The Abo Blood Group System

Published Date: 02 Nov 2017

Group: B (1-4)

Instructor(s): Dr. Sonia Richards- Malcolm

Ms. Daphne Davis

Course: Immunohaematology 1- Practical (MET2012)

Date: April 26, 2013

Title: ABO, Sub-grouping & Discrepancies, Rh, Weak D Testing, DAT and IAT.

Aim(s):

To prepare a 3-5% red cell suspension from four unknown blood samples labelled 1-4.

To perform ABO blood grouping and Rh typing on samples labelled 1-4.

To carry out the weak- D test on Rh negative samples.

To perform sub-grouping on samples with discrepancies for Group A.

To perform the Indirect Antiglobulin Test on four unknown serum samples labelled 1-4.

To perform the Direct Antiglobulin Test on four unknown blood samples labelled 5-8.

Principle

3-5% red cell suspensions are achieved by washing red blood cells in order to remove the unbound proteins that can neutralize a reaction and then adding an adequate amount of normal saline to bring the samples to a similar concentration in terms of their viscosity. Normal saline is used to wash a minimum of three times Red cell suspensions of 3-5% concentration should be cherry red in colour.

The ABO blood group system is a system of antigens and their naturally occurring antibodies. It is the only blood group system in which the individual inherits a type of antigen or antigens and has naturally occurring antibodies in their serum that are opposite to the antigens present on the red blood cells, without prior exposure to the antigen (D. Harmening p.109). The blood group is determined by the type of agglutination reaction between red blood cells and antibodies. Blood group is determined by using commercially made reagents to detect the presence or absence of antigens for the forward testing and commercially made reagents or pooled cells to detect the presence or absence of antibodies for the reverse. Subgroups of the ABO blood group system can cause discrepancies when carrying out ABO blood grouping. Some sub-groups of A, are A1 and A2 which may not give the expected results in the forward or reverse testing. When this occurs, special lectins have to be used (Dolichos biflorus for A1 antigen) to identify the type of antigen present.

Red blood cells can also be typed as Rh positive or Rh negative. When Rh typing is done in the lab, commercial reagents are used to detect the presence or absence of the D-antigen on red blood cells. Rh typing is based on agglutination of red blood cells with an anti-D reagent. If there is agglutination with the anti-D reagent, the patient’s cells are D- positive. If a person’s Rh status appears negative, a weak D test is performed in case there is a weakened expression of the D-antigen. Individuals with a weakened expression of the D-antigen produce the antigen but it may not be detected after the immediate spin because the expression is altered and must be taken through the antiglobulin phase to be detected (D. Harmening p.140).

The Indirect Antiglobulin Test is used to detect unexpected antibodies that may cause a reaction and/or complement components in vitro. Serum is incubated with red cell antigens to allow antibodies to attach to red cells antigens. The AHG reagent is used to agglutinate the cells. Albumin is used to accelerate the agglutination by bringing the cells closer together. The antibodies are usually IgG in nature which are warm- reacting antibodies and need to be incubated at 37oC for attachment to the surface antigens.

The Direct Antiglobulin Test is used to detect the sensitization of red cells with unexpected antibodies. AHG reagent is used to agglutinate cells that have been sensitized in vivo. A potentiator is not need since the antibodies are already attached to the red blood cells. There is also no need for incubation at 37oC because incubation occurred in the body.

Method

Preparation of 3-5% Red Cell Suspension

The normal saline provided was used to fill the four unknown blood sample tubes ¾ -way.

The tubes were balanced in the serofuge and spun for 1 minute.

The clear supernatant was poured out of the tubes until the cell button was dry.

The bottoms of the tubes were tapped to displace the cell button.

The washing and decanting (steps 1- 4) were repeated twice.

After the final wash, the cell buttons were displaced and an adequate amount of saline was added to the tubes to form a cherry red suspension.

The colour of the suspensions made were observed and recorded.

Blood Grouping and Rh Typing (Tube Method)

Seven clean test tubes were collected in a test tube rack for each sample; Samples 1-4.

The tubes were labelled for each sample (E.g. #1A, #1B, #1AB, #1A cells, #1B cells, and #1Rh and #1O cells).

One drop of Anti-A was dropped in tube #1A.

One drop of Anti-B was dropped in tube #1B.

One drop of Anti-AB was dropped in tube #1AB.

One drop of A-cells was dropped in tube #1A cells.

One drop of B-cells was dropped in tube #1B cells.

One drop of Anti-D was dropped in tube #1Rh.

One drop of O-cells was dropped in tube #1O cells.

A plastic pipette was used to re-suspend the patient’s serum.

One drop of #1 serum was added to tubes #1A cells, #1B cells and #1O cells.

The pipette was used to re-suspend the patient’s cell suspension.

One drop of sample 1’s 3-5% cell suspension was added to tubes #1A, #1B, #1AB and #1Rh.

