| Abstract|| |
Background: Cold agglutinins (CA) are benign naturally occurring low titer autoantibodies present in most individuals. Those with moderate strength are found in infections, malignancies or autoimmune conditions with diagnostic importance. Aim: Present report deals with CA that brought spontaneous hemagglutination in blood units stored at 2-6°C. Study design: Over 32 months period between July 1993 and December 1995, blood units were inspected for spontaneous cold auto-hemagglutination (SpCA) phenomenon. The plasma from these units was separated and investigated for serological specificity using in house red cell panel and standard serological methods. Results: Among 51,671 blood units, 112 units showed SpCA phenomenon. A rising trend seen in first half of study period significantly fell in remaining half. Specificities of the antibodies detected include anti-I (27), anti-i (53), anti-Pr (21) with remaining few being undetermined specificity. Absorption of serum using enzyme-treated red cells revealed a presence of anti-Pr among the cases, the two of which with new specificities that reacted preferentially with red cells from either new-born or adults and were tentatively named as anti-Pr Fetal and anti-Pr adult , respectively. While 9 cases showed optimum reaction at neutral pH of 7, 68 (62%) cases reacted at pH 5.8 through 8.0, 28 (26%) cases preferred an acidic pH 5.8 and 4 cases opted an alkaline pH 8. Of 28 cases with antibodies preferentially reacting in acidic medium, 17 (60%) cases were anti-i and 7 (25%) cases were anti-Pr. Conclusion: Unique SpCA phenomenon observed in blood units stored under blood bank conditions seems to be due to CA developed in response to vector-borne infectious agents. Majority of the cases displayed their specificities, otherwise are rare to be encountered.
Keywords: Blood donors, spontaneous cold agglutinins, blood group specificities
|How to cite this article:|
Joshi SR, Naik RA, Gupte SC. Unusual spontaneous cold auto-hemagglutination phenomenon in blood units stored under blood bank condition: A retrospective analysis. Asian J Transfus Sci 2015;9:141-4
|How to cite this URL:|
Joshi SR, Naik RA, Gupte SC. Unusual spontaneous cold auto-hemagglutination phenomenon in blood units stored under blood bank condition: A retrospective analysis. Asian J Transfus Sci [serial online] 2015 [cited 2019 Aug 25];9:141-4. Available from: http://www.ajts.org/text.asp?2015/9/2/141/162705
| Introduction|| |
The cold agglutinins (CAs), preferentially agglutinating red cells at the low temperatures (0°C), are present in the serum of almost all individuals as low titer (<1:10) auto-antibodies. , While CAs with very high titer and with higher thermal amplitude are associated with idiopathic condition called cold hemagglutinin disease, those with moderately high titer found in patients with auto-immune hemolytic anemia secondary to certain cancers  and infections caused by bacteria, ,, virus ,,,, or parasite infestations. ,, We present here our observational study on spontaneous cold auto-hemagglutination (SpCA) phenomenon in blood units kept under blood bank storage condition.
| Materials and Methods|| |
Over a 32 months period between July 1993 and December 1995, the blood units stored at 2-5°C were inspected for SpCA. Plasma specimen from blood units showing SpCA was separated and preserved at −20°C for serological investigations. Repeat donations by the donors were excluded from the calculation for the epidemiological purpose. Serological specificity of the cold reacting auto-agglutinins is mainly confined to anti-I, anti-i, and anti-Pr. Serological specificity was determined using in house red cell panel. Ordinary group O red cells are the source of I+ while new-born baby's red cells (umbilical cord blood) served as the source of i antigen. Ordinary group O red cells served as a source of Pr positive antigen while the same red cells treated with a proteolytic enzyme, like papain, served as the source of Pr negative antigen. These red cells were prepared on the day of the testing. No commercial reagents were used but serum samples with anti-I, anti-i, and anti-Pr specificities identified previously in our laboratory were used as controls. Standard serological methods were used as per Bhatia.  Chi-square with Yates correction was calculated online with the help of GraphPad Software Inc (San Diego, California, USA). 
| Results|| |
A total of 51,671 blood units were inspected over 32 months period with 112 blood units showing SpCA phenomenon [Figure 1], [Figure 2], [Figure 3]. The phenomenon was not of a seasonal occurrence. However, it showed a rising trend during the first half of the study period with a sharp decline in remaining half [Table 1].
|Figure 1: Blood unit with spontaneous cold auto-hemagglutination phenomena (vertical holding)|
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|Figure 2: Blood unit with spontaneous cold auto-hemagglutination phenomena (inverted holding)|
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|Figure 3: Blood unit with spontaneous cold auto-hemagglutination phenomena (closer look)|
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|Table 1: Incidence of SpCA during the study period between July 1993 and December 1995|
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The city of Surat, where this study was carried out, had experienced an epidemic of plague in September 1994 while this study was on its way. The data were analyzed from an epidemiological angle, to elucidate whether that has any bearing on the occurrence SpCA. Interestingly, there were 100 cases of SpCA found among 28,750 blood units collected before the epidemic while only 12 cases were observed in the subsequent period. This difference was statistically significant (P < 0.0001) [Table 2].
