Rh-fan

Our institute makes anti D (just as Rhogam) form plasma of immunised donors. Although they are immunised with cells that are D pos, C neg (mostly E pos, further compatible for K, Fy,Jk, Ss) they almost all make also an anti C (or anti G), So there is anti C present in the final product. The titer of the anti D is between 8.000 and 16.000 and the amount of anti C (and other antibodies) must be below 1:32. When you compare these amounths then there is a little bit of anti C, but it is present (just like some anti E, anti K, but mostly weaker than C) and also anti A and anti B will be present. The methode of production is the same as for every IgG product (like IVIg), they just concentrate all the IgG antibodies. When you want random IgG they use random donor plasma, if they want Hepatitis A IgG they use donors with a high titer of anti Hep A, and for the anti D product they use donors with high titers of anti D. There is no purification step to remove other antibodies. Peter

That is what I suspect, Shilly. Although the auto controle and the Direct antiglobulin test is negative we sometimes see anti D (and anti G) antibodies in the eluate made of the patient cells (also when they have not been transfused) in these kind of cases.

Yes and no In samples with weak D type 1 an auto anti D is rare, but in a type 2 it is more common. (type 1 is stronger and mostly expressed with C, while type 2 is weaker and expresses an E) Because of the weaker expression of type 2 the pressence of auto anti D is a little bit more common (not normal, but shown more than in type 1). The weak reaction you mention do fit with an antigen with a weak expression, but tells you nothing about the fact if the antigen is normal or missing epitopes. I Assume that patient expresses the E and no C antigen. The strengthe you mention (weak at IS and 3+) is what we see with type 2 weak D (but still no proof). Partiela DIII variants have normal a stronger expression of the RhD antigen that is the reason that it is not matching your findings, but there is no monoclonal anti D that is not reactive with DIII variants, we normaly only find these patients if they have made anti D (confirmation is done by the use of a polyclonal anti D (made by an another pateint with an RhDIII)). So it is possible that the ALBA kit is total reactive. Only 3 exons is deffinatly not enough you have to do more on DNA. The ALBA kit is recognising the most common (although they are all rare) RhD variant antigens, in the fact that one of the monoclonals is not reactive. The conclusion on the name givving of the variant is not always correct but it is very good in making a difference between a variant and a normal (weak) RhD antigen. On the serological findings I think you are dealing a weak D type 2 with an auto anti D, auto anti G and maybe allo anti C. My advice give C, D neg blood. (If it is allo or auto anti D, makes no difference (as long as the patient is c positive)). Peter

Maybe the low temp (4oC) is also a reason you find a lot of cold antibodies. I have alsways learned that at 4oC you will find cold antibodies in almost everyone, specialy when you incubate for 15 or 30 mins. Antibodies reactive at 4oC have no relation with the patient and with the lab test you perform (20 to 37oC). It is better to test at 16oC, when there is something wrong with the patient (infection etc.) you will find them at 16oC. Antibodies reactive 16oC can also cause problemes in the lab (most test start at 20oC). I think it is not right to blame a cold antibody (at 4oC) for a reaction in your screening perfomed at 37oC (and started at 20oC). It is nice to have a explanation but dont fool yourself. Peter

We were dealing with the same problem, but now we receive (mostly) an EDTA tube, draw true the same needle. When you have a sample with a lot of white cells in it we find that a tube test is most sensitive when you centrifugate, then dissolve (maybe wait 5 minutes) and than centrifugate again, then read the agglutination. But also as you stated only performed by very experienced technicians. Good luck, Peter

When you drive 150 mile in the Netherlands there is a big change you left or almost left the country. We are a lot more dence, in this little space we are with 17 milion people. So we have the smal hospitals but there are always bigger hospitals neerby. I agree with you that patient have an anti E (or other clinical relevant antibody) and are c negative, it is better to give them c negative blood to prevent the formation of anti c. In case of emergancy you will not delay the transfusion because the antibody ID will be easier.

(Why plan ahead if you can give the Blood Bank a heart attack asking for antigen typed blood RIGHT NOW!?)

