Topic: Optimisation and comparison of sensitivity and specificity for detection of clinically significant blood group antibodies using LISS-IAT, Alb-IAT and PEG-IAT (tube versus CAT)

Topic: Optimisation and comparison of sensitivity and specificity for detection of clinically significant blood group antibodies using LISS-IAT, Alb-IAT and PEG-IAT (tube versus CAT)

Pages: 18, Double spaced
Sources: 25

Order type: Research Paper
Subject: Medicine and Health

Style: APA
Language: English (U.S.)

Blood transfusion dates way back before the world war, but it was more significant during the war due to the number of people who were losing their lives due to blood loss. However, a challenge was evident because of clumping of blood right after transfusion deeming the limited information about the different blood groups (Reisner et al., 1996). Blood grouping and performance of cross checking tests is one of the most important tests performed during a surgical procedure period. Automation of the process was inevitable to avoid any complications during the blood transfusion process. It’s imperative to understand the clinical significance of the different blood groups, their typing and cross matching tests. A total of almost thirty blood groups has been recognized by the International Society of Blood Transfusion (ISBT) (Lamba et al., 2013).In order to decide whether or not an antigen-negative blood is needed for transfusion, the extent of being specific by the antibodies is a prerequisite and several other factors are also determinants of the same including the strengths and thermal amplitudes of the antibodies involved, the clinical diagnosis of the patient and the functioning of the bone marrow.
Serological testing
Most clinically significant antibodies are those that operate at a temperature of about 37 degrees Celsius and by the indirect antiglobulin test (Makroo et al., 2014). The clinical importance of antibodies to red cell antigens is also grounded on their capability to truncate survival in the red cell by commencing reaction caused by hemolysis after transfusion (HTR) or through their connotation with hemolytic disease of the newborn (HDN). Cross-matching encompasses fraternization of donor RBCs with the recipient serum to detect fatal reactions. This project is aimed at studying and comparing the different techniques used for the uncovering of the clinically important antibodies. These methods include the low-ionic-strength saline solution and indirect antiglobulin test (LISS_IAT), Albumen test and indirect antiglobulin test (AIb_IAT) and polyethylene glycol indirect antiglobulin test (PEG-IAT) in a bid to determine which method is better in sensitivity and specificity.

