Comparison of role of alternative anticoagulant (MGS[O.sub.4]) in EDTA-induced pseudothrombocytopenia.
Ethylenediaminetetraacetic acid (EDTA) is a well-known anticoagulant since early 1950s and it has certain advantages over other anticoagulants.  EDTA dependent pseudothrombocytopenia is a rare phenomenon (i.e., around 0.1% in general population) which is mostly due to the presence of EDTA-dependent antiplatelet antibodies that react optimally between 0 [degrees]C to 4 [degrees]C, recognise the cytoadhesive receptors gpUb/IIIa, stimulate the expression of activation antigen, trigger activation of trypsin kinase, platelet agglutination and clumping in vitro. This finally leads to a spuriously decreased platelet count. Five basic criteria should be fulfilled to raise the clinical suspicion of EDTA dependent pseudothrombocytopenia, (i) abnormal platelet count typically <100x[10.sup.9]/L. (ii) Occurrence of thrombocytopenia in EDTA-anticoagulated sample at room temperature. (iii) Time dependent fall of platelet count in EDTA specimen. (iv) Evidence of platelet aggregates and clumps in EDTA-anticoagulated samples with either automated cell counting or microscope analysis. (v) Lack of sign or symptoms of platelet disorders. Spurious thrombocytopenia also called Pseudothrombocytopenia (PTCP), results from low platelet counts due to in vitro platelet clumping. [2-8] Platelet clumping in PTCP (Pseudothrombocytopenia) results in inaccurate platelet concentration which leads to misdiagnosis of thrombocytopenia when analysed with haematology analyser. [8,9] Pseudothrombocytopenia remains a challenge in haematological laboratory. Problem is that platelet tends to easily aggregate in vitro, giving rise to lower platelet counts. In automated electronic cell counting of blood samples anticoagulated with EDTA, PTCP was observed due to in vitro platelet clumping induced by EDTA. It is caused by activation of an abnormal protein identified as an immunoglobulin/agglutinin.  Magnesium was used in the beginning of the last century as anticoagulant for microscopic platelet counts.
MATERIALS AND METHODS
This study was conducted in the Department of Pathology in the Institute of Shyam Shah Medical College Rewa and Sanjay Gandhi Memorial Hospital, Rewa (M.P.). Approximate 110 cases during study period April 2015 to March 2016 were evaluated by EDTA (Ethylenediaminetetraacetate) blood samples and MgS[O.sub.4] blood samples.
1. Patients having platelet count less than 100000/cu. mm. in EDTA (Ethylenediaminetetraacetate), anticoagulated sample.
2. Samples showing flags in automated haematological analyser for low platelet count.
3. Only indoor patients included in study.
4. Patient age > 15 years.
1. Patients with normal platelet count.
2. Patients with higher platelet count.
3. OPD (Outpatient department) patients.
4. Patient Age < 15 years.
5. Patient with h/o of bleeding disorder.
Blood samples were collected in a sterile EDTA containing tube and processed following our established laboratory procedure, then report of each patient generated. Informed consent was taken from all patients that took part in our study during repeat blood samples, collected in MgS[O.sub.4] anticoagulant, for use of their samples for medical research, after doing physician request, investigated and generated the report. EDTA (Ethylenediaminetetraacetate) anti-coagulated whole blood samples for routine haematological analysis selected for this study when suspicious for low platelet count. Both samples were run through the automated haematology analyser (three-part differential, Mindray) and made peripheral blood smear for microscopic examination.
This study included a total of 110 patients, out of which Females-84 and Males-36 aged between 15 to 70 years showing EDTA-induced pseudothrombocytopenia due to platelet aggregation. These patients were not associated with symptoms and history of bleeding disorder. Patients included in study were having diseases like Upper respiratory tract infection (36 patients), Coronary heart disease (11 patients), Diabetes mellitus (10 patients), Acute trauma (08 patients), Hypertension (24 patients), Tuberculosis (15 patients), others (07 patients).
