Options for Pre transfusion Testing – Where Does Tube Testing Fit(Webinar)- Susan T. Johnson

good afternoon my name is Ben Greenfield

I’m the director of marketing at Helmer

scientific I would like to welcome you

to today’s webinar entitled options for

preview transfusion testing

we’re just tube testing fit please feel

free to submit questions during the

webinar using the questions pane we will

have time to answer questions at the

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your information for pace credit I am

very pleased to introduce today’s

speaker sue Johnson sue Johnson is

director of clinical education and the

specialist in blood banking program at

Blood Center of Wisconsin part of the

versity system she’s also director of

the transfusion medicine program at

Marquette University and associate

director of the Indian Amino hematology

initiative at the clinical and the

Clinical Instructor in the College of

Health Sciences at the University of

Wisconsin Madison sue has 25 years of

experience in immuno hematology

reference laboratories and developed

interest in expertise in autoimmune

hemolytic anemia susan shares her

expertise through lectures at both

national and international educational

programs we are thrilled to have sue as

our speaker today without further delay

i’m pleased to turn the presentation

over to sue johnson thank you Thank You

Ben for that kind introduction and thank

you also to Colleen and the group from

Helmer for the invitation to do this

webinar today I always love talking

about pre-transfusion testing and

especially the the tube testing piece of

it right because I you’ll see as I go

through today

that I am still a strong believer that

you need to have a test tube’s available

for for much of your pre-transfusion

testing so objectives for today I’ll be

reviewing the technologies available for

pre-transfusion testing and of course

looking at advantages and limitations of

those different techniques and then talk

about some different scenarios where it

makes sense to utilize test tube methods

I’m a firm believer or I love all the

methodologies available but we have to

be cognizant of you know what they offer

to us and where there might be places

where we need to do a little bit more

testing or problem solving to you know

solve those kinds of problems that we

might run into so let’s start with

Rh typing if we look at it from just a

methodology perspective of course we

have our tried-and-true test tube method

and and we know that it’s quick we can

do it usually in under 10 minutes it’s

you know been the gold standard right

for typing for years we also have

available to us microplate testing now

if we look at our different

methodologies that we have available

some of the automation actually for

example the automation that has solid

phase testing actually utilizes a fluid

based type method which is the

microplate method so the anis area anti

a anti B anti D is added to the cut to

the well instead of a test tube and then

incubated agitated and then red and then

finally we have column agglutination

technology that’s available either gel

or glass beads outside the US and again

really great methodology for detecting

and to the determining type so it’s

great we have options but one of the

challenges is when we have an

discrepancy and I think that’s where

again where I see two testing

absolutely fits in so if we just look

quickly at reviewing an discrepancy

right we all know the standard is that

if the forward doesn’t match the reverse

type where we should see an eighth if we

see an A in the forward type that

reverses should also reverse as an A

meaning that the patient has an T B if

those don’t correlate of course there’s

a discrepancy we need to go into a

problem-solving mode another discrepancy

discrepancy is where our present

type what we just did what we saw in the

testing is not matching what we have in

our history so we have a record now of a

patient that is a no as a no but we just

typed them as an A for example so that

is definitely I would consider a

discrepancy although not by the classic

forward doesn’t equal reverse definition

and finally also I like to classify as a

discrepancy those results that don’t

meet the expected results so for example

when we do typing we like to see you

know three to four plus positive

reactions and if we see only a weak

positive or we see mixed filled

reactions that is important to note

because that gives us information about

the patient and maybe they had a um cell

transplant maybe it’s a subgroup there’s

lots of different scenarios where

unusual reactivity will should flag that

this is a discrepancy of sorts now

that’s I think where the challenge is

for on some of the different

methodologies so for example if we look

at a patient that has mixed field two

cell populations for example a group a

patient who’s received a group all

hematopoietic stem cell transplant or

what we used to call a bone marrow

transplant the instrument might actually

call it as a question mark or an NTD or

no type determined

so what it’s calling us to do right as

the laboratory scientists is to look at

that and and try to determine what is it

right we could visually look at it but I

think that’s difficult to do on that

little micro plate while right so a

place where the tube testing can really

fit in as to in this kind of scenario is

if we repeat the testing where we have

this know type determine or question

mark in the tube we could see oh there’s

