics Evaluation and Research (CBER) – FDA guidelines, quality control evaluation of polyspecific AHG-containing anti-C3d and anti-IgG requires freshly collected “O” type blood group cells which are generally sensitized with IgG and C3d because they do not contain cell surface antigens. The National Institute of Biologicals carries out the quality control testing of polyspecific AHG reagents for batch release. For this purpose, the laboratory has to collect fresh “O” blood samples from the Indian Red Cross Society (IRCS), New Delhi, and sensitize them on the same day to carry out the evaluation. To overcome this problem, a comparison with respect to potency testing of polyspecific AHG-containing anti-C3d and anti-IgG was done using freshly sensitized red cells and cryopreserved red cells sensitized with IgG and C3d.
Aim and objective
Sensitization is the process in which there is a specific binding of antibody to its antigenic receptor on RBCs without agglutination or lysis. As per the CBER-FDA guidelines, it has been established that potency testing of anti-C3d component and anti-IgG of polyspecific AHG requires coated cells (sensitized) and fresh plasma of the same donor. The collection, sensitization, and potency testing of AHG take 5–6 h which is not possible to complete on the same day. To overcome this problem, a study was designed in which freshly collected red cells were cryopreserved and stored at a temperature of −70°C for 30 days and then sensitized with C3d and IgG after deglycerolization. Then, their viability was checked by potency testing using polyspecific AHG-containing anti-C3d and anti-IgG.
| Materials and Methods|| |
Anonymous left over fresh whole blood samples were collected from the IRCS with CPDA as an anticoagulant, and the samples were treated within 1 h of collection. The RBCs were washed three times with normal saline (pH 7.0) at 2500 rpm for 3 min. ABO and Rh (D) phenotyping was performed by test tube method. RBCs and plasma from the same donor were used throughout the study.
For sensitization of O +ve RBCs with C3d, 0.5 ml of washed packed red cells were added to 19.8 ml of buffered sucrose solution (pH 5.1) in ice bath (0°C) with gentle stirring. A volume of 0.5 ml of plasma of the same O +ve RBC and 0.1 ml of magnesium chloride (0.4 M) was added to the above mixture and the solution was incubated for 30 min at 0°C with gentle stirring. Then, the coated cells were washed four times with excess of isotonic saline. C3b-coated cells (0.5 ml) were converted to C3d cells (sensitized) by adding 1 ml of 0.1% trypsin. They were mixed and incubated for 30 min at 37°C. The red cells were washed four times with an excess volume of normal saline, and a 2% red cell suspension was prepared.
For sensitization of O +ve RBCs with IgG, in-house calibrated standard anti-D (IgG) was used to coat the O +ve RBCs. An equal volume of diluted anti-D (IgG) –1:16 was added with an equal volume of O +ve packed red cells. The mixture was incubated at 37°C for 30 min. The red cells were washed with excess volume of isotonic saline, and 2% red cell suspension was prepared.
For glycerolization of O +ve RBCs, two volumes of glycerolyte (40% w/v) are added to one volume of freshly washed RBCs and mixed gently by rotating the tube. Fifteen aliquots of glycerolized O +ve RBCs were kept at 2–8°C for 30 min and then shifted to −20°C for 30 min and finally at −70°C for testing to be done on 1st, 3rd, 7th, 15th, and 30th day, respectively.
For deglycerolization  of cryopreserved O +ve RBCs, 12% normal saline is added to the cells (half of the volume of cryopreserved cells) and incubated at room temperature for 3 min, equal volume of 1.6% normal saline was added to the mixture and then equal volume of 0.9% normal saline was added to the above mixture. The mixture was centrifuged at 2500 rpm for 3 min. The cells were washed twice with a mixture (1.6% NaCl and 0.9% NaCl in 1:1 ratio) at 2500 rpm for 3 min. The red cells were washed with normal saline till the supernatant is clear. The percent (%) recovery of the cells was noted. Finally, the washed cryopreserved cells were used for sensitization with C3d and IgG.