Several studies have been done regarding the different methods that are used in the identification of clinically significant. The discovery of the ABO group is the most important part of the pre-transfusion, this is done to detect the antibodies present in the blood as the wrong grouping could lead to a fatality caused by the antibody-antigen clumping when the donor and the receiver are incompatible. Clumping can give rise to an immune mediated hemolytic transfusion reaction. Automation in blood blank serology was introduced in the developed countries and is rapidly being adopted worldwide as there is an improved quality of testing and a reduction of human errors as compared to the previously used methods like the Conventional tube techniques (Bhagwat et al., 2015). Pre-transfusion compatibility testing is a precarious constituent of the whole transfusion procedure to augment vein-to-vein care. The Rh and ABO blood group of the receivers and the givers, and the cross-matching between the recipient and the donor in indirect antiglobulin (IAT) stage to perceive clinically significant antibodies are crucial phases of the compatibility testing.
CAT Method
Pre-transfusion of blood must always include an antibody monitoring that can detect the possible clinically significant red cell antibodies. However at any given instance, anti-A, -B and -A, B must always be regarded as clinically significant (Joyce, 2011). Column agglutination technology [CAT], solid-phase and liquid-phase approaches are all valid for the testing of IAT. Chary contemplation must, however, be taken into place when choosing the method to use because there are dissimilarities in the heftiness or dependability of each method of detection. The outcome of the screening process may either be negative or positive. Negative is an indication that the patient’s plasma does not have the antibodies being unexpected antibodies while the positive is a sign of the presence of a red cell antibody. The ABO is not direct products of genetics but is formed in the body. ABO mismatched transfusions lead to more severe clinical outcomes than any other blood group (Powell, 2017).
Antiglobulin trials are grounded on the statistic that the anti-human globulins (AHGs) obtained from immunized non-human species impasse on human globulins such as IgG or complement system, either unrestricted in serum or bound to antigens on red blood cells (RBC).There are two major types of anti-human globulins including the monospecific and polyspecific AHGs. Polyspecific AHG contains both antibodies to human IgG and to the C3d component of human complement while the monospecific AHGs contains only one antibody specificity: either anti-IgG or antibody to specific elements such as C3b or C3d (Green et al., 2005). Antiglobulin tests, therefore, distinguish IgG and complement-sensitized RBCs (Green et al., 2005). In 1945, Coombs and colleagues termed the use of globulin serum that is anti-human in sensing antibodies that are bound in red cells and are non-agglutinating. Hare, (2010) depicted that any antibodies existing within the antiglobulin content were geared against individual constituents of complement. Reagents used for antihuman globulin identify non-agglutinating antibody fragments and fragments of complement close to erythrocytes after in cell or out cell antigen-antibody responses (Green et al., 2005). IgG antibodies are termed as agglutinating because their monomer arrangement is too insignificant to clump sensitized RBCs unswervingly and before the discovery of the antiglobulin test only the IgM antibodies were able to be discovered. Therefore the development of the antiglobulin test was significant in the detection of the IgG antibodies and also the discovery and characterization of the different blood groups. Agglutination takes place in two stages, the first one being Sensitization followed by Haemagglutination (Joyce, 2011).
Sensitization in the antiglobulin tests occurs in two different ways, in vivo, and in vitro. In-vivo sensitization is identified by a one-phase technique, the direct antiglobulin test (DAT) while the in-vitro sensitization of RBCs takes place in two-stages and is referred to as the indirect antiglobulin test (IAT).The first stage involves the antibodies binding to the red blood cells during incubation and the second stage, after washing an Antihuman Globulin (AGH) is added to the test tube, and a lattice is formed. In the DAT the when the anti-human globulin is added to the cells having globulin, agglutination occurs but when there is no globulin, no agglutination is observed in the test area (Green et al., 2005). Antibodies that display the strong reaction by the IAT may be more vigorous in vivo than if the same antibody demonstrated weedier responses.
LISS testing
In the serological tests for blood groups, ionic measure of response media is fundamentally reliant on the intensity of sodium and chloride ion contributed by isotonic saline. A low amount of ionic saline (LISS) is used to enhance the initial phase of antigen-antibody feedbacks (sensitization). LISS normally has reduced number of ions that reduce the protection effect of cations which in turn will harbor anions to protect the positive ions on antibodies and negative ions on antigens, thus averting the negative and positive charged opposite sides from clumping together. LISS increases the sensitivity of most clinically sensitive antibodies. LISS has two general methods of being carried out including the cells being suspended in saline solution, then adding of serum and two drops of LISS solution, or just the use of the LISS solution on its own. LISS affects the initial phase of agglutination. Indirect antiglobulin test using the ionic strength concentration.
Albumen testing using bovine serum started being used when it was discovered that the rhesus ant-D would not agglutinate in the saline environment. Bovine albumin was used to enhance the activity of the antibodies of the different blood groups as agglutination of red blood cells coated antibodies depends on the dimensions of the antibodies and their ionic strength. Bovine is mostly used to potentiate the agglutination of the Red Blood Cells and IgG. It mainly influences the second stage of agglutination by affecting the water of hydration and reduction of the zeta potential. Albumin tests are also done at times in the diagnosis of pulmonary edema. Polyethylene glycol (PEG) is a long water soluble chain that is used in the enhancement of interaction between the red blood cells and the antibodies. It does this by competing with water on the surface of the red blood cells and in so doing makes the concentration of antibodies around the face of the red blood cell to be increased (Shirley et al., 2004).
PEG testing
PEG has proved n various studies to be more efficient as compared to the Low ion saline solution and the albumin tests. It’s easier to prepare and store and even gives better results In a study conducted in a single center in a Caucasian-dominant population, albumin (Alb-IAT) was used for eight years between January 1989 and December 1996 and polyethylene glycol (PEG-IAT) for twelve years between January 1997 and December 2008 (Reisner et al., 1996).The Alb-IAT test involved close to thirty-two thousand patients who were screened for antibodies and out of these four thousand, six hundred and fifty recipients blood transfusion with RBC s was carried out. The RBCs had been derived from whole blood. For the PEG-IAT, slightly over forty thousand patients were put through screening for antibodies and out of the number, 8036 were transfused with RBCs derived from unabridged blood. Delayed hemolytic transfusion reactions were then determined by the analysis of the patient’s transfusion-associated clinical signs, test results and blood transfusions carried out during the period and same antibodies used for the trial before and after the transfusion. In the Albumin indirect ant-globulin test bovine was used and after incubation Rabbit monospecific anti-human globulin used. In the PEG-IAT test, polyethylene glycol was used and Rabbit monospecific anti-human globulin. Panel cells were utilized for the screening of the antibodies. The outcome carried out using Fischer’s test was that PEG-IAT detected the clinically significant antibodies as compared to the Alb-IAT test (Reisner et al., 1996).
Among the Japanese, the rate of usage agglutination technologies was little in comparison to the use of test tube technology. Surveys in the area have given an insight of the better reliance of the PEG-IAT method both before and after transfusion as compared to the albumin. Delayed hemolytic transfusion reactions in indirect antiglobulin tests. Thus the use of PEG-IAT is a better bet in the prevention of the (Reisner et al., 1996). A study using papain to evaluate the sensitivity and specificity o PEG showed that polyethylene glycol had higher sensitivity and thus increased detection of antibodies (Reisner et al., 1996).Albumin does not provide the commonly accepted level of sensitivity( American Red Cross).
Comparison studies
When compared against the low ionic strength solution, PEG-IAT displayed more sensitivity in detection of the clinically significant antibodies. In a study carried out by Shirley et al., (2004) involving a blinded comparison of the low ionic strength solution and PEG before transfusion took place. The PEG and LISS potentiate used in a sample of five hundred patients. Out of the study sample, one hundred were of a known antibody while the rest was unknown. The outcome depicted that the PEG antiglobulins reaction was greater than the LISS antiglobulin responses. When the antibodies present are weak, sodium and chloride ions in the LISS solutions may interpose with the binding of the antibody to the antigens present on the red blood cell membrane. The PEG did not have any adverse outcome, compared to the LISS which had one negative result. PEG identified a total of sixteen antibodies in the sample with unidentified antibodies and out of these, seven were of clinical importance. LISS, however, may enable the singling out of antibodies indicating the ability to react at thirty-seven degrees Celsius phase of testing
Ono et al., carried out a study on the detection of sensitivity of erythrocytes alloantibodies by the use of micro tube column systems of agglutination (2016), aimed at comparing the gel systems and the PEG tube method on two hundred and seventy samples indicated that columns with gel as compared to cells had less sensitivity in exposure of clinically important antibodies. In the screening of unexpected antibodies, using the two stage papain test and LISS direct agglutination, there was a result of a high number of unwanted positive reactivates which depicted these methods as inappropriate for the pre-transfusion screening of unwanted antibodies.
The adoption of PEG-IAT compared to the Abl-IAT has led to the increased proficiency in the result of the detection rate of clinically substantial irregular antibodies and decline in that of irrelevant antibodies. The post-transfusion trends also show significant favor in the tests carried out using the PEG-IAT tests as compared to LISS and the Abl-IAT tests. To evade identifying fewer substantial autoantibodies, many hospitals offering transfusion avoid the using of an autologous test control with auto red blood cells and serum.
Aim of study
This study is aimed at exploring the different methods that have been used in potentiation for clinically significant antibodies and compare the superiority of each based on previously done tests.
Polyethylene glycol (PEG-IAT) test is superior to both the LISS_IAT and AIb_IAT tests.
LISS_IAT test is superior to both the Polyethylene glycol (PEG-IAT) test and AIb_IAT tests.
AIb_IAT test is superior to both Polyethylene glycol (PEG-IAT) test and LISS_IAT test.