Out of which 51 patients found raised platelet count >150x[10.sup.3]/[micro]l in MgS[O.sub.4] anticoagulated blood samples. Mean platelet count in EDTA anticoagulated blood sample of 110 patients was 71.67 (67.08-76.26)x[10.sup.3]/[micro]L and in MgS[O.sub.4] was 153.63 (44.96-169.84) x[10.sup.3]/[micro]l. (Table 1). Comparison of two anticoagulants was performed using paired t-test. p<0.0001 (software GraphPad for statics), which shows statistically extremely significant. Mean platelet volume in EDTA was 11.45 (11.09-11.08) fl and in MgS[O.sub.4] was 10.32 (9.35-11.3) fl (Table 2). In peripheral smear examination of EDTA-PTCP patients, 80% of patients showed platelet aggregation and clumping (Table 3). We also compared other parameter of complete blood count in EDTA and MgSO4 anticoagulated blood sample. On comparison of Red blood cell count and Total leucocyte count in both anticoagulants that is EDTA and MgS[O.sub.4] in 110 blood samples, mean [+ or -] SD of EDTA was 3.68 ([+ or -] 4.04)/[micro]L and of MgS[O.sub.4] was 3.49 ([+ or -] 1.00)/[micro]L, EDTA was 8.66 ([+ or -] 8.58) x[10.sup.3]/[micro]L (Table-4) and of MgS[O.sub.4] was 8.43 ([+ or -] 8.44) x [10.sup.3]/[micro]L (Table-5) respectively. Table-6 Shows Mean [+ or -] SD in EDTA and MgS[O.sub.4] blood samples of Neutrophil was 67.58 ([+ or -] 15.16) % and 67.09 ([+ or -] 15.16) %, Lymphocyte was 24.88 ([+ or -] 14.17) % and 25.72 ([+ or -] 15.55) %, Mid was 7.61 ([+ or -] 3.77) % and 7.90 ([+ or -] 4.17) respectively. No significant difference was found in mean of RBC count, Total leucocyte count and Differential leucocyte count in both anticoagulated blood samples.
Ethylenediaminetetraacetic acid (EDTA) is commonly used as an anticoagulant for estimation of blood cell counts. EDTA-PTCP is an in vitro phenomenon due to antiplatelet antibodies that cause platelet clumping in blood that had been anticoagulated with EDTA. 
Aim of this study is to demonstrate that Magnesium sulphate can effectively prevent platelet aggregation in whole blood samples from patients showing PTCP in the presence of EDTA. Platelets are not stabilised in EDTA anticoagulated blood samples. They undergo changes in shape and size, thus leading to time dependent changes of the mean platelet volume (MPV). [12,13]
In this study, EDTA-PTCP was confirmed by microscopic examination of peripheral blood smear for platelet clumping and aggregates that shows low platelet count on Mindray three-part differential automated analyser.
Peter Schuff Werner et al, estimated correct platelet count in EDTA-induced pseudothrombocytopenic patients by using MgS[O.sub.4] anticoagulant, found higher platelet count in MgS[O.sub.4] anticoagulated sample. Nakamoto K et al also found raised platelet count in MgS[O.sub.4] anticoagulated blood sample than sample anticoagulated with EDTA. Peter Kohlschein et al also found magnesium sulphate anticoagulated samples prevent platelet aggregation in individuals with EDTA associated pseudothrombocytopenia.
In this study, we found that sample anticoagulated with magnesium sulphate do not show platelet aggregation in peripheral blood smear when examined in light microscope. It shows significantly higher mean platelet count in MgS[O.sub.4] anticoagulated blood sample than EDTA anticoagulated sample.
In this study, we also compared other parameter of complete blood count (CBC) in EDTA blood sample and MgS[O.sub.4] blood sample in patients with PTCP. Magnesium Sulphate normalised platelet counts without interfering with other blood cell counts; shows no significant correlation in mean of Red blood cells, Total leucocyte count and Differential leucocyte count in EDTA anticoagulated blood sample and MgS[O.sub.4] blood sample.
