As the name suggest, this test is used to detect cold antibody. Not only do we detect whether there is cold antibody, we also detect the cold antibody titre.
Firstly, we serial dilute the antibody of the patient. Then we test the different concentrations of antibody against 3-5% patient cells and control cells. The titre value is determined by the highest dilution that can still cause a positive reaction. Titration value can help quantify the relative antibody concentration in the plasma.
The most common type of cold auto-antibody is IgM. This antibody is capable to cause cold haemagglutin disease (CHD). The most common specific cold antibody seen is auto anti-I.
CHD can be acute or chronic. Acute condition is usually caused by mycoplasma pneumoniae infection. For chronic condition, it usually occurs in elderly suffering from chronic hemolytic anaemia.
As a control, a segment group O cells will be taken from a random blood unit that has no blood antigen listed on the unit of blood. (The pints of blood have a sticky label on top of it. Should there be any antigen, it'll be listed on the pint of blood) For example, some units are (K-). Since the units are selected at random, we must also note that should this test be repeated with a segment from another group O cells, we expect similar results, but never the same results.
Procedure:
1. Label 2 sets of tubes, first set with "own cells followed by the dilution", e.g. OC 1, second set with "control cells followed by the dilution, e.g. CC 1024.
2. We pipette 6 drops of saline into tubes from: OC 2 to OC 1024.
3. Then, 3 drops of patient's serum into OC 1 and CC 1, 6 drops into OC 2.
4. From OC 2, we mix the saline and serum well, then pipette 6 drops into OC 4, and 3 drops into CC 2. This ensures that the OC and CC concentration are the same.
5.From OC 4, we pipette 6 drops into OC 8 and 3 drops into CC 4.
6.From OC 8, we pipette 6 drops into OC 16 and 3 drops into CC 8.
7. This process is repeated until we reach OC 1024. 3 drops are pipetted into CC 1024 and 6 drops are pipetted out into the waste container.
8. We should obtain 3 drops in every tube, and we must be careful as to not introduce any air bubbles in the process of pipetting as it may influence the readings.
9. Add 1 drop of 3-5% patient's own washed cells to each of the OC tubes
10. Add 1 drop of 3-5% control own cells into each of the CC tubes
11. Incubate the tubes at 4 degrees for at least 2 hours.
12. Read the tubes and rate them from 4+ to +w(where the w is in superscript), +w represents plus weak, i.e a very very weak agglutination reaction where you see numerous agglutinates in a background of free cells.
13. To check for microscopic agglutination reaction, cold glass slides are are in the refrigerator is used.
Agglutination reaction at 1:32 dilution or lower is regarded as clinically insignificant while agglutination reaction at 1:64 or higher is regarded as clinically significant.
Results are expressed as the reciprocal of the highest serum dilution able to cause a macroscopic reaction with group O cells. Reactions in both sets of tubes should appear very similar unless there is a mistake somewhere. It is also important that we read the results inside the fridge, that way, the positive results will not become negative reactions
Yanhong
0703979E
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Hi!
ReplyDeleteWhy do you need to use cold glass slide? Is it that if we use the glass slide of room temperature will interrupt the agglutionation?
thanks!
eriko
0700477C
hie yanhong!
ReplyDeletehow do you serial dilute the antibody of the patient?
nyzah
@Eriko.
ReplyDeleteYup. That's why we do that. Reading those 11 tubes takes time, esp when you have to view macroscopic neg results or unclear results under the microscope, so we don't want to heat up the tubes.
@Nyzah.
Erm, basically, follow what i said in the procedures steps 1 to 8. That's how we do it. Patient's antibody is in the serum, so when we do serial dilution with the serum, we're actually diluting the antibody level.
Eriko,
ReplyDeleteWhat is cold Ab? Is there "hot" Ab??
Ooopss....the question I asked should be directed to Yan Hong, not Eriko. Apologies!
ReplyDelete@Dr. Alex Lee
ReplyDeleteThere's warm antibody, which is basically IgG.
Cold antibody are antibody reactive at colder temperature, i.e they are likely to cause a reaction, e.g. haemolysis/agglutination at cold temperatures(4deg-22deg). Like IgM.
For warm antibody they'll be more reactive at higher temperatures, at about 37deg. (body temp)