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Re:Antibody Titres


NAN47

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Hi there I am interested in hearing people's opinions on which antibodies required to be titred during pregnancy, and Also any other info which is considered to be important in the antibody titre process, ie running against a standard or previous sample , and also any important points which should be considered in the reporting of titres. Many thanks

Tricia

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Hi NAN47,

Any antibody that reacts at STRICTLY 37oC should be titrated during pregnancy (except, of course, for anti-D and anti-c, which, in the UK, are measured by quantification against NIBSC standards).

The titration should take place at first identification and then again at 28 weeks gestation. If the titre is less than 32, then the antibody is unlikely to cause clinically significant alloimmune haemolytic anaemia and disease of the foetus and newborn. Only rarely is an antibody titre high enough to warrant further titration after 28 weeks gestation.

The obvious exception to this is anti-K (and other Kell-related antibodies). These should be titrated when first identified, and then every four weeks to 28 weeks gestation, and then 2 weekly thereafter, until delivery. Indeed, women with anti-K who are pregnant, and who have a partner who is K+k+ or K+k- should be referred to a Specialist Foetal Medicine Unit, so that the pregnancy can be monitored by experts in the field.

It is always recommended that the previous sample is titrated in parallel with the present sample (or quantified, in the case of anti-D or anti-c), as this will show up genuine rises in the titre, as opposed to rises due to operator differences and errors and changes due to the different expression strengths of an antigen from one red cell sample to another. Where possible, the red cells used should have heterozygous expression of the antigen, although, in certain cases, this can be difficult to do (e.g., if the antibody is anti-U, it is not possible to tell whether the red cells used for the titration are U+/U+ or U+/U-).

In rare cases, titration of the previous sample, in parallel with the present sample, may show up a second specificity. For example, if the pregnant woman is known to have an anti-E, with a titre of, for example, 4 against r"r red cells, and then all of a sudden the titre goes up to 128, but ALSO shows as 128 with the previous sample run in parallel, it may be that the r"r red cells used on this occasion also express an antigen against a low prevalence antigen, against which the woman has also produced an antibody (for example, an anti-Wra).

The end point of a titration is usually given as the reciprocal of the last dilution in which agglutination can be seen with the naked eye.

We are actively re-writing the BCSH Guidelines (technical guidelines) as I post, and the complementary RCOG Guidelines (clinical guidelines) are also in their final draft.

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  • 4 weeks later...

Hi there I am interested in hearing people's opinions on which antibodies required to be titred during pregnancy, and Also any other info which is considered to be important in the antibody titre process, ie running against a standard or previous sample , and also any important points which should be considered in the reporting of titres. Many thanks

Tricia

Titre: The reciprocal of the highest serum dilution that causes macroscopic agglutination when serial dilutions of an antibody are tested against selected red cells.

Applications:  

Prenatal testing,                                                                                                                                                                    IdentificatioofHTLA,                                                                        Complex antibody identification,                                                                                                                                    Differentiation of pathological and harmless autoanti- I  and  Procurement of antisera Quality assurance of reagents.

 

Limitations:

Titrations are only semiquantitative estimates of antibody reactivity due to several variables that affect their performance. Three main variables are the technologist, the red cells, and the method.

Ways to Minimize Variables:

  1. technologist: experienced with proven technique
  2. red cells: ideally when titres of samples are to be compared, use fresh red cells (antigens deteriorate on storage) from the same donor (same number of antigenic sites present). If this is not possible, use commercial red cells of the same apparent genotype.
  3. method: when sequential samples are examined for change in titre, store samples frozen and run new samples in parallel with the immediately preceding sample. This is the most practical way to control that an increase in titre is real.

Prozone Phenomenon:

This may cause reactions to be weaker in the first tubes than in higher dilutions and is believed to be caused by an antibody excess in which all antigenic sites are sensitized with antibody leaving none free to form cross-links

Significant Difference in Titres:

When comparative studies are done, such as in prenatal testing, a difference in titre of at least 2 tubes is required to be considered a significant difference. For example, if the titre changes from 32 to 64, this is not considered to be significant (difference of only 1 tube); however, a change from 32 to 128 would be significant (2 tube difference).

These are the important points to know about AntibodyTitration, in case you may need more, let me know.

Edited by Abdulhameed Al-Attas
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