Yanxia

Interesting update. Received a very small amount of additional sample and performed a manual GEL (BioRad) crossmatch and it was compatible. Decided to try a 3% 3 cell screen (Immucor) converted to 1% for GEL testing and it was also negative. Very different results since we had been seeing pan reactivity in manual GEL previously (about a week ago). Possible preservative antibody that is neutralized (?) or non reactive in LISS tube? Hoping for more sample to perform repeat testing, maybe whatever it was has gone below detectable limits?

We were thinking an anti-I initially as well but we didn't find much literature about a premature baby forming an anti-I at birth since it appears to be IgM. We were also wondering about other possible antibodies that would be non-reactive or weaker with cord cells. We requested Immunoglobulin tests and received the results today so I will post them for additional information. IgM is double the upper value for the range??
IgM= 45 mg/dL (range for <1 month old is 4-20 mg/dL); they repeated to confirm IgM= 47 mg/dL
IgG= 519 mg/dL (range for <1 month old is 649-1627 mg/dL)
IgA= <6 mg/dL (range for <1 month old is 0-0 mg/dL)

With a syringe and cannula I trust My Good Sir!!!!!!!!!!!!!!!!

Most assuredly!!  She's a nurse and would not allow such activity unless performed with the upmost professionalism!  

I confess that I am no expert on the chemistry of this, but, as I understand it from the late Prof. Patrick Mollison's book, it is purely to do with differences in the equilibrium constants of weak and strong antibodies.

In the Reference Laboratory I worked in (in the UK, NHSBT-Tooting Centre in London), we would freeze any useful samples from both patients and donors (although the latter were quite rare), but we also belonged to a scheme named SCARF (Serum, Cells And Rare Fluids).  I must confess that, having retired in late 2016, I'm not certain that SCARF is still going, or, come to that, how much it costs to join.

One thing that I would caution against, and that is diluting a "strong" antibody to make a "weak" antibody (although this is far more important when trying to make a "weak antibody" to use as a control (for example, for an IAT), as a "strong" antibody has a completely different equilibrium constant (although this may not be too important if you just use them for teaching).

Vis-a vis Sc:-3 blood, I remember when I was working as a VERY junior member of staff in the IBGRL Red Cell Reference Laboratory of Dr Carolyn Giles and Joyce Poole, we did a family study following the relatives of an Sc:-3 female in a small village in Papua New Guinea (PNG), and we found six others.  This was at least 40 years ago now, but it may be worthwhile contacting the PNG Blood Service to see if any of them are still donors, or, indeed, if they have found any other such donors.

I don't think the AABB comments are evidence based.  Washing with 37 degree saline is extremely unlikely to cause false negatives with clinically significant antibodies,  and I'm unaware of any evidence that this is so.  Any such antibody would be very low affinity to be washed away by saline at any temperature, and unlikely to have in vivo/clinical significance. 
As argued persuasively above by Malcolm Needs, anything that doesn't react at 30 degrees or above in typical serologic testing isn't going to cause clinical problems.  Patients are neither at 30 degrees nor centrifuged :).  Our serologic techniques are overly sensitive,  in general,  for clinically insignificant agglutinins. 
No need for cold panels ever, with rare exception, and more for intellectual curiosity than clinical decision making.  Perhaps a mini-cold screen someetimes just to confirm you are indeed detecting a weak cold agglutinin in 37 degree testing, which disappears with prewarm technique. 
Like Malcolm, I've never seen a patient with an hemolytic reaction due to an antibody that disappears with prewarming, in close to 50 years of clinical practice.  I know there are in vitro examples of clinically significant antibodies that weaken or disappear with prewarm, but I've never seen any clinical consequences.

Most certainly, you need to have a thorough transfusion history on the patient, as my good friend Yanxia says above, but it also depends upon the condition of the patient.  If the patient is exsanguinating, the old adage comes into play that it is not a medical triumph to give perfectly compatible blood to a corpse, when, in very many cases these days, a haemolytic transfusion reaction can be treated.  HOWEVER, it is ALWAYS a decision to be made by a medically qualified person, rather than a laboratory qualified person to make, as to how urgent the transfusion may be.

