Case Series
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Discrepancies in Immunohaematological Testing-Importance of Use of Multiple Modalities of Testing |
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Soumee Banerjee, Parimala Puttattaiah, Anupapa Jacob, Sitalakshmi Subramanian 1. 2nd Year Postgraduate Trainee, Department of Transfusion Medicine and Immunohematology, St John’s Medical College, Bangalore, Karnataka, India. 2. Lecturer, Department of Transfusion Medicine and Immunohematology, St John’s Medical College, Bangalore, Karnataka, India. 3. 3rd Year Postgraduate Trainee, Department of Transfusion Medicine and Immunohematology, St John’s Medical College, Bangalore, Karnataka, India. 4. Professor and Head, Department of Transfusion Medicine and Immunohematology, St John’s Medical College, Bangalore, Karnataka, India. |
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Correspondence
Address : Dr. Sitalakshmi Subramanian Department of Transfusion Medicine and Immunohematology, St John's Medical College, Bangalore, Karnataka, India. E-mail: slvbs@yahoo.co.in |
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ABSTRACT | |||||||||||||||||
Two cases are being reported here which shows that in case of immuno-hematological discrepancies, the standard mode of testing should always be supplemented by additional methods to gain an insight into further work-up. Case 1: A 28-year-old multigravida without history of co-morbidities, transfusion or drug intake, showed pan-agglutination with all commercial cells- three reverse grouping cells, Indirect Antiglobulin Test (IAT) and antibody screening on automated and manual gel-card and tube methods. Auto-control and Direct Antiglobulin Test (DAT) were negative on both platforms. Forward grouping was B positive on both platforms. Using in-house cells, automated and manual gel-card and tube methods, IAT was negative and reverse grouping was B positive. Suspecting reagent dependant reactivity and washing the commercial cells resolved all discrepancies on all platforms. Case 2: A 20-year-old lady without any history of any major illness or drug intake came for a routine check-up. Forward grouping showed 4+ reaction with Anti-A and Anti-D on automated and manual gel-card methods, but Anti- B showed double population with multiple commercial and donor-derived anti-B antisera. By tube method, it showed mixed field reaction. Reverse grouping was consistent with AB positive and Coomb’s minor cross-matches with AB positive units were compatible, with B and O units cross-matching was incompatible and with A positive units, it showed double population. DAT was negative and saliva testing confirmed secretor status for A and B antigens. Suspecting AB and A mosaic, cells were agglutinated with anti-B, the agglutinates allowed to settle and free cells re-suspended. This was done repeatedly to get a population of completely free cells that showed A positive on forward grouping. Both patients lacked any significant relevant history and did not want further testing. Counselling was done for them and their physicians about the discrepancies and safety of future transfusions. Hence in discrepant cases, repeat testing with atleast one additional modality is highly recommended as a first step to sculpting out a patient-specific line of management. | |||||||||||||||||
Keywords : Antiglobulin test, Automation, Mosaic | |||||||||||||||||
INTRODUCTION | |||||||||||||||||
Automation has advantages like high through-put, facilities for data storage and retrieval, better standardisation, accuracy and reproducibility (1). However, ancillary methods are required to resolve discrepancies identified by automated testing platforms. In non-automated settings, Column Agglutination Technology (CAT) and Conventional Tube Test (CTT) should be used to supplement discrepant findings in immuno-hematology. Two cases are presented here. Although examples of both cases have been reported previously in the literature, the novelty of these case reports presented lies in the interpretation of the significance of discrepant and similar findings, respectively, on different modalities of immuno-haematological testing. These reports also illustrate a step-wise and patient-specific approach to discrepancies and the importance of prioritisation, that can be particularly relevant in resource limited settings. | |||||||||||||||||
CASE REPORT | |||||||||||||||||
