Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 
  • Users Online:166
  • Home
  • Print this page
  • Email this page

 Table of Contents  
Year : 2020  |  Volume : 5  |  Issue : 2  |  Page : 197-201

Immunohematological characterization of direct antiglobulin test-negative autoimmune hemolytic anemia through simple, sensitive methods: Experience from a tertiary care hospital in India

Department of Transfusion Medicine, Apollo Gleneagles Hospitals, Kolkata, West Bengal, India

Date of Submission10-May-2020
Date of Decision12-May-2020
Date of Acceptance12-Sep-2020
Date of Web Publication13-Nov-2020

Correspondence Address:
Sudipta Sekhar Das
Department of Transfusion Medicine, Apollo Gleneagles Hospitals, Kolkata, West Bengal
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/GJTM.GJTM_65_19

Rights and Permissions

Background and Objectives: A negative DAT does not rule out AIHA. The incidence of this clinical entity known as DAT negative AIHA has been reported to be 2-4%. Sensitive techniques like enzyme linked antiglobulin test (ELAT), flow cytometry (FC), complement-fixation antibody consumption test etc. have been described for diagnosis of DAT negative AIHA. Majority of blood bank laboratories lack these advanced methods. Here we share our experience of investigating DAT negative AIHA using simple, sensitive methods which are otherwise less practised. Methods: The prospective study included 377 anemic patients clinically suspected of AIHA. Blood samples received in blood bank were subjected to polyspecific DAT using both conventional tube test (CTT) and column agglutination technique (CAT). Polyspecific DAT negative results were further evaluated using sensitive but simple methods. Hematological and biochemical parameters of all patients were obtained from hospital information system. In vivo hemolysis was categorized as per the criteria established by previous workers. SPSS statistical software (version 13, USA) was used for all statistical evaluation. Results: Of the final 353 clinical AIHA patients evidence of autoimmunization by CTT was observed in 335. Where DAT negative AIHA was observed in 18 (5.1%) patients, 14 showed evidences of autoimmunization by extended sensitive methods. Four patients responded well to AIHA therapy despite DAT negativity by available methods. The mean Hb, Retic, S. Bil and LDH were found to be 6.9 g/dL, 3.25%, 2.8 mg/dL and 512 IU/mL respectively. Conclusion: We conclude that DAT negative patients with clinical suspicion of AIHA and positive laboratory evidences should be evaluated for the presence of autoantibody by alternate sensitive methods which are otherwise less practiced. Blood banks may establish these useful simple techniques and stick to the defined protocols to diagnose DAT negative AIHA.

Keywords: Autoimmune hemolytic anemia, Coombs test, direct antiglobulin test, elution, hemolysis

How to cite this article:
Das SS, Chakrapani A, Bhattacharya S. Immunohematological characterization of direct antiglobulin test-negative autoimmune hemolytic anemia through simple, sensitive methods: Experience from a tertiary care hospital in India. Glob J Transfus Med 2020;5:197-201

How to cite this URL:
Das SS, Chakrapani A, Bhattacharya S. Immunohematological characterization of direct antiglobulin test-negative autoimmune hemolytic anemia through simple, sensitive methods: Experience from a tertiary care hospital in India. Glob J Transfus Med [serial online] 2020 [cited 2021 May 7];5:197-201. Available from: https://www.gjtmonline.com/text.asp?2020/5/2/197/300632

