Laboratory Methods

Question 1

List three categories of patients in which antibody screening must be carried out on.

Answer 1

Pregnant women, blood donors, and infants suffering from Haemolytic Disease of the Newborn (HDN)

Question 2

State the two scenarios in which an antibody would be considered ‘clinically significant’.

Answer 2

If the individual were to receive a blood transfusion, with cells that express the antigen against which the antibody is directed, pr if
the antibody is capable of reacting at 37°C

Question 3

What are the only two possible results of an antibody screen?

Answer 3

‘No antibody(ies) detected’ (negative), or ‘antibody(ies) detected’ (positive)

Question 4

True or false? Antibody screening confirms the identity of the antibody.

Answer 4

False

Question 5

Name two unexpected antibodies.

Answer 5

Anti-D and Anti-K

Question 6

True or false? On rare occasions, antibodies may arise without a known stimulus, such as a transfusion, or a pregnancy.

Answer 6

True

Question 7

The IAT is used to detect __________ sensitisation.

Answer 7

In-vitro

Question 8

IAT is the acronym for the _____ ______ ______.

Answer 8

Indirect Antibody Test

Question 9

State the principle of the IAT.

Answer 9

The principle of the test relies on the fact that, due to antibodies being gamma globulins, an antibody to gamma globulin can form ‘bridges’ between red cells sensitised with antibody, causing agglutination. The antibody to gamma globulin is called Anti-Human Globulin (AHG); this reagent is generally prepared from rabbits or goats, and contains an antibody to human globulin, such as monospecific Anti-IgG, as is the case in routine pretransfusion testing, or contains polyspecific antibodies to human globulin and human complement (anti-C3), for detection of in-vivo (in-body) sensitisation.

Question 10

The IAT enhances the detection of antibodies of the _____, Rh, Kidd, and Duffy systems, and can also detect Anti-Lea, Anti-Leb, Autoanti-I, and Anti-P1.

Answer 10

Kell

Question 11

Which type of screen cells are used in the IAT?

Answer 11

Group O, chosen to provide a wide range of common antigens across the three cells

Question 12

In the IAT, the _______ control is generally weak IgG anti-D reagent, added to RhD-________ cells (although other antibodies can be used); the control is treated in the same manner as any other tube.

Answer 12

Positive; positive

Question 13

Negative control is generally weak IgG anti-X reagent, added to cells that are negative for the X antigen; the control is treated in the _____ manner as any other tube, and must be negative.

Answer 13

Same

Question 14

For quality control, if a tube appears to be negative, ____-sensitised cells (Coombs’ control cells) are added; provided the previous steps were correctly executed, false negatives should become positive.

Answer 14

IgG

Question 15

The ________ ________ _______ is used to detect in-vivo sensitisation by IgG or C3.

Answer 15

Direct Antiglobulin Test

Question 16

The DAT can be used in which circumstances?

Answer 16

The investigation of haemolytic disease of the newborn (HDN), haemolytic transfusion reactions, autoimmune haemolytic anaemia (AIHA), drug-related haemolytic anaemias

Question 17

If the DAT is negative, what can be added?

Answer 17

IgG-sensitised cells

Question 18

False-_______ results can stem from inadequate washing, interrupted testing, loss of activity of AHG, omission of AHG, improper centrifugation, number of RBC, etc..

Answer 18

Negative

Question 19

False-_______ results can be due to already-agglutinated RBC, contaminated saline, dirty glassware, excessive centrifugation, contaminated AHG, for IAT, using cells with a positive DAT will give a false-positive result, or for DAT, bacterial contamination of the sample.

Answer 19

Positive

Question 20

_________ coats RBC by attaching to complement-binding antibodies on the RBC, or non-specific binding, due to the presence of immune complexes in the plasma may occur

Answer 20

Complement

Question 21

Outline the procedure and features of the saline test.

Answer 21

The saline test involves the incubation of screen cells with patient serum/plasma in a suspension of saline (the formula being two drops of patient sample, and one drop of a 3-5% suspension of screen cells) for between thirty to sixty minutes, at 4°C, room temperature, and 37°C. The results are then interpreted and graded. With respect to the advantages of the saline test, it is a simple exercise, and it can detect IgM antibodies over a range of temperatures; however, it is generally lacks proficiency in alerting the scientist to the presence of IgG antibodies, and, when performed at temperatures lower than 37°C, has a tendency to detect clinically-insignificant IgM antibodies. This technique can pick up Anti-M, Anti-N, occasionally Anti-S, Anti-Lea, Anti-Leb, Autoanti-I, and Anti-P1

Question 22

Outline the method and principle of the Low Ionic Strength Solution (LISS) test.

Answer 22

Enhances the sensitisation step in antibody-antigen reactions, thus improving agglutination, and, as it causes a declension in ions, there is a decrease in ‘shielding’ effects. There are two forms of the LISS test: LISS addition (LISS is added to screen cells and serum) and LISS suspension. The LISS is beneficial in that it is a more rapid test, with an incubation period of 10-15 minutes, and it offers more sensitivity in detecting most clinically-significant antibodies. In terms of disadvantages, reagents used must be brought to room temperature prior to the commencement of the exercise, as there is also increased sensitivity for cold, clinically-insignificant antibody detection, and non-specific complement binding may occur if the stringent ratio of 2:1:2 (serum:cells:LISS) is not adhered to

Question 23

‘Enhances the detection of IgG antibodies via the use of screen cells that have been treated with an enzyme, such as papain (derived from the papaya fruit), ficin (obtained from figs), or trypsin (taken from the lining of hog stomach). The enzyme is capable of stripping of negatively-charged ions from the surface of RBCs, thus causing a decline in zeta potential, and also reducing steric hinderance by removing proteins adjacent to antigens, thus furnishing antibodies with better access to the antigens. It can be a one- or two-stage technique, depending on whether or not the screen cells were pre-incubated with the enzyme. The enzyme technique is somewhat hindered by its denaturation of certain antigens, such as those of the MNSs and Duffy blood group systems, and therefore cannot be the sole method of antibody detection, but it is exceptionally capable in detecting Rh blood group system IgG antibodies, rapid, and can be useful in identifying antibodies when their is a combination, by denaturing some of the corresponding antigens, such as Anti-c and Anti-Fya. It also detects Kidd and some cold-reactive, clinically-insignificant IgM antibodies, such as Anti-Lea and Anti-Leb. Many laboratories do not use the enzyme technique as part of antibody screens’. To which method does the passage refer?

Answer 23

Enzyme test