|Year : 2019 | Volume
| Issue : 1 | Page : 22-29
Establishing reference ranges and normal values for coagulation screening in healthy Indian male volunteers enrolled for a longitudinal study
Rehan Ahmed1, Prosenjit Ganguli2, Natwar Singh1, Surinderpal Singh3, Umesh Das Gupta4, Yogesh K Jaiswal5, Zahid Asharaf6, Prasanna Reddy7, Velu Nair8
1 Department of Clinical Hematology, Centre for Stem Cell Therapy and Research, Army Hospital (Research and Referral), New Delhi, India
2 Department of Pathology, Command Hospital (Eastern Command), Kolkata, West Bengal, India
3 Administration, DADMS (Deputy Additional Director of Medical Services), Tajganj, Agra, India
4 Ex-Director, Scientist G, National Jalma Institute of Leprosy and other Mycobacterial Diseases-ICMR (NJIL & OMD-ICMR), Tajganj, Agra, India
5 Faculty of School of Studies, Biochemistry, Jiwaji University, Gwalior, Madhya Pradesh, India
6 Department of Biotechnology, Jamia Milia Islamia University, New Delhi, India
7 Department of Cardiorespiratory, Defence Institute of Physiology & Allied Sciences, Defence Research & Development Organization, New Delhi, India
8 Ex-DGMS-Army (Director General Medical Services) Palam Vihar, Gurgaon, Haryana, India
|Date of Submission||22-Nov-2018|
|Date of Decision||26-Dec-2018|
|Date of Acceptance||03-Jan-2019|
|Date of Web Publication||13-Mar-2019|
Prof. Prosenjit Ganguli
Department of Pathology, Command Hospital (Eastern Command), Kolkata, West Bengal
Source of Support: None, Conflict of Interest: None
Background: The study was designed for establishing reference ranges and normal values for coagulation screening in healthy Indian male volunteers. At present, there are no standard parameters established for coagulation screening assays of Indian population. The parameters used as a reference in the coagulation assays are of Western origin. We know that ethnicity of the western population is different from the Indian population which may result in a different set of reference ranges of the coagulation assay. It is necessary to determine the mean normal values for the coagulation assay, namely prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen in an Indian population. The aim of the study was to establish the reference ranges of coagulation assays for all future experiments on the cohort. Methods: Six hundred healthy male volunteers were sampled and underwent coagulation testing at a seven hundred-bedded hospital at Jammu (Jammu). Fresh normal pool plasma (FNPP) was prepared concurrently and investigated (n = 50 volunteers). In the study population, the arithmetic means of the coagulation assay were as follows: PT: 13.00 s, APTT: 34 s, TT: 17.3 s, and fibrinogen: 298 mg/dl (19 s), and in the FNPP, it was 12.8 s, 33.2 s, 17 s, and 298 mg/dl (19 s), respectively. Results: The reference range of coagulation screening in our study was established as the following values: PT: 10.7–15.3 s, APTT: 26.8–41.2 s, TT: 12.8–21.7 s, and fibrinogen: 223–372 mg/dl. This study has set a reference range of normal values for coagulation assay screening in longitudinal studies where these tests were repeated on the same set of individuals at six-month interval for the subsequent 3 years. Conclusion: We see no effect of age on Coagulation cascade in our study. Overall mean values resembled with various age groups in coagulation cascade. These parameters of Coagulation cascade set a standard for high altitude studies where these tests are in normal procedure.
Keywords: Activated partial thromboplastin time, coagulation, fibrinogen, prothrombin time, thrombin time
|How to cite this article:|
Ahmed R, Ganguli P, Singh N, Singh S, Gupta UD, Jaiswal YK, Asharaf Z, Reddy P, Nair V. Establishing reference ranges and normal values for coagulation screening in healthy Indian male volunteers enrolled for a longitudinal study. Biomed Biotechnol Res J 2019;3:22-9
|How to cite this URL:|
Ahmed R, Ganguli P, Singh N, Singh S, Gupta UD, Jaiswal YK, Asharaf Z, Reddy P, Nair V. Establishing reference ranges and normal values for coagulation screening in healthy Indian male volunteers enrolled for a longitudinal study. Biomed Biotechnol Res J [serial online] 2019 [cited 2019 Jul 22];3:22-9. Available from: http://www.bmbtrj.org/text.asp?2019/3/1/22/254095
| Introduction|| |
The mechanism of blood coagulation is a complex and dynamic interaction of platelets, plasma, and blood vessel endothelium. Blood coagulation is an important part of hemostasis process. It is usually initiated through damage to the vessel wall and subsequent activation of protease enzymes and ends with the transformation of soluble fibrinogen into insoluble fibrin. A common model used to describe the mechanism of coagulation is the cascade system, which is separated into 3 areas. The intrinsic system commonly measured by the activated partial thromboplastin time (APTT) is activated by surface contact. The extrinsic system, commonly measured by the prothrombin time (PT) test, is activated by vascular injury. The common pathway leading to clot formation is activated by the intrinsic and/or extrinsic pathway. The coagulation screening test such as PT, APTT, thrombin time (TT), and fibrinogen are important for the basic assessment of hemostasis.
