Immediate spin crossmatches

[slc7067]

I work for a hospital that is part of a small group of hospitals and clinics. Our blood bank does antibody screens by gel, and immediate spin crossmatches when there is a negative gel screen and negative history. When we implemented this policy, we justified it to our very concervative medical director with the increased sensitivity of gel over tube antibody testing. I recently realized that one of our newer, more remote hospitals also started doing immediate spin crossmatches when they became part of our system. I'm all for consistency across the system, but this hospital doesn't have gel. I'm concerned that immediate spin crossmatching isn't adequate without a more sensitive antibody detection technique. Anyone have any thoughts?

TIA,

Sandi

[Malcolm Needs ☆]

I work for a hospital that is part of a small group of hospitals and clinics. Our blood bank does antibody screens by gel, and immediate spin crossmatches when there is a negative gel screen and negative history. When we implemented this policy, we justified it to our very concervative medical director with the increased sensitivity of gel over tube antibody testing. I recently realized that one of our newer, more remote hospitals also started doing immediate spin crossmatches when they became part of our system. I'm all for consistency across the system, but this hospital doesn't have gel. I'm concerned that immediate spin crossmatching isn't adequate without a more sensitive antibody detection technique. Anyone have any thoughts?

TIA,

Sandi

Hi Sandi,

It rather depends on what technology they do use, and also on the "homozygosity" of the screening cells.

Could you shed some light on these matters please?

:confused:

[slc7067]

Hi Malcom,

Good questions. They do tube method with Immucor's Panascreen screening cells. They are a small community hospital, with relatively little blood banking workload. They don't do any antibody identification or antigen testing. If they get a patient with a positive screen they have to send out the patient specimen and segments from the units they want crossmatched for antibody ID and unit antigen testing. Alternatively, they send the whole patient out for transfusion at another facility.

[Malcolm Needs ☆]

Hi Malcom,

Good questions. They do tube method with Immucor's Panascreen screening cells. They are a small community hospital, with relatively little blood banking workload. They don't do any antibody identification or antigen testing. If they get a patient with a positive screen they have to send out the patient specimen and segments from the units they want crossmatched for antibody ID and unit antigen testing. Alternatively, they send the whole patient out for transfusion at another facility.

Sorry to be a pain (I live in the UK and so am not familiar with the Immucor Panascreen screening cells. Do they have one as S+s- and another as S-s+, and the same for the Duffy and Kidd antigens?

This is quite crucial to the answer I would give.

[David Saikin]

We performed ISxm using tubes with LISS for our antibody screens. This is a change that actually happened in the mid-1980's. There is no need to be concerned about the sensitivity of your methodology - gel, tubes, capture. what you need are good screening cells, like Malcolm has said - homozygosity with the Duffy's, Kidd's, MNSs.

[Malcolm Needs ☆]

We performed ISxm using tubes with LISS for our antibody screens. This is a change that actually happened in the mid-1980's. There is no need to be concerned about the sensitivity of your methodology - gel, tubes, capture. what you need are good screening cells, like Malcolm has said - homozygosity with the Duffy's, Kidd's, MNSs.

David is absolutely correct.

When Boral and Henry first mooted the immediate spin cross-match following a negative antibody screen in 1977, Lapierre et al had not dreamed of the gel technique, which they did not describe until 1990, and this technique did not become universally accepted for some time. The immediate spin cross-match, following a negative antibody screen was, in those days, performed by tube techniques, and did not result in an avalanch of transfusion reactions.

As long as the tube technique being used is validated and the screening cells do express the "homozygosity" for these antigens, the technique should be quite safe.

:)

[slc7067]

Thanks everyone, for your answers. You've set my mind at ease considerably. I asked them to fax me the antigram for their screening cells, and it does show good "homozygosity" so I think I can go back to sleeping well at night.

[Malcolm Needs ☆]

Thanks everyone, for your answers. You've set my mind at ease considerably. I asked them to fax me the antigram for their screening cells, and it does show good "homozygosity" so I think I can go back to sleeping well at night.

Sleep well and sleep soundly!!!!!!!!!!

:D:D:D:D

[RR1]

I thought that it was difficult to standardise the immediate spin technique, and that's why it's not ideal to use.

[Malcolm Needs ☆]

No, it isn't that difficult to standardise, as long as EDTA saline is used.

