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 Table of Contents  
Year : 2019  |  Volume : 4  |  Issue : 1  |  Page : 96-100

Severe hemolytic crisis due to cold agglutinins associated with Mycoplasma pneumoniae infection that complicated the compatibility tests

1 Lok Samarpan Regional Blood Center, Surat, Gujarat, India
2 Pathology Laboratory, Ayush Multi-Specialty Hospital, Doctor House, Surat, Gujarat, India

Date of Web Publication22-Apr-2019

Correspondence Address:
Dr. Sanmukh R Joshi
Lok Samarpan Regional Blood Center, Surat, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/GJTM.GJTM_29_18

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The cold agglutinins (CAs) associated with Mycoplasma infection may give rise to autoimmune hemolytic anemia (AIHA), and in rare cases, results in severe crises requiring hospitalization. The present case was this kind with severe hemolytic crises due to clinically significant CA that had caused the life-threatening AIHA in association with the infection by Mycoplasma pneumoniae. The blood specimens were collected in the warm environment to obviate spontaneous autoagglutination. Autoimmune nature of the CA involved was established by the direct antiglobulin test (DAT) using reagents obtained from commercial sources. The serological specificity of the CA was ascertained by titration by saline tube test against the red blood cells (RBCs) from the adults and the newborn infants as well as the hemagglutination inhibition study using human milk as a source of the soluble I antigen. The patient's clinical and other details were obtained from the hospital records. A 20-year-old male admitted to the hospital with a severe hemolytic crisis that required blood transfusion. DAT on his RBCs was positive due to the presence of C3d complement fraction, in concurrence with a high-titer CAs with anti-I specificity and positive serological test for M. pneumoniae. A severe hemolytic crisis was evident by sudden drop in hemoglobin, presence of reticulocytes and erythroblasts in circulation. The patient was conservatively treated with antibiotics, steroid, and blood transfusions with improvement in condition and was discharged after 7 days of hospitalization. He was followed up for the next 3 months through which he continued to be in good condition. The investigations showed a rare case of the CA, with anti-I specificity, causing severe hemolytic crisis in association with M. pneumoniae infection that required hospitalization.

Keywords: Anti-I, cold agglutinin, Mycoplasma pneumoniae infection, severe hemolysis

How to cite this article:
Joshi SR, Sheladiya A, Dhanani H, Godiwala P. Severe hemolytic crisis due to cold agglutinins associated with Mycoplasma pneumoniae infection that complicated the compatibility tests. Glob J Transfus Med 2019;4:96-100

How to cite this URL:
Joshi SR, Sheladiya A, Dhanani H, Godiwala P. Severe hemolytic crisis due to cold agglutinins associated with Mycoplasma pneumoniae infection that complicated the compatibility tests. Glob J Transfus Med [serial online] 2019 [cited 2020 Aug 13];4:96-100. Available from: http://www.gjtmonline.com/text.asp?2019/4/1/96/256746

  Introduction Top

The low level of autoantibodies reacting strictly at cold temperatures is present in every individual, the precise role of which is not clear yet.[1] However, the cold agglutinins (CAs) with high titer and with wide thermal amplitude are associated with idiopathic CA disease and may give rise to occlusion of microcapillaries that lead to cyanosis and necrosis of the extremities, the clinical sequel akin to Raynaud's phenomenon.[2] The CA in moderate strength, have also been reported in various viral infections,[3],[4],[5] and with anti-I specificity, often found in association with Mycoplasma pneumoniae infection, the feature was used as the diagnostic tool before the specific serological tests were developed for the infection.[6] The association has seldom caused hemolytic episodes yet on rare occasions, it was implicated as cause for severe hemolysis following the infections.[7],[8] The present case seems to be one of the rare examples of its kind to yield severe hemolytic episode in association with M. pneumoniae infection.

  Materials and Methods Top

Standard serological methods were employed. As the patient's initially collected blood specimen showed gross autoagglutination, the subsequent samplings and processing were done by keeping warm environment. For this, the blood specimen was aspirated using prewarmed syringe, transferred into prewarmed container and incubated at 37°C to avoid spontaneous autoagglutination. The red cells were washed with and suspended in warm saline for blood grouping and direct antiglobulin test (DAT). The serological tests were performed using conventional saline tube method. The immunoglobulin (IgG) type was determined by treating his equal volume of serum with 20 mmol dithiothreitol (DTT 15.48 g/l) in saline at room temperature for 15 m).[2] Antisera, including antiglobulin reagents used were of commercial origin (Tulip diagnostic, Goa, India). The antiglobulin tests were carried out using polyspecific (IgG + C3d) and monospecific antihuman globulin reagents. Specificity of the CAs was determined using RBCs from the adults and the newborns and autologous and papain enzyme-premodified RBCs. Titration of antibody was carried out at different temperatures and the strength of positive reactions were graded as 4+, 3+, 2+, 1+, and weak+ as per size of the hemagglutination clumps. The negative reactions, noted as the absence of hemagglutination, were confirmed through microscopic examination. Antibody titer values were expressed as reciprocal of the highest dilution at which the hemagglutination was visible.[9] Hemagglutination inhibition test was carried out using human milk. For this, one volume of the patient's serum was mixed with equal volume of diluted milk (1:2 in saline) and left at room temperature for 20 min following which an equal volume of 3% saline suspension of RBCs from the adult was added and further incubated at 4°C for 1 h. An absence of hemagglutination was interpreted as the antibody was being inhibited. Normal saline, in place of milk, was used as dilution control in the test. The test for anti-Pr CA was carried out by titration of antibody using untreated and papain enzyme-treated RBCs. The patient's clinical and other details were obtained from the hospital records.

