Life's Blood
Table of
Contents
CLASS NOTES
LABORATORY TECHNIQUES: REAGENTS, REACTION
GRADING; CELL WASHING; SAMPLE COLLECTION
BLOOD BANKING REAGENTS
The techniques used in Blood Bank involve mixing antigens, usually on red blood cells
with antibodies. The environment where this reaction occurs can range in temperature
from 4oC to 37oC. With the most common being room temperature for ABO and the
initial Rh(D) testing and 37oC when screening and identifying other clinically significant
antigen-antibody reactions. In a number of situations we are looking for particular
antigens on the red cell such as looking for A or B antigens to determine a patient's
ABO type. Other times we may be looking for particular antibodies that may cause
transfusion reactions or hemolytic disease of the newborn. Depending on whether we
are looking for a particular antigen or antibody will determine what reagents we are
going to use. If we are looking for an A antigen on a patient's red cells, we will use
known anti-A reagent that will cause agglutination of the A antigens on the red cells. If
the patient has on B antigens or no ABO antigens, as in the case of an O individual,
their cells will not agglutinate with anti-A reagent.
Sources of Antigen Testing:
In almost all blood bank techniques we have red cells with antigens present. These red
cells may either reagent red cells with known antigens, patient red cells, or donor red
cells. The reagent red cells are commercially prepared and have all the red cell
antigens identified.
When we use red cells where the antigens have already been determined, we can
identify the possible antibodies present. For example: Anti-A and Anti-B are expected
antibodies in patient's who lack that particular antigen. Therefore if a patient or donor
has only the A antigen on their red cells, then they should have anti-B in their
serum. When we test their serum with A1 and B cells, agglutination will occur with the B
cells and not with the A1cells since they have an anti-B. The reagent cells used for
blood banking include the following:
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A1 and B cells for confirmation of the ABO type in all patients and donors other
than newborn babies
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Antibody screening cells are O cells that have been studied to determine the
presence of a number of antigens for specific antibodies that are known to cause
transfusion reactions and hemolytic disease of the newborn. The antibody
screening technique is part of all compatibility tests done before blood is
transfused. Some of the more common antibodies detected are anti-D, anti-E,
anti-K.
Antibody identification cell panel are again O cells with the specific antigens
known. Usually there are between 8 and 12 different cells in a cell panel. The
pattern of positive and negative reactions helps identify the antibody.
Sources of Antibody for Testing
Antibody is found in serum. If it is the patient's serum that is being tested, we do not
know what antibody may be present so we are using one of the 3 types of reagent cells
listed about. If the serum is commercial reagent, the specific antibody present is
already known. The commercial serum reagent is referred to as antisera. Therefore,
we use Anti-A antisera to determine if a patient or donor is Type A. If we are trying to
determine if the patient is Rh + or Rh -, we will use anti-Rho (D) antisera. Table 1 is a
summary of known and unknown sources of both antigens and antibodies.
Known Source with known
components
Antigen Reagent Red Blood Cells
Antibody Commercial Antisera
Table 1
Unknown components - The source is
either the patient or the donor
Patient or Donor red blood cells
Patient or Donor serum or plasma
Testing Procedures Routinely Done in Blood Banking
In a transfusion service there are a number procedures routinely done. The ones noted
in red are those done even in small hospitals whereas the rest are more likely done at
larger hospitals and reference laboratories:
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ABO/Rh(D) typing
Antigen typing from other blood group systems such as Rh antigens other than
D, Kell, Kidd, and Duffy
Antibody screening for antibodies form to blood group antigens other than A
and B
Antibody identification to determine the specific antibodies detected in the
antibody screening
Crossmatch, or compatibility testing, which determines whether donor blood
can probably be safely transfused to the recipient
Table 2 summarizes the sources of both the antigen and antibody (Modified from Basic
& Applied Concepts of Immunology, Blaney and Howard, lst ed., p. 39
Procedure
ABO/Rh typing
Purpose
Source of Antigen
Detects A, B, and D Patient's RBC's
Source of Antibody
Commercial anti-A,
Antigen typing
Detects antigens of
other blood group
Patient's RBC's or
systems (examples: Donor RBC's
K, E, C, Fya, Jka)
Detects antibodies
Commercial
Antibody screening with specificity of
Screening Cells
RBC antigens
Identifies the
Antibody
Commercial Panel
specificity of RBC
identification
Cells
antibodies
Determines serologic
compatibility between
Crossmatch
Donor RBC's
donor and patient
before transfusion
anti-B, and anti-D
Commercial antisera
to the specific
antigens (examples:
anti-K, anti-E, anti-C,
anti-Fya,
anti-Jka)
Patient's serum
Patient's serum
Patient's serum
GRADING REACTIONS
Grading agglutination reactions gives an indication of the relative amount of antigen or
antibody present. All tubes tests should be graded. The technique used in the
resuspension of the cells will affect the grading of the reaction. The correct procedure
for resuspending and grading reactions follow:
1. Resuspension Procedure:
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use lighted agglutination viewer
read only one tube at a time
hold tube upright
position cell button so it is facing you
in the mirror
very gently shake the tube and
observe how the cells come off the
cell button
2. Grading Reactions
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swirling off = negative
coming off in chunks = positive
continue shaking till all cells resuspended
tilt tube, read and grade reaction
3. Grading system:
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4+ = solid clump
3+ = several large clumps
2+ = small to medium sized clumps; clear background
1+ = small clumps; cloudy background
+w = tiny aggregates; cloudy background
+ micro = positive upon microscopic examination only
MF = mixed field. Small clumps amidst many unagglutinated cells. Can be
confused with 1+
hem = hemolyzed (a positive reaction)
neg = negative, no agglutination (Never use - for negative; either write neg or 0)
WASHED CELL SUSPENSIONS
3% Red Blood Cell Concentration in Saline
Between 2-5% cell suspension provides optimum antigen concentration for the tube
method for red blood cells typing. To make sure your suspension is within this range
use reagent red cells for comparison.