nice mix field here there’s a classic in

this case four plus one agglutinate with

lots of free cells in the background and

then it it totally makes sense with our

patient history patient was in a we’re

seeing their a cells but they’re

developing into an old so they’re

starting to produce their own cells and

thus we see that cloudy background so

those are the old cells that are unag

Lewton aidid so classic example of where

to test to method could absolutely show

us that now for you column users you

know also that mix field will also show

very nicely and in a column and a gel

tester or a column gluten ation test

with beads you’ll see the gluten aidid

line in the unincluded native line or

the unincluded cells that fall to the

bottom so i just don’t have an image of

it to show you but you know gel would do

also provide that information for you

another scenario where we might see may

be unusual question mark or no type

determined type reactivity is when we

have missing or weak antigen so for

example here if we see this might be

suspicious they back type as an a/b they

forward type with a questionable

reactivity with an ta i’d be thinking

it’s weak a antigen based on the back

type right so i think the back type is

correct so something is unusual is going

on with the typing with the anti a so

again

if we can go to a test tube method

sometimes all it takes is a little bit

different methodology and when we do a

test tube method of course or

centrifuging and that sometimes it’s

just enough to give you a nice stronger

positive where you feel confident in the

typing that yes this week’ antigen is

actually weak I agree but now it’s

within that realm of Yap this patient

looks like a maybe there’s no question

mark there anymore

and then another place where it can

become helpful and when we look at tubes

versus other methods is one of the

things we know about column

agglutination is that it is not the best

at detecting anti a and a and tbh I mean

we just know that about the method and

so it’s not surprising to see two plus

reactions with X with say for example

b-cells now in this example I’m only

showing a weak positive but two plus is

not that unusual but when you get a weak

positive that makes you question it

right or even the instrument may

question that so we again we know this

to be true and you know gel is wonderful

a column is wonderful for lots of things

but for it’s sometimes they’ll

that’s one of the limitations of the

method and if you go ahead and repeat it

in a test tube method just a regular

test tube method with a cells and B

cells now you see that the B cells are

three plus and we feel much better about

that reaction and we can now say yep my

patient is an A and I really don’t have

an e Bo discrepancy so it’s just another

place again where I think test tubes can

really be valuable I mean in solving

those kinds of issues

and then what about this example here is

another discrepancy and now it looks

like we have what we might say extra

antibody so if I take a look at this

what you can see here is we’re forward

typing as a B and in Reverse typing as

an O and but the thing is is that these

these b-cells are two plus so forward as

a B reverse as at all that means I have

a discrepancy Hobbit’s obviously so what

do I think is correct I usually look at

where I have a clear negative and a

strong positive so I believe this is

correct my patients really a B so my

extra reactivity the extra antibodies

with these b-cells now you look at your

antibody detection tester your

antibodies screen and it was performed

in a either column or solid phase and

it’s negative and then you’re like hmm

okay

it looks like I have extra activity here

but and there’s no reactivity here but

what we have to be remember is that our

antibody detection methods is especially

column or solid phase are designed to

detect IgG antibody it may not detect an

immediate spin antibody that is reacting

unexpectedly with these b-cells so again

if we go to a test tube method that

allows us to look at different phases of

reactivity and you can see now here and

when we run our panel you can see that

there’s nice reactivity at immediate

spin

there’s reactivity at 37° and they know

all the blood bankers they’re like

trying to eyeball out and figure that

out right but what it looks like is you

know a nice anti out because all the m+

cells are positive and the m- cell is

negative so this just is a nice example

to show that the different methodologies

are wonderful for dumb IgG antibodies

but if we have an ABR discrepancy due to

an IgM we may not see it until we go to

using

I test tube method where we can look at

immediate spent to identify that IgM

antibody and then of course you know to

finish off this case if we look at our

forward type as a B reverse type as a oh

we still have this discrepancy so we

have to do something to resolve that we

could pre-warm it warmed the a cells and

the B cells at 30 degrees and then

senator flee and read or we could select

cells that are M negative and verify

that the reactivity is there only with a

with the a cells showing that is what

exactly what we want and that the B

cells are negative so we can see now

that my patient forward type says a are

B excuse me and back type says a B so

now we have our discrepancy resolved and

again all this kind of testing most most

likely it’s going to be done in test

tube methodologies although you could do

this as well as in a column okay so