Percentage of red cell recovery
The percentage recovery was calculated using total packed red cell volume measurements before glycerolization and after deglycerolization. The percentage of recovery was calculated using the following formula:
| Results|| |
The cryopreserved cells were deglycerolized, and the percentage recovery of the cell was noted. After deglycerolization, the packed red cell volume was estimated on day 1, day 3, day 7, day 15, and day 30 by taking total packed red cell volume measurements before glycerolization, after deglycerolization, and total volume of the vials into account. The percentage recovery was found to be 85.0% on day 1, 85.0% on day 3, 84.50% on day 7, 84.0% on day 30, and 84.0% on day 15. It was observed that all the samples stored at −70°C gave an average recovery of 84.5% for 30 days [Table 1]. Upon deglycerolization, an average loss of 15.5% was observed due to the lysis of RBCs during washing after 1 month of storage at −70°C (±1°C). The percentage cell loss remained constant (between 15% and 16%) throughout the month [Table 1]. This increased fragility of cells did not appear to affect the quality of the patterns and the definition of end points throughout the entire period of testing. The potency obtained with sensitized washed cryopreserved red cells for 30 days was compared with freshly sensitized RBCs (day 1).
|Table 1: Cryopreservation of red cells and their recovery using potency testing of anti human globulin|
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Further, it was found that the sample stored at −70°C gave a potency titer of 1:64 for anti-C3d for the first 3 days [Table 2], which is equal to the titer observed on the 1st day of collection with fresh cells whereas the titer decreased by 1 dilution, i.e., 1:32 on 7th day and further titer reduced to 1:16 on 15th and 30th day [Table 2].
|Table 2: Quality control comparison of anti human globulin in fresh red blood cells and cryopreserved cells|
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Potency testing for anti-IgG in the same batch of AHG for the blood sample stored at −70°C gave a titer of 1:64 for the first 7 days [Table 2], which is equal to the titer observed on the 1st day of collection with fresh cells whereas the titer decreased by 1 dilution, i.e. 1:32 on the 15th and 30th day respectively [Table 2].
There was a gradual decrease in the titer of anti-C3d present in polyspecific AHG which may be attributed to the loss of activity of C3d sensitized RBCs upon storage at temperature −70°C [Graph 1] and [Graph 2].
| Discussion|| |
Polyspecific AHG contains antibody to human IgG and to the C3d component of human complement, whereas anti-complement reagents, such as anti-C3b-C3d reagents, are reactive against the designated complement components only and contain no activity against human Igs. Commercially available polyspecific AHG (C3d, IgG) contains antibody activity to kappa and lambda light chains which is common to all Ig classes.
The rate of heat release during freezing is also an important contributor to the post-thaw quality of RBCs. Traditional freezing of RBCs at −25°C in a mechanical freezer normally requires high glycerol concentrations to minimize ice formation. However, high concentrations of cryoprotectant can exert detrimental effects on the RBC.
AHG or anti-IgG reagent can be tested against a selection of week antibodies to determine the optimum potency. Antibody preparation should not be diluted, and the use of single-donor antibody preparation is preferred. Specificity of AHG (polyspecific - anti -IgG, -C3d) reagent test should be evaluated by the cells coated with IgG, C3b, C3d, C4b, and C4d, and for the coating of these cells, normal-clotted or anticoagulated whole blood samples should be collected and treated within 1 h of collection.
In this study, we checked the viability of cryopreserved cells stored at −70°C temperature over a period of 30 days and used the same cells for sensitization with C3d and IgG. The sensitized RBCs were then used to check the potency of anti-C3d and anti-IgG present in polyspecific AHG. As per the established standard procedure, it is recommended  to use freshly collected O +ve cells and serum to check the potency of anti-C3d and anti-IgG. The RBCs maintained their viability throughout a period of 1 month of testing (1st day, 3rd day, 7th day, 15th day, and 30th day). Potency titer of 1:64 for anti-C3d using cryopreserved cells matched with the titer obtained with fresh cells for 3 days whereas anti-IgG gave a titer of 1:64 for 7 days. This shows that the results were clearly reproducible for 3 days using cryopreserved cells sensitized with C3d and 7 days for IgG-sensitized cells.
It was observed that the titer of anti-C3d decreased by 1 dilution (1:32) on the 7th day and further dropped by 1 dilution (1:16) on the 15th day and remained constant till the 30th day of the study. The titer of anti-IgG in polyspecific AHG decreased by 1 dilution on the 15th day and remained constant for a further period of 15 days.
Although the activity of C3d and IgG decreased for 30 days from 1:64 for anti-C3d to 1:16 and 1:64 to 1:32 for anti-IgG, it falls within the international acceptance criteria for the quality control of polyspecific AHG which is ≥1:4 for anti-C3d and ≥1:32 for anti-IgG activities.