  Introduction Top

In vivo hemolysis with a positive direct antiglobulin test (DAT) is the hallmark for the diagnosis of autoimmune hemolytic anemia.[1] The conventional tube technique (CTT) using polyspecific Coombs reagent is still considered as the gold standard method to investigate DAT due to its high specificity.[1],[2] However, due to low sensitivity of CTT, false-negative DAT may result in patients with clinical suspicion of autoimmune hemolytic anemia (AIHA).[2] The incidence of DAT-negative AIHA has been reported to be 2%–4%.[3],[4] Previous authors have suggested performance of more sensitive tests such as column agglutination technique (CAT), enzyme-linked antiglobulin test (ELAT), flow cytometry (FC), complement-fixation antibody consumption test, immunoradiometric assay (IRMA), and mitogen-stimulated DAT to diagnose DAT-negative AIHA.[5],[6],[7] In addition to these sensitive methods, the authors also described simple techniques such as cold wash DAT, DAT using monospecific (anti- immunoglobulin (Ig) G, anti-IgA, anti-IgM, anti-C3c, and anti-C3d) antisera, DAT using anti-IgG subclass (anti-IgG1, anti-IgG3) antisera, and red cell elution methods to detect DAT-negative AIHA.[8],[9],[10],[11] Despite numerous technical advancement, diagnosis of DAT-negative AIHA is still challenging and needs extensive laboratory investigation. Diagnosis of DAT-negative AIHA only on the basis of clinical response to conventional therapy and excluding other causes of hemolysis has also been described.[9]

Aims and objectives

The objective of this study is to share our experience of investigating DAT-negative AIHA using simple, sensitive methods, so that they can be implemented routinely.

  Materials and Methods Top

Ours being a tertiary hospital with clinical hematology facility, we encounter patients of AIHA frequently. This prospective study that included 377 AIHA suspects was carried over a period of 5 years as a part of immunohematological investigations performed in the hospital blood center.


Due ethical clearance was obtained from the hospital Ethics committee.

Routinely, the treating physicians advise DAT once they suspect AIHA on the basis of clinical and laboratory evidence. The DAT result is reported immediately to the physician. Further DAT evaluation is ordered by the physician to characterize the antibody and classify AIHA. The current study included those patients who were AIHA suspects but were DAT negative by CTT. For these 18 patients, the physician requested elaborate immunohematological investigations using possible sensitive methods. Details of patients and their laboratory parameters were obtained from the patient file and hospital information system. As described by previous workers,in vivo hemolysis was documented when three or more than three of the following four laboratory parameters were deranged.[12]

  • Hemoglobin (Hb) <12.3 g/dL
  • Reticulocyte count (Retic) >2.06%
  • Total serum bilirubin (S.bil) >1.5 mg/dL
  • Serum lactate dehydrogenase (LDH) >220 U/L.

Fresh blood samples with “DAT evaluation” request were sent to the blood center. As per the departmental standard operating procedure, DAT was repeated on the fresh by CTT using polyspecific Coombs antisera (Ortho diagnostics, Johnson and Johnson, USA) followed by CAT using polyspecific Coombs gel cards (BIO-RAD, Cressier s/Morat, Switzerland). Irrespective of the result obtained by polyspecific CAT, the blood center performed extended DAT evaluation that included monospecific (anti-IgG, anti-IgA, anti-IgM, anti-C3c, and anti-C3d) DAT using gel cards (BIO-RAD, Cressier s/Morat, Switzerland), DAT using IgG subclass (anti-IgG1 and IgG3) gel cards (BIO-RAD, Cressier s/Morat, Switzerland), cold acid elution, and cold wash DAT as described before.[10],[11] Briefly, in cold wash DAT patient's red cells were washed with ice cold saline and subjected to DAT using Coombs antisera. None of the DAT-negative patients received any treatment including blood transfusion before being referred to our center.

Statistical analysis

The data were expressed as mean, median, and range using SPSS statistical software (version 13, IBM, Armonk, New York, USA) for statistical calculations.

  Results Top

The current study initially screened 377 clinically suspected AIHA patients. Twenty-four patients were diagnosed as nonimmune hemolytic anemia. Of the remaining 353 patients, DAT was positive by CTT in 335 patients and negative in 18 patients. Of the 18 (5.1%) DAT-negative AIHA patients, red cell bound autoantibody were observed in 14 using sensitive methods. Autoantibody could not be detected in the remaining 4 patients by available methods in the blood center. Once the physicians finally excluded other causes of hemolytic anemia in these 4 patients they initiated specific AIHA therapy. All 4 patients responded well to the therapy. Out of these 18 DAT-negative AIHA, 6 were males and 12 were females (M:F = 1:2). The median age of patients was 37 years (range: 27–51 years).

