Antibody Detection and Identification

According to AABB antibody screen must be done on:
1- Serum of donor (to avoid passive transfer of antibody).
2- The serum of obstetric patients to identify women with alloantibodies that:
a- Might cause hemolytic disease of the newborn.
b- Might cause compatibility problems if transfusion is needed.
Antibody screen is useful:
1- Detect unexpected antibodies due to transfusion or pregnancy.
• Only 0.3 – 2% of the general population have positive antibody screen.
2- Clarify laboratory results or clinical observations:
a- A discrepancy exists between results of serum and cell tests for ABO group due to antibody.
b- The direct antiglobulin is positive and the patient has recently been transfused or has autoantibody.
c- There is increased destruction of red cells and immune mediated destruction is suspected.
• The antibody screening test cannot detect all antibodies of potential clinical significance . Some antibodies will be missed such as:
1- Antibodies reactive with low incidence antigens will be missed.
2- Antibodies that react only with cells homozygous for particular antigen.
@ The degree of positive reaction generally indicates the amount of antibody present not its significance. For example an anti-D that reacts weakly will have lower titer than one reacts +4 but they are equally significant.

Screening Cells:
• They are commercially prepared group O cell suspension obtained from individual donors that are phenotyped for the most commonly encountered and clinically important RBC antigens.
• Group O cells are used so that naturally occurring Anti-A or anti-B will not interfere with detection of unexpected antibodies.
• The cells are selected so that the following antigens are present on at least one of the cell samples, C, E, c, e, M, N, S, s, P1, Lea , Leb, K, k, Fya, Fyb, Jka, Jkb.
• It is important that the lot number on the screening cells matches the lot number printed on the antigram because antigen make up will vary with each lot.
• The ideal screening cells have RBCs with homozygous of as many antigens as possible and preferred to contain heterozygous expression of antigens which show dosage effect ( like Jk and M) to enable to detect weak antibodies.
• Screening cells are available in three forms:
1- A single vial of no more than two donors pooled together.
2- Two vials each with different donors.
3- Three vials representing three different donors.
• Pooled cells are less sensitive but may be used for detection of antibodies in donor units because weakly reacting antibodies may be missed, but low levels of antibody in the plasma of a donor unit will not harm a recipient.
• Detection of very low levels of antibody in recipient serum is important because transfusion of antigen positive RBCs may result in a secondary immune response with rapid production of antibody and subsequent destruction of transfused RBCs.
** There is no requirement for the use of enhancement reagents in antibody detection tests.

Interpretation of Antibody Screen:\
The investigator should consider the following questions
1- In what phase (s) did the reaction(s) occur?.
* Antibodies of IgM class ( anti-N, anti-I, and anti-P1) will react best at low temperature and are capable of causing agglutination of saline suspended RBCs ( immediate spin phase).
* Antibodies of IgG class ( anti-Rh, anti Kell, anti kidd, anti duffy.)will react best at the AHG phase.
* Anti Lewis and anti M antibodies may be IgG, IgM or mixture of both.
* Potent cold antibody binding complement reacting in AHG phase ( not detected in immediate spin).
2- Is the autologous control negative or positive?
* Autologous control is negative and positive antibody screen - alloantibody.
* autologous control is positive and positive antibody screen  autoantibody , antibody to medication or it may be idiopathic.
* If the patient is recently transfused , the positive autocontrol may be due to autoantibody coating circulating donor cells.
3- Did more than one screening cell react and if so, did they react at the same phase and strength?
* Single antibody specificity should be suspected when all cells react at the same phase or strength.
* Multiple antibodies are most likely when cells react at different phases and strength.
4- Is hemolysis or mixed field agglutination present?
* Anti (Lea , Leb, P, P1, Pk and Vel) are known to cause in vitro hemolysis.
* Anti Sd a and Luthera antibodies make mixed agglutination.
5- Are the cells truly agglutinated or rouleaux present?.
*Rouleaux will not interfere in the AHG phase, because the patient serum is washed away prior to addition of the AHG reagent.

Antibody Identification:
Antibody identification is performed in the same manner as the antibody screen except that a panel of reagent RBCs is used in place of screening cells.
• Panels are expanded versions of antibody screening cells consisting of 8 to 16 group O RBCs suspensions.
• Rare phenotypes include cells lacking high frequency antigens( U,Vel, Yta)or cells having low frequency antigens(Wra Cw,Cob).
• A well- designed panel will identify most commonly encountered antibodies and eliminate or rule out the presence of antibodies that are not present.