[trisram]

I am sorry, can somebody explain homozygosity for me? It's been a long time since I have been out of school. Thank you.

[YorkshireExile]

How critical is it to use EDTA saline for the immediate spin XM?

I know there is one paper from 1988 that warns about prozone and steric hindrance effects, but that is all I have ever managed to find concerning ISXM problems. Could you use normal saline? If you collect the patients sample in EDTA is this a help?

Could you even use LISS? It just seems a nuisance to make a cell suspension in LISS for use with our Diamed Gel grouping cards and then another suspension in saline for the ISXM.

[Malcolm Needs ☆]

How critical is it to use EDTA saline for the immediate spin XM?

I know there is one paper from 1988 that warns about prozone and steric hindrance effects, but that is all I have ever managed to find concerning ISXM problems. Could you use normal saline? If you collect the patients sample in EDTA is this a help?

Could you even use LISS? It just seems a nuisance to make a cell suspension in LISS for use with our Diamed Gel grouping cards and then another suspension in saline for the ISXM.

Hi,

I presume you are talking about the paper by Judd WJ, Steiner EA, O'Donnell and ObbermanHA? There is not an awful lot else in the literature to be honest, but our National External Quality Assessment Scheme produced a document on the same subject in late 1995, and it is also mentioned in the BCSH Guidelines for compatibility in hospital blood banks.

I see no reason why you should not add EDTA to your LISS (although I am far from being an expert on this, and just wonder whether it would affect the rather delicate ionic strength, which is the whole point of using LISS).

As the problem appears to be associated with the steric hinderance of the C1 component of human complement, and the fact that EDTA is a "complement poison" (because of the chelation of Ca++, Mn++ and Mg++ ions), I suppose that collecting the samples into EDTA would help; but I don't know for certain.

[YorkshireExile]

Yes Malcolm, thats the paper I was referring to. The AABB technical manual also refers to using the patients sample collected in EDTA as an alternative approach to prevent the steric hindrance complication. But it doesn`t give any of the science behind it.

So... if I use EDTA samples can I use normal saline for my ISXM?

Is using EDTA samples and making my suspension in LISS definitely not recommended?

If I really have to use EDTA saline, do I have to make it myself or can I purchase it commercially?

What do others do?

[Malcolm Needs ☆]

Yes Malcolm, thats the paper I was referring to. The AABB technical manual also refers to using the patients sample collected in EDTA as an alternative approach to prevent the steric hindrance complication. But it doesn`t give any of the science behind it.

So... if I use EDTA samples can I use normal saline for my ISXM?

Is using EDTA samples and making my suspension in LISS definitely not recommended?

If I really have to use EDTA saline, do I have to make it myself or can I purchase it commercially?

What do others do?

From what I understand of the science behind this (which is not a lot I might add) as long as your samples are in EDTA, you should be okay.

If you samples are clotted, or in any other anticoagulant, I "think" you do need to use EDTA saline.

[huntilla]

I believe that if the staff at the satellite facilty follow their policies and procedures they will detect just as many antibodies as gel technique or Capture. The overiding concern with quality of tube testing is a non-issue as most of the transfusions prior to the mid-90's were all conducted using the tube technique for antibody screening in most small to mid-sized hospitals. Tube testing or gel testing are only as good as the person performing and reading the results.

[huntilla]

zygosity is the state of inheritance for the specific antibody system. If the donor/reagent cells are homozygous they express one allele(Jka= Jkb +) which leads to a stronger reaction when screening or performing antibody I.D. Heterozygous reflects inheritance of both alleles (Jka + Jkb+) and can lead to weakend or non-existent reactions when performing screening or I.D.

[Malcolm Needs ☆]

I believe that if the staff at the satellite facilty follow their policies and procedures they will detect just as many antibodies as gel technique or Capture. The overiding concern with quality of tube testing is a non-issue as most of the transfusions prior to the mid-90's were all conducted using the tube technique for antibody screening in most small to mid-sized hospitals. Tube testing or gel testing are only as good as the person performing and reading the results.

I agree with a lot of what you say, but I am fast becoming convinced that nothing will detect as many "antibodies" as Capture.

