| Abstract|| |
Autoimmune hemolytic anemias (AIHAs) are not very common, and autoimmunity is an important cause of hemolytic anemia, especially in female population. Some primary AIHAs are cold or warm agglutinin mediated anemia. Autoimmune reaction leads to macrophage activation as well as conversion of normal RBCs into spherocytes leading to their destruction and causing hemolysis leading to anemia. Rarely autoimmune phenomena can lead to agglutination however Cold agglutinin disease or Warm agglutinin disease have specific tests which are diagnostic for them. Here, we highlight a known case of Grave's disease who was admitted to the emergency and on sampling the peripheral blood repeated agglutination/clumping of RBCs occurred on glass surface but nothing happened in the plastic EDTA tube.This to the authors was extremely puzzling phenomena which occurred even on repeat sample for the case,Hence the authors warranted a need to discuss this.
Keywords: Agglutination, agglutinins, Grave's disease, red blood cells, unexplained phenomena
|How to cite this URL:|
Tiwari N, Tiwari AK, Bansal R, Tomar S. A unique case of red blood cell agglutination reaction on glass surface in otherwise normal sample. Asian J Transfus Sci [Epub ahead of print] [cited 2023 Jan 28]. Available from: https://www.ajts.org/preprintarticle.asp?id=363216
| Introduction|| |
Autoimmune hemolytic anemias (AIHAs) are not very common, and autoimmunity is an important cause of hemolytic anemia, especially in female population. Some primary AIHAs are cold or warm agglutinin-mediated anemia. Approximately one-half of AIHAs are idiopathic, while the nonidiopathic etiologies include lymphoproliferative disorders, autoimmune disease, and medication-induced AIHA.
Cold antibody autoimmune anemia (CAD) is characterized by the presence of antibody IgM which is reactive at cold temperatures (3°C–4°C) and leads to immune responses by coating the RBCs. There is mild anemia with reticulocytosis, positive direct Coombs test (DCT), elevated levels of lactate dehydrogenase (LDH), low levels of serum haptoglobin, and high titers of cold agglutinin.,, It can present as chronic CAD or secondary to paraproteinemia such as Waldenstrom's macroglobulinemia, B-cell type malignant lymphoma, systemic lupus erythematosus or transiently in Epstein–Barr virus infection or Mycoplasma pneumoniae infection., The IgM (monoclonal kappa chains) antibodies in cold agglutinin disease are specific for the I-antigen expressed on the surface of RBCs.,, Primary CAD is most often seen in the elderly (range 30–92 years) with an incidence rate of 1 per 1 million people per year and may develop in association with various hematological/immunological diseases.
Warm agglutinin anemia caused by IgG antibodies attaching to the protein–RBC complex at body temperature and the complex is transported to the spleen. This autoimmune reaction leads to macrophage activation as well as conversion of normal RBCs into spherocytes leading to their destruction and causing hemolysis leading to anemia.
Here, we highlight a known case of Grave's disease who was admitted to the emergency with altered sensorium, anemia, and shortness of breath. Her sample that sent for complete blood count (CBC) after 24 h of admission exhibited a strange phenomenon of agglutination of RBCs on glass but not on plastic surface. The sample in Ethylenediamine tetraacetic acid (EDTA) vial was with adequate sample to anticoagulant ratio.
| Case|| |
A 53-year-old female, known case of Grave's disease on the treatment for 8 years, presented with complaints of altered sensorium, weakness fatigue, and difficulty in breathing. Her Thyroid Peroxidase (TPO) (IgG) levels were raised, and Thyroid stimulating Hormone (TSH) levels were reduced on admission, and liver function test was mildly deranged. Her blood group was tested to be B positive.
The patient was admitted to the hospital and a stat CBC sample was sent on January 4, 2022 The CBC parameters were within normal range with reduced hemoglobin levels only. Smears showed normocytic normochromic picture with few microcytic and macrocytic cells. However, the sample repeated on January 6, 2022, and the CBC counter showed bicytopenia (hemoglobin and platelet). On preparing General blood picture (GBP) smears of the same, the sample would agglutinate on glass surface making it difficult to spread. Her coagulation profile was Prothrombin time (PT) of 14.0 s and Activated partial thromboplastin time (APTT) of 30 s (previous outside report).