  Results Top

The case

A 20-year-old male was admitted to the primary care hospital with the complaints of abdominal discomfort, breathlessness, and giddiness developed for 2–3 days following the initial symptoms of fever, body ache, and headache for 5–6 days. On admission, his temperature was 100°F; pulse rate was 118/min; blood pressure was within normal range, that is, 110/70 mmHg; respiratory rate was increased as 40/min; and peripheral O2 saturation was 96%. X-ray photograph [Figure 1] showed the chest congestion during the onset of infection and the CT scan revealed a bilateral basal consolidation suggestive of pneumonitis with minimal pleural effusion.
Figure 1: X-ray photograph showing chest congestion during the onset of infection

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The basic investigations showed hemoglobin (Hb) value of 4.4 g/dl as was drastically reduced from 12.6 g/dl that was recorded just 4 days before hospitalization. Total bilirubin level was marginally high (6.8 mg/dL), with direct being 4.9 mg/dL and indirect as 1.9 mg/dL. While ALT value of 33 U/L was well within normal range, the AST level was high (72 U/L). The reticulocyte (Retic) count was 9%, hematocrit value as 12.8%, and with that, the corrected Retic count was derived as 2.3%. Based on these, Retic Production Index (RPI) was worked out as 0.77. A high leukocyte count of 15700/cmm was further raised to 24400/cmm within a day but then on was in a declining mode in the next few days. Malarial parasites were not seen and level of G6PD enzyme was within normal range. BM examination, primarily carried out to rule out underlying possibility of lymphoma, revealed an erythroid hyperplasia with adequate myeloid and megakaryocytic series of cells, the findings that favored the diagnosis of hemolytic anemia. No evidence of parasite, malignant cell deposit, dysplasia, focal increased blasts, or granuloma was seen.

The patient's RBCs in blood specimen were in agglutinated form so was difficult to interpret the results on forward grouping. Also his serum, with a presence of autoantibody, reacting with both A and B RBCs, had misled in confirmation of group by reverse grouping. However, he was grouped A1 RhD positive by forward grouping performed on his RBCs washed with warm saline (40°C) that had corroborated with reverse grouping performed on his autoabsorbed serum carried out at 4°C in three consecutive rounds of absorptions. The autoagglutination feature persisted through his hospitalization while under treatment but not during the follow-up period.

Immunohematological workup showed the DAT as strongly positive (4+) with polyspecific (anti-IgG + C3d) antiglobulin reagent but negative with monospecific anti-IgG. Indirect antiglobulin test was also positive (+3) at 37°C with the polyspecific antiglobulin reagent [Table 1]. The patient's serum showed the presence of CAs with the titer values of 1:256 at 4°C, 1:8 at 22°C, and 1:2 w at 37°C by saline tube method. The CA titer was higher (1:256) against RBCs from adults as compared to those from the newborns (1:8) suggesting its specificity as anti-I. Hemagglutination inhibition test on the antibody using human milk confirmed its specificity as anti-I [Table 1]. Mycoplasma IgM test on the patient's serum by ELISA showed a ratio of 12:1 that was much higher than the cutoff value of 1.1 for the positive results, thus establishing the laboratory diagnosis of the infection.
Table 1: Titer values of the cold agglutinins in the patient's serum at different temperatures [Figure 2]

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The patient was conservatively treated with steroid (solumedrol and methylprednisolone), antibiotics (zostrum, azithromycin, and meropenem) and blood transfusion that brought recovery within a week of hospitalization. He was discharged with an increment of Hb to 9.4 g/dl.