If PTCP in EDTA sample is not identified in laboratory for clinical purpose, then falsely low platelet count may suggest unnecessary, expensive and even invasive diagnostic or therapeutic procedure. To avoid this, a safe and practical alternative should be used. Magnesium sulphate anticoagulated blood samples can be used for platelet counting, when platelet count in EDTA blood sample do not match with clinically, or flagging in haematology analyser, as suggestive of platelet aggregates. This can also be confirmed by examination of peripheral blood smear.
Before EDTA was introduced as the anticoagulant of choice for haematology testing, magnesium salts were used as anticoagulants for sampling capillary blood for platelet enumeration.  Present study has been attempted to determine the role of MgS[O.sub.4] as anticoagulant in EDTA-induced pseudothrombocytopenic patients. Our observation confirms MgS[O.sub.4] as an anticoagulant inhibits platelet aggregation in blood sample. No aggregation seen in peripheral smear. Magnesium sulphate normalised the platelet counts without interfering with other blood cell count or differentiation result. To avoid this clinical misinterpretation of platelet count, we suggest the use of MgS[O.sub.4] as an anticoagulant if blood count does not match with clinical expectation and flagging in automated analyser.
 Gordan HG, Larson NL. Use of Sequestrene as an anticoagulant. Am J Clin Pathol 1955;23:613-18.
 Shreiner DP, Bell WR. Pseudothrombocytopenia: manifestation of a new type of platelet agglutinin. Blood 1973;42(4):541-9.
 Wilkes NJ, Smith NA, Mallet SV. Anticoagulant-induced pseudothrombocytopenia in a patient presenting for coronary artery bypass grafting. Br J Anaesth 2000;84(5):640-2.
 Mori M, Kudo H, Yoshitake S, et al. Transient EDTA-dependent pseudothrombocytopenia in a patient with sepsis. Intensive Care Med 2000;26(2):218-20.
 van der Meer W, Allebes W, Simon A, et al. Pseudothrombocytopenia: a report of a new method to count platelets in a patient with EDTA- and temperature-independent antibodies of the IgM type. Eur J Haematol 2002;69(4):243-7.
 Yoshikawa T, Nakanishi K, Maruta T, et al. Anticoagulant-induced Pseudothrombocytopenia occurring after transcatheter arterial embolization for hepatocellular carcinoma. Jpn J Clin Oncol 2006;36(8):527-31.
 Lichtman MA, Buetlar E, Seligsohn U, et al. Hemostasis and Thrombosis-Thrombocytopenia. Williams Hematology. 7th edn. New York: McGraw-Hill 2007; p. 2.
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Hemlata Bamoriya (1), S. K. Mishra (2), S. K. Sutrakar (3), Ashutosh Garg (4), Kamlesh Sutrakar (5), Kali Gandhi (6)
(1) Junior Resident, Department of Pathology, S. S. Medical College, Rewa, Madhya Pradesh.
(2) Associate Professor, Department of Pathology, S. S. Medical College, Rewa, Madhya Pradesh.
(3) Associate Professor, Department of Pathology, S. S. Medical College, Rewa, Madhya Pradesh.
(4) Demonstrator, Department of Microbiology, S. S. Medical College, Rewa, Madhya Pradesh.
(5) Medical Officer in District Hospital, Tikamgarh, Madhya Pradesh.
(6) Junior Resident, Department of Pathology, S. S. Medical College, Rewa, Madhya Pradesh.
HOW TO CITE THIS ARTICLE: Bamoriya H, Mishra SK, Sutrakar SK, et al. Comparison of role of alternative anticoagulant (MGS[O.SUB.4]) in EDTA-induced pseudothrombocytopenia. J. Evolution Med. Dent. Sci. 2017;6(38):3065-3068, DOI: 10.14260/Jemds/2017/661
Financial or Other, Competing Interest: None.
Submission 04-04-2017, Peer Review 29-04-2017, Acceptance 04-05-2017, Published 11-05-2017.
Dr. Hemlata Bamoriya, Junior Resident, Department of Pathology, Shyam Shah Medical College, Rewa-486001, Madhya Pradesh.
