Case 60 – summary

Panel:
In this case we interpreted an antibody panel to establish this lady was A RhD positive and had an anti K antibody.

When a possible antibody is detected it is important to phenotype the patient’s cell for the corresponding antigen to assist in confirmation (this is not reliable if transfused in last 3 months). Further panels may also be needed to comprehensively exclude other antibodies in difficult cases.

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The patients red cells reacted with Anti A and Anti Rh D suggesting A and Rh D antigen on the patients red cells. Her serum reacted with B cells that confirmed the presence of Anti B in the patient’s serum. Hence group A RhD positive was confirmed.
Panel interpretation shows only two reactions in line 2 and line 6, these reactions are uniform which may be suggestive of a single responsible antibody. All the other antigens can be excluded on the basis that there is a negative reaction when the antigen is present with homozygous expression in the cell panel. (Note: Anti K, Anti C and Anti E can be excluded using heterozygous cells) (3). The reaction was unchanged by Enzyme papain is also consistent with Kell system.
Enhanced reactions papain treatment: Rh, Kidd, P1, Lewis
Negative reactions papain treatment: MNSs and Duffy
Unchanged reactions in papain treatment: Kell and Lutheran
History:
History is critical in HDFN to establish how the patient acquired the antibody and its possible implications.
In this case we established, she has never been pregnant and has no history of IV drug use. She was transfused abroad after a motorcycle accident. In this case like 80% of  HDFN due to Anti K antibodies were acquired through previous transfusion rather than a prior pregnancy (2).
This case highlights the importance that females under 50 without a known K type always receive K negative units to prevent alloimmunisation.
Risk assessment:
When any antibody is identified during pregnancy it should be quantified (2). All women with a recognised alloantibody need four weekly monitoring up to 28 weeks and two weekly monitoring from then onwards (1,2). The reason for the monitoring is that there could be development of further alloantibodies and a sudden change in titre may alert the clinical team to a sensitisation event.
It should be recognised in that in the case of Anti K titre the degree of correlation between significant HDFN compared to the antibody titre is varied. Hence, any woman with Anti K detected regardless of titre needs referral to the fetal medicine team (1,2).
The risk to the fetus can be calculated through paternal phenotyping and if required cell free fetal DNA testing to guide further management (2). In our case the father was a hetrozygote for the K antigen, hence fetal DNA testing was performed.
Paternal phenotype testing is important for any clinically significant maternal antibody. It should always be remembered that the pitfall in relying on this alone could be that any partner may not be the biological father. Indeed the RCOG suggests it may be valid to go straight to Fetal DNA testing (1).
If the paternal testing confirms paternal homozygote for the antigen concerned, in this case K then no further testing should be performed as the child is at risk. If the father is a hetrozygote then a maternal blood sample should be taken for cell free fetal DNA testing.

It is clear the accuracy of cell free fetal DNA testing improves at later gestations and initial samples should be sent around 20 weeks for Kell typing (16 weeks for Rh testing) with need for further confirmatory testing around 28 weeks. The exact timings should be discussed with the laboratory concerned (1,2). Fetal DNA testing has a false negative rate of <1% 2. Cell free fetal DNA testing can be performed for RHD (D) RHCE (c and or E) or KEL 01 (K) at present (1).
An at risk pregnancy will have MCA Doppler assessment weekly from 20 weeks looking for signs of fetal anaemia. Signs also detected through USS are skin oedema, polyhydramnios and cardiomegly that may also indicate anaemia (1). MCA dopplers detect anaemia by increasing MCA peak systolic velocity. This technique can predict moderate to severe anaemia with 88%-100% sensitivity and with false positive rates of 12-18% (1,4). After 36 weeks the sensitivity of this test is reduced 4. In cases of elevated Doppler reading >1.5 Multiples of Median (MoM) fetal blood sampling and Intrauterine transfusion should be considered (4).
BCSH guidelines suggest the benefits of ongoing testing of antibody titre is doubtful if regular MCA monitoring, hence at the discretion of the fetal medicine team this can be omitted as long as there is close monitoring (2).
Intrauterine Transfusion (IUT):
IUT should be performed every 2-3 weeks aiming to keep Hct >0.45 (4). IUT units have special requirements, hence early notice is needed to organise the units they also have only a 24 hour shelf life post irradiation. The units should be less than 5 days old (lower potassium content), they should be suspended in CPD (citrate, phosphate and dextrose) rather than usual SAG-M (saline, adenine, glucose and mannitol) this is because of possible risks of hypoglycaemia and renal tubular damage and fluid shifts in brain of fetus that may be caused by SAG-M (4). Blood should be CMV and HEV negative and be only from returned donors. Hct 0.7-0.85 in an IUT is required to maximise benefit from IUT. In this case it should be Kell negative, sickle negative and group O (as baby group not yet known). It should also be compatible with maternal serum on IAT crossmatch.
The procedure is only performed in specialist centres using direct USS visualisation of the cord. Complications include death 1% (upto 20% mortality if hydrops present), preterm labour, fetal bradycardia (particular issue if blood is not warmed), cord haematoma, bleeding and vessel spasm (4).
Delivery:
For affected HDFN babies delivery is planned based on the decision of the obstetric team. Gestation should not proceed beyond 37-38 weeks in cases of clinically significant antibodies, even if MCA dopplers are normal as the risks of higher titre antibody formation from sensitisation events increases towards term.
All babies who need IUT will require admission to neonatal unit for monitoring and possible phototherapy/exchange transfusion. Early discharge post-delivery is not advised.
In the case of Anti K it can cause profound anaemia, but is not usually associated with hyperbilirubinemia. This is because uniquely Anti K Antibodies can directly suppress the K positive erythroid progenitor cells and can even promote their immune mediated destruction (5). This explains why disease can be significant even at lower antibody titres.
DAT, Hb, Bilirubin and reticulocytes should be checked at delivery. A positive DAT should lead to an eluate to detect any maternal antibodies that may bound to fetal cells.
If the baby is profoundly jaundiced and does not respond to phototherapy then an exchange transfusion may be considered and it has similar requirements to an IUT. A top up transfusion may be used if anaemia alone, see table below for special requirements summary.
In the case of the lady with Anti-K she delivered a baby boy who required a single top up transfusion during his stay on the neonatal unit. He was discharged home and is doing well.
Pre-transfusion testing of babies upto 4 months requires (4):
Maternal ABO, RhD group and antibody screen.
ABO forward group, RhD group, DAT. Antibody screen only if no maternal sample available.
Careful selection of compatible blood group for mother and baby needed, hence often group O is given. If all antibody screening is negative then the top up transfusions can continue to 4 months without any further sampling.

Summary of blood product requirements for fetus and neonates (4):


References:
1) The management of women with red cell antibodies during pregnancy. RCOG Green Top Guideline No 65. May 2014.
2) White et al. Guideline for blood grouping and red cell antibody testing in pregnancy. BCSH guideline 2016.
3) Milkins et al. Guidelines on pre-transfusion compatibility procedures in blood transfusion laboratories. BCSH guideline 2012.
4) New et al. Guidelines for transfusion in foetuses, neonates and older children. BCSH Guideline 2016.
5) Vaughan et al. Inhibition of erythroid progenitor cells by Anti Kell antibodies in fetal allo immune anaemia. NEJM 1998; 338:798-803.

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