On inspecting the EDTA vial, the sample was unremarkable. Considering the low ambient temperature in winters, the sample was incubated at 37°C for 30 min. Repeated attempt to make slide yielded similar results [Figure 1], [Figure 2], [Figure 3]. We put a drop of blood in a plastic Petri dish and saw that the blood did not agglutinate in the plastic Petri dish [Figure 5]. This made us rule out a possibility of cold agglutinin disease. The cold agglutinin assay on the same sample was negative. DCT was also negative.
|Figure 2: Slides preincubation (marked as O) and postincubation (marked I) showing clumping of red blood cells|
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|Figure 4: Sample received in EDTA after 48 h for routine processing for complete blood count showed no clumping|
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|Figure 5: Plastic Petri dish showing no clumping in the same sample which shows agglutination on glass [Figure 3]|
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The patient was clinically critical and had a history of transfusion of 1 Packed red blood cells (PRBC) postadmission with no transfusion mismatch event reported. The sample sent to us on January 6, 2022, was not immediately posttransfusion and was collected with all aseptic precautions by trained phlebotomist.
A repeat sample was collected immediately the same day in a fresh EDTA vial and gave same results. Since a definite cause could not be elicited for the case, we repeated the sample for CBC and GBP after 48 h and found that the samples were normal with moderate anemia and a picture similar to the first CBC sample sent on January 4, 2022. Since then, the patient has been on follow-up and is clinically stable [Figure 4] and [Figure 6].
|Figure 6: Sample after 48 h showing no clumping of red blood cells on glass surface|
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| Discussion|| |
The incubation of samples in CAD helps resolve the clumping. These cases may show a negative Coombs test if not in active hemolytic phase. In a case study, it was mentioned that in patients with M. pneumoniae-related CAD, the incidence of patients with a positive DCT was shown to fall with time., However, in our case, no resolution postincubation was seen, and the cold agglutination assay turned out to be negative. Few cases of CAD patient showed associated megaloblastic anemia.,, In our case, there were mild changes; however, no established diagnosis of cold agglutinin disease could be made. There have been cases with reduced complement levels (C3, C4, and CH50) observed in CAD;, however, we did not perform the studies. About 20%–25% of cases of Common variable immunodeficiency (CVID )have associated CAD.
75% of all AIHAs in adults are warm AIHA. Its signs and symptoms are mostly weakness, dyspnea, jaundice, and tachycardia. Laboratory findings of patients with warm AIHA include positive direct antiglobulin testing (Coombs test), spherocytes on peripheral blood smears, reticulocytosis, elevated LDH, indirect hyperbilirubinemia, and decreased haptoglobin. None of the above relevant findings were seen in our case. The first documented case of amoxicillin-induced AIHA was reported in 1985.
An extensive search of literature cannot clearly explain the cause behind this unusual agglutination activity of this patients blood. The case has a history of Grave's disease; hence, we hypothesize that this could have been an idiosyncratic autoimmune event during the patient's hospitalization. There was no marked deranged coagulation profile, so there was no suspicion of Disseminated Intravascular Coagulation (DIC), etc., To the best of our knowledge, our case is the first documented case in which a patient sample agglutinates (not clot) on touching the glass slide not resolving on incubation.
Phosphatidylserine is found in the inner monolayer of the cell membrane. Destruction of the RBC membrane by autoantibodies leads to alteration of RBC membrane organization, leading to thromboembolisms., Another hypothesis is that this type of phenomena could have been taking place in the patient's body at that point in time, leading to activation of alternate complement pathway affecting extrinsic coagulation factors, leading to a supposed increase the zeta potential on the glass surface which would cause the blood to aggregate and clump. A secondary reaction to some drugs which resolved after 48 h could also be there. Chances of some sort of sampling error were minimum since repeat sample was called for.
| Conclusion|| |
We could not find a definite cause of these phenomena; however, this is a truly interesting feature which as per the authors needs to be reported and may be studied upon further to understand the nuances of autoimmune processes going on in our body.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
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Conflicts of interest
There are no conflicts of interest.
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Department of Pathology, Subharti Medical College, Meerut, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]