that’s a B oh one more thing though just

to touch on with is what if there’s

an urgent need for transfusion our

instrumentation the different

methodologies are wonderful but they

take some time there’s a long

centrifugation phase there’s a

processing time so I think here’s

another situation if we don’t have a

historical Rh the test test tube

method for RH typing is is much

faster right again like we can do that

in in ten minutes right versus waiting

for that centrifugation or processing

time so another place where I think

tubes or twos definitely have an

advantage all right on to RH or typing

with aunt Edie

so again most of the time when we’re

doing our testing with different

methodologies it’s not a problem but we

need all know we’re all

challenged by those individuals that

have a weak expression of the RHD

antigen and today I mean there’s four

RHD gene alone there’s greater than 200

alleles that have been identified that

cause weak expression of the Rh D

antigen there’s those individuals that

are weak D types over 135 that those are

generally the people that have a

quantitative difference there’s much

less antigen expressed on the red cells

there’s many different types of partial

DS that have been described now where an

individual is missing an epitope of the

D protein so therefore they can make an

antibody to that and then they’re the

individuals that are very very weak that

probably no methodology will detect but

definitely part of all these different

alleles that can cause weak an

expression of RHD antigen that we’re

trying to detect with our our normal

typing so you know we’ve all seen this

if you work in blood bank transfusion

service at all the frequency is

estimated to be about 2.9 percent among

a mixed population in the u.s. also

meaning if you have a diverse patient

population

you’re gonna detect about 3% or so of

individuals will have this serologic

weak D type if your if your patient

population is mostly of a European

background then it’s probably more

around a half a percent to one percent

but it is enough right to cause

consternation and challenges sometimes

in our typing and if we look at how do

we define or how do we detect these

unusual phenotypes or D RHD phenotypes

is usually by definition that was

defined in by a work group that came

together at the C AP and AABB divided by

dr. Sandler where we defined a serologic

weak D phenotype and that would be an

anti D reagent that gives no or weak

reactive

less than or equal to two plus an

initial testing but agglutinating

moderately or strongly with an ax

globulin reagent

okay so anti-human globulin is you know

most often right applied in that test

tubes or column methodology and the

other place that serologic greek d

phenotype would be detected as if you

have discordant typing results so you

have a history of a patient being rh d

positive for example and now you’re

typing them is d negative by whatever

methodology you’re using and so those

are challenges for us right those are

things that we have to deal with and

again when we look at our different

methodologies available microplate

methodology may call it question mark or

no type determine a gel or column again

questionable or even a less than or

equals a to plus you could consider that

a questionable RHD typing and again if

it was discordant with your your

historical typing it would be another

place where you’d be like ok now what

what do i do okay on this slide just

shows one example of an rh discrepancy

algorithm that I think is used quite

often in one form or another but

basically what it’s saying here is that

if your auntie D is greater than 2 plus

then you’re good and it must matches

your history your reporting your patient

is aren’t reach positive if it’s not

greater than 2 plus there and your

standard test anti D testing your

standard methodology is negative you

repeated it used and you still have

inconclusive results then you would go

on to test with different anti D

reagents definitely add immediate spent

and probably doing an ANA globulin test

it’s tedious to do right and most of us

probably don’t have more than one or two

anti D reagents available but that will

help us in resolving these

kinds of serological discrepancies and

in fact today at last count I have a

student we’re doing a project and there

are actually 17 different anti D

reagents that are available on the

market now summer for gel summer for are

used on different automation auto

automated platforms but there’s a lot of

different anti D reagents and for the

most part most of them can be used in

tube testing as well and in fact um

there’s one paper that was published

actually in May of this year where they

they actually used that to their

advantage to determine when when would

we consider doing our HD genotyping for

example and in this particular paper

then just to point out that they used a

gel NTD and a test tube aunt Edie and so

they looked at this as a clue for one to

do molecular typing and you can see here

they talked about if the gel was to plus

or greater then the test tube result Ora

was less than or equal to two plus with

both methods or less than or equal to

two plus with gel or less than one plus

with test tubes only or there was an

anti D they would actually go ahead to

do our HT genotyping and interestingly

in the 49 patients that they tested

there in forty nine patients they saw 39

partial DS and 10 weak D types so using

both test tubes in jail they were able