[Figure 1] describes the serological investigations performed in the current study to diagnose DAT-negative AIHA. Among 335 DAT-positive AIHA patients, 218 (65.1%) were Warm AIHA, 88 (26.3%) Cold AIHA and 29 were Mixed AIHA. Out of 18 DAT-negative AIHA cases, 8 showed DAT positivity by polyspecific CAT with agglutination strength ranging from 1+ to 2+. Two patients showed DAT positivity by monospecific CAT, 1 by IgG subclass CAT with IgG3 specificity, 2 cases were DAT positive by cold wash DAT method and 1 patient was confirmed DAT positive after performing cold acid elution. The remaining 4 DAT-negative patients responded well to AIHA therapy. [Table 1] depicts the clinical, laboratory, and serological characterization of the 18 DAT-negative AIHA patients. Secondary AIHA was observed in 3 patients (16.7%) who had underlying rheumatoid arthritis, and glomerulonephritis. All the 8 polyspecific CAT-positive cases showed IgG specificity in monospecific CAT with IgG1 specificity. While all patients complained of fatigue on routine activity, 5 presented with clinical icterus. The mean Hb, Retic, S. Bil, and LDH were found to be 6.9 g/dL, 3.25%, 2.8 mg/dL, and 512 IU/mL, respectively. In one older female with glomerulonephritis, DAT was only positive (IgG3: 2+) by IgG sublass CAT. Evidence of DAT positivity by elution technique was observed in another female with underlying rheumatoid arthritis. Serum antibody was not observed in any of the 18 patients under study.
Figure 1: Immunohematological methods to diagnose direct antiglobulin test-negative autoimmune hemolytic anemia

Click here to view
Table 1: Clinical, laboratory and serological characterization of direct antiglobulin test.negative AIHA (n=18)

Click here to view

  Discussion Top

Various factors causing DAT-negative AIHA have been described by previous authors. These factors included IgG sensitization below the threshold of detection by the anti IgG reagent or low affinity IgG antibody sensitization or due to IgA or other immunoglobulins coating the red cells.[1],[4],[5],[11] The authors in the current study observed that unavailability of more sensitive techniques such as ELAT, FC, and IRMA in developing countries including India makes diagnosis of DAT-negative AIHA challenging. To an extent, such challenges can be confronted by adopting simple and sensitive methodologies available in blood banks as discussed in the study. Where discussions on high-end sensitive costly methods are immense in the literature, but utility of simple techniques to diagnose DAT-negative AIHA are scarce.

In most cases of DAT-negative AIHA, the number of red blood cells (RBCs) bound IgG molecules are below <400 molecules, which is below the detection level of CTT.[13] More sensitive but simple techniques such as CAT and solid-phase antiglobulin test help in diagnosing these patients. The present study used polyspecific CAT to detect low level of IgG molecules (120–400 molecules/RBC) to confirm DAT-negative AIHA.[13] Eight of the 18 patients who were DAT negative by CTT were positive by polyspecific CAT. All these patients showedin vivo hemolysis as evidenced by the laboratory parameters.

The present study observed the utility of monospecific Coombs technique. Red cells of 2 patients who were otherwise DAT negative by polyspecific CAT were found to be sensitized with IgG or IgG + IgA. Previous authors described AIHA caused by IgA bound red cells. Usually these IgA autoantibodies are warm-reacting and almost always associated with IgG and/or IgM antibodies and may act synergistically with these immunoglobulins and complement in producing red cell destruction.[14],[15]

Interestingly, a 47-year-old female patient, diagnosed with glomerulonephritis and who was DAT negative by both polyspecific and monospecific CAT was found to be positive by IgG subclass CAT. Her red cells were coated with IgG3. Previous workers found that IgG3 antibodies have higher affinity for mononuclear phagocyte receptors as compared to IgG1, IgG2 and IgG4. As low as 230 molecules of IgG3 per red cell can cause monocyte reactions, greater and rapid rosette formation and early initiation of erythrophagocytosis.[16]

Garratty described cold wash DAT in patients who were DAT negative by conventional CTT. The cold wash method was found to prevent dissociation of low affinity autoantibodies.[11] Two patients who were DAT negative by other available methods were positive (1+ to 2+) using cold wash DAT method. Blood bank without CAT facility may establish simple cold wash DAT to diagnose DAT-negative AIHA.

