Transfusion Medicine, Blood Bank IT, Pathogen Inactivation, Plasma Fractionation, COVID-19 Convalescent Plasma Production
Category: Patient
Includes compatibility testing (AHG, electronic, immediate-spin), antigen typing, antibody screening and identification, direct antiglobulin testing, elution, transfusion reaction and drug reaction workups, component typing tests upon receipt in hospital blood bank, release and return of components
When I was affiliated with National Guard Health Affairs in Riyadh, my staff and I gave weekly new-arrival nursing orientations. The attached PowerPoint file from 2004 shows the manual system in effect at the time, but it is still illustrative to acquaint new staff on the hospital blood bank as it relates to their nursing duties, including specimen collection, pick up and transfusion of blood components, and adverse effects of transfusion.
I wish to give full credit to Mr. Abdullah Al Khashan, who prepared this file in conjunction with Ms. Editha Durante, the Transfusion Clinical Resource Nurse. The three of us used to rotate giving this lecture. This is my version of their presentation (with some minor formatting changes).
I cannot emphasize enough proper technique in doing the washing during the elution process. We are usually concerned about too little washing and thus possibly residual reactions in the last wash. However, aggressive overwashing may remove the bound antibody resulting in a negative result.
Here is an example of anti-PP1Pk (alias anti-Tja). The mother’s panel shows an antibody to a high prevalence/incidence antigen with negative autocontrol and no lability at enzyme phase:
The neonate’s DAT was weak positive at polyspecific and IgG monospecific phases. An eluate was performed. Here is the result after washing four (4) times:
Since 2 cells in the last wash were very weakly positive, the washing was continued for a total of 9 times with the following results:
Even then there was very weak positivity in one cell, but the eluate was negative. We had washed away the attached antibodies.
The direct antiglobulin test DAT may be the first or last place that a delayed hemolytic transfusion reaction can be detected. In my practice, I always performed it the first time I encountered a patient with a positive DAT. If the subsequent DAT testing is of the same strength and there is no change in the clinical status of the patient, then I might empirically repeat the elution after 14 days, i.e. just when new antibody emergence may be detected.
In summary, my criteria for elution after a positive DAT are:
First patient encounter with a positive DAT
At least 14 days since the previous elution
DAT strength has increased since the previous exam (at least 1+ increase in strength in either IgG or C3)
Patient shows evidence of hemolysis
Suspected cases of drug-related hemolysis
Transfusion Medicine physician specifically orders it otherwise
In the software processes, the following parameters were recorded:
Technique of elution (acid glycine, dichloromethane, digitonin, ether, etc.)
Number of washes (too many may remove the antibody from the RBCs)
Last wash result (must be negative to accept conclusion)
Manufacturer of the elution kit (if any)
Elution result based on panel testing
Note:
All indeterminate or positive eluates would trigger an antibody identification.
All results would be reviewed by me or the covering Transfusion Medicine physician. A comment would be entered into the results that could be viewed by the clinician either directly from Medinfo or through the hospital information system.
If the DAT was only positive for complement, it was up to the Transfusion Medicine Consultant to decide whether to proceed with elution. Personally, I would usually proceed because the eluate is more concentrated and may occasionally detect an antibody that was not noted in the patient’s plasma.
The attached Medinfo workflow shows the process designed by me for use at HMC Doha.
A 31 year old Indian female’s prenatal testing results follow:
ABO Group B—unremarkable pattern
Rh(D) positive by both DVI+ and DVI- reagents by multiple manufacturers, both with gel and tube methods
Extended Rh and Kell:
No reactions for C, c, E, e; Kell negative
(These results were confirmed by multiple manufacturer’s tube and gel reagents)
Extended Antigen Typings:
From the phenotypic data, we could already rule out anti-H, k, PP1Pk, U, and some unusual MN system variants found in the region.
DAT: Polyspecific and monospecific IgG, C3b/C3d were all negative.