to determine those individuals that we

should call Rh positive the weak D types

most likely or Rh negative and treat

them as Rh negative which would be the

partial D types Oh

so again just showing the value of where

test tubes could come into play and of

course all of our the week D tests that

are done along with it okay moving on to

our antibody detection test or antibody

screen as we all call it here’s you know

where there’s actually lots of different

possibilities we all know that our we

want our antibody screen to

detect antibodies to red cell antigens

in an individual in most cases our

patient but also in our blood donors we

know it has to detect IgG antibody that

that’s the standards and it might detect

IgM antibody and we I gave you that

example showing where we had negative

antibodies screen but we detected the IG

eminent in the type and of course

our goal is to detect as many clinically

significant antibodies as possible while

minimizing the detection of

insignificant antibodies always our goal

because we don’t want to get those extra

positives that we don’t know what to do

with so if we look at our different

different methodologies of course our

first it and you know the gold standard

I still think is our test tube methods

course that’s my opinion but we’ve done

lots of things over the years with our

test tube methods right to ensure that

we’re detecting antibodies if our

standard is a two to one two drops a

serum two one drop of cells but we know

we can enhance our test tube method by

adding more drops of serum or plasma we

can incubate our incubation time that we

can increase excuse me increase our

incubation time from say 30 minutes to

60 minutes

we have additives and these there’s

benefits to using all of these different

additives list increases the speed of

antibody uptake so it doesn’t take as

long to do the test peg increases

antibody uptake and also agglutination

in enzymes we all know testing enzyme

treated cells will give us lots of

different hits right as to is the

antigen destroyed to which the antibody

is specific for and we can sort things

out using those and of course when we’re

looking at detecting antibodies we

always want to have red cells that have

a nice double dose or homozygous

expression of the antigen as much as

possible so that we can detect all

antibodies

and you know we all know and of course

we all of these methodologies again we

like tubes because we can read that

immediate spin we can read at 37 degrees

and we also know we’d like to be able to

do our in a globulin testing and of

course you know there’s actually some

wonderful cell washers available now one

pictured here that I’ve had the

opportunity to use and they’re really

they’re really cool compared to the old

like my original cell washer was a green

looked like r2d2 and actually I have the

rotor on my desk as a pencil holder

right now so some of you can visualize

this I know so the new cell washers are

pretty slick and of course it’ll give

you nice results so when we’re doing

tube testing it’s important to remember

then right if we read out all the phases

that this slide just lists all the

antibodies IgM type antibodies that we

would see most likely at immediate spin

a room temperature and remember that

there are antibodies that will react

best or maybe only after the 37-degree

incubation and spend time and that’s

usually IgM type antibodies we’ll go

through that more in a bit and then you

come to your Anna globulin test and of

course you see everything all of that AG

G’s and including anti cd38 or Dara

which is pretty yeah a popular topic

right now but again we can we can see

all these different antibodies reacting

at these different phases now if we move

on to column agglutination as I

mentioned it can be gel or beads just

have two listed or shown here on this

slide one has got three columns and the

other half excuse me had six columns

can’t count the other has eight but

these are nice antibody detection

methods where they utilize this so the

red cells are suspended in a list

solution and then

incorporate it into the screen into the

cards as a anti jeje so these methods

absolutely will detect IgG antibody so

of course we know it’s automatable it’s

a standardized method we don’t have to

wash like we do with our our tube tests

but we do need special incubators and

centrifuges and pipettes and as I

alluded to before there is a long

centrifugation time about 10 minutes for

most of the methods that are available

or I should say exactly 10 minutes

depending on the manufacturers

directions for use there are some unique

other unique characteristics just in

reactivity so cold reactive antibodies

may show that mixed cell or mixed field

agglutination so here is a classic mixed

field type reactivity where you see the

agglutinated cells at the top on

agglutinated cells at the bottom if

you’re doing an type you would say

as an example I showed you you would say

that’s two cell populations it’s an A

and all for example but if you’re doing

an antibody screen you shouldn’t be

seeing mixed field per se by the true

definition there aren’t really two cell

populations and in your screening cells

at least you hope not

so if you see that kind of reactivity

that’s actually characteristic also of a

cold agglutinin type antibody could be

an anti I’m a cold auto antibody and

what you see is that the antibody the

IgM antibody probably binds right away

when you mix the plasma with the red

cells and then during that 37 degree

