Asian Journal of Transfusion Science
Home About Journal Editorial Board Search Current Issue Ahead of print Back Issues Instructions Subscribe Login  Users: 1965 Print this page  Email this page Small font sizeDefault font sizeIncrease font size 

Previous Article  Table of Contents  Next Article  
Ahead of print publication
A method for selecting P antigen-negative red cell units for patients with paroxysmal cold hemoglobinuria

 Blood Bank, District General Hospital, Monaragala, Sri Lanka

Click here for correspondence address and email

Date of Submission28-Jun-2019
Date of Decision05-Apr-2020
Date of Acceptance20-Jun-2020
Date of Web Publication12-Dec-2022


In the absence of specific antisera and molecular typing technique, selection of P antigen-negative red cell unit is a challenge. This article explains a new technique that can be used in emergency situations to screen P antigen-negative red cell unit for the transfusion of patients with paroxysmal cold hemoglobinuria tested positive for Donath-Landsteiner antibody. The technique is based on the theory behind the Donath-Landsteiner test, and it can be performed even in a transfusion laboratory with minimum facilities.

Keywords: blood transfusion, P antigen-negative blood, paroxysmal cold hemoglobinuria

How to cite this URL:
Manchanayake GS. A method for selecting P antigen-negative red cell units for patients with paroxysmal cold hemoglobinuria. Asian J Transfus Sci [Epub ahead of print] [cited 2023 Jan 28]. Available from:

   Introduction Top

Paroxysmal cold hemoglobinuria (PCH) is a form of immune hemolytic anemia mediated by biphasic IgG autoantibodies directed against erythrocyte P antigens. These antibodies trigger complement-mediated intravascular hemolysis. The biphasic nature of the antibody can be demonstrated by Donath-Landsteiner test.[1]

Although most cases of PCH are acute and self-limiting, some patients need blood transfusions during acute phase of illness due to severe and rapidly progressive anemia. A patient with PCH was investigated for developing episodes of acute intravascular hemolysis following blood transfusions, for which P antigen-negative red cell units were indicated. Due to lack of facilities, P antigen-negative blood pack selection was a challenge, and ultimately, a technique was developed using the theory behind Donath-Landsteiner test.

   Case Report Top

A child admitted with fever, loose stools, and dark color urine was diagnosed with PCH after having positive Donath-Landsteiner test. Since hemoglobin level was 4 g/dL, three blood units was ordered. Her blood group was A Rh D-positive and antibody screening was negative. The patient was transfused with group-specific cross-match–compatible blood in three occasions using a blood warmer. Following each blood transfusion, she developed episodes of hemoglobinuria.

Transfusion reaction investigations excluded the presence of erythrocyte alloantibodies and ABO incompatibility of the patient with transfused units. Therefore, the exacerbations of hemolysis following blood transfusions were considered as triggered by Donath-Landsteiner antibody present in the patient's serum. P antigen-negative red cell transfusion is an option for such situations to avoid further hemolysis.[1],[2] However, with the absence of specific antisera and molecular typing methods, selecting this type of blood unit was a challenge.

In Donath-Landsteiner test, Donath-Landsteiner antigen (probably P specificity)-positive red cells are hemolyzed by Donath-Landsteiner antibody in the patient's serum. The hemolysis is detected in the supernatant compared to the color of the patient's serum. I tried to apply the same principle to test the P antigen status of donor red cell packs. When a set of group-specific donor red cell suspensions are mixed in 1:9 ratio with the patient's serum (containing Donath-Landsteiner antibody) and incubated at 0°C followed by 37°C, supernatant of the tubes containing P-positive red cells should appear darker than the patient's serum due to the hemolysis. Necessarily, the tubes without a color change following incubation need to carry P-negative red cells.

Ideally, positive and negative controls should be run in parallel to the test using P-positive and P-negative red cells against the Donath-Landsteiner antibody-positive serum. Since P-negative red cells were not available, only test control was performed as follows. In positive control, P-positive red cells are mixed in 1:9 ratio with the patient's serum expecting hemolysis and a color change of the supernatant following antigen-antibody reaction. In negative control tube, P-positive cells are reacted with fresh serum of a healthy person with the same blood group. Its supernatant color should be similar to the patient's serum. P-positive red cells can be prepared by pooling three randomly selected group-specific blood units.