Antibody Screen: 4+ panreactive
Antibody Panel: 4+ panreactive, autocontrol negative, also 4+ by enzyme
Clinical Course:
At the time of the prenatal specimen, we informed the clinicians that we did not know the significance of the panreactive antibody. We recommended screening any blood relatives and autologous blood collection. We also recommended genotyping of the mother.
Several months later the patient presented in labor with severe fetal hydrops. The previous workup was repeated and confirmed. Neither genotyping or autologous collection had been done. No relatives had the same phenotype, and none were compatible with the mother.
We now knew that the antibody was highly clinically significant and very dangerous. With the permission of the treating obstetrician, the mother’s blood was used for transfusion of the newborn (washed, irradiated). Both mother and baby were group B positive.
To date of my departure from HMC Doha, the antibody had not been characterized, but we continued to recommend genotyping and autologous collection from the mother.
This is a presentation from my time at NGHA Riyadh on a strategy for rare RBC types in the Gulf/Middle East region. This is my practical method for dealing with these antibodies.
As regards prophylactic antigen matching, my remarks are based on the antibodies observed in my region. I know that some would match at least Duffy antigens, but emergence of anti-Fya and anti-Fyb have not been common in my practice.
Based on the previous software post, I use the antiglobulin test algorithms to construct the antibody screen ABS, namely an INDIRECT antiglobulin test, i.e. incubate reagent red cells with the donor or patient’s plasma and then perform the DAT. At HMC Doha and at NGHA in Riyadh, we mainly used three cell screens (no pooled cells for donors). The reactions could be machine-read (we had the interface) but the interpretation of the specificities found was done manually.
Donor ABS:
We screened all donors for irregular antibodies. I disqualified most donor with non-negative screens from donation (my personal preference). However, for nonspecific antibodies, I might elect to discard the current collection and then reassess the donor’s status at the next encounter. Not everyone would agree with this most restrictive approach but I would not use the platelets or plasma in such situations. I also did not use the donor RBCs, even though they were in SAGM—but I could elect to override this decision in Medinfo if there were a rare blood group (e.g. r’r’).
Example #1 (following) shows a sample donor ABS process:
Patient ABS:
Excluding emergency release, if the patient did not qualify for the electronic (computer) crossmatch, then the AHG crossmatch was required. A three-cell antibody screen including R1R1, R2R2, and rr RBCs was used that usually included cells homozygous for the Kell, Duffy, Kidd, MNS antigens. For any case with a non-negative antibody screen, either donor or patient, an antibody identification was performed.
Note that I did not use any other software to make antibody identifications. I required my technologists to be proficient in identifying basic and intermediate-level testing. The actual antigen makeup of each lot number of antibody screen or identification panel was NOT stored in Medinfo. We scanned the antibody screen and panel workups and could store those in Medinfo.
Example #2 (following) shows a sample patient ABS process:
Patient ABS Fix for Interface Regression Caused by HIS:
Regretfully, with a software update from the hospital information system HIS vendor, a major regression occurred. It would not discard results from Medinfo if more than one antibody result (e.g. anti-E and anti-c) was sent back. We could see the results in Medinfo, but those outside Transfusion Medicine could not see antibody results. The entire interface for antibody identification had to be rewritten so that the HIS would see each result uniquely and thus all results could be sent back to it.
Regression Example:
Anti-Kell sent from Medinfo: one antibody result, HIS would store and show this result.
Anti-E and anti-c sent from Medinfo: No antibody results shown, both results kicked out.
Regression Fix:
Anti-E sent as Method Type1-Antibody 1 and anti-c sent as Method Type 1-Antibody 2: both results displayed in HIS.
This presentation is from my time at National Guard Health Affairs Riyadh and suggests an algorithm to assess the clinical significance of the reaction. In my opinion, the essence of immunohematology testing is the antiglobulin test so I would spend much time with technical and medical staff, including trainees/fellows (even those not based in transfusion medicine) to make certain they understood how to interpret it.
