Kleihauer Betke

[RR1]

Hi scodina,

The Kleihauer test detects the presence of fetal haemoglobin (HbF) not the presence of the RhD antigen, so can be used for detecting bleeds from any baby to mum. It is a fairly difficult test to standardise...after all, if you calculate the bleed are you actually using the mums 'real' blood volume based on her current weight- or just the assumed 'constant' used?

This therefore also means that the dose of RhoGAM that patients receive is not really that accurate and should also possibly be based on their weight (but this would be far too complicated!). The main thing to check is if a large bleed has occurred to perfom a repeat Kleihauer test (48-72 hrs post Ig dose) to ensure the fetal cells have cleared.

Please could someone tell me the doses you use in the US . In the UK we use the value of 125iu of RhoGAM per ml of fetal bleed. Also an antenatel Ig dose is given to all our Rh D- pregnant patients of 1500iu at 28 weeks gestation.

Thanks!

[Mary**]

:clap:There is a Rhogam Dose calculator on the College of American Pathologists web site(www.cap.org) that you can download to calculate the number of doses based on height and weight. Click on References Resources, then Topic Centers/Pathology, then Transfusion Medicine. It is great and easy to use.

[Kimster]

Does anyone have a reference for the need for doing them ante-partum?

For a significant FMH, needing more than 1 vial of RhIG, the bleed must be greater than 30 mLs.

At what point during pregnancy is the fetus large enough to have a blood volume capable of a FMH. How many weeks gestation?

For those doing KB's for trauma, do you also do a Fetal Screen first to determine who needs a KB, or do you go directly to KB?

Fetal blood volume is about 150 mL/kg. The average fetal hematocrit is 50% so 30 mL of circulating RBCs would correspond to 60 mL of whole fetal blood. Using the 150 mL/kg rule, 60 mL would roughly be the circulating volume for a 400 gram fetus. 400 grams is approximately the 50th centile for fetal weight at 20 weeks.

[RR1]

Thanks Mary,

Is it common practice in the US to ask for height and weight for each dose given ?

[Mabel Adams]

The usual dose in the US is 300 micrograms of RhIG. There is a smaller dose (I think 150 micrograms) that can be used for miscarriages before 12 weeks gestation. The full dose is given at 28 weeks gestation and again after delivery if the baby is Rh pos.

[hmcmahon]

Hi there,

We do Kleihauers 24 hours a day 7 days a week and it is performed in the Blood Bank department. We do get requests for Kleihauers from physicians for those pregnant patients suffering trauma or if they are not sure if the bleeding is baby or mom.

Heather

[pluto]

We have found that washing the specimen and removing any buffy coat layer helps to diminish the complication of white cells in the final smear. As a final assurance, we always place the pippette at the bottom of the tube of washed cells to obtain our specimen for the stain. We have noticed a huge improvement. By the way, they are performed on all shifts by generalists.

Would you not lose foetal cells in the washing process ??

also would the foetal cells not be at the top just underneath the buffy coat , I may be wrong but I thought with centrugation the old cells go to the bottom and the new cells go to the top ?

A correctly mixed sample is hence essential or you will get falsely low or negative results

ie under reported foetal bleeds

[Kimster]

The usual dose in the US is 300 micrograms of RhIG. There is a smaller dose (I think 150 micrograms) that can be used for miscarriages before 12 weeks gestation. The full dose is given at 28 weeks gestation and again after delivery if the baby is Rh pos.

Microdose is 50mcg per syringe.

[AMcCord]

Asking maternal weight is not universal, but is a desirable practice and not uncommon in the US.

[Mary**]

We ask for each for that information on each KB Stain that is ordered. The calulator can be used without it, but it is not as accurate.

[RR1]

Thanks AMcCord and Mary** for the info !

[Mabel Adams]

I agree with Pluto. There is a procedure in the Technical Manual for separating reticulocytes from other red cells in a sample by centrifugation in microhematocrit tubes. This is possible because the retics are lighter and end up on top of the other cells. By this logic, Kleihauer and fetalscreen samples should not be centrifuged because those big early fetal cells will all be at the top causing either false pos or false neg results depending on where you pipet from and how many of them you decant during washing.

[RR1]

Just to add to this differential cell layering. If the fetal cells were additionally sensitised (DAT Positive) with maternal antibody where would these cells then end up in a spun sample?

[Mabel Adams]

Hmmm. Good SBB research project.

[RR1]

I suppose the safest way for Kleihauers and DATS is to ensure the sample is thoroughly mixed before performing the test.

[GilTphoto]

Interesting. Isn't someone at your facility the least bit concerned that the fetus might be bleeding out when a pregnant woman falls or has other abdominal trauma. For those patients RhIG was not the reason we did KBs.

I think that since we're a small hospital that doesn't handle trama, it hasn't come up. Most cases are just episodes of bleeding with or without abdominal pain, some ectopic or misscarriage

Trama patient's are taken to Jackson Memorial Hospital in Miami.

At what point of pregnancy is the blood volume of the fetus large enough for a FMH to be detected?

