TOPIC: ANTIBODY DETECTION AND IDENTIFICATION TEST FROM A LEUKAEMIA PATIENT’S SAMPLE

INTRODUCTION
In today’s world where science are ruled by the ever changing clinical laboratory, few mishaps and problems are still at arising which requires deep understandings and solutions to overcome the problems. Some of the problems do required immediate attention and some are just occurring problems which eventually a solution must be provided. However no problems can be more severe than problems and issues arising from a specific department which is one of the most important sections in a clinical laboratory. That department is called the transfusion service department. At times, nothing is more confounding to a laboratory scientist then the solutions to antibody detection and identification problems. Errors from this antibody detection and identification should be resolved and dealt in a organized and logic mannered and immediate effect should be implemented as fast growing of elderly and oncology population receiving supportive transfusions are increasing as time develops.
Some antibodies issues can take up to hours for a solutions to be implemented, and when this occurs, pretransfusion testing will be delay and this will definitely caused many severe damages including costly hospital extensions. Antibody detection and identification testing is one of the most complicated testing ever conducted in a clinical laboratory. This is such as many areas of error and problems can occur during testing period or even during result reading and analyzing period. This study provides an example of a scenario where a resolving some of the problems encountered daily in the blood bank laboratory. A case study will be included in this study and based from the case study, this study of antibody detection and Identification testing on leukaemia patient’s sample will be developed and further studied.

A 63-year old white man was hospitalized with palpable splenomegaly. Medical history revealed that several years earlier, the patient has been diagnosed with sideroblastic anaemia. On admission, he presented with leukopenia (2700/mmз) and thrombocytopenia (55,000/mmз). Notable blood chemistries showed an increase in uric acid and vitamin B12. Leukocyte alkaline phosphatise was increased. Bone marrow examination revealed moderate cellular hyperplasia with marked increasing myeloid elements. Megakaryocytes were normal. A splenectomy was schedule and four units of RBCs were ordered for the splenectomy procedure. While performing the preoperative ABO detection and identification test, a discrepancy was resulted. The problem is then analyzed and further evaluated into this study. i) Antibody detection
In antibody detection phase is where presence of antibody are detected and observed. There are two type of test method that can be carried out in determining the presence of immunoglobulin or complement or commonly known as antibody. There are the Direct antiglobulin test to detect in-vivo sensitization and Indirect antiglobulin test to detect in-vitro sensitization.
The direct antiglobulin test which shortly known as DAT is conducted to show the presence of an antibody that has caused in vivo sensitization of red blood cells (RBCs). In order this sensitization to occur, many sources of anti-human globulin or AHG serum are commercially available. This AHG serum is usually polyspecific serum that consists of anti-IgG and anti-C3d.This serum causes the formation of agglutination of saline-washed red blood cells that have been sensitized. And vice versa goes to the unsensitized red blood cells where no agglutination formation will occur.
A positive results of DAT can occur in cases such as haemolytic disease of the newborn (HDN), transfusion reactions, autoimmune haemolytic anaemia and drug-induced haemolytic anaemia. Furthermore, when a positive result of DAT is gathered, after using polyspecific AHG serum, a monospecific AHG serum should be used where either anti-IgG single or anti-complement (C3b + C3d). This will ensure the end results of very specific proteins sensitizing the red blood cells’ surface and more accurate and reliable result can be compromised. The direct antiglobulin test has wide ranges of applications in antibody detection and identification procedure.
The indirect antiglobulin test or commonly known as IAT is specially designed to demonstrate the in vitro reactions of red blood cells being sensitized by the antibodies. This antibody detection test should only be performed with plasma or serum sample with a set of reagents group O cells. However, serum is the more preferable sample as antibodies present are enhanced due to complement activations and the strength binding to the red blood cells. While in plasma, there are few disturbances in the form of fibrin clots which gives a false results and not accurate readings. Apart from antibody detection and identification testing, IAT is also commonly used in red blood cell antigen typing, weak D testing and the important compatibility testing process.
In IAT, reagents cells consist of blood group O cells are used to enhance the sensitization of antibody reactions towards red blood cells. These reagent are usually in two types which as a pool of two donors in a single vial where is implemented for donor antibody screening test. Another type is a matched set of two or three different group O red blood cells used singularly, which gives greater sensitivity during the test.
Since reagents are widely used in laboratories, for the past 5 years, various methods, procedures and process have been developed throughout and what was discovered to be an enhancement media or potentiate antigen-antibody reactions. These enhancement media can promote both direct agglutination and indirect agglutination tests. Examples of routine enhancement media are bovine serum albumin (BSA) and low-ionic-strength saline (LISS) which is the most common enhancement media used in all major clinical laboratories.