There are many variations for this most important test. In the very least, this consists of:
Screening polyspecific AHG for detecting IgG and/or complement
If positive, reflex monospecific tests to distinguish IgG from C3 reactivity
There are many kinds of reagents:
Immunoglobulin: whole molecule IgG, monospecific gamma heavy chain, monospecific alpha heavy chain, monospecific mu heavy chain
Complement: C3d, C3c, C3b, C5, etc
Regretfully, some of the monospecific are not really monospecific (e.g. whole molecule IgG detects both heavy and light chains and thus may detect IgG and give weaker results for IgM or even IgA.)
In 1984 effective with the 13th Edition AABB Standards, the requirements for performing a direct antiglobulin test and autocontrol for compatibility testing were eliminated. The DAT is very important to detect delayed hemolytic transfusion reactions, certain autoimmune conditions, and drug-related hemolysis.
Since that time, the immediate-spin crossmatch and now the electronic computer paperless crossmatch may be used for most compatibility testing in place of the classic, antiglobulin-phase (indirect antiglobulin test) crossmatch.
If an antiglobulin phase (indirect antiglobulin test–IAT) crossmatch is performed, a donor unit with a positive DAT will cause a false-positive reaction. Since most crossmatching does not include the IAT, it will not be affected by the DAT status of a donor unit.
Policy:
Donor RBC units will NOT be routinely tested for DAT as part of component processing.
The type of compatibility testing selected for a particular patient should be the technically simplest one (no need to do extra work unless so instructed by the transfusion medicine consultant/designate):
Do a full antiglobulin-phase IAT crossmatch if ANY of the following applies:
There are no two independent ABO/D typings on the patient during the current admission.
The ABO/D type of the current admission does not match the historical information.
The patient has a detectable antibody at 37C
The patient has a history of a clinically significant antibody but no current antibody
Whenever the consultant, transfusion medicine/designate requests it.
Whenever the Medinfo HIIG record so indicates (in comment section)
Do the immediate-spin crossmatch if ALL of the following apply:
Only one determination of the ABO/D type
The historical ABO/D type agrees with the current type.
There are no antibodies reacting at 37C AND there is no history of antibodies at 37C.
Use the computer/electronic crossmatch if ALL of the following apply:
There are two determinations of the ABO/D type and they both agree with each other.
The historical ABO/D type agrees with the current type.
There are no antibodies reacting at 37C AND there is no history of antibodies at 37C.
When to do a DAT on a donor unit:
Patient antibody screen is negative but the full AHG crossmatch is incompatible.
Part of a transfusion reaction workup where the AHG crossmatch of donor cells and patient serum is incompatible.
Whenever the consultant, transfusion medicine/designate requests it.
If a donor unit is found with a positive DAT, perform:
Do polyspecific and monospecific (IgG and C3) antisera
Perform an acid-elution.
Send the results to the transfusion medicine consultant/designate for review.
The reviewer will enter his review in the Medinfo HIIG in the Donor Consultation Section both as global donor comment and a result-specific comment against the antibody screen result.
Use of the DAT-positive donor unit:
Select another RBC unit for the transfusion.
The final decision to use the unit will be made by the Transfusion Medicine consultant/designate.
Important: Don’t do a classic AHG/IAT phase crossmatch unless you have to do it (see conditions above.) A donor unit with a DAT is unlikely to be clinically significant and may be transfused safely to the patient in most situations. Patients receiving electronic-crossmatch and immediate-spin crossmatch are receiving units with positive DAT without incident.
References:
Standards for Blood Banks and Transfusion Services, Current Edition, AABB, Bethesda, MD, USA
Guidelines to the Preparation, Use, and Quality Assurance of Blood Components, European Committee (Partial Agreement) on Blood Transfusion (CD-P-TS), Current Edition
Technical Manual, Current Edition, AABB, Bethesda, MD, USA
This is a presentation I gave while working as Head of Transfusion Medicine for Saudi Arabian National Guard Affairs, King Abdulaziz Medical City at Riyadh. I used this strategy there and later at HMC Doha.
Fortunately we had access to rare antisera such as anti-Tja (anti-PP1Pk) which we could purchase from Diamed AG and its successor Biorad. I emphasize that we only sparingly used these rare antisera AFTER ruling out more common high-incidence antibodies.
Dr. Zeyd Merenkov 