A novel method for negAting cold AgglutinAtion interference by dithiothreitol during complete blood count And peripherAl blood smeAr : A cAse study

cold agglutinin disease is an autoimmune disorder that is characterized by antibodies attacking polysaccharide antigens on one’s own erythrocytes. In cold agglutinin disease, hemolysis occurs due to degeneration of erythrocyte membranes and autoagglutination of erythrocytes. Both autoagglutination and hemolysis alter many laboratory test results of the patients, especially complete blood count (cBc) and peripheral blood smear analyses. In our laboratory, we have encountered a blood sample of a 65-years-old man, who was independently diagnosed with cold agglutinin disease, and failed to produce meaningful cBc or peripheral blood smear analysis. Fresh blood samples were taken from the patient, and cBc and peripheral blood smear analyses were repeated with and without water bath incubation at 37 °c. also, varying amounts of dithiothreitol (dtt) were added to fresh samples without heat treatment prior to blood analysis. Heat treatment at 37 °c for varying lengths of time failed to improve cBc and peripheral blood smear analysis. on the other hand, addition of DTT into a blood sample of 2 ml in a K3-EDTA tube was sufficient to negate cold agglutination interference during both cBc and peripheral blood smear analyses. the presented method supports the potential for dtt to be used in negating cold agglutination interference during cBc and peripheral blood smear analyses of patients with cold agglutinin disease. the method we describe is very easy and quick with remarkable results.

cold agglutinin disease is an autoimmune disorder that is characterized by antibodies attacking polysaccharide antigens on one's own erythrocytes.In cold agglutinin disease, hemolysis occurs due to degeneration of erythrocyte membranes and autoagglutination of erythrocytes.Both autoagglutination and hemolysis alter many laboratory test results of the patients, especially complete blood count (cBc) and peripheral blood smear analyses.In our laboratory, we have encountered a blood sample of a 65-years-old man, who was independently diagnosed with cold agglutinin disease, and failed to produce meaningful cBc or peripheral blood smear analysis.Fresh blood samples were taken from the patient, and cBc and peripheral blood smear analyses were repeated with and without water bath incubation at 37 °c.also, varying amounts of dithiothreitol (dtt) were added to fresh samples without heat treatment prior to blood analysis.Heat treatment at 37 °c for varying lengths of time failed to improve cBc and peripheral blood smear analysis.on the other hand, addition of DTT into a blood sample of 2 ml in a K3-EDTA tube was sufficient to negate cold agglutination interference during both cBc and peripheral blood smear analyses.the presented method supports the potential for dtt to be used in negating cold agglutination interference during cBc and peripheral blood smear analyses of patients with cold agglutinin disease.the method we describe is very easy and quick with remarkable results.

K e y w o r d s: cold agglutinin disease, dithiothreitol, autoimmune hemolytic anemia.
C old agglutinin disease is an autoimmune disorder characterized by usually IgM type, and rarely IgA or IgG types, of antibodies directed against polysaccharide antigens on erythrocytes [1].These antigens are mostly part of the Ii blood system, which are linked to membrane glycosphingolipids, or on the Pr glycophorin in rare cases [1].Even though mycoplasma infection, lymphoma or an autoimmune disease is the usual suspect, its etiology is idiopathic in general.Clinically, in addition to typical anemia symptoms, clotting of peripheral vessels, such as acrocyanosis in response to cold, livedo reticularis and skin ulcers, and difficulty of and pain during swallowing cold liquids and foods could be observed.Jaundice and splenomegaly could be a part of the clinical manifestation.While the prevalence of cold agglutinin disease is speculated to be about 10 cases per million in Europe, reliable epidemiologic statistics are missing.A study conducted across Norway has found an incidence rate of 1.0 case per million per year [2].As such, accurate identification of such a rare disorder is crucial for prognosis [3,4].
In cold agglutinin disease, hemolysis occurs due to degeneration of erythrocyte membranes by antibodies activated in cold, as well as autoagglutination of erythrocytes [1,4].Both autoagglutination and hemolysis alter many laboratory test results of such patients.During complete blood count (CBC) analysis of these patients in particular, all parameters doi: https://doi.org/10.15407/ubj89.02.116 are affected except for a few [5][6][7].Peripheral blood smear is also of limited use due to autoagglutination.Heating in a water bath is utilized to overcome this interference, but this is neither generally effective nor very practical.Here, we present a remarkably practical method for negating such interference in this study.