Using PEG-IAT in detecting clinically substantial antibodies has proved to be the best method as compared to the utilization of the LISS_IAT and AIb_IAT tests. It has an easy way of preparation, concentrates the antibodies and lowers the number of false reports in the antibody detection tests. Future studies could be directed towards totally decreasing the number of false positives for the efficiency to be dependable.


Bhagwat, S, J Sharma, J, H, Jose, J and Modi, C, J (2015) The Antiglobulin Test.
Green, B and Hughes, V. (2005). Comparison between Conventional and Automated Techniques for Blood Grouping and Crossmatching: Experience from a Tertiary Care Centre.
Hare, V. (2010) Blood Bank-Reviewing the basics. American Red Cross Southern Region
Douglasville, Georgia
Joyce, A. (2011). Immunohematology. AIMS NZIMLS SouthPacific Congress
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Lamba, R, Kaur, Rand Sabita B (2013). Clinically Significant Minor Blood Group Antigens amongst North Indian Donor Population.
Makroo, R., J Arora, B, Bhatia, A, Chowdhry, M and Nakamatathil, L, R.(2014) Clinical significance of antibody specificities to M, N and Lewis blood group system.
Ono, T, Hikichi, R .Kawabata, K and Ohto H. (2016). The detection sensitivity of red cell alloantibodies using micro tube column agglutination systems. International Journal of Blood Transfusion Immunohematology.
Powell v. (2017) Blood Group Antigens and Antibodies. Blood Group.
Reisner R, Butler G, Bundy K, Moore SB. (1996) Comparison of the polyethylene glycol antiglobulin test and the use of enzymes in antibody detection and identification.
Shirey, R.S., Boyd, J.S., and Ness, P.M. (2004), Polyethylene glycol versus low-ionic-strength solution in pre transfusion testing: a blinded comparison study.

Final Project Report:
To be submitted by the end of Week 13 (Sunday 22nd October, 2017 by 2359 hours via Turnitin in Blackboard).
For Clinical Biochemistry project students a Hard Copy of your report is NOT required. For all other disciplines, please submit a hard copy to the “Academic Services 201.2.10” assignment box by Monday 23rd October, 2017.
You have already done a background introduction as part of your literature review. Do NOT add your literature again to this report for the turnitin submission. You should add the literature review to the hard copy report for completeness. Note however it will not be remarked.
Following on from your literature review you should have a one paragraph introduction to include your specific project aims and a hypothesis if relevant. The project report should be similar to a scientific manuscript which is published in a scientific journal. The main part of the report should be 5000 words (or approx. 7,000 words with your literature review embedded in the hard copy) and consist of:
1. Introduction.
2. Methods and Materials.
3. Results.
4. Discussion.
5. Conclusion.
The 5,000-7,000 words are approximately 20-22 pages (12 pt Times New Roman, 1.5 line spacing) and does not include the tables, figures, figure legends, and the following:
Also included in the report should be:
1. Title page and declaration
2. Abstract (250 words), on separate page.
3. Table of Contents (list of figures, list of table, list of abbreviations …….. ect) on separate page.
4. List of references; ˷30-50 references in Vancouver format.
5. Acknowledgements.