Follow-up study

The patient remained in good health through the follow-up with a gradual rise in hemoglobin values to 13.5, 14.8, and 15.8 g/dl in the next 3 months vis-à -vis the Retic count reducing to normal value of 1.5% [Table 2]. Likewise, the antibody level in serum was also dropped to 1:16. However, the DAT remained positive, with the mixed field appearance after 1 month and much weaker by the 2nd month [Figure 2] and completely negative in the 3rd month. The leukocyte count had remained within normal range through the convalescent period [Table 2].
Table 2: Results on hematological parameters during onset and recovery period of the infection

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Figure 2: Direct antiglobulin test during the follow-up period

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  Discussion Top

The development of CA in association with M. pneumoniae infection is a known phenomenon, the feature was used in diagnosis for the infection before its specific tests were developed.[8] The presence of CA in association with M. pneumoniae infection may not be a specific diagnostic test for the infection as they are detected only in 60% of the patients with the infection. Furthermore, the CAs are found in association with other infectious organisms, such as Epstein–Barr virus, Cytomegalovirus, and Klebsiella pneumoniae besides being found in lymphoproliferative diseases.[8] While most reported cases with M. pneumoniae infection had CA with anti-I specificity, it had seldom caused hemolytic episodes.[6] On rare occasions, however, it gave severe clinical hemolysis requiring hospitalization as was the case in our patient.[7],[8],[10]

Retics are the measure of adequacy of bone marrow erythropoietic activity and generally increased among the patients showing signs of clinical hemolysis, blood loss, or in some other pathological or physiological conditions. In our case, the patient showed higher than normal count of Retic suggesting to a hyperactive bone marrow. However, the RPI is more reliable than Retic percentage because it corrects the degree of anemia as well as increased the peripheral circulation time of immature Retic. A lower RIP in the present case vouch for an inadequate erythropoiesis,[11] evident by a presence of enumerable erythroblasts in peripheral circulation in the face of an acute blood loss in a short period of time and with fever, it did not allow the bone marrow to respond swiftly and adequately.

Marked leukocytosis in our patient, as was observed by others in such cases,[8] may be in response to the ongoing infection or aimed at getting rid of the red cell debris generated through hemolysis. The level of G6PD enzyme was within normal range that ruled out the enzymopathy as a cause of drug-induced acute hemolysis. So also the malarial infection, that is an endemic disease in this part of region, was ruled out by an absence of parasitemia.

Clinical hemolysis in vivo by CA depends on in vitro antibody titer, its thermal amplitude, and its complement-biding nature.[10] However, the cardinal point is the reactivity at warmer temperatures, and not the titer, that correlate well with severity.[12] In the CA disease, the IgM autoantibody combines with antigen on RBC at lower temperatures in the peripheral area in the body, but the complement pathway triggers at warm temperatures in the inner parts of the body that cleaves the C3 complement molecule to C3d. While the antibody molecules dissociate from the RBC surface upon reaching to inner warm environment, the C3d fraction remains tagged to the RBC membrane, thus showing a positive DAT with anti-C3d.[10] In the present case, the DAT was positive with polyspecific (anti-IgG + C3d) antiglobulin reagent, but not with monospecific (anti-IgG) reagent clearly suggested to a complement-mediated event occurred in the patient.

Anemia in CA disease in association with Mycoplasma infection is variable and usually associated with marked pulmonary involvement.[13] In the present case, the CT scan revealed a bilateral basal consolidation suggestive of pneumonitis with minimal pleural effusion that corroborated with the condition.

It is difficult to diagnose the causative agent in this condition. The organism is too fastidious to grow in culture medium. Serological approach, based on specific ELISA kit for M. pneumoniae, is more reliable index for routine laboratory diagnosis,[6] though a variety of tests are developed in recent times that involve molecular technology. In this context, the present case was adjudged to CA disease, secondary to M. pneumoniae infection as it showed an ELISA-positive result.

Most patients of CA disease in conjunction with M. pneumonia infection recover with supportive care.[14] It is imperative to keep the patient warm throughout and to manage for underlying conditions, for example, infection, malignancy, etc.[15],[16],[17] The patients having Mycoplasma infection and treated with appropriate antibiotics showed rapid resolution of the hemolytic process.[14] Multipronged treatment may prove useful in primary CA disease. A patient with corticosteroid-resistant hemolytic crisis was successfully managed by plasmapheresis and cyclophosphamide.[18] However, corticosteroid seems to be useful in treating hemolytic anemia as secondary condition to Mycoplasma infection.[15],[19],[20] So also is the intravenous IgG administration as to control hemolysis in AIHA and the packed red blood cell transfusion to provide oxygen-carrying capacity.[7] However, while administering blood transfusion in patient with CA, one should bring the blood unit to warm temperature to obviate in vivo immunological sequel during transfusion. Our patient was successfully treated with steroids, antibiotics and blood transfusions and was recovered within a week of hospitalization. He improved through follow-up period as was seen with gradual rise in Hb values vis-à -vis reduction in Retic count as well as antibody level. However, the DAT continued to remain positive for 2 months, suggesting that the C3d-coated cells were still present in blood circulation till then, though he did not show any sign and symptom of the disease.

  Conclusion Top

The present case depicts a rare instance of its kind as the CA with anti-I specificity that has caused severe acute hemolytic crisis in association with Mycoplasma infection that required hospitalization. The patient was successfully treated by conservative treatment of antibiotics, steroids, and blood transfusions.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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