incubation some of the antibody falls

off and falls to the bottom of the micro

tube so you actually get this kind of

characteristic the gluten ation and it

can give you hints as to what the

antibody is and then vs. rule all which

kind of is more stringy you kind of see

the cells kind of hung up in the column

versus here where it’s a little bit more

clear-cut cells at the top cells at the

bottom

so it’s a nice clue when you see that

kind of reactivity other unique

characteristics we know column

agglutination is wonderful at detecting

an TD there are some other antibodies

that have been reported to be weaker

anti biggie and anti big k and and more

previous days I haven’t heard that as

much now and it’s not not as sensitive

at detecting anti JK and anti JK be

especially as compared to solid phase

methods we know it enhances warm Auto

antibodies and it may not detect the

gluten aiding antibody IgM types and of

course there’s and I say that of course

because we always have that there are

some unidentified positive reactions and

you don’t see him Alice’s as quite as

much and speaking of those unidentified

type reactions one of the classic

reports came out in 2013 by Lou Ann

Grossman where they looked at the

antibodies of undetermined specificity

30 months of antibody detection tests

are over 138 thousand patients and they

found that 18 percent or over 1,400

tests had undetermined specificity so

positive reaction where they weren’t

able to identify an antibody and most of

these were weak to 1 plus and actually

were the most reported reason for a

positive antibody screen and when they

went to the peg tested method they

showed knowing antibody present and

interestingly sub 7 developed into new

antibodies so definitely there’s

antibodies of undetermined specificity

in using gel methodology now as far as

looking at the steerer logic behavior of

antibodies I showed this this this slide

previously looking at test tube methods

and for column it’s similar if you’re

using the buffered card you’d be able to

see the IgM antibodies if you use your

buffer card and incubated at 37 degrees

you’d also see these IgM type antibodies

and as well as of course all the IgG

antibodies now the thing is most of us

don’t use the buffer cards routinely so

you have to remember that again if you

don’t if you aren’t looking you won’t

see some of these IgM antibodies and

most the time that’s fine

we don’t care except for of course like

I showed him that ABR discrepancy

example moving on to solid phase red

cell adherence this methodology is

totally different it’s it’s working with

a micro well wear red cell stroma of the

screening cells is actually a face to

the wall and you really can’t see it

with the bare eye and so what you do is

you add your patient this is a negative

patient antibody in the this column and

you can see that if the antibody is

specific for an antigen on the stroma

it’ll bind and the way that we visualize

it is by adding IgG coded indicator red

cells that will bind to the patient

antibody so a positive the cells are all

effaced and negative all the cells fall

to the bottom the other on solid phase

methodology is one where protein a is

coated on the wall and it’s here we

actually add directly a drop of patient

plasma a drop of the screening cells and

some lists patient antibody will bind to

the red cells you wash the well and a

and T IgG and then again the IgG can

preferentially because of the pH of the

buffer can bind to the protein a and

then the antibody coated cells screening

cells will then bind and again a

positive will be all effaced and the

negative the cells will fall to the

bottom again it’s not available

standardized method but of course you

need special incubator centrifuges etc

absolutely Saliva’s methods are

enhancing an TD been nice reports and I

think almost everybody that is used

solid-phase red cell adherents have seen

an increased detection of an T JK and

an CJK be sensitivity is high for all

antibodies war models of course also are

enhanced unfortunately though there’s a

little bit less variability in a

positive reaction so for those of us

that are used to solving antibodies and

looking at for variability and positive

like ones a one plus and one’s a three

plus you don’t see that quite as much

using the solid phase methods you may

not detect the gluten aiding antibodies

those IgM types probably less so even

than with column you would just wouldn’t

see that but there’s an increased rate

of unidentified positives so of course

with the increased sensitivity also

comes a decrease a bit in specificity

and then the other thing that’s unique

is that few people test the auto control

because you have to prepare your own

monolayer of the patient red cells on

the bottom of the well and now what

about antibodies of undefined

unidentified specificity in solid-phase

red cell in here it’s this is a paper

that that we did in my facility and we

looked at six years of antibody

detection tests so four years of using a

PEG tube method versus two years of

using the solid phase red cell adherence

and unidentified specificity was

reported using PEG tube as well but only

at 1.8 per 10,000 tests versus using

solid phase where it was about it was we

calculated at 20 per 10,000 tests so

definitely more unidentified

specificities now just like with column

we have to remember that we’re only

looking for IgG antibody and so again

these are the antibodies that we would

most likely see in if we’re using a

