A frightening case of blood typing results

Preamble

Correct identification of ABO blood group system is the primary prerequisite to ensure the safety of clinical blood transfusion, and it is also the most important blood group system in safe blood transfusion, so many hospitals have listed ABO blood group system identification as the first inspection item
for patients.
However, in actual work, we often encounter the phenomenon of blood type inconsistency due to various physiological or pathological reasons, which requires our staff to be vigilant, carefully find the cause, and verify it in multiple ways, in order to provide patients with an accurate identification result
.

Case history

On a normal working day, near noon, a blood group was done, and the patient was typed according to the usual steps as usual, and after 5 minutes, the centrifugation was completed, and the blood type identification card was taken, and the results were shown
in Figure 1.

Fig.
1 Patient's blood grouping results

From Figure 1, we can see that the patient's blood grouping has inconsistent positive and negative stereotypes, positive as AB/RHD positive and reverse as A/RHD positive
.
In the usual work, I often encounter the phenomenon of inconsistency in the positive and negative stereotyping of blood type, generally in Figure 1, this situation will be considered that the anti-stereotype has agglutination that should not occur, and the patient has been reviewed with the polycoagulamine method
.

Microscopy found that strong agglutination of positive pore anti-A, anti-B, and anti-D appeared, scattered red blood cell agglomeration appeared in the pore of reverse pore A, and large cell agglutination mass
appeared in B cell well.
Looking at the patient's blood sample, it was found that fine sand-like particles appeared on the wall of the tube, and the patient's blood sample was made into a blood smear, and the blood cells were observed to show a large aggregation under the microscope, and the results are shown
in Figure 2-3.

Fig.
2 Appearance of the patient's blood sample

Figure 3 Patient erythroscope below

Since the control hole did not agglutinate, I secretly determined in my heart that there was a certain amount of cold autoantibodies in the patient's body, and the titer of the antibody was not too high, and it was not enough to produce agglutination with standard red blood cells and own red blood cells [1], but only interfered with the anti-stereotype
.
At this time, I was already sure that the patient's blood type was AB, and only the last step was verified
.

50ul of patient serum was pipetted into three test tubes, 50ul standard A cell suspension, B cell suspension and O cell suspension were added to the three test tubes, incubated in a 37 °C water bath, and removed
after 30min.
It was thought that the agglutation of the A and B cell tubes disappeared after incubation, but as shown in Figure 4, there is still a clot
visible to the naked eye in the B tube.

Fig.
4 Anti-stereotype results after incubation

Seeing such a result, I was a little confused at the time, which is the same as the anti-stereotype result of the cassette method, and there is still no progress
after going around in a circle.
How should this patient's blood type be reported? Without resolving the inconsistency between positive and negative stereotypes, type AB must never be reported
.

Since anti-stereotyping can't find flaws, let's start with positive stereotyping, could it be that non-specific agglutination has appeared in the anti-B hole? Some patients with liver disease, infectious diseases, tuberculosis, kala-azar, multiple myeloma, macroglobulinemia, etc.
tend to have increased globulin; Myocardial infarction, infection, trauma, tumor and other diseases can cause fibrin to increase; Some drugs are mainly plasma dilators such as dextroside, povidone, hydroxyethyl starch, etc.
, which are easy to cause the accumulation of red blood cells [2].

The patient's red blood cells were washed three times with normal saline, and 50ul of anti-A, anti-B, and anti-D reagents were added respectively, fully mixed, and observed after a period of reaction, and the results were shown
in Figure 5.
We can see obvious clots in anti-A and anti-D tubes, weak agglutination in anti-B tubes, not very clear to the naked eye, and smaller cell clot clumps
can still be seen under the microscope.

Fig.
5 Positive setting results after normal saline washing

I didn't get the desired result, and this blood sample has been sent for a long time, and the ward will be urged if it doesn't come out of the result, how is this good
.
Click on the patient's electronic medical record, hoping to find clues to answer my doubts
.