What is wrong with serology? A bottle of anti c is not that expensive, and it is very easy. Also for card methodes there are cards (Diamed/Biorad). If the donor centers start with DNA typing it is cheaper to test al the antigens (or genes). In the Netherlands we have one blood supplier and they perform an Rh fenotyping on all donations.

For mabel and Malcolm (and others who are interested). Transfusion 2008; 48 (5): 941-952 This is the tekst I was refering to page 948-949 Severe HDFN in screen-undetected cases The coverage validation study identified eight cases of severe HDFN, all with a negative screen at the first trimester; seven of eight infants needed an exchange transfusion because of anti-c (n = 3), anti-c and anti-E (n = 2), or anti-E (n = 2), respectively (Table 4). One of the children (anti-c plus anti-E) suffered from kernicterus with a maximum bilirubin level of 1109 mmol per L and showed permanent severe brain damage at 1 year. Another child (anti-c) had an intracerebral bleeding, caused by asphyxia, possibly related to the severe prenatal anemia (Hb 2.4 mmol/L = 3.8 g/dL). At 1 year, the prognosis was still unclear. One child only needed a top-up transfusion because of anti-Miltenberger, a low-incidence antigen. In this child other unrelated morbidity contributed to the observed anemia. The prevalence of severe HDFN with negative antibody screens was 2 in 100,000, leading to a total prevalence of 9 in 100,000 (Table 4). [ATTACH]596[/ATTACH] As you can see mostly anti c(+E), some anti E and one Miltenberger (you never will find during screening. (I hope the attachment worked) Peter

Mabel, Yes it is 80-82% in our population. Beside the anti c we also found a few anti E (mostly together). But for more details I have to look into the publication. I will try to find it and send you a copy. Peter

A few years ago a large study is performd to follow women with klin relevant antibodies during pregnancy (OPZI study). During this study we also looked foor childeren with severe HDN (need of a (exchange)transfusion) while the antibdy screening at week 12 was negative. There were 6 of 7 childeren with severe HDN (even one with kern-ictarus) all were anti c antibodies. This is why we (the netherlands) started in july 2011 to type women at weak 12 for the presence of the c antigen (beside ABO RhD and antibody screening) and repeat the antibody screening for those that are c negatieve at weak 27 (just as the RhD neg women). We expect to find more than 6 women per year because we have only looked at those who became sick. Peter

you have found it, therefore it is possible. In serology eberthing is possible we say. Did you make an acid eluate (elu-kit Immucor). With this eluate we see this very often, the presence of an allo antibody in the eluate while there is no transfusion in the last 3 months (most often with anti K). I think we are dealing with a combination of thing first of all the fact that the specificity of the anti K (in this case) is not htat narrow. Or as Malcolm says "mimick specificity" in his first reply. And the fact that in elu-kit bovine albumine is added in the buffer. This is used to enhance the reaction strength of antibodiesin the aluate but also from antibodies yoy dont expect in the eluate. Peter

Malcolm has given most of the info but as an Rh fan I have some aditions. This sounds most like a weak D type 2 (ccDEe) with auto anti D. The "anti C" can be an allo anti C or an auto anti G. The only way to make the difference is by absorption/elution. If the "anti C" is in the eluate than it must be an anti G (auto). You have no real prove that the anti D antibody is an auto antibody, except the fact that the anti D is in the eluate (en thare was no transfusion. We see these cases almost once a year (weak D type 2 with auto anti D), and most of the times the RhD antigen is weaker because of blocking by the auto antibodies.

It is not only the ponit of chosing but also what do have to chose from. I think that this are the HFA antigens that they can test, but also the once you expect the first to find. Although rare they are a little more commen than other antibodies to HFA. Mostly you can not buy these reagents so you mostly use cells and sera from patients you already have found. Peter

Anti P can be present without transfusions or pregnancy. Malcolm, What do you mean with "Indirect Donath-Landsteiner Test"? Is that a DL test with the adition of complement or do wash somewere? I would not expect these strong reactions with a PCH

We rather do the K1 after 18 weeks but sometimes we try it sooner (before 16 weeks) in strong cases of HZFN because that is the time they can start with IUT. Peter

