Abdulhameed Al-Attas

YES, 24 hours, at Room temperature and without agitation.

Just read this month's AABB News and saw Heather Vaught's name, a member of PathLab Talk:
 
Heather Vaught, MLS(ASCP)SBB, director of technical operations at Indiana Blood Center in Indianapolis, was recently recognized as one of the "Top Five" in the 40 under 40 list from the American Society of Clinical Pathology.
 
Congrats from your PathLab Talk friends!

I agree with both David and Anna for their respective suggestions of an enzyme pretreated panel in gel and extended phenotype on the pre-transfusion sample.
You have done an elution on the patient's post-transfusion red cells, and the resulting
eluate tested for antibody specificity.
Note  that in this case, even though the antibody elutes from the patient's red cells, it is NOT an autoantibody as it actually eluted from the donor's red cells now in the patient's circulation.

My heart goes out for all the people affected by this earthquake.                                Nothing is more beautiful than seeing people from different countries and cultures showing so much love for one another.

I have always trained my staff with these words, "Record what you see, not what you think it should be." In other words, proceed based on observations, not expectations.

For those taking the temp, do you validate (presumably annually) the device you take the temp with?
 
We chose to validate our 30 minute rule.
 
Validation Design60 red cell units will have their temperature monitored for 40 minutes after their release from the Blood Bank. Units released from the Blood Bank will be taken to defined locations where they will be placed near the floor's receptionist. Prior to the lapse of 30 minutes, the products will be returned to the Blood Bank. Temperature monitoring will continue for an additional 10 minutes before the temperature monitor will be stopped. TempTale Preparation-Set TempTale to monitor 1-100C for 2 days. Unit Preparation-A unit of blood will be split into 2 equal parts.-The 2 parts of the unit of blood will be rubber banded together with an activated TempTale placed in the middle. Minimize as much air between both halves of the unit as possible.-Place unit in a monitored refrigerator in Coordinating for at least 24 hours.-Record date and time unit is placed into refrigerator on form created for this validation. Monitoring-After 24 hours, unit prepared for this validation can be used for temperature monitoring.-Prepare a timer for 30 minutes.-The release of the unit must coincide with the release of an actual blood product to be transfused on the floors - this will be used to reflect the time it takes to issue a product. Unit must be placed in a white plastic bag used for transport of blood products.-Document the day and time of issued on the form created for this validation. Start timer and proceed to one of the locations defined by this validation.-Once at the defined location, place the bag with the unit near the receptionist's station. Remember to leave this location in enough time to be back in the Blood Bank by 30 minutes from release of the product. Record the time the 30 minute timer goes off.-Once back at the Blood Bank, place the bag on the counter and wait an additional 10 minutes until stopping the TempTale.-Record the time you stop the TempTale on the validation form.-Download TempTale data. Enter time of issue, 30 min, and 40 min intervals on TempTale temperature graph. Prepare unit for next observation.

4.  Or possibly another antibody that reacts with the expected screening cells, but is a different specificity, that is not detected in the IAT cross-match because, unlike the screening cells, which are in a preservative that is designed to "keep antigenicity", but not "oxygen carrying capacity", the unit's red cells are kept in a preservative that is designed to "keep oxygen carrying capacity", but not "antigenicity", and, in any case, may only have heterozygous expression of an antigen, whilst the screening cells have homozygous expression of an antigen.  Why take the risk with a patient's life, and your own future in the profession?

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 

Identification of HTLA

Comlex 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:

technologist: experienced with proven technique
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.
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 that case Kelly, there is even less to worry about.
 
If your husband does not carry the RHE gene, he cannot pass it on to your baby, and so the baby will not express the E antigen.  If there is no E antigen present on the baby's red cells, your own anti-E cannot "attack" the baby's red cells.
 
Both anti-E and anti-Cw can be, what is termed "naturally occurring".  By this, we mean that they can be stimulated by something other than exposure to red cells by either transfusion or pregnancy.  Of course, in reality, they cannot be "naturally occurring", and must have been stimulated by something, but the stimulus is most probably something like a bacterium, many or which carry proteins and sugars on their surface that mimic humanblood group antigens closely enough to stimulate the immune system to produce antibodies against these proteins and sugars, but these antibodies are almost always clinically insignificant, and nothing whatsoever to worry about.
 
Have a great pregnancy and enjoy your new baby when he/she arrives into the big wide world!

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 

Identification of HTLA

Comlex 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:

technologist: experienced with proven technique
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.
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.