As a Reference Laboratory we are increasingly receiving numbers of samples from hospitals who have recently gone over to this technology, and we can find absolutely nothing in them. This could be down to us of course, but none of the patients have undergone the slightest haemolytic transfusion reaction (acute or delayed) when the hospitals have acted upon our findings, and none have sent in a further sample, post-transfusion, suggesting that the "antibody" has been further stimulated by the transfusion.

Capture may, I suggest, be a little too sensitive?

:confused::confused:

[Malcolm Needs ☆]

zygosity is the state of inheritance for the specific antibody system. If the donor/reagent cells are homozygous they express one allele(Jka= Jkb +) which leads to a stronger reaction when screening or performing antibody I.D. Heterozygous reflects inheritance of both alleles (Jka + Jkb+) and can lead to weakend or non-existent reactions when performing screening or I.D.

Again, I agree with most of what you say, but zygosity is the inheritance, in this case, of genes leading either directly (e.g. Rh) or indirectly (e.g. ABO) to antigens. It has nothing to do with the antibodies in the plasma, but the rest of your post is correct.

:):)

[TimOz]

I will attempt to explain the importance of screening with homozygous cells.

Many blood group antigen come in pairs called antithetical antigens. They are genetically linked and in many cases, the number of antigens present on cells depends on the zygosity. This is true for many but not all antigens. We used to say that homozygous cells were “double doseâ€. That is a poor and inaccurate name but it serves to indicate that the cell has a dose of Jka from the mother and a dose of Jka from the father and no Jkb so it is therefore is only Jka positive. Think of it this way, if a cell can only have a certain number of Jk antigens on the surface, say 14,000, then IF the cell is Jk(a+b+) the cell will have 7,000 Jka antigens and 7,000 Jkb antigens. If the cell is Jka homozygous, Jk(a+b-) the cell will have 14,000 Jka and 0 Jkb.

From this we can explain the effect we used to call "antibodies that show dose". This means that many antibodies will show a stronger serological reaction when they react with a homozygous cell compared to heterozygous cell. Antibodies do not really show “dose†at all but the serology we see is due to the "dose" or antigen number on the cells we are testing. As an example, here are some reactions you may see with an anti-Jka antibody against the 4 common Jk phenotypes:

Score phenotype Name

0 Jk(a-b-) Jk null

0 Jk(a-b+) Jkb homozygous

3 Jk(a+b-) Jka homozygous

1 Jk(a+b+) Jka heterozygous, Jkb heterozygous

So we could also see a weaker anti-Jka that would give a score 1 with Jka homozygous cells and a 0 with heterozygous. In this situation, we could do a crossmatch and randomly select a ABD group matched unit that was Jk(a+b+) and get a compatible test. This unit would be very likely the cause of a delayed transfusion reaction. BUT, if we screened with a panel that had a Jka homozygous cell we would get a score 1 positive antibody screen, do an ID panel, find an anti-Jka and test and issue Jka negative units and prevent the reaction.

In summary, antibody screening with homozygous cells increases the sensitivity of the test. For this reason it is usual for manufacturers of screening cells to ensure their panels have homozygous expression of C, c, E, e, Fya, Fyb, Jka, Jkb, M, N, S and s.

And now Immediate Spin Crossmatching

A plea! Please STOP using this hopeless name!!!!!! – for 3 reasons.

1. An Immediate Spin crossmatch is not a crossmatch. A crossmatch must include an IAT phase to detect IgG class antibodies.

2. If you do an immediate spin crossmatch immediately, a significant number of tests will fail. A very large study performed in 2005 at Westmead Hospital in Sydney tested 543 patient samples tested against donor units of a range of ABO types. This study looked at the effects of using unwashed vs washed donor cells. This showed that 1.8% of IS XMs failed to detect an ABO mismatch. 75% of these were resolved when washed donor cells were used. This still leaves a failure rate of 0.44%. Further testing also showed that the faster an IS XM is done, the higher the failure rate. They suggested a 2 minute RT incubation helped.

So, if you are doing a few IS XMs and take your time, it works OK but if you are in a hurry, the test is urgent and you do not let the cells and serum stand for a few minutes it is far worse. If you do not wash the cells it fails 4 times as much.

3. I often run into Asian labs who screen with Caucasian screening cells (which means they fail often) and then use an IM XM because it is in the AABB Technical Manual. They actually think it is a real crossmatch because it is called one. They miss many IgG alloantibodies in the antibody screen and then miss them in the crossmatch.

