Uncorrectable red blood cell agglutination, what exactly is the result?

Preface

The phenomenon of red blood cell condensation set, everyone may not be unfamiliar, through the general warm bath can solve more than 90% of the red blood cell agglutination problem, but the case of red blood cell condensation set encountered today is not "cold", ordinary warm bath can not be corrected, so what to do? Let's learn together
.

Case history

One day I was summarizing the quality control of the month, and a teacher came to me and said that there was a patient's condensation bath that could not be treated, let me see, I checked the patient's test results as shown in Figure 1 below

Figure 1

The patient's RBC count is 1.
36×1012/L, HGB 70g/L, HCT 16.
1, experience tells me that RBC and HGB are disproportionate (generally the relationship between the two is about 30 times), MCH and MCHC are abnormally increased, RDW-SD and RDW-CV parameters are not out (indicating abnormal red blood cell histogram), check the red blood cell histogram and alarm information
.
It was found that there was a arched back phenomenon in the right part of the histogram, indicating the presence of a large red blood cell population, and at the same time the alarm message prompted: red blood cell agglutination? Platelet aggregation, Figure 2
below.

Figure 2

So find the specimen, observe the specimen traits, and find that obvious fine sand-like particles can be seen on the tube wall, but there is a certain difference from the condensation collection specimen we usually observe, and this specimen has no obvious wall phenomenon, as shown in Figure 3
below.

Condensation sets in the past

In this case, red blood cells accumulate

Figure 3

So is it a condensation set? We can see under the microscope that the blood smear can indeed see the phenomenon of red blood cell agglutination, as shown in Figure 4

Figure 4

After the sample was bathed at 37°C for 30min, there was not much improvement, red blood cells were still very low, MCV and MCHC were still high, as shown in Figure 5

Figure 5

This result can not be sent ah, suddenly remembered that I read an article on red blood cell agglutination treatment before, bedside sampling immediate detection can solve some problems, so immediately call the clinic, let the patient come to the laboratory window, I personally drew blood, immediately on the machine for testing, I thought that this method could work, but did not expect that the patient's red blood cell agglutination is too tenacious, even bedside testing can not solve the problem
.

So what exactly to do? I suddenly thought of a stupid method, that is, the "gold standard" of RBC counting, the modified abalone counting plate uses a diluent of Herma to count manually, there is no dilution and normal saline instead, but before doing manual counting, you need to replace it with plasma
.

So, we perform plasmapheresis 3-5 times of blood and then count it under a microscope according to manual counting
.
Although RBCs have been replaced and diluted, there are also a small amount of aggregation under the microscope as shown in Figure 6 below, but they are still clearly countable
.
In this way, we count about 256 RBCs in the 5 Chinese squares, RBC=256×5×200×10×106/L=2.
56*1012/L
.

Figure 6

The RBC results are there, and the HGB results are also available, but if you want to get accurate MCV, MCH, MCHC you also need to know HCT, because the HCT of the sysmexXN instrument is the result of the instrument measurement, so we put the replaced blood on the machine again to detect HCT, and the measured HCT is 20.
6
.

MCV=HCT/RBC=20.
6/2.
56=80.
4fl

MCH=HGB/RBC=70/2.
56=27.
3pg

MCHC=HGB/HCT=70/20.
6=339.
8g/L

After such a correction, the three parameters of RBC basically returned to normal, and then smeared to see the picture under the microscope, and the agglutinated red blood cells were not so obvious, and finally the report
was sent according to the corrected results.

Figure 7

Case study

The phenomenon of red blood cell agglutination has a great influence on the results of blood routine, which can lead to serious errors in the results of many parameters, especially the ratio of RBC and HGB is seriously inconsistent, HCT is seriously low, and MCV, MCH, MCHC are abnormally increased
.
However, our work also found that agglutination phenomenon not only affects RBC, but also produces a certain agglutination effect on white blood cells and platelets, and we have found that white blood cells and platelets have also increased
after replacement after repeated testing.

Most of the red blood cell aggregation phenomena encountered in our daily examination work are caused by cold agglutinins, which can be corrected
by a warm bath at 37°C.
Cold set refers to the phenomenon
that red blood cells spontaneously coagulate into clumps when exposed to cold (generally below 30 °C) due to the presence of autoantibodies.