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 

Identification of HTLA

Comlex 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:

technologist: experienced with proven technique
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.
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.

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 

Identification of HTLA

Comlex 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:

technologist: experienced with proven technique
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.
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.

As Anna, mentioned above ''You need to use a control that is equivalent to the reagents you are controlling''.
that is to say, from the same manufacturer of your Anti-D. The Rh control contains everything that is present in the anti-D typing sera except the anti-D.
If Rh control is not available then you can use from 6-8% Albumin Because the Albumin added to the Rh control is about 6-8%. And as David said above,The control is necessary (especially in apparently group AB positive patients).
Another application is to use as a negative control when testing a Du or Weak D.
 
 

You need to use a control that is equivalent to the reagents you are controlling. Most of the time, companies sell a reagent control. This is actually NOT a waste of money. The reagents contain lots of things in their buffers in addition to albumin and you can occasionally get patients who react with these other things too, giving you a false positive result

Yes, Malcolm, you are absolutly right But we in Third countries use ONLY serology Techniques, Molecular is still a dream for us.

Happy Lab Week to all of my PathLabTalk friends and fellow Blood Bankers and laboratorians

Our Hospital consider CMV seronegative and leukocyte reduced blood products equivalent and would consider them equivalent for all patients. This includes low birth weight neonates.
We use these components interchangeably for all transfusion recipients including neonates.

Anti-Complement in your antiglobulin reagent.

I am a student of Malcolm,but I can help; just double click the small graph and it will turn big enough to be useful.

I am a student of Malcolm,but I can help; just double click the small graph and it will turn big enough to be useful.

I am a student of Malcolm,but I can help; just double click the small graph and it will turn big enough to be useful.

Thanks Malcolm,for the great explanation as usual.

Hi Sara,
The first clue that the antibody may be anti-G, or anti-C+G, rather than anti-C+D, is indeed that the titre of the anti-C is higher than that of the anti-D, but the fact that the anti-C titre is higher than that of the anti-D is only a clue. It could be that you actually have an anti-C+D where, coincidentally, the anti-C titre is higher than the anti-D. It is absolutely essential, therefore, that you can prove that no anti-D is present.
One way of doing this is to divide the patient's plasma sample into two.
The first sample is adsorbed using Ro red cells treated with a proteolytic enzyme, such as papain or ficin, which would adsorb out any anti-G and any anti-D present, but would leave any anti-C present. At the end of the adsorption process (about 4 cycles), this plasma is tested against r'r red cells by IAT. If there is a positive reaction, then the original plasma contained anti-C and, possibly, anti-G. If there is a negative reaction, then the original plasma contained, possibly, anti-G and anti-D, but not anti-C.
The second sample is adsorbed using r'r red cells treated with a proteolytic enzyme, such as papain or ficin, which would adsorb out any anti-G and any anti-C present, but would leave any anti-D present. At the end of the adsorption process (about 4 cycles), this plasma is tested against Ro red cells by IAT. If there is a positive reaction, then the original plasma contained anti-D and, possibly, anti-G. If there is a negative reaction, then the original plasma contained, possibly, anti-G and anti-C, but not anti-D.
So, if there are no reactions with the plasma adsorbed with Ro red cells when tested with r'r red cells, and no reactions with the plasma adsorbed with r'r red cells when tested with Ro red cells, then the original plasma contained only anti-G.
If there is a reaction with the plasma adsorbed with Ro red cells when tested with r'r red cells, and also a reaction with the plasma adsorbed with r'r red cells when tested with Ro red cells, then the original plasma contained both anti-C and anti-D (and may also have contained an anti-G).
In all cases, however, you would give cross-match compatible C Negative, D Negative blood (you would still have to perform a serological cross-match, because there are some EXTREMELY rare donors around who are C Negative, D negative, but G POSITIVE.
As far as obstetric patients are concerned, both anti-C and anti-G usually cause far less severe haemolytic disease of the foetus and newborn (unless they have an unusually high titre), than does anti-D. However, it is important that the pregnant lady is offered prenatal and postnatal anti-D immunoglobulin prophylaxis, so that they do not get immunised against the D antigen.
I hope that this rather long and complicated post helps in some way, but, if you need to know more, please do not hesitate to ask more questions.

We are very saddened to hear to your loss and would like to express our sincere condolences to you and family.
Remember that we ( in PathLab Talk ) love and care about you.Our thoughts and prayers are with you and family.

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.