The patient was an elderly man who was admitted to the hospital with low back pain and painless hematuria for 3 days, and there were no other special abnormalities
.
I picked up the patient's blood sample again to observe carefully, somehow I just identified the patient as a condensation syndrome, but this time I changed my mind, although the patient's serum can not make the standard O-type red blood cells agglutinate, I still used the test tube method to add 50ul anti-A, anti-B, anti-D reagents to the patient's red blood cell suspension, fully mixed, 37 °C water bath incubation for 30min, and at the same time re-used the blood group card to do positive and negative setting, and put the gel card into a special incubation incubator for 30min and then centrifuged, The results are shown
in Figure 6-7.

Fig.
6 Positive setting results of test tube method after 37°C incubation

Fig.
7 Positive and negative shaping results of cassette method after incubation at 37°C

From the results of incubation, we can see that the positive and negative stereotyping discrepancies are indeed caused by non-specific agglutination of red blood cells caused
by cold agglutinins in the patient.
Condensation hormone has the strongest ability to respond at 4 °C, and loses the ability to aggregate red blood cells at 37 °C, so the pseudoagglutination in the pores of positive B cells disappears after incubation, so that the positive and negative stereotypes are consistent, and the patient's blood group is positive for type A RHD, and the results will be successfully reviewed
.

Case studies

In this case, the blood group is inconsistent, the reason for the inconsistency of the positive and negative is that the cold agglutinin makes the patient's red blood cells appear non-specific agglutination, and the 37 °C water bath makes the cold agglutinin in the patient's body lose its vitality, and finally the blood group is consistent with the positive and negative stereotype, and the correct typing result
is obtained.

Cold aggregation refers to the phenomenon of agglutination of red blood cells in a cryogenic environment caused by autocold antibodies, and the cold hormone usually refers to the IgM
of anti-red blood cells.
In general, healthy people will contain a small amount of cold agglutinins in their serum, but the titer of cold agglutinins in normal human serum is usually very low
.

Cold aggregates are only active below 4 °C, while cold agglutinins usually do not agglutinate at temperatures of 20 °C and above, and when the temperature reaches 37 °C, the agglutinated red blood cells will reversibly and completely disperse without causing non-specific agglutination
.

Cold agglutinins are mainly present in serum, and more interference is anti-stereotyped, and in this case, more cold autoantibodies are absorbed on the red blood cells, and there is obvious agglutination under room temperature conditions, while the control well and anti-setting are not affected
.

For this phenomenon, I personally understand that the titer of cold agglutinins in the patient's body is not high, which can be seen from the above first positive and negative shaping results, the red blood cells in the anti-B well do not appear completely agglutinated, showing the phenomenon of cell stratification, indicating that the red blood cells are not completely sensitized
.

The low-potency cold agglutinins are adsorbed to their own surface by the patient's red blood cells, and there are very few free cold agglutinins in the serum, which is not enough to agglutinate control O cells and standard anti-stereotyped cells, so the control wells show negative, and the anti-training is not affected in any way
.
For this explanation, I have not found favorable evidence to prove it, which teacher knows the reason, please comment in the message area, welcome to criticize and correct!

For this case, I have another doubt, there is no doubt that the patient has cold hormone syndrome, but the patient was admitted to our hospital with a diagnosis of nephritic syndrome with mild glomerular abnormalities
.

We all know that cold aggregates are most common in mycoplasma pneumonia, autoimmune anemia, paroxysmal hemoglobinuria, infectious mononucleosis, leukemia, acute cirrhosis, viral pneumonia, schizophrenia, and advanced maternal age can also cause a short-term increase in cold agglutinins [3], and for the cold aggregates syndrome caused by nephritis I have seen for the first time, is this patient not only nephritis but also other diseases? Is the patient's nephritis caused by an autoimmune disorder?