The IS MX is a good test for the right reasons under the right conditions but it cannot be done immediately and it is not a crossmatch. May I suggest we call this test the “Saline phase ABO checkâ€, because that is what it does – and all it can do.

[YorkshireExile]

Tim,

Thanks for your comments about the, erm, saline phase ABO check. I agree its not really a crossmatch, but every piece of literature and guideline calls it that, so I suppose we are stuck with it. I suppose you could say the same thing about the electronic crossmatch. All that basically is is just issuing blood by the computer. What should we call that?

I was interested in the Westmead hospital study. At my last place, before we implemented ISXM, we did an in-house test of 100 patients against various donor types using cells suspended in normal saline, not washed, and with a RT incubation of 2 minutes. Our failure rate was 0%. Every ABO incompatibility was detected. In over ten years of doing ISXM we have not, to the best of my knowledge, missed an ABO incompatibility.

In the Westmead study what did they suspend the cells in - normal saline, EDTA saline, or LISS?

[TimOz]

The Westmead study used serum (not edta plasma), cells at 3-5% suspended in buffered saline. No formal incubation period at all and a maximum of 4 tests performed at a time.

Note that the AABB method is:

Procedure

1. Prepare a 2% to 5% suspension of donor red cells in normal saline or EDTA saline. Some serologists using serum for testing prefer to suspend the donor red cells in EDTA saline because high-titer anti-A or anti-B can initiate complement coating, which can cause steric hindrance of agglutination.

2 The use of a patient’s sample collected in EDTA is an alternative approach to prevent this phenomenon.

2. Label a tube for each donor red cell suspension being tested with the patient’s serum.

3. Add 2 drops of the patient’s serum or plasma to each tube.

4. Add 1 drop of the suspension of donor red cells to the appropriate test tube.

5. Mix the contents of the tube(s) and centrifuge according to the calibration of the centrifuge.

6. Examine the tube(s) for hemolysis, gently resuspend the red cell button(s), and examine for agglutination.

7. Read, interpret, and record test results.

There is no requirement in the procedure for washing of donor cells or any RT incubation time. I think it is one of the problems with the method in that the cells and serum can be in contact for seconds before centrifugation or maybe minutes.

Borocliff. You mention the 2 minute RT incubation. Why? And where did this come from? Did you use washed cells? If you the test is quite reliable BUT you are actually not following the published procedure but a modified (and improved!) version.

By the way. I was in Abu Dhabi 3 weeks ago. Just passing throught the airport but they confiscated my laser pointer!

[Malcolm Needs ☆]

Tim,

Thanks for your comments about the, erm, saline phase ABO check. I agree its not really a crossmatch, but every piece of literature and guideline calls it that, so I suppose we are stuck with it. I suppose you could say the same thing about the electronic crossmatch. All that basically is is just issuing blood by the computer. What should we call that?

I was interested in the Westmead hospital study. At my last place, before we implemented ISXM, we did an in-house test of 100 patients against various donor types using cells suspended in normal saline, not washed, and with a RT incubation of 2 minutes. Our failure rate was 0%. Every ABO incompatibility was detected. In over ten years of doing ISXM we have not, to the best of my knowledge, missed an ABO incompatibility.

In the Westmead study what did they suspend the cells in - normal saline, EDTA saline, or LISS?

That's an easy one; electronic issue (which is what it should have been called from day 1).

[YorkshireExile]

Borocliff. You mention the 2 minute RT incubation. Why? And where did this come from? Did you use washed cells? If you the test is quite reliable BUT you are actually not following the published procedure but a modified (and improved!) version.

By the way. I was in Abu Dhabi 3 weeks ago. Just passing throught the airport but they confiscated my laser pointer!

[Malcolm Needs ☆]

Borocliff. You mention the 2 minute RT incubation. Why? And where did this come from? Did you use washed cells? If you the test is quite reliable BUT you are actually not following the published procedure but a modified (and improved!) version.

As for your airport incident - Did they say why they took it? Maybe they thought it was a lethal weapon:). Seems strange though - normally they are very lazy at customs.

In the UK, we are having reported increasing episodes of laser pointers being shone into the eyes of pilots landing and taking off from airports. This may have something to do with it????????????????