solid phase mother now when would just

use two tests regardless right when

you’re using gel or solid phase well one

is inconclusive results right we switch

methods we look for a way to

determine is this real reactivity is it

an antibody we need to worry about will

also switch methods when we look where

we see cells that are more the patient

reacts with almost every single panel

cell we test in the method that we’re

using solid phase or gel or our beads or

if there’s an IgM antibody we suspect or

even a newly developing IgM antibody

we’ll look at each of these a little bit

more so one of the things that both

reports the home that I talked to you

about Lulu or Miller they looked at what

about transition of unidentified

reactivity to rely on a body and you can

see interestingly when you compare the

two gel showed 31% of these

unidentifieds transition to an owl or an

auto in the PEG tube method 29%

transition to an alloy an auto and the

solid phase method only 16% which again

shows us that it was a morte it’s a more

sensitive method but also more sensitive

to interfering substances so again

leading us to looking at having to do

some tube testing or another methodology

or Kuby gel gel or solid phase positive

with most red cells again have in most

cases if everything is positive you’re

going to be going to rare cells testing

antigen negative selected cells and you

know two tests are generally were where

we’re using what we’re using for those

kinds of methods although you could

again use column warm Auto antibody you

have a patient that has a strong

positive dat it’s react the panel’s

reactive with everything we all like I

already mentioned both solid phase and

column is good at detecting warm Auto

antibodies so many people in the IRL

like to go to a saline or a list Hana

globulin test because it’s less

sensitive it’s still a good method it’ll

detect a low antibody but it won’t

detect the warm Auto antibody

cold auto antibody kind of similar

except here now it’s reactive with a

strong positive dat may or may not be

positive in your column or gel but we

know that if we do a media it’s been in

test tubes we’re gonna get a good handle

on what’s causing the positive in the

other methodology in anti cd38 or Dera

or the other drugs now there on the

market we there are some labs that are

taking the approach that if it’s

positive in my automation I’m going to

use my test tubes next to see if the

antibodies still reactive again the test

tube method is kind of have them in the

order you’re sailing less pag they’re

least sensitive or less sensitive to the

anti cd38 so you may actually have

negative screen which is great because

then you can still roll out a low

antibodies and then what about those

inconclusive antibody IDs

remember your IgM antibody if it’s there

it’s going to react best at immediate

spin your 37-degree reactive alemana by

could be a newly forming antibody that’s

just transitioning from IgM to IgG and

again this allows us to test different

phases of reactivity so as an example of

course here’s another antibody that

reacts best at immediate spin incubation

it gets a little bit weaker but there is

one positive in the ANA globulin test

and if you look closely at this one this

is consistent with an intp 1 which we

know classically can vary greatly and

reactivity between different donors and

you will get that unusual result but you

wouldn’t see it by doing just an ANA

globulin test so here’s a nice anti p1

that’s reactive and immediate spend in

37 degrees and then as I mentioned

there’s those antibodies that are just

being developed the primary the

patient’s been exposed and we all have

to remember that if the patient’s been

transfused and it’s well then say 10 to

14 days

it could be the primary immune response

and the first antibody that shows up is

the IgM class antibody so if we catch a

patient and we’re doing our testing at

the right time that is where we might

see the best reactivity is right here

when they’re developing the antibody and

what that can look like if we go – we

might see some weak positive that

doesn’t make sense and whatever method

that we’re using but if we go to do a

test tube method and read for gluten

ation at 37°

we might see perfect reactivity and in

this case classically anti Biggie is

famous for doing that the other antibody

that potentially is kind of famous for

doing that is anti BK and again might be

one that we wouldn’t see or wouldn’t be

able to figure out until we test it at

that phase of reactivity okay that’s all

about antibody screens and antibody

detection and we’re test tubes can come

in handy another place is when we’re

doing our cross matching right using

cross match you know most of us use an

immediate spin cross match or an

electronic rest matches probably the

best methodology for a cross match but

you might come across a case where

you’re doing tests to immediate Sven

cross match where you’ve done either

column or solid phase and you have a

negative screen but your cross match

isn’t compatible again we’re testing it

at a different phase of reactivity and

actually we’re looking right in a cross

match for incompatibility so we

should expect to see reactivity at with

a method that’ll detect an IgM antibody

at immediate spin now this incompatible

cross match rate can be Judah and

incompatibility of course we’re all

gonna check that right away but again it

could be due to an IgM antibody could be

an antibody to a low prevalence antigen