ii) Antibody identification
After antibody are detected using either through DAT or IAT method, the identification of the types of antibodies presence during the DAT or IAT tests are carried out in Antibody identification testing process. There are two methods which are the antigrams and routine panels. With these two methods, antibodies which are detected can be identified with sensitivity assured.

As mention earlier, during antibody detection tests, reagent used red blood cells matched set of two or three blood group O donor via single vial are used. This has been used to gain significant antigens profiles which will be identified through antibody identification testing. This is where usage of antigrams and routine panels are implemented. The antibody identification procedure includes a panel of 8 to 16 groups of O red cells. Reactions of positive and negative are compared with the antigen makeup of the panel cells. This is where detected antibodies are identified and given their identity for further investigation.
The antibody screening cells must present the following examples of antigen profiles, D, C, E, c, e, M, N, S, s, Lea, Leb, P1, K, k, Fya, Fyb, Jka and Jkb. The screening cells are commercially prepared and manufactured from blood bank reagents dealers and manufacturer. For some of the weaker antigens which contains low-frequency antigens such as Lua, Cw or V, are given special request from some laboratories.

Complete antibody detection and identification testing consist of few test combined. The test comprises of ABO grouping test, Rh grouping test, antibody screening test, and lastly antibody identification test where antigram is used.

i) ABO and Rh grouping tests

ABO grouping test determines the type of blood group of the patients and this decreases the chances for blood transfusion errors, for instance transfusing the wrong type of blood group will cause complications as adverse effect in blood transfusion. To conduct ABO grouping test, patient’s information and medical history from the worksheet should be checked and validated with the sample taken from the patient. There are two types of method will be conducted, which the forward ABO grouping and reverses ABO grouping methods.

In forward ABO grouping method, sample will be centrifuged to remove the serum. The concentrated red blood cells will then diluted with normal saline to prepare a 3 % suspension red cells which then will be used during the forward ABO grouping method. Reagents used in this method are Anti-A, Anti-B, Anti-AB, and Anti-D with control. One or two drops of the washed 3% suspension red cells will be mixed with one drop of all the reagents separately and respectively.

In reverse ABO grouping method, only serum which was separated during the centrifugation will be used with two reagents of A1 cells and B cells with same method as forward ABO grouping test.

To carry out these two methods, few ways can be done, example are tile method, tube method or the modern technology way using Diamed blood grouping cassette. Most major laboratories uses the Diamed blood grouping cassette as it saves time, energy and its 98% accuracy approved by the manufacturers and furthermore it is more user-friendly will less complications to handle.

ii) Antibody screening test

There are few reasons why antibody screening test is carried in a daily basis in a blood bank department. The main purpose of this test is to ensure there are no presences of unexpected antibodies which will be transfused during transfusion process. For instance, patient’s serum is tested with antibody screening test before transfusion to ensure no unexpected antibodies reacting with the donor cells. In another phrase, this test avoids a cause of positive direct antiglobulin and transfusion reaction as these antibodies present could attach to the patient’s cells.
Antibody screening test is also a mandatory test for pregnancy women. A maternal serum sample will be taken from a pregnancy woman and tested to make sure pregnant mother has no antibodies to react with their fetal cells. This is a common test to avoid the famous disease for pregnancy, haemolytic disease of the newborn or known as HDN. HDN is caused when mother’s immunoglobulin IgG antibodies crosses the placenta and attaches to the foetus’s red blood cells, this can be fatal to the foetus. Therefore it is very important to know the antibody presents and the rhesus grouping of every pregnant woman.
In antibody screening test, O cells or specific antibody screening reagent red blood cells are assorted with serum sample. Adding enzymes or albumin or any enhancement media can be combined with the mixture in order to increase the bonding and help to promote the interaction of the antibodies with the red blood cells for a more accurate result. A positive result will be shown where presence of alloantibody or autoantibody in the serum is indicated. According to antibody screening test procedure, there are three main phases involved which are the phase one immediate spin, phase two 37°c incubation and phase three coombs AHG or AGT.

