Andrea Pointer

Discarding your tips is a "best practice" from molecular assay pipetting techniques (my work history is in molec genetics before I went to BB). It may truly not make a difference on the macro scale for most blood banking, but we opted to retain the practice of fresh tips to avoid any potential carryover.
My facility is a Safety Net hospital with a HUGE OB service line, compete with high-risk preg center, and they use rising titers as a benchmark to determine whether invasive procedures or invasive monitoring of the pregnancy is required. I'm also in the US where maternal mortality is very high, and most of my SOPs have extra protections for pregnant people or people who have the potential to become pregnant.

Discarding your tips is a "best practice" from molecular assay pipetting techniques (my work history is in molec genetics before I went to BB). It may truly not make a difference on the macro scale for most blood banking, but we opted to retain the practice of fresh tips to avoid any potential carryover.
My facility is a Safety Net hospital with a HUGE OB service line, compete with high-risk preg center, and they use rising titers as a benchmark to determine whether invasive procedures or invasive monitoring of the pregnancy is required. I'm also in the US where maternal mortality is very high, and most of my SOPs have extra protections for pregnant people or people who have the potential to become pregnant.

Hi! My facility performs antibody titers in Ortho pre-buffered anti-IgG cards and Ortho workstation using Universal Gel Protocol. I am not permitted to straight up share my procedure per facility rules, but I can copy+paste the language for the actual recipe. Note that we use double-dose antigen cells when possible, and ALWAYS maintain the dose of reagent cell throughout preg. I recommend purchasing Ortho's Panel B to always keep a double K. Even though Kell doesn't dose, until it does 
 
Principle:
Antibody titration is a semi quantitative method of determining antibody concentration. Serial twofold dilutions of plasma are prepared and tested for antibody activity. The reciprocal of the highest dilution of serum or plasma that gives a 1+ reaction is referred to as the titer (i.e. 1 in 128 dilution; titer=128). In pregnancy, antibody titration is performed to identify women with significant levels of antibodies which may lead to HDFN, and, for low-titer antibodies, to establish a baseline for comparison with titers found later in pregnancy. The titer and the antibody specificity (in the absence of more invasive tests) guide the obstetrician’s decision to deliver the fetus to avoid fetal complications.
 
Process:
Prepare doubling dilution.
1. Prepare 11 tubes for the master dilution by labeling with the titer "2 4 8 16 32 64 128 256 512 1024 2048"
2. Add 250 uL saline to each tube using a calibrated pipette.
3. To master dilution tube #2, use a calibrated pipette to add 250 uL patient plasma.
4. Discard the pipette tip.
5. With a clean tip, return to master dilution tube #2 and use the pipette to mix contents. Depress and release the plunger in a slow and controlled manner (do not cause frothing) 8-10 times.
6. Express any residual fluid back into the tube and retrieve 250 uL from that tube to be placed in the next tube (tube #4).
7. After expressing the contents of the pipette into tube #4, discard the pipette tip. 8. Using a clean pipette tip, repeat the mixing step in tube #4, and transfer 250 uL of this mixture to tube #8. Discard the pipette tip. Get a clean pipette tip and continue this pattern until you have mixed the contents of tube 2048.
9. Visually inspect the volumes in the tubes. Tubes 2-1024 should have equal volumes (250 uL). Tube 2048 should have a double volume (500 uL). Perform the gel test
10.Label 2 gel cards with the patient’s identifying info.
11.Label 12 wells as follows "neat 2 4 8 16 32 64 128 256 512 1024 2048"
12.For each well, use a calibrated pipette to add 50µL 0.8% reagent red cell. See above for choosing red cell.
13.In the “neat” well, use a calibrated pipette to add 25µL plasma.
14.In the remaining wells, use a calibrated pipette to add 25µL of the master dilution corresponding to each well’s label.
15.Incubate at 37±2°C for the 15 minutes, but no longer than 40 minutes.
16.Centrifuge the gel cards at the preset conditions of 1032±10 RPMs for 10 minutes.
17.Read the front and the back of each microtube macroscopically and record reactions as described in the interpretation section of the corresponding MTS Gel Card package
 
Results: The titer is reported as the reciprocal of the highest dilution of serum at which 1+ agglutination is observed. A titer ≥64i (anti-D) is considered significant and may warrant monitoring for HDFN by cordocentesis, high-resolution ultra-sound, or examination of the amniotic fluid for bilirubin pigmentation.
 