Caption: Figure 1.100X Peripheral Blood Smear of EDTA Blood Samples Showing Platelet Aggregation
Caption: Figure 2.100x Peripheral Smear of MgS[O.sub.4] Blood Sample
Caption: Graph 2. Comparison of Mean Platelet Volume in Blood Samples Anticoagulated with EDTA and MgS[O.sub.4]
Table 1. Comparisons of Mean Platelet Counts in Blood Sample Anticoagulated with EDTA and MgS[O.sub.4] Parameter EDTA MgS[O.sub.4] Mean Platelet Count x [10.sup.3]/[micro]L 71.67 153.63 Standard Deviation ([+ or -] SD) 24.27 65.79 Standard Error of Mean (SEM) 2.31 6.27 Range 67.08-76.26 44.96-169.84 Table 2. Comparison of Mean Platelet Volume in Blood Samples Anticoagulated with EDTA and MgS[O.sub.4] Sl. No. Parameter EDTA MgS[O.sub.4] 1. Mean in 11.45 10.32 2. [+ or -] SD 1.893 5.179 3. Standard error of mean 0.1805 0.493 4. Range (lower-upper) limit 11.092-11.80 9.350-11.30 Table 3. Causes of Pseudothrombocytopenia Sl. No. Causes Total No. of % Patient Patient 1. EDTA- platelet 110 88 80% aggregation and clumping 2. Partial clotting 110 10 9.09% of specimen 3. Platelet satellitism 110 05 5.54% around WBC 4. Giant platelet 110 07 6.36% Table 4. Comparison of RBC Count in Blood Samples Anticoagulated with EDTA and MgS[O.sub.4] Sl. No. Parameter EDTA MgS[O.sub.4] 1. Mean of RBC /pL 3.68 3.49 2. Standard Deviation [+ or -] 4.04 1.00 3. Standard Error of Mean (SEM) 0.38 0.09 4. Range (lower-upper limit) 1-44 1.2-6.8 Table 5. Comparison of Total Leucocyte Count in Blood Samples Anticoagulated with EDTA and MgS[O.sub.4] Sl. No. Parameter EDTA MgS[O.sub.4] 1. Mean of TLC x [10.sup.3]/[micro]L 8.66 8.43 2. Standard Deviation 8.58 8.44 3. SEM 0.81 0.80 4. Range (lower-upper limit) 2.2-66.8 2.2-89 Table 6. Comparison of Differential Leucocyte Count in Blood Samples Anticoagulated with EDTA and MgS[O.sub.4] Sl. No. Parameter Neutrophil EDTA MgS[O.sub.4] 1. MEAN 67.58 67.096 2. SD 15.16 15.34 3. SEM 1.445 1.46 4. MINI LIMIT 64.7 64.19 5. MAX LIMIT 70.5 69.99 Sl. No. Parameter Lymphocyte EDTA MgS[O.sub.4] 1. MEAN 24.88 25.72 2. SD 14.17 15.55 3. SEM 1.352 1.48 4. MINI LIMIT 22.20 22.78 5. MAX LIMIT 2.56 28.66 Sl. No. Parameter MID EDTA MgS[O.sub.4] 1. MEAN 7.61 7.90 2. SD 3.77 4.17 3. SEM 0.3602 0.3977 4. MINI LIMIT 6.90 7.11 5. MAX LIMIT 8.33 8.69 Graph 1. Comparison of Mean Platelet Counts in Blood Samples Anticoagulated with EDTA and MgS[O.sub.4] EDTA MgS[O.sub.4] MEAN 71.57 155.65 SD 24.27 65.79 Note: Table made from bar graph. Graph 3. Causes of Pseudothrombocytopenia % of pt aggregation 80 satellitism 9.09 giant platelet 5.54 partial clotting 6.36 Note: Table made from bar graph.
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|Title Annotation:||Original Research Article|
|Author:||Bamoriya, Hemlata; Mishra, S.K.; Sutrakar, S.K.; Garg, Ashutosh; Sutrakar, Kamlesh; Gandhi, Kali|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||May 11, 2017|
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