These results suggest that, in case of nonavailability of fresh cells, cryopreserved RBCs stored at −70°C for 30 days can be used for quality control evaluation of anti-C3d and anti-IgG activities of polyspecific AHG. As with all tests, antiglobulin results must be viewed in light of clinical diagnostics in most common clinical situations where the antiglobulin test can help classify the causes of cross-matching, hemolytic disease of the fetus/newborn, hemolytic transfusion reaction, autoimmune and drug-induced hemolytic anemia, and antibody screening for antibodies.
| Conclusion|| |
It is concluded that, in case of nonavailability of blood cells, the performer can use cryopreserved RBCs for 30 days for sensitization of C3d and IgG to check the potency of anti-C3d and anti-IgG activities in polyspecific AHG.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Coombs RR, Mourant AE, Race RR. A new test for the detection of weak and incomplete Rh agglutinins. Br J Exp Pathol 1945;26:255-66.
Moreschi C. Neue Tatsachen uber die Blutkorperchen – Agglutination. Aentbl Bakt 1908;46:49-51.
Rosen F, Geha R. Case Studies in Immunology. A Clinical Companion.4th
ed. New York: Garland Science; 2004. p. 173.
Garratty G, Petz LD. The significance of red cell bound complement components in development of standards and quality assurance for the anti-complement components of antiglobulin sera. Transfusion 1976;16:297-306.
Engelfriet CP, Pondman KW, Wolters G, Borne A.E.G. Kr. von dem, Beckers D, Misset-Groenveld G, et al.
Autoimmune haemolytic anaemias 3. Preparation and examination of specific antisera against complement components and products, and their use in serological studies. Clin Exp Immunol 1970;6:721-32.
Plunkett RW, Cunningham RK, Lichtenberg SH. Cryopreservation of complement-coated erythrocytes. Transfusion 1980;20:569-73.
Council of Europe. Guide to the Preparation, Use and Quality Assurance of Blood Components, Recommendation R (95) 15. 14th
ed. Strasbourg: Council of Europe Publishing; 2008.
Kakaiya R, Aronson M, Julleis J. Whole blood collection and component processing. In: Roback J, Combs M, Grossman B, Hillyer C, editors. American Association of Blood Banks technical manual. 16th ed., Ch. 6. Bethesda (MD): American Association of Blood Banks. 2008.
Peyrard T, Pham BN, Le Pennec PY, Rouger P. Transfusion of rare cryopreserved red blood cell units stored at -80 degrees C: The French experience. Immunohematology 2009;25:13-7.
Smith AU. Prevention of haemolysis during freezing and thawing of red blood-cells. Lancet 1950;2:910-1.
Lelkens CC, Noorman F, Koning JG, Truijens-de Lange R, Stekkinger PS, Bakker JC, et al.
Stability after thawing of RBCs frozen with the high- and low-glycerol method. Transfusion 2003;43:157-64.
Valeri CR, Ragno G, Pivacek LE, Srey R, Hess JR, Lippert LE, et al.
A multicenter study of in vitro
and in vivo
values in human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4 degrees C in AS-3: Assessment of RBC processing in the ACP 215. Transfusion 2001;41:933-9. [Erratum in: Transfusion 2002;42:1618].
Brecher ME, editor. Technical Manual. 14th
ed. Maryland: AABB; 2002.
Kim H, Tanaka S, Une S, Nakaichi M, Sumida S, Taura Y. A comparative study of the effects of glycerol and hydroxyethyl starch in canine red blood cell cryopreservation. J Vet Med Sci 2004;66:1543-7.
Valeri CR, Ragno G, Pivacek LE, Cassidy GP, Srey R, Hansson-Wicher M, et al.
An experiment with glycerol-frozen red blood cells stored at -80 degrees C for up to 37 years. Vox Sang 2000;79:168-74.
FDA Draft Docket, Docket No: 84S-0182, March 1992. Recommended Methods for Evaluation Potency, Specificity, and Reactivity of Anti-Human Globulin; 1992. Available from:
Valeri CR. Frozen red blood cells. In: Rossi EC, Simon TL, Moss GS, Gould SA, editors. Principles of Transfusion Medicine. 2nd
ed. Baltimore, MD: Williams and Wilkins; 1995. p. 61-6.
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[Table 1], [Table 2]