Notes:
1. Titration studies should be performed upon initial detection of the antibody.
2. When the decision has been made to monitor the pregnancy by an invasive procedure such as amniocentesis, no further titrations are warranted.
3. For antibodies to low-incidence antigens, consider using paternal RBC’s having established that father carries the low-incidence antigen.
4. Failure to obtain the correct result may be cause by a. Incorrect technique, notably, failure to use separate pipette tips for each dilution. b. Failure to mix thawed frozen plasma.
5. The gel technique is more sensitive than earlier traditional tube methods and typically results 2 dilutions higher than the tube method. The historic literature describing the clinical importance of different titers tacitly assumes the tube method, so caution should be used when referencing texts that do not specify “by gel technique”.

Hi! My facility performs antibody titers in Ortho pre-buffered anti-IgG cards and Ortho workstation using Universal Gel Protocol. I am not permitted to straight up share my procedure per facility rules, but I can copy+paste the language for the actual recipe. Note that we use double-dose antigen cells when possible, and ALWAYS maintain the dose of reagent cell throughout preg. I recommend purchasing Ortho's Panel B to always keep a double K. Even though Kell doesn't dose, until it does 
 
Principle:
Antibody titration is a semi quantitative method of determining antibody concentration. Serial twofold dilutions of plasma are prepared and tested for antibody activity. The reciprocal of the highest dilution of serum or plasma that gives a 1+ reaction is referred to as the titer (i.e. 1 in 128 dilution; titer=128). In pregnancy, antibody titration is performed to identify women with significant levels of antibodies which may lead to HDFN, and, for low-titer antibodies, to establish a baseline for comparison with titers found later in pregnancy. The titer and the antibody specificity (in the absence of more invasive tests) guide the obstetrician’s decision to deliver the fetus to avoid fetal complications.
 
Process:
Prepare doubling dilution.
1. Prepare 11 tubes for the master dilution by labeling with the titer "2 4 8 16 32 64 128 256 512 1024 2048"
2. Add 250 uL saline to each tube using a calibrated pipette.
3. To master dilution tube #2, use a calibrated pipette to add 250 uL patient plasma.
4. Discard the pipette tip.
5. With a clean tip, return to master dilution tube #2 and use the pipette to mix contents. Depress and release the plunger in a slow and controlled manner (do not cause frothing) 8-10 times.
6. Express any residual fluid back into the tube and retrieve 250 uL from that tube to be placed in the next tube (tube #4).
7. After expressing the contents of the pipette into tube #4, discard the pipette tip. 8. Using a clean pipette tip, repeat the mixing step in tube #4, and transfer 250 uL of this mixture to tube #8. Discard the pipette tip. Get a clean pipette tip and continue this pattern until you have mixed the contents of tube 2048.
9. Visually inspect the volumes in the tubes. Tubes 2-1024 should have equal volumes (250 uL). Tube 2048 should have a double volume (500 uL). Perform the gel test
10.Label 2 gel cards with the patient’s identifying info.
11.Label 12 wells as follows "neat 2 4 8 16 32 64 128 256 512 1024 2048"
12.For each well, use a calibrated pipette to add 50µL 0.8% reagent red cell. See above for choosing red cell.
13.In the “neat” well, use a calibrated pipette to add 25µL plasma.
14.In the remaining wells, use a calibrated pipette to add 25µL of the master dilution corresponding to each well’s label.
15.Incubate at 37±2°C for the 15 minutes, but no longer than 40 minutes.
16.Centrifuge the gel cards at the preset conditions of 1032±10 RPMs for 10 minutes.
17.Read the front and the back of each microtube macroscopically and record reactions as described in the interpretation section of the corresponding MTS Gel Card package
 
Results: The titer is reported as the reciprocal of the highest dilution of serum at which 1+ agglutination is observed. A titer ≥64i (anti-D) is considered significant and may warrant monitoring for HDFN by cordocentesis, high-resolution ultra-sound, or examination of the amniotic fluid for bilirubin pigmentation.
 
Notes:
1. Titration studies should be performed upon initial detection of the antibody.
2. When the decision has been made to monitor the pregnancy by an invasive procedure such as amniocentesis, no further titrations are warranted.
3. For antibodies to low-incidence antigens, consider using paternal RBC’s having established that father carries the low-incidence antigen.
4. Failure to obtain the correct result may be cause by a. Incorrect technique, notably, failure to use separate pipette tips for each dilution. b. Failure to mix thawed frozen plasma.
5. The gel technique is more sensitive than earlier traditional tube methods and typically results 2 dilutions higher than the tube method. The historic literature describing the clinical importance of different titers tacitly assumes the tube method, so caution should be used when referencing texts that do not specify “by gel technique”.

We are a Level 1 adult and Peds trauma center.....we don't have a limit............
We will give any MTP patient O POS (leuko reduced) LTWB until our supply runs out.  We will give Oneg to peds - but if we run out of Oneg - they will get Opos.  Our facility is currently involved in several studies using WB in the trauma setting.
In the words of our Medical Director and manager..........."They have to live to have a problem"  Might sound crass to many - but, it's true.  For all the patient's we have transfused out of group WB to - we have had VERY FEW delayed reactions - maybe 2 anti-A's in the eluate and a few anti-D's - but all were males.
Our 1st concern is saving the patient.........