If a woman is only 4-6 weeks pregnant is the fetus developed enough to even have a vascular system with blood? At what week gestation has cellualr division specialized to form all the different systems?

[Mabel Adams]

John, I always felt that if they really thought the baby was in trouble they would get a faster answer with a non-stress test or ultrasound. The KB may help answer why the baby is in trouble, but isn't going to give a timely answer to 'if".

[scodina]

We are a trauma center and run many K-B's on pregnant women who have had trauma. Our routine protocol is to monitor mom for 4 hours then discharge as long as there is no other reason to keep her in the hospital. Per our OB department, there is a very strong correlation between fetal demise and a positive K-B. Our physicians do not use this as their only monitoring method. However, they do increase the amount of time and level of monitoring when the K-B is positive. I admit K-Bs are not the most accurate test, but NSTs and ultrasounds can also yield false negatives with small bleeds.

[RR1]

I think that since we're a small hospital that doesn't handle trama, it hasn't come up. Most cases are just episodes of bleeding with or without abdominal pain, some ectopic or misscarriage

Trama patient's are taken to Jackson Memorial Hospital in Miami.

At what point of pregnancy is the blood volume of the fetus large enough for a FMH to be detected?

If a woman is only 4-6 weeks pregnant is the fetus developed enough to even have a vascular system with blood? At what week gestation has cellualr division specialized to form all the different systems?

You may be able to detect FMH from 8weeks+ ....but why would you want to do this, why not just give a specified Ig anti-D dose to prevent sensitisation? If a fetus <18-20weeks is bleeding- what can actually be done about this- apart from monitoring by ??doppler so an IUT could be given if there were signs of anaemia from about 20-25weeks.

The Rh antigens must be formed fairly early on in red cell development as they are structural proteins .I think the figure for amount of red cells needed to cause RhD sensitisation is 0.1ml red cells, so presumably a fetus from about 8-12 weeks may be capable of this. These figures however do not take into account twin etc..pregnancies.

We only perform Kleihauers from 20weeks +, anything under just gets a specified Ig dose. ...though thinking this through- may be we need a modified protocol for twin/ multiple pregnancies???

[Kimster]

On day 38 the fetus has developed RBC's. FMH is one of the largest reasons for fetal demise. That is why a KB should be done on all Rh negative moms with a Rh positive fetal death to determine how much RhIG should be given. I recently asked a maternal fetal specialist regarding multiple births and he does not treat any different than a single fetus when it comes to covering with RhIg.

[RR1]

Thanks for the info Kimster. It's very interesting learning about the different practices between countries.I suppose the most important thing is to ensure that we comply with our own national guidelines.

[John C. Staley]

John, I always felt that if they really thought the baby was in trouble they would get a faster answer with a non-stress test or ultrasound. The KB may help answer why the baby is in trouble, but isn't going to give a timely answer to 'if".

Mabel, most of the time it was used as a screening test to help determine if they needed to look much further or watch closer. It's also relatively inexpensive compared to other testing and could be positive before the baby really started getting into trouble.

:imslow:

[Eloise]

Hi to all,

I was wondering whether anyone who is doing KB currently has looked into doing Flow Cytometry instead KB.

I was speculating that perhaps labs were still doing KB because they don't have access to Flow or maybe Flow wasn't available within a sufficient TAT and that might be why people are still using KB.

I'd be interested to know also how many labs are using the CAP calculator also. (Our doctors are willing to use that calculator, but not many labs in my area are doing that yet.)

Thanks for any feedback.

Eloise

[rravkin@aol.com]

We do K/B staining on all shifts and on all traumas and post partum per doctor's orders. Whole blood (EDTA) specimens are used and the control consists of well mixed cord blood and adult male whole blood. One drop of cord blood to nine drops of adult male blood. We prepare the whole blood specimen and control for testing by adding two drops of each sample to their respectively labeled tubes and three drops of saline. The smear is then made as a we usually make a push smear. This ratio of whole blood specimen to saline almost always results in a monolayer of cells. But before we proceed to stain we check the smear under the microscope to ensure a monolayer. Then it's 5min of fixative, wash, dry, 10min of buffer, drain, and 3min of stain, wash, and let dry. View microscopically. The main difficulty in this procedure is simply the lack of practice. As John has stated earlier, but not this way, this procedure is based on staining fetal hemoglobin in an adult maternal system (where the fetal screen is based on detecting Rh positive cells in an Rh negative system). Therefore, many times this procure is ordered for any traumatic experience and any Rh type because, like we all said, the docs are interested in the extent of fetal bleed into the maternal system. Admittedly, the many times I've performed this procedure there have been only a very rare positive result. Once, we actually calculated a volume that was well above what the fetus' circulation could even support. We suspected that the mother may have had a subpopulation of RBC's containing fetal hemaglobin. Our TAT is approximately one hour but sometimes it can take longer. Before reporting any result a second MT will review the smears and any calculations. Both techs will initial the completed form and the result is always called to the floor. I personally enjoy performiing this procedure becuase I like the technical challenge, the microscopic work, and the mathmatical calculations, if needed.

[KtVl]

Is the CAP calculator available on line? I try to search for it but got lots of hits for taxes.