The extra blood left in the dropper was squeezed back into its original test tube.

Steps 3-14 were repeated for the remaining samples in the rack.

All the tubes were placed in serofuge for 15 seconds.

All the tubes were observed for agglutination reactions using an Rh view box.

The reactions were graded and the results were recorded in a table.

Typing for Weak D

An additional drop of the anti-D reagent was added to the samples that appeared negative for the Rh tube in the previous method (#2 and 4).

The tubes were placed in the serofuge for 15 seconds.

The cell buttons displaced and the tubes were observed for agglutination.

The samples were incubated in a 37oC water bath for 15 minutes.

After incubation, the tubes were centrifuged for 15 seconds.

The cell buttons were displaced and the tubes were inspected for agglutination and the results recorded.

The samples were washed three times and a dry cell button was achieved.

After the third wash, two drops of AHG reagent were added to each tube and the tubes were centrifuged for 15 seconds.

The cell button was displaced and the tube was observed for agglutination.

Two drops of Check Cells were added to the tube(s) that still appeared negative.

It was centrifuged for 15 seconds and the reaction was graded and the interpretation recorded.

Sub-Grouping

Two clean test tubes were collected and labelled for sample 1which showed discrepancies for group A. (E.g. #1A, #1H)

One drop of the patient’s 3-5% suspension was added to the test tubes.

One drop of the anti-A lectin was added to the 1A tube and one drop of the anti-H lectin was added to the 1H tube.

The tubes were centrifuged for 15 seconds.

The cell buttons were displaced and the tubes were examined for agglutination.

Indirect Antiglobulin Test

Four patient serum samples were obtained (samples 1-4).

Four test tubes were labelled 1-4 to correspond with the four patient samples.

Two drops of the patient’s serum were dropped in its respective test tube.

Two drops of pooled O cells were added to each tube. And the suspension was gently mixed.

Two drops of 22% bovine albumin was added to each tube.

The four tubes were spun in a serofuge for 15 seconds.

The agglutination reactions were graded and recorded.

The four tubes were incubated at 37oC in a water bath for 15 minutes.

After incubation, the tubes were spun in a serofuge for 15 seconds.

The results were recorded.

The four tubes were washed three times with normal saline.

After the final wash and achieving a dry cell button, two drops of AHG reagent was added to each test tube.

The tubes were spun in a serofuge for 15 seconds.

The agglutination reactions were observed and recorded.

Two drops of check cells were added to the tubes that had no agglutination.

Those tubes were spun for 15 seconds and the reactions were graded and recorded.

Direct Antiglobulin Test

Four patient samples (red cells) labelled #5-8 were obtained.

Eight test tubes were collected to be used for the four samples and a control for each sample.

Two drops of the patients’ suspension received were dropped its respective test tube and its respective control tube. (One drop of Sample 4 was dropped in tubes labelled Test 4 and Control 4)

All the tubes were washed with normal saline three times.

After washing and achieving a dry cell button, 2 drops of AHG reagent was added to each tube labelled ‘Test’ and 2 drops of 22% bovine albumin was added to each tube labelled ‘Control’. The tubes were gently shaken to mix the reagent and samples.

All tubes were serofuged for 15 seconds.

The cell buttons were dislodged and the reactions were observed using an Rh view box.

All the tubes were incubated at room temperature for 5 minutes and spun in the serofuge for 15 seconds.

The tubes that had no agglutination were mixed with 2 drops of check cells.

Those tubes were spun for 15 seconds and the reactions were observed and recorded.

Results

3-5% Red Cell Suspension

The 3-5% red cell suspensions made from samples1-6 were cherry- red in colour.

Blood Grouping and Rh Typing (Tube Method)