During phase one which is the immediate spin phase, 3 tubes will be labelled as recipient serum with saline suspension Screening Cell I, Screening Cell II, and the recipient's own cells acts as the auto control. The three tubes will then be centrifuged and observed for agglutination reaction in those three tubes. The reason for phase one immediate spin is to detect IgM antibodies which are usually considered as ‘troublesome’ antibodies. This phase also gives the extra edge in identifying whether the agglutination reaction is due to presence of IgM antibodies istead of any IgG antibodies. In addition, immediate spin phase is also known to detect cold antibodies.
Next is phase two which is the 37°c incubation. This phase is a mandatory step and proves as a vital step in antibody screening test. The three tubes will be placed in a water bath of 37°c temperature for 15 minutes. This is where IgG antibodies are detected as there are warm-acting antibodies. 37°c phase detects only warm antibodies. Furthermore, it is not necessary to observe for agglutination at this point of stage of testing, directly to phase three can be implemented.
However, there is an extra step that can be included in this phase to shorten the period time of the tubes inside the water bath. This can be achieved by adding enhancement media into the tubes. Most common enhancement media use is the low ionic strength solution or shortly known as the LISS which actually speeds up the antigen-antibody reaction. Some laboratories use albumin or certain enzymes such as papain cystein where it is added to lower the potential of the cells so that it could agglutinate at a higher rate without Coombs phase.
LISS media is composed of NaCl, glycine and phosphate buffer with sodium as the preservative. The only downfall of this LISS media is that it enhances not only the reaction of antigen-antibody but also the ‘troublesome’ antibodies. Therefore, LISS should be added into the tubes right after phase one is completed.

An important step in antibody screening test and where a number of presence antibodies may be detected only at this point of phase is the third phase which is the coombs phase (AHG or AGT). At this phase also IgG antibodies are detected. The reason for having two phases to detect IgG antibodies is because IgG antibodies are clinically significant antibodies which are the main cause of few fatal diseases such as the Hemolytic Disease of the Newborn and the Hemolytic Transfusion Reactions.
This phase is carried out right after removing the tubes from water bath. The cells are washed up to three or four times and at the last wash, the saline was removed and AHG or AGT reagent is added into the mixture. The solution will then be centrifuged for agglutination reactions will be observed after that. If a negative result was shown, Coombs Control Cells will be added to reconfirm the negative results.

RESULTS i) Personal details Name : Case Study 2 | Hospital : # | Date : 17 April 2002 | Age: 63 | Sex: Male | Diagnosis : Splenectomy | Patient’s blood type:? |

ii) ABO and Rh grouping Specimen date: 17 April 2002 | Anti- | Cells | Anti- | | | A | B | A,B | A1 | A1 | B | D | C | c | E | e | Rh control | | Results | 4+ | 0 | | 0 | 2+ | 4+ | 4+ | 4+ | 4+ | 0 | 4+ | 0 | |

iii) Antibody Screening Tests

Antibody Screening Test | Cells | LISS | Albumin | Cold Screen | | IS | 37°c | AHG | | | | | | | I | 0 | 0 | 0 | | | | | | | II | 0 | 0 | 0 | | | | | | | III | 0 | 0 | 0 | | | | | | | Auto | 0 | 0 | 0 | | | | | | | A1 | 2+ | | | | | | | | | A2 | 0 | | | | | | | | | B | 4+ | | | | | | | | | Cord | | | | | | | | | |

iv) Antigram Result Donor | S | Rh-Hr | Kell | MNSs | Duffy | Kidd | Lewis | Lutheran | P | Xg | Test Method | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 1 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 2 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 3 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 4 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 5 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 6 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 7 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 8 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 9 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 10 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 11 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |