Guidelines for the Estimation of Fetomaternal Haemorrhage

[MinerJ]

I was reading the BSH Guidelines for the Estimation of Fetomaternal Haemorrhage, and I had a few questions:

7.2 Slide Preparation

Quote

Thin films are best achieved by diluting an aliquot of the maternal whole blood sample before making the film. This is typically a 1:2 or 1:3 dilution with phosphate buffered saline, but might depend on the maternal PCV.

 

 

In my current workplace, we do not dilute the sample, and my previous workplace did. I could not see a significant difference on the slide that was made and found them both equally adequate, but I was wondering if other have any input on them

7.3 Controls

Quote

The samples may be stored at 4°C for a maximum of 4 days but fresh slides should be made each time a batch of tests is performed.

RECOMMENDATION 5

Controls should be performed with each batch of slides stained and should be treated in exactly the same way as the maternal sample.

 

 

We currently prepare the control slide a week in advance and not fix or stain them, until required for batch testing. I have checked all the slides after it had undergone AE test, and all of them had been adequate. I was wondering if there is something which we are overlooking?

These were the only things that were nibbling at the back of my mind after going through the guidelines. If someone could put it to rest, I will be grateful.

Bonus Question

I was wondering if transfusing Rhesus D Negative pregnant women have any implication on the FMH screening/ quantification, and what they would be?

Cheers,

Jermin

Edited August 25, 2017 by Jermin

[Malcolm Needs ☆]

2 hours ago, Jermin said:

Bonus Question

I was wondering if transfusing Rhesus D Negative pregnant women have any implication on the FMH screening/ quantification, and what they would be?

I am unable to answer your questions about the staining of Kleihauer slides, as this most definitely NOT my area of expertise, but I will have a crack at the bonus question (but with a comment about terminology thrown in!).

Yes, a transfusion certainly will have implications on the FMH screening/quantification, and it will lower the results, but only if the transfusion is given for an ante-partum haemorrhage very close to birth, or a post-partum haemorrhage soon after birth, but before the sample has been taken to estimate the FMH.

If you think about it, the only reason for the woman to have a transfusion at these times is if she, herself, has had a haemorrhage.  The ratio of "adult" red cells to foetal red cells she loses will be identical to the ration in her circulation, but as soon as she receives a transfusion, the ratio of "adult" red cells to foetal red cells in her circulation will rise.  The reason for this is that those red cells being transfused to her would contain no foetal red cells, and so, in effect, these transfused "adult" red cells will "dilute" the mixture of maternally-derived "adult" red cells and foetal red cells in her circulation.  Of course, and also in effect, this means that the amount of "adult" red cells will be concentrated (maternal red cells + transfused red cells), meaning that the ratio of foetal red cells to "adult" red cells in the circulation will be lower (if you like, it is a bit like the more saline you add to an antibody, the weaker will be the reaction, as is the case with an antibody titration).

Now, it may be argued that the ratio of foetal red cells to maternal red cells that are bled out during the haemorrhage will be the same as that of the original blood in the maternal circulation, which means that the actual volume of foetal red cells in the maternal circulation (as opposed to on the Labour Ward floor) is reduced, and I would find it difficult to gainsay this, so it may well be that the calculated amount of anti-D immunoglobulin required to prevent sensitisation to the D antigen, and so the dilution of the foetal red cells remaining in the maternal circulation, versus the actual volume of the foetal red cells in the circulation is irrelevant, and will still be adequate, but I would err on the side of caution, and give a slightly higher dose of anti-D immunoglobulin.

Now for my comment!

There is no such thing, and never has been such a thing as the Rhesus Blood Group System.  Rhesus with an upper case "R" was an ancient King of Thrace.  rhesus with a lower case "r" is a monkey.  As I have said many times, neither Rhesus, nor rhesus worked (directly) in blood transfusion.

The correct terminology for the Blood Group System is Rh.  However, the gene is RHD, the protein carrying the red cell antigen is RhD, but the antigen itself is just plain D (not Rhesus D, not rhesus D and not Rh D), and so the pregnant women of which you are talking are D Negative.

The wrong name has come about because Landsteiner and Wiener injected rhesus monkey red cells into various animals (rabbits and guinea pigs) in an effort to discover more antibodies against antigens expressed on human red cells.  They managed to do so, and the antibody they found was thought to be identical to the anti-D described in a human by Levine and Stetson, but in the early 1960's it was found that the antibody described by Landsteiner and Wiener, and that described by Levine and Stetson were far from identical (and, indeed, the genes encoding the antigens are found on different chromosomes).  As a result, the antibody described by Landsteiner and Wiener was designated as anti-LW, and this particular Blood Group System is now designated as LW.

[MinerJ]

Hi Malcolm,

As always I am always glad to see your replies. Thank you for educating me about the Rh blood group system. I have since read the BBTS chapter on Rh Blood group system and made some amendments to my specialist portfolio. I have always been good at being told what to do, but never thought to ask why, an issue which I aim to rectify.

As to answering my FMH estimation query, I am again grateful for you comprehensive explanation. It has been beneficial, and I feel more prepared if I come across this scenario.

Cheers,

Jermin

[gagpinks]

Hi Jermin 

Your question regarding dilution,  you should follow manufacturer instruction because if you do anything outside manufacturer instructions you might have to validate process again. 

Second question regarding staining and storing sample 4 C  for 4 days.  

Why would store sample for 4 days. Because Rh neg lady should have her antiD dose within72 hours. 

Secondly I would suggest if you prepare slide and don't fix them then there might EDTA changes on your slide. If you fix your slide with different lot and when you stain patient slide it might be different batch no. You are not really controlling process. Control should be done the exactly the same way the way you run your patients sample or run parellal with patient. Otherwise check ISO 15189 manual 

I would suggest check with QMG staff.

 

[MinerJ]

Hi gagpinks,

Thank you for your reply. I think I may need to clarify my queries

14 hours ago, gagpinks said:

Your question regarding dilution,  you should follow manufacturer instruction because if you do anything outside manufacturer instructions you might have to validate process again. 

 

I was referring to the guidelines set by BSH. We do not use a manufacturer supplied foetal spiked red cell reagent. We create our positive controls, and the reagents used for fixing and staining Kleiharuer films do not mention how to go about making the slide, just how to stain them.

14 hours ago, gagpinks said:

Why would store sample for 4 days. Because Rh neg lady should have her antiD dose within72 hours

Again, I was referring to the BSH guidelines on storing controls, not maternal sample. 

15 hours ago, gagpinks said:

You are not really controlling process. Control should be done the exactly the same way the way you run your patients sample or run parellal with patient. Otherwise check ISO 15189 manual 

 

I see what you mean. I will check the ISO manual and see what I can derive from that.

Thank you,

Jermin

 

[Emily Brock]

I am looking at validating and introducing alkali denaturation into our practice to distinguish between maternal and foetal cells. I am however struggling to find a method to reference or manipulate for our standard operating procedure, we currently have a 0.1M NaOH and was hoping to keep this however will look into purchasing a 1M NaOH if required.  

 

Sorry this is my first post and unsure if I have put it in the correct place

Edited December 4, 2018 by Emily Brock
first post

[Malcolm Needs ☆]

I would have a look at the insert in the product (there must be one, if your kit is commercial), and there should be a reference given in this insert.  If not, then, if I were you, I would write to the company for the citation.