| How many percent is the A1 and A2 | • A1 (80%) and A2 (20%) |
| What is Lectin and examples | • Lectins: seed extracts that agglutinate with human cells with a degree of specificity.
Dolichos biflorus (only agglutinates with A1 or A1B cells)
Grifonia simplicifolia (B cells)
Ulex europaeus (O cells) |
| Difference between the A1 and A2 | The difference between the A1 and A2 subgroup is that they are both qualitative and quantitative. A1 has an increase quantity compared with A2. |
| A cell subgrouping reagents | 3-5% Type A Red Cell Suspension
Physiologic Saline (0.85-0.90% NaCl) in Wash Bottle
Anti-A reagent serum
Anti-A1 Lectin |
| PROCEDURE: A cell subgrouping | 1. Make a 3-5% Type A RBC Suspension (RBC suspension procedure) 2. Prepare 2 clean tubes and label with A and A1 respectively. 3. Place 2 drops of Anti-A Reagent Serum in tube A and 2 drops of Anti-A1 lectin on tube A1. 4. Add 2 drops of freshly prepared 3-5% Type A RCS to both tubes. Follow the tabulated procedure below. 6. Mix the solution and spin for 15seconds at 3,400 rpm. Gentle mixing is recommended. Grade the reaction, record andinterpret the results |
| Why we need to add the clear solution first | In every tube method, always add first the clear solution. This is to ensure that all needed solutions are added. |
| Interpretation: A cell subgrouping | SUBGROUP A1. Agglutination in tubes A and A1
SUBGROUP A2. Agglutination in tube A only
[0] No agglutination
[+] With Agglutination |
| Why subgroup A2 yield negative in Tube A1 | Subgroup A2 does not have the capacity to identify A1 as part of the A subgroup. |
| SALIVA TESTING FOR SECRETOR STATUS | SALIVA TESTING FOR SECRETOR STATUS |
| What is secretor | there is presence of Antigen in the Body fluids including the Saliva |
| Why saliva is the specimen used? | • Saliva is used because it is easier to extract. It has the same level |
| REAGENTS: | -Saliva
-Human polyclonal anti-A and anti-B serum
-Anti-H lectin from Ulex europaeus
-Physiologic Saline
-3% to 5 % washed group A, B, and O cells |
| Secretor Substances | • Considered as a secretor if ABO gene and Sese gene is inherited (Ex. The type A antigen can be detected in the body fluids)
• Synthesized on Type 1 precursor chain
• Secretion of A, B, and H Ag in body fluids.
• 78% can be found in saliva and gastric juices
• Readily or easily detected
• Capable of Hemagglutination and if presented with an antibody it will agglutinate |
| A, B, and H Soluble (means Secretor) Substances | • Secreted substances are glycoprotein
• Synthesized on Type 1 precursor chain
• On the Beta 1-3 linkage
• The responsible glycosyltransferase for the formation of soluble substances is the A-2-L flucosyltransferase enzyme |
| Fluids in which A, B, and H substances can be detected in Secretors | Saliva
Tears
Urine
Digestive juices
Bile
Milk
Amniotic Fluid
Pathological fluids: Pleural, Peritoneal, Pericardial and Ovarian Cyst |
| PROCEDURE:
A. SALIVA COLLECTION AND PREPARATION: | 1. Collect about 2 to 3 mL of saliva in a test tube.
2. Centrifuge at 900 to 1000 rpm for 8 to 10 minutes.
3. Transfer supernatant to a clean test tube, and place stoppered tube in a boiling water bath for 10 minutes. This inactivates enzymes that might otherwise destroy blood group substances.
4. Recentrifuged at 900 to 1000 g for 8 to 10 minutes.
5. Collect clear supernatant into a clean tube. |
| B. TEST FOR SECRETOR STATUS | 1. Dilute saliva with an equal volume of saline (undiluted saliva contains nonspecific glycoproteins that can inhibit antisera and lead to incorrect results).
2. Add one drop of diluted antiserum to an appropriately labeled tube (anti-A, anti-B, anti H).
For dilution, titrate anti-H, anti-A, and anti-B, testing against appropriate cells at immediate spin. Select the dilution giving 2+ agglutination, and prepare a sufficient quantity to complete the test.