Case 1 A 28-year-old multigravida (G3P3L1A1) presented at 36+3 weeks of gestation with pain in the abdomen. There was no associated co-morbidity, drug or transfusion history. One unit of Packed Red Blood Cells (PRBC) was requested in anticipation of a caesarean section. Investigations Step 1: ABO-Grouping and RhD Typing The forward grouping on our automated platform (IH-500, Biorad, Cressier, Switzerland) with commercial cells (ID-Diacell ABO, Biorad, Cressier, Switzerland) and grouping gelcards (ID-card, DiaClon ABO/D+reverse grouping, Biorad, Cressier, Switzerland) was ‘B Positive’. In reverse grouping, there was panagglutination (4+) with A, B and O cells as seen in (Table/Fig 1). By CTT with inhouse cells, the grouping was ‘B Positive’ as seen in (Table/Fig 2). Step 2: Evaluation of Panagglutination (Indirect Antiglobulin Test and Antibody Screen) Indirect Antiglobulin Test (IAT) with commercial cells (ID-Diacell Pool, Biorad, Cressier, Switzerland) on IH500-positive (4+). With inhouse cells, IAT by manual gelcard (ID card- LISS/Coombs, Biorad, Cressier, Switzerland)-negative. Antibody screen with commercial 3-cell panel (ID-Diacell I-II-III, Biorad, Cressier, Switzerland) by manual gelcard method was panreactive (4+) with negative autocontrol. Step 3: Reaffirming Discrepancy- Interchanging Platforms With commercial cells the reverse grouping and IAT were positive by CTT and manual gelcard methods. With inhouse cells, the reverse grouping showed “B” and the IAT with pooled O cells was negative on IH500. This is highlighted in (Table/Fig 3),(Table/Fig 4). On IH500, The grouping control and DAT were both negative. Step 4: Identifying the Discrepancy Since positive reactions were seen only with commercial cells and not donor cells at the same phase of testing, reagentdependent reactivity was suspected (2),(3). Antibodies have been described against various components of the reagents (4) e.g., bacteriostatic agents. Step 5: Resolving Discrepancy and Establishing Safety of Transfusion The grouping was done on IH500 and ID Card- NaCl, enzyme and cold agglutinins (Biorad, Cressier, Switzerland) and IAT on gelcard with saline washed commercial reagent cells. There was resolution of both issues. The grouping was ‘B Positive’ and IAT was negative. This is shown in (Table/Fig 5). Coomb’s Major cross-matches with 2 B Positive units were compatible both by automated and manual gelcard methods. On repeating the complete work-up with a fresh sample, same results were obtained. Management The patient did not require any transfusion during the current admission. Her treating physicians were informed of this serological issue. Due to her financial constraints, it was decided to limit the work-up to resolving her serological issue and assessing safety of future transfusions rather than further characterising the implicated antibody. She was grouped as “B Positive”. Case 2 A 20-year-old female had asked for a blood grouping as part of a routine check-up. Investigations Step 1: ABO-Grouping and RhD Typing ABO Grouping and RhD typing showed double population with anti-B on DiaClon ABO/D+ reverse grouping (Biorad, Cressier, Switzerland) gelcards in both IH-500 and manual methods. When done by manual tube method, there was mixed field reaction with anti-B antisera (Monoclonal, Eryclone, Tulip Diagnostics, Goa). There was 4+ agglutination with anti-A and anti-D. Reverse grouping was consistent with AB. This is shown in (Table/Fig 6),(Table/Fig 7). Step 2: Confirming the Discrepancy The forward grouping showed the same pattern with antisera from 2 different manufacturers (Combined ABO Monoclonal Antibodies, J.Mitra, New Delhi and Span Clone ABD agglutinating antisera, Span Akrey Healthcare, Surat) and with sera obtained from donors. Step 3: Assessing RBC antigenic and antibody expression patterns Her DAT by manual gelcard was negative. With Anti-AB and Anti-H lectin as highlighted in (Table/Fig 8). Step 4: Documenting Secretor Status Her secretor status by a saliva test was as illustrated in (Table/Fig 9). Step 5: Attempting to Resolve the Discrepancy After a thorough literature search, suspecting a AB Positive and A Positive mosaic (5), agglutination of cells with anti-B antisera was done, followed by separation of the agglutinated cells by repeated sedimentation and resuspension (6). With the free cells, forward grouping by CTT was A Positive. Step 6: Assessing Safety of Transfusion Since this seemed to be a RBC-related issue, to assess the safety of any future transfusions, Coomb’s minor cross-matching with sera from A, B, O and AB donors was tried. There was compatibility with all 3 AB Positive donors and 1 out of 3 A positive donors. With the other 2 A positive donors, there was double population. The findings are summarised in (Table/Fig 10). Management After discussing the findings with the patient it was elicited that she had no significant past history of illness, transfusion or hematopoietic stem cell transplantation. She has two elder siblings and she was unaware of their blood groups. She did not want any further testing on herself or her family. She was grouped as AB Positive and in view of compatibility with “AB Positive” donors she was advised that for future transfusions, AB PRBCs would be safe for her. | |||||||||||||||||