IF there is time, it is always worthwhile doing a few more investigations.  For example, is the patient DAT Positive, and, if so, is it IgG, IgM, IgA (rare), complement or a combination?  Is the reaction seen in the auto-control due to a "cold" auto-antibody, or something else.

To repeat what I wrote above, it MUST always be a decision for a medically qualified person, rather than a "lab rat" (HATE that term, but I hope you know what I mean, without taking offence - being a retired "lab rat" myself), but, if it was a case with which I was dealing, apart from doing a few basic tests (see above) I would be happy to give the blood - and more importantly, receive the blood, if I were the patient.

Thanks for your input.  I was hoping you might respond.
The Daniels book says that "No case of HDFN caused by anti-Lua or -Lub and requiring any treatment other than phototherapy is reported, although raised bilirubin or a positive DAT may be detected."  Does this description equal "clinical significant HDFN" by your definition or is there newer information on more severe HDFN from these since Daniels published the 3rd edition?  My thought is that, if there is no evidence of any case needing any early intervention, then there is no point in running titers to determine when to begin early intervention.
 

I wouldn't bother, to be honest.
Apart from the fact that the Lutheran antigens vary in strength of expression, making it difficult to ensure that the recorded titres would "match up" one to another, but the expression of the Lutheran antigens on foetal and cord erythrocytes is known to be weak.  On top of that, of course, there is the problem of finding a regular source of adult erythrocytes with heterozygous expression.
In addition, anti-Lua and anti-Lub can be either IgG or IgM but are more commonly IgM.  It might be worth your while treating the maternal plasma/serum with a reducing agent such as 0.01M dithiothreitol, 2-mercaptoethanol or ZZAP to see how much, if any, IgG is present.
Even if the antibodies are IgG, they are thought to be adsorbed on to foetal Lutheran glycoprotein on the placental tissue.
Lastly, as you so rightly say, clinically significant HDFN caused by anti-Lub is incredibly rare, and so, all in all, you could be giving yourself an awful lot of work for very little return.  If you do decide to test the maternal plasma/serum with reducing agent, and you find that there is an element of IgG present, it might be worthwhile just performing a titre once, in order to see that you have not got one of these incredibly rare examples that might cause clinically significant HDFN, and, as lone as the titre isn't massive. I would rest easy.

If you want, I can cite references to back up what I have written above, but I haven't done so straightaway, as actually finding some of these papers to read is equally hard work!!!!!!!!!!
I hope that helps.

In the UK, NHSBT stopped performing a DAT routinely on donor units some time ago (when I was still working).  If a unit was found to be DAT positive through, for example, an incompatible cross-match, and the unit was returned to the supplier, the unit was tested, and then discarded, and the hospital reimbursed.  If considered necessary, the donor's GP was informed.

However, of course, it is almost certain that many DAT positive units were not discovered, and were transfused to a patient as a result of electronic issue.  I have NEVER heard of a patient having any serious clinical sequalae as a result of this practice.

A bit late for hopping in but. Another possibility suggests from the 2+ strength typing. Yes, it might just be heterozygous expression, but some techs will call a mixed field 2+. I know you said you didn't have transfusion history, but that's where I'd go first in workup before genotype. Try retic separation and retest.
If your patient had a low titer Jka, received unmatched units elsewhere either from this MVA as uncrossmatched units before transfer, or at an outside hospital between july and now, then came to you. I think it is feasible  you'd see this presentation.  DAT positive in c3d first as the Jka re-activates, an anomalous Jka+ result due to transfused cells but a clearly demonstrated anti-Jka. They could also have a weak expression variant of Jka, where they test positive but can form it. Or an auto Jka. But those are way less common than I think my boring scenario :-)
 

The simple answer is gagpinks, but this is the answer I have just received from my friend at the IBGRL (who shall remain anonymous for now).

The question I put was as follows:

"Sorry to bother you yet again, but I have had a query from a friend. I think I know the answer, but I wanted to check with an expert. If a pregnant lady has an allo-anti-D, can this affect cffDNA harvesting from the mother's circulation? I don't think it does unless the anti-D knocks out all of the foetal red cells. Best wishes from this bloody nuisance, Malcolm"
Answer below.

"Hi, that's right, anti-D makes no difference to the cffDNA test. The two biggest problems are false negatives due to insufficient RHD gene in the test sample and mums with a RHD gene (despite pheno typing as D-) leading to strong positive results. Take it easy."