3. Add one drop of supernatant saliva to each tube. Mix and incubate at room temperature for 8 to 10 minutes.
4. Add one drop of the appropriate indicator cells (A, B, or O cells) to the properly labeled tube.
5. Mix and incubate at room temperature for 30 to 60 minutes.
6. Centrifuge for 15 to 30 sec at 3600 rpm
7. Observe for macroscopic agglutination. |
| CONTROL | CONTROL: to ensure the process IS correct
1. One drop of saline is used in place of dilute saliva. Test in parallel with the saliva.
2. Test saliva from a known secretor and a nonsecretor in parallel with test saliva. |
| Interpretation: | NONSECRETOR. Agglutination of RBCs by antiserum-saliva mixture; control tube positive.
SECRETOR. No agglutination of RBCs by antiserum and saliva mixture; control tube positive. |
| What is the reason why there is no agglutination if positive | The antiserum has been neutralized by the soluble blood group substances or antigens in the saliva, which react with their corresponding antibody. Therefore, no free antibody is available to react with the antigens on the reagent RBCs used in the testing
This negative reaction is a positive test for the presence of ABH-soluble antigens and indicates that the individual is a secretor. |
| Procedure Notes: | 1. Dilution prevents zonal reactions due to either excessive amount of antigen (post zone) or excessive amount of antibodies (prozone).
2. The procedure follows the hemagglutination inhibition or neutralization reaction, which provides a means of evaluating the relative strength or potency of the water-soluble blood group substances. |
| Rh TYPING REAGENTS: | Anti-D Typing Sera
3-5% Known Red Cell Suspension
Physiologic Saline (0.85-0.90% NaCl) in Wash Bottle |
| Rh blood type | • Identified before blood transfusion
• IgG
• Reacts optimally at 37C
• Can cross placenta
• Rh (+): D or weak-D test is positive
• Rh (-): Neg for both slide/tube and IAT testing
• Immunogenic
Determining the D status of an RBC sample is required when testing donor bloods. Blood for transfusion is considered Rh positive if either the D or the weak-D test is positive. Any donor blood sample that is typed Rh0 (D)-negative by the slide or rapid tube method must be tested further by an indirect antihuman globulin technique. If both tests are negative, the donor sample is considered Rh-negative. If the donor sample tests positive in any phase of Rh0 (D) testing, the sample is considered Rh-positive. Blood for transfusion is considered Rh-positive if either the D or weak-D test is positive; if both the D and weak D tests are negative, blood for transfusion is considered Rh-negative. Most Rh antibodies are IgG immunoglobulins and react optimally at 37C or following antiglobulin testing; exposure to less than 0.1 mL of Rh-positive RBCs can stimulate antibody production in an Rh-negative person. |
| Presence and absence of D antigen | Presence of D- Rh positive
Absence of D- Rh negative |
| PROCEDURE:
A. Slide Method | 1. Prepare 1 clean slide and label with “Anti-D”
2. Perform capillary puncture. Remember to wipe out first before placing a drop of blood to the slide. The first drop of blood may contain capillary proteins that may interfere the reaction.
3. Place a drop of anti-D typing sera just beside but not touching the blood.
4. Carefully mix the two solutions by using a clean applicator stick
5. Record the result.
Agglutination- Rh positive
No agglutination- Rh negative |
| B. Tube Method | 1. Make a 3-5% RCS of both Patient (Px) and Donor (Do
2. Prepare 2 clean tubes and label with PX, DO respectively
3. Place 1 drop of anti-D typing sera on to each tube.
4. Add 1 drop of Patient RCS to tube PX and Donor RCS to tube DO.
5. Cover the tubes with Parafilm.
6. Mix the solution and spin for 15 seconds at 3,400 rpm. Gentle mixing is recommended.
7. Carefully dislodge the cell button by gently mixing it and examine for agglutination or hemolysis.
8. Grade the reaction and record the results. |
| D CONTROL | If anti-D tube is negative OR all tubes in the forward type are positive (patient appears to be AB positive) a negative D (Rh) control tube must be run. |
| MAKING D-CONTROL TUBE: | a. Label a tube with patient initials and “D ctrl” below initials.
b. Place 1 drop of patient RBC suspension in the tube
c. Add 1 drop of Rh control reagent to the labeled tube and mix
d. Centrifuge for 15-20 seconds
e. Shake gently to resuspend
f. AB Positive - If tube is negative, it confirms the patient is AB Pos; however, if agglutination is observed, further testing is required before a blood type can be determined. STOP HERE for AB Pos.