I am not mabel but I think I know it. There is methode in wich you can isolate DNA from the plasma of the mother. The plasma contains also a percentage of fetal DNA. The mother is lacking the K allel so if you find a K allel in this DNA it must be from the mother. If you do not find the K allel you have to look for other DNA markers to confirm the presence of fetal DNA. You need a lot of EDTA blood (30ml) and a very specific PCR test for the test. This test is developed for RhD but can now alos be performed for C, c, E, K and HPA1a. Peter

I do not like "cold" antibodies when the auto controle is negative. In these cases I want to know the specificity before I change to a different methode and give negative Xmatch. Peter

Although the numbers are not as big as in the UK, we see the same kind of HDN as Malcolm stated. We see childeren at 16 weeks pregnacy in need of a transfusion and as Malcolm also stated with different titers (and als the lower titers). We also do ADCC testing during pregnancy (a test with monocytes to predict the severity of the HDN, expressed in a percentage). Above 50 there is a great change of HDN, this is largely validated for anti D antibodies (1980's). But because of the lower number of anti K pregnacies a good review of those is only made a few years ago and there we see that above 30 almost every (K+) child is in need of IUT. Peter

No problem, good idea for the new thread

On my original indated panel(s) I was not able to rule out anti-E. I select cell #5 from on expired panel in order to rule anti-E out. I must test cell #5 with a known anti-E to prove that the E antigen is still there, and I must test cell #5 with serum known to NOT contain anti-E as a negative control. Before phone call, we ran a positive control if we were using the cell to rule OUT, or a negative control if we were using the cell to rule IN. CAP did not relent when I explained the logic of this. Outdated cells must have both positive and negative control run. The only problem is that the anti E reagent you use will mostly be strong reactive (3+/4+), so you only rule out strong antibodies, weak antibodies you can miss. I like you study and hope you can publish it, maybe we can then the panel firms will stretch the expiration date, that would be nice. From the things I hear from the USA, the rules are very thight (specialy the FDA). So I am surprised that the CAP (I do not know what it stands for) is accepting the use of a product that is past the expiration date. I know that a reference lab has more possibilities and must be more secure, but for every hospital counts that patient care is patient care, if the hospital is very smal or very big, that should be the same. Peter

Maybe it is not the good location but I want to continue on the anti K in OB patients. In the Netherlands we are scared for an anti K from a titer of 1:2 (titer in IAT, 30’ 37oC, no additives). We do not have a lot of cases with problems and such a low titer but we seen them. Also we are afraid for a rise in titer later in pregnancy. When we find these antibodies at week 12 we do ADCC and type the father*, and if father is positive (mostly Kk) we do fetal DNA typing. We hope these test are done before week 16 and send patient and results to an special OB clinic that can perform IU transfusions. When we find these antibodies later in pregnancy we send the patient to this clinic and than do the tests. Maybe we are a little to scared but we have seen to many early problems with K antibodies. * In the Netherlands we are giving K neg blood to woman below 45 for quite some years now, so most anti K antibodies formed now a days are made during pregnancy, so we see a lot more K positive man in these Group. Peter

We do not use outdated red cell for the exclusion of clinical relevant antibodies, sometimes we use them as an extra positive cell (mostly in LFA cases). The problem is that you use these cells when you have more than 1 antibody present (otherwise you don’t need them). When a patient has 2 or more antibodies than they will make also a new antibody (if that is possible), and for those patients you are going to work less sensitive (these cells are not outdate for no reason). You can better screen with outdated cells than exclude a third antibody with an outdated cell. In a patient with no antibodies that change that there is an antibody is very low. And nobody will do that. When you do use outdated cells a positive negative control is not enough. The antigen you want there to be is not gone in one day, it will be present but the expression can be lesser than you need. The only good control is an antigen strength determination (titration), where you compare the outdated cell with an not outdated cell. If the expression is the same you have prove the expression of the antigen is enough. I know that working in a reference lab is easier, we have more than 100 cell from which we can pick. But also for a hospital situation I think that the risk you take is too high for these group of patients. Peter

Knops is a system but HTLA is not. Don't forget the JMH and Csa

Yes and no. Glad to have a solution, but sorry to hear that there is already a paper on it. It must be hard, no serology for 2 weeks:cool:, have a nice Chrismas and hope to hear something in the new year. Peter