Wastage with plates for solid phase depends on what platform you are going to use. With manual testing and the Echo you use antibody screen strips that test 2 patient samples per strip. When I last looked at the Galileo (5 years ago), you could run 4 patient per test plate. That may have changed - I don't know. I also don't know what the Neo uses. Early on with the Echo, you had to use 2 test strips (which would run 4 patients) per run, sacrificing 3 antibody screens if you ran 1 patient. Now if you have a run that is for 1 or 2 patients, you use a balance strip so that large waste factor is gone. With 1 patient runs, you will still lose 1 antibody screen. If you never, ever run more than one patient at a time, you will lose 50% of your total antibody screens.
When cost per test is calculated, what you need to look at is how often you can 'batch' patient samples in runs of 2 (or 4 or whatever Neo uses) to determine waste. That percentage of waste is plugged into the reagent use formula and included in the cost per test figures. The Immucor sales people do that as part of your cost per test calculations (and very happily, too - naturally ;>). We calculated very conservatively and said we would have 40% waste and we still came out money ahead over gel for our contract with Immucor (3 years ago). Our actual useage pattern is better than 40% waste, as we are able to batch many of our runs with non-urgent tests like prenatal antibody screens, pre-surgical testing, next day transfusion patients and the like. So consider your work/patient order patterns when comparing automation methods. That should help make things clearer.
We have been satisfied with our Echo's performance and the technical support we get for it. I was concerned with how far away our service person is - about a 5 hour drive. BUT...we are in a rural area and that is not uncommon for service with any of the companies we do business with for hematology, chemistry, etc. The plus with the Echo is that it has been extremely reliable and we've only needed 1 service call in 3 1/2 years. All the other problems (and there have not been that many of them) were fixed by me with telephone assistance from technical support and a little box of parts that come with the instrument. **Disclaimer: I am definitely not a mechanically adept person, but I get alone with the Echo tinkering just fine.** It was designed for parts replacement by users, very simple replacement. Their capability to 'dial in' to the instrument, just like the technical folks do for the big chem analyzers allows for long distance diagnostics and adjustments to camera and centrifuge operation.
As of the first CAP automated survey of 2011, there were 407 Echos, 520 Provues, 130 Galileos, and 39 Tangos reporting. From the survey performance of each platform, I think you will see that they are all performing within acceptable limits. The switch from interpreting patient results in gel and interpreting patient results in solid phase is NOT difficult. None of my techs had a problem with that. (Note: we were manual gel users, not ProVue users.) We do have increased sensitivity, which is great for detecting alloantibodies in our patient population. We do see a few more warm autos that we did with gel, though that is a trade off I'm willing to make for the ability to catch a few more -Jk-a, Jk-b, Fy-a, Fy-b, and little c antibodies. We do not have an annoying number of non-specific reactions anymore (software upgrades, and a recent instrument adjustment, have helped that issue significantly).
Would I switch from the Echo to an automated gel system? If the ProVue has some new features that improve it's function over the function of the Echo and the money difference is significant...it's a possibility - gel worked fine for us when we were manual users. Am I satisfied with what I have right now with the Echo, functions and $$s...definitely Yes.

I am going to be REALLY unpopular here, but I'm going to say it anyway (because I am a pedant)!!!!!!!!!!!
 
Antigens CANNOT be either heterozygous or homozygous; only genes can be heterozygous or homozygous.
 
An antigen can be described as either showing homozygous expression, or heterozygous expression.
 
That having been said, is a red cell sample that types as K+k- phenotypically, genotypically K/K or K/Ko, or even K/k, with a mutation within the Kell gene that prevents the k antigen being expressed and detected with all anti-k grouping reagents (just in case anyone doesn't believe me - we had one!).
 
That's got that off my chest.
 
Now then, there is NO doubt that there are some anti-K's around that only react with K+k- red cells (dosage), but they are fairly rare, however, how many people use antibody screening red cells that are K+k-?  I doubt if there are any.  Therefore, we are all ruling out anti-K using red cells with apparent K antigen heterozygous expression on every single sample that (apparently) has no atypical alloantibodies present.  Am I wrong about this?
 
It follows, therefore, that, over the years, there MUST have been occasions when a patient with a very weak anti-K (one that is only detected using red cells that are apparently showing homozygous expression) and who has been transfused with K+ blood (do the maths).  As far as I know, there are no papers within the literature that report a case of either a delayed or an acute transfusion reaction as a result of this.  Yes, this may cause the anti-K to become stronger (and, hence, be detectable using an apparent heterozygous red cell sample showing K+k+ expression), but then, if this happens, you give K- blood.
 