DISCUSSION | |||||||||||||||||
An immuno-haematological work-up ideally should begin with ABO grouping and RhD typing. Many discrepancies can be detected at this step itself. The nature of discrepancy and the results of any attempts made to resolve it can help in deciding the next steps of the work-up. These steps often include- DAT (to detect invivo sensitisation of RBCs by antibodies or complement) and IAT (to detect in-vitro sensitisation of reagent RBCs by antibodies in patient’s serum) (1). Any implicating antibodies thus detected can be identified by antibody screening and identification by multiple cell panels. Two common methods to perform these tests are by CTT with appropriate antisera and pooled cells, where presence/absence of agglutination determines a positive/negative reaction, or gel-based CAT. Here, after a centrifugation step, the gel traps agglutinated RBCs at the top of the column and free RBCs form a cell button at the bottom. Positive and negative reactions are read accordingly. CAT is also available on automated platforms. Antigens of the ABO system are secreted in other body fluids as well, and non-serological tests to detect them, like saliva test can provide helpful supplemental information in resolving immuno-haematological discrepancies. In the first case, the difference in reaction patterns on the two platforms in the same phase prompted consideration of reagentdependant reactivity as a possible cause. This helped rule out other differential diagnoses like antibodies against High Frequency Antigens (HFA) (7). Discrepancies in reactions seen only with commercial cells and not donor cells at the same phase of testing may be due to reagent-dependent reactivity. For the second case, confirming the pattern of agglutination with three modes- automated gelcard, manual gelcard and conventional tube method with different commercial and donorderived antisera helped ascertain the uniformly aberrant pattern of reactivity with anti-B antisera and come-up with possible explanations for the same. Relying on any one modality may have caused attribution of this phenomenon to specific antiserarelated issue or a weak reaction. Performing a simple test like saliva secretor status helped reaffirm the serological findings. Even in resource limited settings, this can prove to be a useful supplement to routine serological tests. Persistent double population with antisera of different manufacturers or donorderived antisera in patients with no history of transfusion or transplant, may indicate a mosaic. All work-up was done as part of our routine protocol of investigating a discrepancy with the ultimate goal of providing safe blood for the patients and were requested by the treating physicians. The patients have given written consent permitting use of their investigation details for publication under the condition of anonymity. However, the antibody in the first case and the suspected mosaic phenotype in the second case required further serological and gene-based testing, respectively, for confirmation. Both patients were, however, unable to afford and unwilling to do any further testing. So the focus was on establishing the safety of future transfusions and counselling them and their physicians accordingly. The biggest challenge for most such discrepancies is that their clinical significance is often ill-defined. They also prolong the time required to provide safe blood products to these patients. So the extent and course of investigation must be tailored to the needs of each specific patient. These logistical issues highlight the need for a high degree of suspicion and a proper investigative protocol in cases of discrepancies encountered in our daily practise so that we know how and when to further evaluate such cases to best suit specific patient requirements. | |||||||||||||||||
CONCLUSION | |||||||||||||||||
Most institutions supplement discrepant findings on one modality with at least one other mode of testing. The cases discussed above highlight the importance of that practice. So, discrepant cases should be approached in a systematic manner and repeating the tests on at least one other modality should be the first step in that approach. Assessing secretor status can also substantiate the findings of routine serological testing. | |||||||||||||||||
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TABLES AND FIGURES | |||||||||||||||||