As I said, the friend will remain anonymous for now, but, suffice it to say, he/she is one of the people who do the test, so I think the answer can be trusted!
 

This is from our ABID SOP (sorry the format is weird): 
a.    Warm Autoantibody Guidelines –(Expert judgment is required on a case-by-case basis to supplement these guidelines.  Contact supervisor or Reference Lab for advice) [return to top]
                        i.        Clues it’s a Warm Auto
1.    Reacts with most or all cells tested—usually at a fairly consistent strength.
2.    Positive DAT.
3.    If it is severe enough that there is hemolytic anemia, the patient would be anemic with a high LDH or bilirubin and high reticulocyte count.  The patient’s plasma sample may appear hemolyzed or icteric.  Haptoglobin would be low.
4.    Contact Blood Bank Supervisor for further consult if uncertain. 
5.    Patient has not taken anti-CD38 (daratumumab/Darzalex/DARA) or anti-CD47 drugs in prior 6 months.
                      ii.        Clues it’s NOT a Warm Auto
1.    Reactivity with most or all cells in gel, usually 2+ or less, varying strength could be HTLA-like antibody or antibody to gel diluent. It’s not uncommon for patients to have a positive DAT without having a detectable warm auto antibody so the positive DAT could be present coincidentally.
a.    To confirm antibody to gel diluent, convert 3% screen cells to 0.8% to run in gel. If negative, turn out the 3-cell gel screen and document situation in the patient record.
b.    An HLTA-like antibody will usually remain detectable (although sometimes weaker) in PEG and even saline techniques.
2.    Alloantibodies:
a.    If patient transfused in the prior 3 months, confer with the most expert person available before calling it a warm-auto.
b.    Elution may produce both a panagglutinin and another specificity or may show only a new alloantibody. The panagglutinin may be so strong a weak allo is undetectable.
c.    If the DAT looks mixed field (repeat IgG DAT in gel for clear-cut mixed field), it may be an allo.
3.    Drug-induced antibody:
a.    Research patient’s diagnoses for multiple myeloma, amyloidosis, or other autoimmune diseases. Look for recent surgeries or infections as a clue for cephalosporin treatment. Research their drug history for cephalosporins and anti-CD38 or anti-CD47 drugs.  There is variability whether these present with only a positive DAT (more common with cephalosporins) or with a negative DAT and positive screen or with both positive.
b.    Rarely, other drugs cause what looks like a warm autoantibody.
                     iii.        When the Auto control (AC) or DAT is positive, first check transfusion history.  The AC could be positive due to a delayed serologic/hemolytic transfusion reaction and NOT a warm auto.  See Positive autocontrol.
                     iv.        If not already done, perform a DAT (Direct Antiglobulin Test Procedure).  Warm autoantibody patients can have RBCs coated with IgG only or IgG and complement both.  Occasionally, only complement may be present.
                      v.        If not already done, perform an antibody panel in gel (or other primary testing method).
                     vi.        People who make warm autoantibodies are more likely to make allo-antibodies as well. We need to identify any allo-antibodies in the sample so we want to avoid detecting the autoantibody if possible but still be able to detect allo-antibodies (at least strong ones).
                    vii.        If reactivity in gel is < 1+ or there are some negative reactions, start a 3% tube PEG antibody screen.  If reactivity in gel is ≥ 1+, start a 3% Saline (no enhancement) 30-minute tube antibody screen.  If amount of specimen is minimal, skip PEG screen and only do saline or, if some negatives, rule out with negative reactions found in gel and run selected cells needed to complete ruling out the usual antibody specificities.
                   viii.        If patient has not been recently transfused and usual specificities can’t be ruled out in tube testing, a PEG Autoadsorption should be considered.  PEG adsorptions should not be attempted with patients who have a strong complement coating.
                     ix.        If can’t rule out usual specificities in tube testing and the patient has been transfused in the past 3 months, send the specimen to the Reference Lab for allo-adsorptions to determine the presence/absence of underlying alloantibodies. See Red Cross (ARC) BloodHub/Connect--Standard Work.  If the patient is too critical to wait for the workup, contact the on-call Pathologist and Blood Bank supervisor.  Phenotypically matched units may be indicated. See Increased Risk Transfusion Release Form. 
                      x.        Turn out ABID results as Warm Autoantibody plus any other specificities detected.
                     xi.        Approach to crossmatching in the presence of warm auto-antibodies:
Situation
Pt Hemolyzing*
XM Methods
XM Results
Enough negs in gel to rule out all usual Ab specificities
Yes
Gel
Incompatible
Enough negs in gel to rule out all usual Ab specificities
No
Gel
Compatible
Can rule out all usual Ab specificities in PEG
Yes
Gel
Incompatible
Can rule out all usual Ab specificities in PEG
No
PEG
Compatible
Can rule out all usual Ab specificities in Saline
Yes
Gel
Incompatible
Can rule out all usual Ab specificities in Saline
No
Saline
Compatible
Alloantibodies identified or can’t rule out some Ab specificities in Saline
Yes
Saline** to assess compatibility with allos, then gel to turn out results
Incompatible
Alloantibodies identified or can’t rule out some Ab specificities in Saline
No
Saline
Compatible
Autoadsorption required and able to rule out usual Ab specificities
Yes or No
Gel with neat plasma
Incompatible
Autoadsorption required and alloantibodies identified or can’t rule out usual Ab specificities
Yes or No
With adsorbed sample** to assess compatibility with allos, then gel with neat sample to turn out results
Incompatible
 