materials and methods
On a routine day at the laboratory, a technician informed the laboratory specialist that a particular CBC analysis produced abnormal and clinically unexpected results, even though analysis was performed twice in two different instruments of the same model and that the blood sample itself appeared clearer than normal with dark speckles.Consequently, the laboratory specialist contacted the patient and invited him back to the laboratory so that a second blood sample could be taken.
The patient was a 65-year-old male military veteran.The medical history of the patient revealed that initially cyanosis on toes and hands appeared after he had swum in a cold pond.He was then diagnosed with cold agglutinin disease by a hematologist at a different health facility by the cold agglutinin titer method.The patient suffered a severe infection of the respiratory tract afterwards and visited our hospital with complaints of said infection, where blood tests were ordered.
The blood samples were collected from the patient into 2-mL K3-EDTA tubes (BD Vacutainer®, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with a vacutainer system that utilizes vacuum for collecting blood, eliminating the use of syringes.The tube that was used for heat treatment was warmed in the water bath to 37 °C prior to sample collection and the sample was not allowed to cool before analysis.The dithiothreitol (DTT) (Sigma-Aldrich, Missouri, USA) solution (0.01 M) was prepared according to the manufacturer's instructions and was kept at 4 °C away from direct light.DTT was not dispensed directly into the sample tube in fear of cross-reaction with EDTA, and was added immediately after blood collection.Afterwards, the tube was gently mixed on a tube rotator for a few minutes until speckles (agglutination) in the blood sample disappeared.CBC analysis was performed on two Beckman Coulter LH780 Hematology Analyzers (Beckman Coulter, CA, USA).
Written informed consent was obtained from the patient prior to sample collection.

results and discussion
The patient was called and invited back to the hospital, and two fresh blood samples were collected for further analysis.One of the tubes was incubated in a water bath at 37 °C.The other tube was used for CBC and peripheral blood smear (Table ;   The laboratory specialists searched the literature on cold agglutinin disease subsequently.We then realized that we could utilize DTT in this case, which is used in our laboratory to eliminate immune complexes that occasionally arise during immunofixation electrophoresis.Furthermore, Arndt, et al have reported their use of 0.01 M DDT solutions in serologic tests of cold agglutinin disease patients [8].A solution of 0.01 M DDT was prepared in our laboratory.The patient gave four fresh 2-ml blood samples that were collected in K3-EDTA tubes.5, 10, 25 or 50 μl of 0.01 M DDT solution was added to each tube.These samples were then used for periphe ral blood smear and CBC analysis.The hemoglobin concentration of the patient was used as referen ce during CBC analysis, in order to compensate for the dilution effect.This is because hemoglobin is measured by optical density and thus not affected by cold agglutination interference [5,6].As a result, we found that the optimal additional DTT amounts were 5 and 10 μl of the solution.Nonetheless, when the peripheral blood smears were observed, local agglutination of erythrocytes and bridging between numerous erythrocytes were recorded in the sample supplemented with 5 μl of DTT solution.In conclusion, an addition of 10 μl of 0.01 M DTT solution into a blood sample of 2 ml in a K3-EDTA tube is sufficient to negate cold agglutination interference during both CBC and peripheral blood smear analysis of patients with cold agglutinin disease (Table ; Figure B).
CBC is a frequently used test for assessment of general health status.Leukocyte, erythrocyte and platelet counts, and corresponding indexes are determined by CBC analysis, which provide clinicians crucial information for the diagnosis and follow-up of anemias, acute or chronic diseases, hemorrhage propensity, blood cell anomalies and malignancies.Due to antibodies formed against erythrocyte antigens in cold agglutinin disease patients, CBC cannot be performed accurately, and many parameters of these patients, including white blood cell count, red blood cell count, mean corpuscular volume and mean corpuscular hemoglobin concentration in particular, cannot be determined [5][6][7].Unfortunately, peripheral blood smears of these patients also fail to produce valid results.Incubation in water bath at 37 °C is commonly used for negating cold agglutination interference in such patients, but this process, as in this case, is often ineffective, not to mention time-consuming and tiresome.
DTT (also known as Cleland's reagent) is a small redox molecule with the chemical formula C 4 H 10 O 2 S 2 .It is generally used for reducing intramolecular and intermolecular disulfide bonds found in proteins.DTT impairs immunoglobulin function by disrupting their disulfide bonds.Because of these properties, DTT can be utilized for negating cold agglutination interference seen in cold agglutinin disease patients [9,10].Another substance that depolymerizes immunoglobulins by disrupting disulfide bonds is 2-mercaptoethanol (2-ME).It has been shown that both DTT and 2-ME could be used for negating IgM agglutination in human sera during Brucella antibody screens [11].Thus, in case DTT is absent in the laboratory, 2-ME may be added to samples instead prior to blood analysis of cold agglutinin patients.However, this was not tested in our study.
Even though cold agglutinin disease is a rare disorder, preventing the interference of cold agglutination for CBC and peripheral blood smear analysis is difficult, time-consuming and tiresome.As presented in this report, we have used DTT to negate cold agglutination interference in such patient during CBC and peripheral blood smear tests.This novel method is remarkably practical and has the potential for allowing the clinician to assess the results of CBC and peripheral blood smear with ease and reliability.
The main limitation of this study is that only a single patient was involved, which is due to the low incidence of cold agglutinin disease cases.Further samples with cold agglutination need to be treated as described here in order to confirm the efficacy of our method.Although DTT is used for elimina ting immune complexes in a number of established biochemical procedures, its use for negating cold agglutination during blood tests is described for the first time in this study.Because of this novelty, technical concerns arise, such as whether DTT in samples affects readings in a hematology analyzer, which should be addressed in a future more extensive study.Finally, we have not established whether, and if so, when the negating effect of DTT dimini shes after a certain period, which is a valid possibility.

Fig.
Fig. Peripheral blood smears of the patient.a -Smear without dithiothreitol (dtt); B -Smear with added DTT.Magnification, 400X Figure a).