g. D Negative – continue with Weak D test (Indirect antiglobulin testing) |
| Interpretation: | Blood Type of the patient is the tube or slide showing AGGLUTINATION. There is no reverse typing (because it is naturally occurring) in Rh since there is no innate or preformed antibody against Rh antigens. Antibody production will only happen when there is exposure to Rh antigen. |
| 1. What group of ABO Discrepancy is this test helpful? Why this phenomenon does cause discrepant ABO Typing? | - It is useful in Group Iv Discrepancy. Unexpected ABO isoagglutinins may cause discrepancy in the patient’s serum when it react at room temperature with the corresponding antigen present on the reagent cells. A2 and A2B individuals are examples of this type of ABO discrepancy which can produce naturally occurring anti-A1, or A1 and A1B, individuals who may produce naturally occurring anti-H. The patient’s RBCs can be tested with Dolichos biflorus to confirm the presence of the ABO subgroup. Dolichos biflorus will agglutinate cells of the A1 but not the A2 phenotype |
| 2. Why is anti-A1 unlikely to cause a transfusion reaction and when is it considered clinically significant? | - It is unlikely to cause a transfusion reaction due to the fact that anti-A1 is a naturally occurring IgM cold-reacting antibody and usually reacts only at temperatures below 37 degrees Celsius. It is considered clinically significant if it is reactive at 37 degrees Celsius. |
| 3. Explain why A antigen has subgroups | - Not all type A gives the same result when tested with anti-A that’s why A antigen has subgroups. Those subgroups are referred to as weak A and it occurs in two common forms: A1 and A2. Individuals exhibiting less antigenic sites are produced in group A2 individuals as the enzyme is less effective in converting the precursor H substance into A antigen. |
| 1. What is the significance for testing secretor status? | - It can be utilized to subdivide A individuals into A3, Ax, Aend, and etc. Moreover, routine secretor studies also detect the presence of only H substance in A3, Ax, and Aend. Additionally, it is also significant in red cell reactions with anti-A and Anti-A, B. If it shows no agglutination, and secretors would demonstrate quantities of A substance in saliva then it is later identified as Am, if secretors contain small amount of A substance in saliva then it is identified as Ay, and if secretors contain only H substance and no A substance in saliva then it is identified as Ael |
| 2. Give rare cases wherein you can observe excessive levels of soluble ABH substances | - Excessive levels of soluble ABH substances are seen in weakly reactive or missing reactions in RBC grouping, which sometimes occurs with certain diseases, such as carcinoma of the stomach and pancreas. |
| 3. An excessive blood group soluble substance causes what group of discrepancy in ABO typing. How can it be resolved | - Too much levels can cause a rare Group II ABO discrepancy. Excess amounts of BGSS substances will neutralize the reagent anti-A or anti-B, leaving no unbound antibody to react with the patient cells and yields a false-negative or weak reaction in the forward grouping. It would be alleviated through washing the patient cells free of the BGSS substances with saline, resulting in correlating forward and reverse groupings |
| 1. What are instances when an accurate Rh type cannot be determined through routine testing? | - False reactions with Rh typing reagents cause inaccurate Rh type determination through routine testing. False positive reactions are likely caused by too heavy cell suspension, cold agglutinins, test incubated too long or drying, rouleaux formation, fibrin interference, contaminating low-incidence antibody in reagent, polyagglutination, bacterial contamination of reagent vial, incorrect selected reagent, too long centrifugation, and too high rpm applied during centrifugation. On the other hand, false negative reactions are likely caused by immunoglobulin-coated cells, Saline-suspended cells, failure to follow manufacturer’s directions precisely, omission of reagent manufacturer’s directions, too vigorous resuspension, incorrect selected reagent, variant antigen used, reagent deterioration, too short centrifugation, too low rpm applied during centrifugation. |
| 2. What is the involvement of Rh blood group system in Hemolytic Disease of the Fetus and Newborn (HDFN)? | - Historically, Rh blood group system has been known to cause hemolytic disease of the fetus and newborn. Anti-D was a frequent cause of fetal death before the implementation of Rh-immune globulin therapy. This was to be expected because while 15% of the population is Rh-negative, 85% of the population is Rh-positive; therefore, the likelihood an Rh-negative woman is carrying an Rh-positive fetus is significant. HDFN caused by Rh antibodies is often severe because the Rh antigens are well developed on fetal cells, and Rh antibodies are primarily IgG, which readily cross the placenta. |
| 3. What are the antigens present in the Rh Blood Group System? Arrange the antigens from the most antigenic to the least antigenic. | - D, C, E, c, and e are the five most common Rh antigens. Most antigenic to the least antigenic is D > c > E > C > e |