*If patient is hemolyzing, no transfused unit will be truly compatible. Use “incompatible” XM result code in STTx, not “least incompatible” for these cases.
**If second XM method (that’s not to be turned out) is required, record on log sheet.
1.    Warm Auto Notes:
a.    The purpose of the PEG or Saline Antibody screen or PEG adsorption is not to be able to call the primary antibody screen negative, but to rule out underlying alloantibodies.  Generally, these tube ABSC’s will NOT be reported in STTx.
b.    Incubation in the presence of enhancement (gel/PEG) reagents may cause reactivity in the AC that is only an in vitro phenomenon. If the DAT is negative and the AC is positive, antibodies to enhancement constituent or autoantibodies reactive only in enhancement medium should be considered.  An Antibody Elution (Eluate) may help determine the presence/absence of warm autoantibody reactivity.
c.    If the patient is demonstrating active hemolysis, use gel or PEG to crossmatch units. The units still may suffer shortened red cell survival in vivo so calling them incompatible is justifiable.
d.    Consult with Blood Bank Supervisor about performing a full phenotype with the available monoclonal (non-AHG) antisera.  Consider giving phenotypically similar RBCs for transfusion.  If alloantibodies are ruled out in a current specimen, units that are only historically antigen-negative are acceptable.   If we must transfuse before alloantibodies can be ruled out, confirmed antigen-matched units are advised if time permits.
e.    Warm autoantibodies can be confirmed in one of two ways: demonstrate that EGA-treated (antibody removed) pre-transfusion autologous cells react with neat plasma or prove that the antibody reactivity is adsorbed out with pre-transfusion, autologous cells.
f.     Patients on daratumumab (Darzalex or DARA or other anti-cd38 drugs) may appear to have a warm auto antibody but it is actually the drug reacting with the cd38 antigens on the red cells. They may have either a negative or positive DAT and AC.  The only effective way to test these patients is to test against DTT treated cells, recognizing that this will miss antibodies to antigens destroyed by DTT like the Kell system.  These patients benefit from having a pre-treatment antibody screen run and possibly antigen typing for K (and if K positive, for k).  In most cases molecular genotyping may be indicated. See Dithiothreitol (DTT) Treatment and Anti-CD38 Drugs (daratumumab/Darzalex)--Blood Bank Testing.
g.    Additional anti-CD38 drug therapeutics are in clinical trials in addition to Daratumumab (Janssen Biotech) include MOR202 (MorphoSys), Sarclisa -Isatuximab (Sanofi-Aventis), and TAK-079 (Takeda) for treatment of systemic lupus erythematosus, Amyloidosis, or other autoimmune diseases. Daratumumab and Sarclisa are approved for treating multiple myeloma.
h.    CD47 is a glycoprotein expressed on all cells including RBCs and platelets, which usually signals to prevent phagocytosis.  Anti-CD47 blocks this signal targeting cells for destruction. Samples from patients taking anti-CD47 drugs (Hu5F9-G4 or avelumab) will react with everything like a warm auto and the reverse type may be affected like a cold auto. Anti-CD47 interferes with all RBC and platelet serological tests performed including ABO reverse typing. False positive reactions can be seen in all phases of testing (immediate spin, 37°C, and IAT) and with all forms of IAT testing (i.e., tube, gel, solid phase). Reactions with D negative cells may be stronger than with other Rh phenotypes. False negative phenotyping test results can occur due to RBCs heavily coated by anti-CD47. DATs may be falsely negative due to a “blocking effect” caused by high levels of antibody present, but eluates are strongly positive. Plasma interference and strong panreactive eluates are observed as soon as 1 hour after drug infusion. CD47 antigens cannot be denatured with DDT or other common denaturing agents.  It is highly recommended to perform pretransfusion testing, including blood type, antibody screen and extended phenotype (either serological or predictive genotype) before initiating treatment. Using monoclonal Gamma-clone Anti-IgG in indirect antiglobulin testing (which does not detect IgG4 immune classes like anti-CD47) may avoid most of the interference in AHG testing.  Giving antigen-matched units may be an option if full phenotyping is available.  [return to top]
i.      Additional CD47 drug therapies are also in clinical trials and include the CD47 targeting antibodies CC9002 (Celgene), which, like Hu5F9, is also an IgG4 antibody, and the human monoclonal SRF231 (Surface Oncology). CD47 agonists are also in clinical trials. These include TT1-621 (Trillium)31 and ALX148 (ALX Oncology), which are fusion proteins with the Fc region of IgG1 antibody fused to the CD47-binding domain of SIRPα with the goal of interrupting the CD47-SIRPα survival signal. Unlike CD47-targeting antibodies, TT1-621 appears to bind only minimally to human RBCs and interference in pretransfusion testing has not been observed or reported to date.