it could be real all probably not a

three plus but it’s possible

again we’re not gonna see that though

unless we look at it and in a test tube

method or the donor has a positive dat

least likely least likely with an

immediate spent positive but all could

cause an incompatible cross-match so

just something to remember I’m not going

to do another show you another panel but

just remember that we need to again be

able to do some immediate spin testing

or a method to detect that IgM antibody

to determine why we have that

incompatible cross-match moving on to

our direct in a globulin test another

place where we consider using tubes so

in places that of course normally you

would do an auto control when you do an

antibody identification panel especially

when you’re doing test tube methods and

if you’re switching methods we always

like to use include an auto control

we’ll do a DAT when the physician orders

it it could be generally a column

agglutination test gel test your beads

or a test tube method and if you don’t

have column you’re probably doing a test

tube method and the other place that

we’ll be doing a direct Anna globulin

test and mostly in test tubes is when

we’re doing solid phase antibody ID

panel again I mentioned that we don’t

normally most people don’t run an auto

control just because of having to

prepare that mono layer of cells so

they’ll go to doing a test tube dat or

you could do a column as well but an

alternative way to do the dat and mostly

in test tubes and of course if your dat

is positive we’re gonna have to test our

a Leawood and again if you’re doing a an

old Louis freeze-thaw lu8 where you are

looking for antibody you lyse the

red cells by putting them in the

refrigerator or freezer you freeze the

red cells and then you thaw them slowly

and you’ll ice them and the eliwood is

very hemoglobin stained so it would be

very difficult to read unless you’re

using test tubes and reading

after you wash the cell you do an

indirect anti globulin test so you’re

gonna wash the reagent cells with the Lu

it wash away most of that hemolyzed

eluent

and add anti jeje so you can read in a

test tube method

rapid acid le what test tubes

the preferred method according to

manufacturer’s directions but you can

validate for use in solid phase and

solid phase methodologies but I think

most will use test tube methods if

you’re using or performing a rapid acid

olliewood so in summary we went through

a lot we went through a lot of different

possibilities or opportunities for using

test tube methods we went through the

technologies available for

pre-transfusion testing including the

column agglutination testing the the

solid phase methodologies and not and as

my solid phase methodologies being a

micro plate methodology we went through

all the antibody detection methods

talked about cross hatches and dat s we

looked at the advantages and limitations

of the different methods where you know

we could see yeah these are definitely

this is an advantage or something I can

see that’s unique to this Martha as well

as the limitations to those methods and

it gave you a lot of different scenarios

of where it makes sense to utilize test

tube methods to help either in the

initial testing or when we go to problem

solving it was that I’d like to say

thank you for listening and I’m gonna

turn it back over to Ben for some

questions thank you sue thanks for

sharing your presentation today with our

audience we are now ready to move to

questions and answers as a reminder for

all our participants you can submit your

questions using the questions pane on

your screen if we don’t have time for

all submitted questions we’ll make sure

to follow up after the webinar again

please use the questions pane on your

screen to submit your question

so sue the first question that we have

is my lab uses mostly generalists is

there still a place for to test in the

transfusion services it’s a great

question

so I think I gave a lot of different

scenarios where there is the value in

the test tube methodologies I guess it

would depend on the your patient

population and what you you know what

your general patient population is and

how often you would come into the

scenarios that I talked about I they I

think there’s always value though I know

many labs will especially a small

laboratory where they have a lot of

generalists might send to another

hospital for example in their system to

do additional work but the one thing you

always have to remember is that there’s

always then the turnaround time issue

and if it’s something that can be

quickly resolved with a test to about

that maybe there is that value to have

that and I know a lot of people say but

I have to keep my laboratory scientists

trained and using the test tube methods

and yes you do I believe you do and I am

happy to report that at least all the

schools that I know still teach the

students you know our seniors learning a

test tube methodology so that they’re

ready to be able to troubleshoot problem

solve

thanks to the the next question that

came in is have you dealt with dtt

treatment of anti cd38 drug coded cells

oh yes

well drug quoted cells are testing in

the presence of an OD tt treated okay so

um yes anti cd38 causing trouble for

everybody and in our laboratory we

actually use a ET treated red cells and

it’s validated to test and test tubes so

it works

well I mean it works well I have heard

some that are validating it to lose it

and column agglutination great the the