Since the patient has painless hematuria with the naked eye, under what circumstances does the cause of his hematuria occur, is it paroxysmal hemoglobinuria? All these doubts have not been found in the case, and because the patient has just been admitted to the hospital, the examinations have not been perfected, and the patient's condition will be followed up and communication with the doctor will be strengthened, hoping to solve this confusion
.

The phenomenon of condensation collection will bring great interference to blood group identification, and it is easy to misjudge the positive type as AB type, while the reverse type is easy to misjudge as O type
.
High-efficiency cold agglutinins still have strong agglutination capacity
at 37 °C or even 43 °C.

Its serological characteristics are: the patient's red blood cells are positive for direct anti-globulin test, the patient's own red blood cells and his own serum reagglutinate at room temperature, and the reaction between serum and other people's red blood cells is stronger
than that of his own red blood cells.

There is an essential difference between agglutination caused by cold agglutinin and blood group antigen antibody reaction agglutination, first of all, the former is mostly weak agglutination, the particles are uniform and fine sand, and the latter is a more obvious lumpy agglutination
.
Secondly, after the 37 °C water bath, the former weakened or even disappeared, and the latter showed irreversible agglutination performance [4].

Therefore, when performing blood grouping, laboratory staff need to pay attention to the following aspects: (1) There should be a suitable environment and temperature during the measurement, and the temperature should be maintained at 37 °C as much as possible to eliminate the interference of cold agglutinins on the blood group identification test; (2) At present, when performing blood group identification, a small number of hospitals with poor conditions only carry out positive typing, and when identifying ABO blood types, it is recommended to carry out anti-stereotyping at the same time as positive typing, and mutually confirm each other to make the final judgment; (3) When the result deviation occurs in the positive and reverse shaping process of red blood cells after washing with normal saline, the sample should be heated in a 37 °C water bath for 15-20min and the results
should be observed again.

Summary

Correct identification of ABO blood group is the most important test before transfusion, and incorrect blood typing can lead to severe hemolytic transfusion reactions and even death
.
However, due to the complexity of blood group antigen antibody response and the influence of various physiological and pathological factors, there is often a situation where the positive and negative stereotypes do not match and the blood group cannot be accurately determined
.

Difficult blood type is only a phenomenon in which blood type is difficult to determine, not a type
.
When we encounter a blood type that is difficult to identify, we must first check the patient's information to rule out whether there is a phenomenon of false sampling; And check whether the reagent is expired, whether there is turbidity precipitation and ensure that the experimental environment is stable, and the equipment and instruments are in good
working condition.

In the case of eliminating personal operation errors, it is also necessary to combine the patient's medical history and strengthen communication with the clinic, and comprehensively identify the correct blood type
of the patient based on various factors.
Clinically, some patients often have positive and negative stereotypes caused by pathophysiological factors, such as weakening of red blood cell antigens, reduction or disappearance of blood group antibody titer in plasma, abnormal or irregular antibodies in plasma protein components, allogeneic peripheral blood stem cell transplantation with different AB0 blood types, autoimmune hemolytic anemia, the presence of cold agglutinins and blood group subtypes in plasma
.
When encountering these situations, we must take each specimen seriously, strive to find the cause of the inconsistency of blood type, and ensure that every report issued from us is accurate based on the principle of respecting every life!

References

[1] Zhou Xiangjing, Chen Bile, Xie Zuoing, et al.
Analysis of the causes of positive and negative stereotyping inconsistencies caused by 37 cases of plasma factors[J].
Chongqing Medical Journal,2006,35(5); 452-454

[2] Ma Haifeng.
One case of positive and negative typotyping of blood group caused by high-potency autocold antibody[J].
Journal of Third Military Medical University,2003,25(7):2.
)

[3] National Clinical Laboratory Practice (Second Edition)[M].
Southeast University Press,1997.

[4] Yu Zhongqing, Hu Lihua.
Blood grouping and transfusion therapy of patients with high-potency cold agglutinins[J].
Journal of Clinical Hematology(Blood Transfusion and Laboratory Edition),2008.