I would most strongly advise you to send a sample, possibly even multiple samples throughout the pregnancy, to a Reference Laboratory.

As the patient is pregnant, there is the possibility that the Jk(a) antigen you are detecting is actually being expressed on the red cells of the foetus, and you are detecting it as a result of a foeto-maternal haemorrhage.  However, the Jk(a) antigen is not necessarily straight forward, as there are weakened forms of the antigen (and the Jk(b) antigen come to that) where there are amino acid substitutions remote from the site usually associated with the Jk(a) and Jk(b) antigens (280 of the mature protein).
In addition though, you have, obviously, to consider the health of the unborn baby who, even if the antibody does turn out to be a maternal auto-anti-Jka, may cause haemolytic disease of the foetus and newborn, albeit this will usually be be very mild.
I attach a PowerPoint which may, or may not help you in your decision to send a sample to your local Reference Laboratory (also tell them the ethnicity of the patient).
 
Interesting case - please keep us informed.
In Depth Lecture on The Kidd Blood Group System.pptx

I would definitely refer this patient's sample to a reference lab for JK sequencing. As my friend Malcolm mentioned above, variants of JK antigens are not uncommon. The most common variant I've seen is caused by c.130G>C, which causes weakened expression of the Jka antigen. Interestingly, some patients with this variant would make anti-Jka, but I don't think we know much about the clinical significance of this antibody.

My motto was "when in doubt, shake it out".  Seemed to work for me.

Need more info - what is your starting volume and hematocrit?  Use formula  C1 x V1 = C2 x V2.  DM if you need more  - jeanne.towery@prismahealth.org

I've always used the C1 x V1 = C2 x V2 formula for such calculations.  My question is why do you want a Hct of 35%??

Sorry,I can't find the English version.The  underlined sentence means the cord blood cells needed to be washed at least once before testing.

I only wish I could know another language anywhere near as well as Yanxia knows English!  She has always impressed me with her blood bank knowledge as well.

My Chinese is a bit rusty, but if I am not mistaken, the underlined sentence states that we need to wash cord blood at least once before testing.  Sorry, I am just kidding, I don't know any Chinese, I just read Yan's explanation. 

Sorry,I can't find the English version.The  underlined sentence means the cord blood cells needed to be washed at least once before testing.