next question the question is what peg

manufacturer do you use at your lab or

what do you recommend okay so I don’t

usually recommend a manufacturer however

this is the easy question for me because

we actually make our own we prepare peg

in-house and we use its twenty percent

peg solution that’s made actually the

procedures from the judge methods book

so it’s really easy and straightforward

and inexpensive but you have to do the

validation and testing so you know just

to verify that it works all the quality

control to verify that it works

appropriately thank you the next

question is what is your current

recommended recommended protocol for

unspecified antibodies on SP dat

negative yeah good question so based on

the study that I showed you the by

Miller at all the you know my side is

this is now my opinion but it’s also

based on that’s the study is that I

would go to a PEG tube method and see

what if you see any kind of reactivity

at all if the pagan tube method is

negative I I am perfectly fine and I’ve

heard many many people now saying that

that’s a methodology that they feel

comfortable with that if there’s no

antibody there in peg you know we’re not

worrying about it

great let’s do the next question is

between the lists and the peg method is

there one you feel that is more

sensitive or preferred yeah guys are

asking me great questions so I totally

totally 100% prefer peg the reason is

that if you look even at the data and

look at studies and just my personal

experience over 20 years of using it peg

enhances agglutination it’ll take an

antibody that’s say a week positive and

lists or saline and make it a 2 plus I

mean it it will enhance agglutination at

least one reaction grade of true

antibodies just a little background on

that list was designed to and even

though it’s called an enhancement

solution it’s not really in an an

enhancement of agglutination it enhances

antibody uptake because by removing the

the ions from around the red saw it

allows the antibody to get to the red

cell faster but if you go back when this

was first introduced everybody was so

excited about this that and the

manufacturers said you can do this in 10

minute incubation we are all using 30

minute incubation so everybody’s like

yes I want 10 minute incubation so we

all went to 10 or 15-minute incubation

so so that’s what we use but that

doesn’t really enhance the agglutination

reaction whereas peg enhances the

agglutination and you know the in-house

prepared peg is a 15 minute incubation

it’s great or if you use the commercial

peg it’s actually a combination of lists

it’s peg diluted endless actually so you

actually get the benefits of both and

you know I so

my fire peg sorry that was a long answer

but I wanted to explain why I believe

that thank you

we have one more question that came in

it’s a longer question okay when there

is a complex patient population for

example mixes of hemopoietic cell

transplants and sickle cell patients

with multiple antibodies is it always

best to keep the tube method as a backup

and the second part of the question is

what type of methodology would be best

in this situation based on your opinion

okay so yeah complex patient population

as you described the hpc the transplants

that’s what I think you’re gonna see

more problems or you know questionable

NTD types in your typing for example

with an automated platform so there I

think test tubes would be super valuable

to be able to you know just you know

have confidence in the type the

other place with multiple with complex

patients is with the sickle cell

patients and lots of antibodies I think

you wanna have test tubes available to

be able to sort out those antibody

problems like like examples I gave where

you might have a newly developing

antibody an IgM type antibody an auto

antibody many of those multiple

transfused patients with sickle cell

have develop auto antibody on top of

their alloy antibody and you know that

you know one of the automated platforms

probably I’ll just call it all positive

and so you’ll need to back off to back

off a bit insensitivity to a test tube

mother and from a test tube method

perspective I would have peg just

because I know it’s I believe that it

would really give you a nice positive

reaction if it’s a weak antibody that

you’re trying to detect and then

actually I would have the ability to

just do a saline indirect and a globulin

test you know just a no additive

just in case it’s an auto antibody that

you don’t want you don’t want to detect

or you could use lists but if you didn’t

want to have the extra cost you could

just go to a sealing method 30-minute

incubation no additive and then wash and

add your anti-human globulin great well

that was our final question sue so thank

you again for the fantastic presentation

and for all all the answers to the

questions that came in I would like to

thank everyone on the line for

participating in our webinar this

afternoon

and Sue if you could advance to the next

slide slide please share there we go

thank you

helmer scientific welcomes everyone’s

input on this program and we actually

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about our webinar program please feel

free to reach out to me Ben Greenfield

FD Greenfield at homer Inc Tom as I

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based on your feedback this concludes

today’s program thanks again and thanks

to you sue you’re welcome thank you

alright thanks everyone