Sample ID#

Anti-A

Anti-B

Anti-AB

A cells

B cells

O cells

Anti-D

ABO/Rh Group

1

4+

4+

4+

3+

-

-

4+

AB pos. Rev B

2

-

-

-

3+

4+

-

-

O neg. Du neg

3

-

-

-

4+

4+

3+

4+

O pos

4

-

4+

4+

4+

4+

3+

-

B neg. Du pos. Rev O

Weak D Test

Patient Name

Immediate Spin

37oC after incubation

After AHG

Check Cells

Results

Interpretation

#2

-

-

-

1+

neg

Du neg

#4

-

1+

1+

N/A

Weak-D pos

Du pos

Sub-Grouping

Patient Name

A1 lectin

Anti-H lectin

Antigens Present

Blood Group

Interpretation

#1

-

-

A

A2

A2B

Indirect Antiglobulin Test

Patient Name

Immediate Spin

After incubation 37oC

After AHG

Check Cells

Interpretation

1

-

-

-

3+

No in vitro sensitization

2

-

-

-

3+

No in vitro sensitization

3

2+

2+

2+

N/A

In vitro sensitization

4

2+

3+

2+

N/A

In vitro sensitization

Direct Antiglobulin Test

Patient Name

After AHG

After Albumin

Room Temp

Check Cells

Interpretation

5 Test

1+

N/A

1+

N/A

In vivo sensitization

5 Control

N/A

2+

2+

N/A

Control positive

6 Test

1+

N/A

1+

N/A

In vivo sensitization

6 Control

N/A

1+

2+

N/A

Control positive

7 Test

-

N/A

-

1+

No in vivo sensitization

7 Control

N/A

-

-

N/A

Control negative

8 Test

-

N/A

-

1+

No in vivo sensitization

8 Control

N/A

-

-

N/A

Control negative

Discussion

Before blood typing or performing an antiglobulin test, red blood cells have to be washed. The cells are washed so that the unbound proteins that may mask a reaction are removed. A 3-5% cell suspension is made up from these washed cells. An appropriate amount of saline is added so that the suspension is not too thick or too thin.

ABO blood grouping involves the identification of the ABO antigen(s) that are present on red cells. They can be the A and/or B antigen or neither. In forward group testing, commercial reagents are used to agglutinate these antigens. They are the anti-A, anti-B and anti-AB reagents. Cells which agglutinate in anti-A and anti-AB are group A. Cells which agglutinate in anti-B and anti-AB are group B. Cells which agglutinate in anti-A, anti-B and anti-AB are group AB. The cells that do not agglutinate in any of the anti-sera have neither A or B antigens and are group O cells. In the reverse group testing, the patient’s serum is used with pooled A and B cells. These pooled cells come from different patients of the same blood group. In this test, antibodies are being detected. According to Landsteiner, a person’s serum should have the antibody opposite to the antigens present (D. Harmening p.109). Sample 3 had no agglutinations in any of the anti-sera giving a forward group of O. According to the reverse, sample 3 had both A and B antibodies in serum. This corresponds with the forward group test. Samples 1 and 4 showed discrepancies to that. Discrepancies can be caused by unexpected antibodies other than A and B causing agglutination or by a weakened or missing antigen. Sample 1’s discrepancy could be because of a subgroup. There can be a case where since the A2 subgroup is present, the antibody for A1 cells is produced. In sample 4, O cells agglutinated which means an antibody not belonging to the ABO system is causing a reaction.

Cells that agglutinate in the anti-D reagent are Rh positive. When donor samples are Rh negative, the weak D test is performed to ensure that the antigen is truly absent in order to avoid sensitization in a patient. The agglutination may not occur after the immediate spin and require incubation because the D antibody reacts at warm temperature. When the samples appear negative after the Anti- Human Globulin reagent it means it does not detect the antigen being tested for. Check cells are added to ensure the AHG reagent is working and the negative reaction at that point was a true reflection that no D antigen is present and the cells are Du negative.

In the IAT, the unexpected antibodies are generally IgG antibodies and are present in the patient’s serum. The serum sample is mixed with O- cells that have the specific antigen for these antibodies. Albumin is used as an enhancement medium to increase the sensitization. Serofuging enhances the agglutination by bringing the cells closer together. The tubes are incubated at 37oC, where complement activation is optimal and to allow the warm- reacting IgG antibodies to bind to the antigen. The incubation period is very important. Sufficient time for incubation has to be allowed for optimal reaction of the antibodies. However, extended times can cause the antibodies to fall off the red cells. The cells have to be washed with normal saline a minimum of three times to remove all unbound protein and decanted of all saline. Residual saline can neutralize the AHG reagent. The AHG reagent agglutinates cells that form the antibody- antigen complex or complement- sensitized cells. When the cells agglutinate, there is an in vitro sensitization. Non- agglutinating cells are mixed with check cells to confirm that the AHG reagent was added and in an active state.

In the DAT, the patient’s red cells are washed with normal saline to remove the unbound protein which can mask a reaction. A dry cell button should be achieved to reduce residual saline which can dilute the AHG reagent and cause a weak agglutination in which case, the reaction can be missed. The test does not include incubation at 37oC because the cells were incubated at that temperature for an appropriate amount of time in the body. AHG is added to cells being tested while albumin is added to cells being used as control. The control occurs without the use of the AHG to detect agglutination. The incubation at room temperature is to give the AHG reagent time to attach to the antibody sensitized cell. If a reaction appears negative, check cells are added. If no agglutination occurs with the check cells, the results would be invalid and means that the cells were not adequately washed or decanted of all saline, or the AHG reagent is not present or it is inactive. Check cells are not added to control tubes because they do not include the AHG reagent. A negative DAT control tube means that no unexpected antibodies are present and no agglutination would be expected with AHG. A positive control tube shows an enhanced reaction to the DAT test tube where the cells agglutinate because of attachment of the AHG with antibodies.

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