Cold agglutinins are caused by red blood cell autoimmune antibodies, most of which are IgM type antibodies, which are the strongest agglutination at 0~4 °C, but agglutination can disappear
after the temperature rises.
Cold set phenomenon mainly occurs in patients with cold agglutinin disease, but may also occur in patients with mycoplasma infection, certain viral infections, lymphoproliferative disorders, macroglobulinemia, and multiple myeloma [1].

After a long bath at 37°C, red blood cells still accumulated, and the results showed that there were no cold agglutinins in the patient, and the patient may have warm antibody autoimmune hemolytic anemia
.
Through clinical communication, it was learned that the patient did have a history of poverty one year ago, and the patient's immunoglobulin test showed that IgG was abnormally elevated
.
IgG is a warm antibody, and the optimal temperature for reaction with red blood cells is 37 °C, so a warm bath at 37 °C does not solve the erythrocyte agglutination caused by IgG, but may agglutinate agglutination
.

This specimen was detected for the first time MCH54.
5pg, MCHC435g/L, and usually the MCHC caused by condensation set is higher, sometimes up to thousands, because the intensity of IgM antibodies in the agglutination reaction is hundreds of times greater than that of IgG, so the abnormal increase in MCH and MCHC caused by IgG warm antibody red blood cell agglutination is not significantly as the condensation set caused by IgM, therefore, special attention should be paid to the erythrocyte agglutination caused by warm antibody IgG antibody, prevention and treatment of missed detection
。

Clinical studies have shown that the selection of 0.
9% sodium chloride solution for the same amount of plasma combined with incubation can effectively eliminate the influence of high-efficiency cold agglutinin on the results of blood routine test [2].

Therefore, in this example, 0.
9% sodium chloride solution was used to wash and replace the plasma, and then manually counted, and at the same time tested by the machine, and the two were combined with each other to obtain more ideal results
.
Analysis of the results of this study found that the specimen can also obtain the ideal effect after the pre-dilution treatment, but the editor did not do the experiment, and the teachers can also test it themselves if they are
interested.

Summary

To sum up, when we find red blood cell agglutination in our daily work, and after a long time of 37 ° C water bath, we still cannot depolymerize red blood cells, we should consider that it may not be red blood cell condensation phenomenon, or it may be the "warm" agglutination phenomenon in this case, which is characterized by not only no improvement after warm bath, but may even aggravate agglutination
.

At this time, we can try to manually count red blood cells using the plasma exchange method, and then test the replaced blood for HCT or HGB, and manually count the red blood cell parameters, MCV, MCH, MCH, MCHC through the formula to obtain the result
.
In addition, the pre-dilution mode of the instrument can also be used for on-machine testing, and more satisfactory test results
may be obtained.

At the same time, the laboratory should formulate the laboratory's erythrocyte agglutination retest rules according to the re-examination rules recommended by the ICSH International Hematology Standardization Committee to avoid and reduce the missed diagnosis and misreporting of test results, and remark the existence of erythrocyte agglutination phenomenon in the test report, remind doctors to note that erythrocyte agglutination phenomenon is related to some autoimmune diseases, which can assist doctors in diagnosis
.

Therefore, as an inspector should also pay attention to it, when the instrument reports "RBCAgglutination", should be combined with the instrument alarm, scatter plot and histogram and other information, the test results should be comprehensively and meticulously analyzed, to find an effective solution, to ensure the reliability of clinical test results, for timely diagnosis and treatment of patient diseases to provide guidance
.

References

[1] LIU Dan, ZHANG Shu, LEI Ting, et al.
Blood-related indexes and activity analysis of systemic lupus erythematosus[J].
Labeled Immunoassay & Clinic,2019,26(5):741-744.
)

[2] WU Xiaojie,WANG Li,DING Fangfang.
Analysis of red blood cell aggregation in blood routine in patients with systemic lupus erythematosus complicated with autoimmune hemolytic anemia[J].
Current Medicine,2021,27(05):68-70.
)