|Table 2: Incidence of SpCA cases 15 months before and after the onset of plague in Surat|
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Serological specificity was tested among 109 cases of the CAs. Direct antiglobulin test was negative among the donors having these cold reacting auto-agglutinins. Immunoglobulin specificity was immunoglobulin M as serum treated with dithiothreitol rendered nonreactive. Titer values ranged from 1:16-1:128 by saline method at +4°C. These autoantibodies never agglutinated red cells at 37°C. Usually, auto agglutination of the red cells is observed when the antibody has high titer and/or high thermal amplitude. An unusual feature of these auto-agglutinins was that in spite of having a moderate strength, and reactive only at a cold temperature, they showed the spontaneous auto-agglutination phenomenon. Anti-I was found in 27 (25%) cases, anti-i in 53 (49%) cases, and anti-Pr in 21 (19%) cases (results are not tabulated). Specificity among the 8 cases was not clear, so cases were classified under the undetermined category and were further tested by absorption of plasma with red cells premodified with enzyme papain. Absorbed serum revealed the presence of anti-Pr specificity that was apparently obscured in its native serum due to a presence of some antibody to crypt antigen. Two of these specimens had hitherto unrecognized specificities. These were tentatively named as anti-Pr Fetal and anti-Pr Adult , as per their reactivity with red cells from new-born infants or adults, respectively [Table 3].
|Table 3: Presence of anti-Pr among the cases with undetermined specificity as revealed by absorption with enzyme treated red cells|
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The pH optima for reactivity of antibodies in these cases showed following feature: 68 (62%) cases reacted through pH ranged between 5.8 and 8.0 irrespective of their antibody specificity, 28 (26%) cases in acidic pH, 4 cases in alkaline pH, and remaining 9 cases reacted at neutral pH. Anti-i was a largest single specificity among the cases exclusively reacting in acidic medium. These results are summarized in [Table 4].
|Table 4: Number of cases with SpCA showing optimum reactivity at different pH|
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In spite of showing a strong agglutination of the red cell in blood unit stored in cold, the red cells agglutinates were dispersed upon keeping the unit at room temperature for 10-15 min and were transfused through transfusion set without problem.
| Discussion|| |
The CAs with low titer and low thermal amplitude are present in almost all individuals as harmless autoantibodies though their precise role has remained unclear.  Moderately, high titer CAs with higher thermal amplitude was associated with certain infections. For example, anti-I with Mycoplasma,  Listeria,  Leishmaniasis;  anti-i with infectious mononucleosis,  and anti-Pr with viral infections like rubella , or Varicella. , One of us (SRJ) had found an anti-Pr a as transient CA in association with falciparum malaria, with the unique feature of being failed to react in the presence of ionized calcium. 
Autoantibodies observed in the present study were found among blood donors with no apparent health problems. However, a possibility of an exposure to infectious agents as antibody stimulant in past was open as the population in the city of Surat was exposed to vector-borne infections such as malaria, gastroenteritis, and infective hepatitis during the contemporary period of this study. This hypothesis draws support from observations that there was a sharp decline in numbers of cases with SpCA coinciding with reduction in number of cases with these infections over a period following extensive intervention by the Municipal authority to control vector-borne infections (as revealed in Surat Municipal Corporation annual Diary, 1998).
Among the CAs, anti-I is common as compared to anti-i and anti-Pr. It is worth noting that, in the present study, an incidence of anti-i had surpassed to that anti-I. An admixture of anti-I and anti-i specificities would misidentify as a nonspecific antibody, that can only be appreciated by differential absorption experiment using red cells from adult and newborn infants. 
In the present study, some cases of SpCA with undetermined specificity showed anti-Pr after absorption of serum using red cells premodified by proteolytic enzyme papain. Interestingly, two of the specimens had unusual anti-Pr being directed against antigens on red cells from newborn or adults and were tentatively designated as anti-Pr Fetal and anti-Pr Adult . CAs with similar but not identical specificities showing preferential reactivity with red cells of adults than that of the newborns and vice versa were reported, though their reactivity was abolished by sialidase but not by protease. 
As regards to study related to pH optima, no specific pattern emerged. Anti-Pr usually reacts better at acidic pH. However, in the present study, it was not only anti-Pr but also anti-i cases preferred to react in the acidic medium in most cases showing SpCA.
| Conclusion|| |
Spontaneous cold auto-agglutination phenomenon observed in blood units stored under blood bank conditions appears to be due to a presence of CA. As these auto-antibodies are strictly reactive at low temperatures, they do not give rise to in vivo hemolysis. As the antibody specificities identified are known to be associated with certain infections, their presence in healthy blood donors could be attributed to exposure to such infections in recent past or present in its subclinical phase at the time of blood donation.
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Dr. Sanmukh R Joshi
Allianze University College of Medical Sciences, Waziria Medical Square, Jalan Bertam2, Mukim 6, Kepala Batas 13200, Penang, Malaysia
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]