Evaluation of Panel Results:
It can be done by answering the following questions:
1- Is the autologous control positive or negative ?.
* The presence of autoantibodies complicates the process of antibody identification.
2- In what phase (s) and at what strength(s) did the positive reaction occur?.
* If all positive reactions occurred at the AHG phase and all were +2. This indicate that the single IgG antibody.
3-What antibodies can be ruled out or eliminated as possibilities?.
* Only cells that gave negative reactions in all phases of testing should be used for ruling out antibodies.
* It is best to rule out antibodies using panel cells from donors that are homozygous for the corresponding antigen because some weakly reactive antibodies may fail to react with cells from heterozygous donors.
*Weak anti-Jka may not react with a Jk(a+ b+) cell because it carries fewer Jk antigens than the Jk(a+ b-) .
4- Does the serum reacting match any of the remaining specificities?.
* The pattern of reactivity will usually exactly match a pattern when there is a single alloantibody present.
*The pattern of reactivity will not always match a specific pattern and there are several reasons for that:
1- Weakly reactive antibodies may only give positive results with homozygous cells.
2- Presence of multiple alloantibodies.
3- Presence of cold reactive autoantibodies.
4- presence of antibodies directed at high and low frequency antigens.
5- Are commonly encountered RBC antibodies ruled out?.
• A patient serum may contain more than one RBCs antibody & the presence of one specificity may mask or interfere with identification of another.
• Antibodies to low frequency antigens are uncommon because of the small chance of being exposed to the antigen, therefore it is not necessary to rule them out.
• If a commonly encountered antibody is not ruled out, it is important to test selected cells that will rule out the presence of the antibody ( positive for the antigens to be ruled out, negative to the antigen for the probable antibody.).
6- Is there sufficient evidence to prove the suspected antibody?
• Conclusive antibody identification requires testing the patient's serum with enough antigen – positive & antigen negative cells ( three cells of each required) to ensure that the pattern of reactivity is not the result of chance alone.
• للتأكد من أن نتيجة الأجسام المضادة التي تم التوصل إليها مقنعة. يجب كذلك اختبار تفاعل سيرم المريض مع ثلاثة خلايا إضافية ايجابية للانتجين وثلاثة أخرى سلبية للانتجين ( يمكن التأكد من ذلك من نفس البانيل الذي تم عليه الفحص أو يكون الاختيار لأنواع جديدة). هند عمل هذا الفحص ويعطي نتائج ايجابية مع الخلايا التي تحتوي على الانتجين ونتائج سلبية مع الخلايا التي لا تحتوي على الانتجين .عندها نقدر أن نقول أن النتيجة صحيحة بنسبة 95% مع وجود نسبة خطأ 5% وان حدوث التفاعل حسب النمط المعين الذي يؤكد وجود الأجسام المضادة حصل لأسباب أخرى غير هذه الأجسام المضادة.
7- Is the patient lacking the antigen corresponding to the antibody?.
• The last step in identification studies is to test the patient's cells for the corresponding antigen.
• A negative result is expected & indicates that identification results are correct.
• If the patient's RBCs are positive for the corresponding antigen, misidentification of the antibody or false – positive typing is the most likely explanation.
• Antigen typing is also useful in the resolution of complex cases because it eliminates many possibilities.
• It is not practical to do extended typing on all patient's with antibodies.
• It is important to know if the patient has been transfused in the last 3 months before using this technique because the presence of donor cells may result in false positive typing.
• مما تقدم نستطيع ان نقول انه يلزم لتحديد نوعية الأجسام المضادة الموجودة عند المريض الإجابة عن سبعة أسئلة. تساعد الخمسة الأولى على التعرف على هذه الأجسام, أما السؤالين الأخيرين تساعد على تأكيد النتيجة التي تم التوصل إليها عن طريق فحص تفاعل سيرم المريض مع ثلاثة خلايا ايجابية وثلاثة أخرى سلبية. وكذلك التأكد من خلو خلايا المريض من الأنتجينات المقابلة لهذه الأجسام المضادة لأنه لا يمكن تكوين أجسام مضادة ضد انتجينات توجد على خلايا جسمه.
Methods of Antibody Enhancement:
1- Extended incubation time ( to 60 minutes detect weak antibodies.).
2- Increase serum to cell ratio( increase the sensitivity of the test and permits detection of weak antibodies.).
• In hemolytic transfusion reaction in which no antibody is detectable, it is useful to increase serum to cell ratio.
3- Enzyme treated cells.
4- Bovine albumin.
5- Manual polybrene treatment.
6- Incubation at temperature below 37C.

Special problems in antibody identification:
1- Multiple identification.
2- Antibody to high frequency antigens ( k 98.8%, Vel > 99.9%, Lub99.8%).
• They should be suspected when the patient auto control is negative but the serum reactsin a uniform manner ( same phase and strength )with all panel and donor cells.
3- Antibodies to low-frequency antigens(Kpa ,Wra).
• They should be suspected when antibody screen is positive but the compatibility test is negative.
4- Cold reactive autoantibodies.
5- Warm autoantibodies.
6- Antibodies to reagents.

بقلم :عادل عبد الحليم السلايمه
أخصائي طب مخبري
مسؤول جودة المياه/ بلدية الخليل