So, my considered answer is that you can exclude using K+k+ red cells.
 
I shall now go and lie down!!!!!!!!!!!!!

For those of who works in transfusion service laboratory and would like to learn more reference cases, I can post some mock-up cases here. If you would like me to do it, please hit the "heart" button on this post. If enough folks want to practice case studies on reference lab cases, I can post mock-up cases here weekly or so.. 

We have an ECHO that is also not interfaced and we must print the results and manually enter. We do not keep our printouts for very long. My reasoning is that we download all the results to DVDs that we have kept since the instrument rolled in here. If someone wanted a result from 5 years ago, I could produce it.
I contacted Immucor about getting the info from the DVDs. There is not proprietary software involved in being able to see the results. The DVDs can be put on any computer and read on Notebook, which I tested to make sure. Do you think the DVDs are good enough?
I did start making my people print the daily QC results because I could see an inspector asking for 2 years of QC records. I wanted to be able to place a box in their hands and not have to shuffle thru DVDs.

My experience, though it was a while ago, was that during those events the vitals are being monitored constantly whether transfusion is occurring or not.  The documentation may or may not reflect that but it is being monitored.  Personally I think the blood bank/transfusion service should start being in the loop concerning vitals when the crisis is over and transfusions are slowing down or stopping.  Then the vitals will be come relevant.  Just my 2 cents worth.

Hi all, this is a late response to the post, but I have some valuable info to add. I searched "vital signs" in the forum so it's still a relevant topic to me.
We just transitioned from CAP to TJC (JCAHO) for lab accred (before you ask why or judge, it was a legit reason; I also was hesitant, but it's been good so far!) and learned that CAP doesn't really care. It's all TJC.
TJC follows AABB guidelines for their standards. AABB published in the 19th Ed Technical Manual (the only one I have; not sure if a more recent version would change the info) indicates a pre-transfusion check, within 15 min of starting, during transfusion "at regular intervals", and after transfusion is done is advised, but there is a lack of published study evidence to actually assign the intervals. TJC will hold you accountable to whatever is in your policy and their transfusion tracers will review charted vitals. 
I'm keenly interested in the topic because we are needing to address our policy, which is a pre, 15 min in, 30 min in, hourly from there, and one hour post. Our initial surveyor is an SBB and said that the majority of facilities she had personally inspected were doing pre, 15 in, hourly, then one hour post, and her suggestion was to adopt that model. Anytime there is a lack of distinct guidance, TJC will direct you to check out the industry best practice. My suggestion is to take all this advice and check with your friends in other hospitals. 
I came here to ask about traumas/emergencies/MTPs and how often THOSE vitals should be taken. Like I said, we have to make a change and it was just suggested by an exec MD that we basically publish our policy to say whatever the hemorrhagic emergency, the patient's vitals will be trash until stabilized so we aren't going to check the vitals until the patient's bleeding event is stabilized. Unstable vitals in an unstable situation will not provide direction for care, per the exec's thinking. TJC will only hold you accountable to your own policy on this until further evidence-based best practice is established. Likely never. 

Hi all, this is a late response to the post, but I have some valuable info to add. I searched "vital signs" in the forum so it's still a relevant topic to me.
We just transitioned from CAP to TJC (JCAHO) for lab accred (before you ask why or judge, it was a legit reason; I also was hesitant, but it's been good so far!) and learned that CAP doesn't really care. It's all TJC.
TJC follows AABB guidelines for their standards. AABB published in the 19th Ed Technical Manual (the only one I have; not sure if a more recent version would change the info) indicates a pre-transfusion check, within 15 min of starting, during transfusion "at regular intervals", and after transfusion is done is advised, but there is a lack of published study evidence to actually assign the intervals. TJC will hold you accountable to whatever is in your policy and their transfusion tracers will review charted vitals. 
I'm keenly interested in the topic because we are needing to address our policy, which is a pre, 15 min in, 30 min in, hourly from there, and one hour post. Our initial surveyor is an SBB and said that the majority of facilities she had personally inspected were doing pre, 15 in, hourly, then one hour post, and her suggestion was to adopt that model. Anytime there is a lack of distinct guidance, TJC will direct you to check out the industry best practice. My suggestion is to take all this advice and check with your friends in other hospitals. 
I came here to ask about traumas/emergencies/MTPs and how often THOSE vitals should be taken. Like I said, we have to make a change and it was just suggested by an exec MD that we basically publish our policy to say whatever the hemorrhagic emergency, the patient's vitals will be trash until stabilized so we aren't going to check the vitals until the patient's bleeding event is stabilized. Unstable vitals in an unstable situation will not provide direction for care, per the exec's thinking. TJC will only hold you accountable to your own policy on this until further evidence-based best practice is established. Likely never.