Procedure was performed as follows.

  • Group A-positive ten blood packs were selected, and 50% red cell suspensions were made from each (P1, P2, P3,…)
  • Ten clean test tubes were taken and labeled (T1–T10)
  • One volume of P1 cell suspension was added to T1 test tube and followed the same procedure for the rest of cell suspensions (e.g., from P2 to T2, P3 to T3,…)
  • Then, nine volumes of 37°C separated patient's serum was added to each T1–T10 tubes
  • Positive control – Nine volumes of 37°C separated patient's serum was added to one volume of 50% cell suspension prepared from pooling three group A blood units
  • Negative control – Nine volumes of 37°C separated fresh serum of a group A healthy person was added to one volume of 50% cell suspension prepared from pooling three group A blood units
  • T1–T10 and control tubes were incubated at 0°C in crushed ice for 1 h and then immediately transferred to water bath at 37°C. After 30 min incubation period, all tubes were centrifuged under low spin for 15 s, and the supernatants were inspected compared to the patient's serum color.

Test results and interpretation

  • The supernatant color of the positive control was darker compared to the patient's serum, while in the negative control, color was similar to the patient's serum
  • Enhanced hemolysis was evident in nine out of ten testing samples. Supernatant of the tenth tube did not show color change compared to the patient's serum, indicating the negativity for P antigen in the red cells of that specific donor
  • Whole test was repeated, and the results were confirmed.

   Discussion Top

This patient had severe anemia requiring blood transfusions. The interesting point pertaining to this case was the aggravation of hemolysis following blood transfusions due to Donath-Landsteiner antibodies. Although P-negative blood transfusion is an option in such situations, its selection was not widely used due to lack of facilities. The method I described can be performed even in a laboratory with minimum facilities.

One limitation of this technique is the subjective nature of result interpretation; thus, trained technicians are required to carry out this procedure. In addition to that, the technique can only be carried out for patients with positive Donath-Landsteiner test. The frequency of P antigen in Sri Lankan population is not reported in the literature. According to the frequencies of other populations, it is a high-frequency antigen. Luckily, I was able to identify one unit out of ten tested donor units. P antigen typing of the identified unit adds an additional value for the technique.

Pk or p-phenotyped red cells have been used to confirm the anti-P specificity of the Donath-Landsteiner antibody. Although it is almost always anti-P, other specificities have also been reported.[1] Therefore, in such occasions, transfusion of P-negative red cells might not enhance the hemoglobin level as expected. As a plus point, this technique has the ability to find a blood pack compatible with the Donath-Landsteiner antibody specificity of the particular patient.

This technique may be useful as a screening test to select a blood unit for a patient with PCH when no other options are available. Therefore, I like to shear my experiences with the community.

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.


I acknowledge Dr. A. J. M. L. S. B. A. Jayasekara, Senior Consultant Transfusion Physician, National Blood Centre, Sri Lanka, and Dr. S. B. Samarajeewa, Medical Officer In-Charge, Immunohaematology Reference Laboratory, National Blood Centre, Sri Lanka, for giving departmental support and encouraging me during this work.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Eder AF. Review: Acute Donath-Landsteiner hemolytic anemia. Immunohematology 2005;21:56-62.  Back to cited text no. 1
Slemp SN, Davisson SM, Slayten J, Cipkala DA, Waxman DA. Two case studies and a review of paroxysmal cold hemoglobinuria. Lab Med 2014;45:253-8.  Back to cited text no. 2

Correspondence Address:
Geethika Sajeewani Manchanayake,
No. 572, Waguruwela, Welpalla
Sri Lanka
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajts.AJTS_65_19


[PREVo] Next Article

     Search Pubmed for
    -  Manchanayake GS
   Article in PDF

   Case Report

 Article Access Statistics
    PDF Downloaded9    

Recommend this journal

Association Contact us | Sitemap | Advertise | What's New | Copyright and Disclaimer | Privacy Notice

2006 - Asian Journal of Transfusion Science | Published by Wolters Kluwer - Medknow
Online since 10th November, 2006