Petz and Garratty detected autoantibodies in elutes of 11 of 27 patients with hemolytic anemia and a negative DAT.[8] The present study used cold acid elution as the last method to confirm DAT status. One female patient with underlying rheumatoid arthritis who was DAT negative by other methods was elute positive. Pan agglutination (1+) with commercial screen panel cells was observed with the elute at 37°C. As elution provides concentrated antibodies, therefore probability of detecting autoimmunization in elutes is higher than autoimmunization evidenced through routine DAT.[17]

Four clinically suspected AIHA patients were DAT negative by available methods. Considering them as DAT-negative AIHA, the physician started specific treatment regimen. All 4 patients responded well to the AIHA therapy. More sensitive method like FC could have detected red cell bound autoantibody in them. Garratty observed that FC can detect as low as 30-40 IgG/RBC and is considered the most sensitive DAT method.[13] Other authors in the past also suggested estimation of RBC-IgG level as a useful tool for diagnosis of DAT-negative AIHA and they opined a cut-off value of 78.5 before treatment.[11],[18],[19]

In the present study 72% of the patients were ≤40 years of age with a female preponderance. In 89% of the cases the presentation was insidious (>12 months). Chaudhary and Das observed occurrence of AIHA more in females than in males; however in children the disease has a male preponderance.[20]

Anemia in AIHA develops when the red cell destructionin vivo is greater than the rate of marrow compensation, which is a gradual process.[1],[21] All patients in the present study presented with symptoms of anemia; however, in secondary AIHA the symptoms of underlying disorders were predominant.

It has been observed that multiple immunoglobulins coating red cells in AIHA more effectively activate classical complement pathway leading to increased hemolysis.[22],[23],[24] In the present study one patient presented with multiple immunoglobulins (IgG + IgA) coating the red cells while the other 9 had single IgG. As reported by Sokol et al. 98% of their AIHA patients had IgG1 as the solitary antibody coating the red cells.[15]