Washing Red Blood Cells Before Making the 3% Suspension
The purpose of washing the red blood cells is to remove plasma, which contains
substance that may interfere with antigen-antibody reaction. The following may be in
the plasma and may interfere with testing:
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Soluble antigens such as A and B may be present and neutralize your reagent.
Interfering proteins such as Wharton's jelly that is seen in newborn cord blood,
cold-acting autoimmune antibodies, and increased levels of immunoglobulins that
may cause either agglutination or rouleaux..
Hemolyzed red blood cells due to a difficult draw will interfere in your grading
interpretation of hemolysis
Fibrinogen can result in fibrin strands forming that makes grading reactions
difficult.
Good Technique when washing and making a 3% cell suspension involves the
following:
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Place 1 to 3 drops of blood in the tube
Aim the tip of the saline bottle towards the center of the tube and forcibly squirt
saline into the tube.
Fill the tube 3/4 full of saline (there will be less splattering in the centrifuge)
Centrifuge long enough spin to pull most of cells into a button in the bottom of the
tube.
Decant the saline completely
Shake the tube to resuspend cell button before washing the cells again. It will
depend on the procedure being done as to how many washings are going to be
done.
COLLECTING BLOOD BANK SAMPLES
Samples for Blood Bank Testing
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Most samples for blood banking are drawn into a red top tube - serum is
preferred. (No clot activation tube should be used since the patient's red
cells may also need to used and no other chemicals should be present)
A few tests require an EDTA sample if complement is not to be activated.
Serum must be tested while fresh to ensure good complement activity.
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Antigens on cells are stable longer (months) in a clot tube.
Patient Identification
The patient MUST be positively identified and preferably banded. Some institutions use
specific Blood Bank arm bands.
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Ask patient to state his/her name.
Responsible party should identify patient if he/she cannot.
Verify information by comparing to ID band.
Resolve any differences before proceeding with the blood draw.
Labeling of Sample
The information on sample MUST match information on ID band, which would also need
to be consistent with the order.
Information on samples MUST include the following:
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Name (last, first, middle initial) and no nicknames.
Unique identification number such as medical record number or possibly social
security number.
Date and time sample drawn along with the signature or unique identifier of
phlebotomist (on sample or on orders)
Gender and birthdate desirable but not mandatory. The date of birth provides
another unique identifier along with the medical record number and full name of
the patient.
Mislabeled Samples
Do NOT accept any sample not properly labeled. The following are what would warrant
an improperly labeled specimen:
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Missing information
Incorrect information
Information on sample not matching information on orders
Improperly labeled samples must be discarded if the problem cannot be resolved. In
the case of an emergency blood draw on a patient who is unidentified at that time, the
blood specimen must also discarded when both name and medical record number have
changed (ex. John Doe, #12240253 becomes Jack Adams # 37859012) unless ID tags
with both sets of information remain in place
OBJECTIVES - GRADING REACTIONS; WASHED CELL
SUSPENSIONS; BLOOD BANK SAMPLES
Identify sources of antigen and antibody used in testing.
Explain why it is important to grade agglutination reactions.
Describe the proper resuspension technique
Explain how the resuspension technique affects the graded result
Describe how each of the following looks: 4+, 3+, 2+, 1+, w+, micro +, hem., MF,
neg
6. State the optimum % cell concentration for blood bank testing
7. Explain why the above concentration is best
8. Describe the proper technique for adequate washing of cells
9. Explain how you can tell if your cell suspensions are the proper concentration
10. Name four plasma (or serum) constituents washing removes, and tell why it is
desirable to remove them before doing blood bank testing
11. Describe the proper procedure for identifying a patient when obtaining a sample
for blood bank testing
12. Explain what should be done if there is a discrepancy in the patient's
identification
13. State the information required on all samples for blood banking testing
14. Explain why gender and birthdate are helpful.
1.
2.
3.
4.
5.
Performance objectives:
1. Correctly prepare washed 3% suspensions of patient or donor red cells for blood
bank testing
2. Utilize the proper resuspension technique to read agglutination reactions
3. Properly obtain a sample for blood bank testing, including use of proper tubes,
identification of patient, and labeling of samples