  Conclusions Top

We conclude that patients with clinical suspicion of AIHA but negative for conventional CTT DAT may be evaluated for red cell bound autoantibody by more sensitive but simple methods. We observed that these methods are less cumbersome, less expensive, and easy to establish in any blood center.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Gehrs BC, Friedberg RC. Autoimmune hemolytic anemia. Am J Hematol 2002;69:258-71.  Back to cited text no. 1
Biagi E, Assali G, Rossi F, Jankovic M, Nicolini B, Balduzzi A. A persistent severe autoimmune hemolytic anemia despite apparent direct antiglobulin test negativization. Haematologica 1999;84:1043-5.  Back to cited text no. 2
WAIHA with a negative DAT. Applied Blood Group Serology. 3rd ed. Miami, Florida, USA: Montgomery Scientific Publications; 1985. p. 536-8.  Back to cited text no. 3
Issitt DP, Gutgseu NS. Clinically significant antibodies not detected by routine methods. IN: Nance SJ, editor. Immune Destruction of Red Blood Cells. Arlington: American Association of Blood Banks; 1989. P 93-9.  Back to cited text no. 4
Barcellini W. Pitfalls in the diagnosis of autoimmune haemolytic anemia. Blood Transfus 2015;13:3-5.  Back to cited text no. 5
Bartolmäs T, Salama A. A dual antiglobulin test for the detection of weak or nonagglutinating immunoglobulin M warm autoantibodies. Transfusion 2010;50:1131-4.  Back to cited text no. 6
Barcellini W, Revelli N, Imperiali FG, Villa MA, Manera MC, Paccapelo C, et al. Comparison of traditional methods and mitogen-stimulated direct antiglobulin test for detection of anti-red blood cell autoimmunity. Int J Hematol 2010;91:762-9.  Back to cited text no. 7
Petz LD, Garratty G. immune haemolytic anemias. 2nd ed. Philadelphia: Churchill Livingstone; 2004.  Back to cited text no. 8
Kamesaki T, Toyotsuji T, Kajii E. Characterization of direct antiglobulin test-negative autoimmune hemolytic anemia: A study of 154 cases. Am J Hematol 2013;88:93-6.  Back to cited text no. 9
Bercher ME. The positive direct antiglobulin test and immune mediated red cell destruction. In: American Association of Blood Banks Edition. Technical manual. 15th ed. Bethesda, Maryland: AABB; 2005. p. 453-82.  Back to cited text no. 10
Garratty G. The clinical significance (and insignificance) of red cell bound IgG and complement. In: Wallace ME, Levitt JS, editors. Current Application and Interpretation of the Direct Antiglobulin Test. Arlington: American Association of Blood Banks; 1988. p. 1-16.  Back to cited text no. 11
Wheeler CA, Calhoun L, Blackall DP. Warm reactive autoantibodies: Clinical and serological correlations. Am J Clin Pathol 2004;122:680-5.  Back to cited text no. 12
Stroncek DF, Njoroge JM, Procter JL, Childs RW, Miller J. A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d. Transfus Med 2003;13:35-41.  Back to cited text no. 13
Garratty G, Arndt PA. Applications of flow cytofluorometry to red blood cell immunology. Cytometry 1999;38:259-67.  Back to cited text no. 14
Sokol RJ, Hewitt S, Booker DJ, Bailey A. Red cell autoantibodies, multiple immunoglobulin classes, and autoimmune hemolysis. Transfusion 1990;30:714-7.  Back to cited text no. 15
Duffy TP. Autoimmune hemolyticanemia and paroxysmal nocturnal hemoglobinuria. In: Simon TL, Dzik WH, Synder EL, Stowell CP and Strauss RG, editors. Rossi's Principles of Transfusion Medicine. 3rd ed. Philadelphia, USA: Lippincott Williams and Wilkins Publication; 2002.  Back to cited text no. 16
Schreiber A, Gill FM, Manno CS. Autoimmune hemolytic anemia. In: Nathan D, Oski F, editors. Hematology of Infancy and Childhood. 4th ed. Philadelphia: WB Saunders; 1993. p. 496-510.  Back to cited text no. 17
Kamesaki T, Oyamada T, Omine M, Ozawa K, Kajii E. Cut-off value of red-blood-cell-bound IgG for the diagnosis of Coombs-negative autoimmune hemolytic anemia. Am J Hematol 2009;84:98-101.  Back to cited text no. 18
Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S, et al. Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood 2009;113:3716-25.  Back to cited text no. 19
Sokol RJ, Hewitt S, Booker DJ, Stamps R. Small quantities of erythrocyte bound immunoglobulins and autoimmune haemolysis. J Clin Pathol 1987;40:254-7.  Back to cited text no. 20
Chaudhary RK, Das SS. Autoimmune Haemolytic Anaemia: From Lab to Bedside. Asian J Transfus Sci 2014;8:5-12.  Back to cited text no. 21
[PUBMED]  [Full text]  
Sokol RJ, Booker DJ, Stamps R. The pathology of autoimmune haemolytic anaemia. J Clin Pathol 1992;45:1047-52.  Back to cited text no. 22
Issit P. Serological diagnosis and characterization of causative antibody. In: Hugh Chaplin JR, editors. Methods in Hematology- Immune Hemolytic Anemia. USA: Churchill Livingston; 1985. p. 1-45  Back to cited text no. 23
Gibson J. Autoimmune hemolytic anemia: Current concepts. Aust N Z J Med 1988;18:625-37.  Back to cited text no. 24


  [Figure 1]

  [Table 1]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Materials and Me...
Article Figures
Article Tables

 Article Access Statistics
    PDF Downloaded44    
    Comments [Add]    

Recommend this journal