PT is a laboratory screening test used to detect disorders involving the activity of Factor I (Fibrinogen), Factor II (Prothrombin), Factor V (Proaccelerin), Factor VII (Proconvertin), and Factor X (Stuart factor) of the extrinsic and common pathways. The PT assesses the function of Factor VII, Factor X, Factor V, Factor II (Prothrombin), and Factor I (Fibrinogen) after the addition of thromboplastin and calcium. The Factor VIIa/tissue factor complex activates Factor Xa and through the action of prothrombinase complex, prothrombin is converted to thrombin. The time in seconds for the conversion of fibrinogen to insoluble fibrin by thrombin is reported as PT. Commercially available thromboplastins vary in their tissue factor source and method of preparation, leading to differing sensitivities to factor deficiencies; therefore, PT results reported using different reagents are not interchangeable. APTT is an assay used to screen for abnormalities of the intrinsic and common clotting systems and to monitor the anticoagulant effect of circulating heparin. It measures the activities of Factors I, II, V, VIII, IX, X, XI, and XII of the intrinsic and common pathways.
TT is a measure fibrin polymerization which forms a clot and the time to form a firm clot in the presence of thrombin is the TT. Thrombin is derived from inactive zymogen prothrombin, which circulates in plasma. When acted on by the prothrombinase complex (Factor Xa, factor Va, Ca2+) assembled on the membrane of activated platelets and other cells, prothrombin is cleaved into thrombin, one of the most potent platelet agonists. The proteolytic activity of thrombin is required for its role as a platelet agonist. Thrombin cleaves a number of biologically important substrates. It removes fibrinopeptides A and B from fibrinogen to form fibrin monomers that spontaneously polymerize to form a fibrin clot.
Fibrinogen is a glycoprotein, present in plasma at a concentration in the range of 2–4 g/l (200–400 mg/dl) that originates in the liver (1.7–5 g/day) and is converted to fibrin during the blood clotting. In fibrinogen assay, diluted plasma is clotted with a strong thrombin solution; the plasma is diluted to reduce/dilute inhibitors. A strong thrombin solution is used so that the clotting time over a wide range is independent of the thrombin concentration. In the PubMed search, not a single Indian study was available on normal range of coagulation assay profile (PT, APTT, TT, and fibrinogen). The normal range for coagulation assay not yet been established in India even though we are second most populated country in the world. Recently in this study, we tried to establish normal ranges of PT, APTT, TT, and fibrinogen in the Indian population. The proposed study was a cohort over a period of time and hence has endeavored to define a normal range of coagulation screen. The population mix in our subcontinent is ethnically varied and we have selected a group which has a proportional mix of ethnicities of North and North West Indian origin.
| Methods|| |
The present study was conducted on human peripheral blood with a cohort (n = 600) of male volunteers those who were healthy donors within the age group of 20–49 years. Six hundred healthy male volunteers were sampled and underwent coagulation testing at a seven hundred-bedded hospital in Jammu at an altitude of 1030 feet from sea level. Informed consent was obtained according to the Declaration of Helsinki. Approval was taken from IEC (Institutional Ethical Committee), Armed Forced Medical College (AFMC), Pune, India. Inclusion criteria were good general health including normal complete blood count, normal biochemistry, and normal physiological parameters. Under the exclusion criteria, normal individuals were excluded, if samples of donors appeared turbid, icteric, lipemic or grossly hemolyzed and any major complication with any organ system.
Samples were collected at 0800 hours after minimum 10 hours fasting. The anticoagulant sodium citrate 3.2% or 3.8% (0.109M) was used for coagulation assay. A ratio of 9 part blood to 1 part sodium citrate was ensured for all samples. The precaution was taken not to delay mixing the blood with anticoagulant, avoiding foaming of the specimen; the samples were taken in plastic Becton Dickinson (BD) vacutainer tubes. Blood sample was obtained by venipuncture in all cases. Venipuncture was nontraumatic, without hemolysis and contamination by tissue fluids. The samples were not included in the study which were not filled properly till the mark on vacutainer provided by the company.
Standardization of preanalytic variables
Coagulation test (PT, TT, and fibrinogen) must be completed within 6 h of sample collection at room temperature (20 ± 5°C). For APTT, plasma remain stable for 4 h at 20 ± 5°C. If sample is preserved at −20°C, coagulation test must be done within 2 weeks of sample collection. If preserved at −70°C, coagulation test must be done within 6 months. For fresh normal pool plasma (FNPP) assay, samples must be fresh. The coagulation tests should be determined with each new lot of reagent. The presence of exogenously added heparin, citrate, oxalate, or ethylenediaminetetraacetic acid from blood collection devices will interfere with test results. Glass syringes or tubes may prematurely activate coagulation, resulting in accelerated clotting times. Venous samples must be collected into plastic syringes or tubes.
Plasma should not be kept at 2-8 degree Celsius for coagulation studies as factor VII may be activated by the kallikrein system affecting prothrombin time. A longer-than-normal PT can mean a lack of or low level of one or more blood clotting factors (Factors I, II, V, VII, or X). It can also mean a lack of Vitamin K; liver disease; or that a liver injury has occurred. Coagulation test results may be affected by commonly administered drugs. A longer-than-normal PT can be caused by treatment with blood-thinning medicines, such as warfarin (Coumadin) or, in rare cases, heparin. Reagent in intact vials are stable until the expiration date, when stored at 2°C–8°C; allow the reagent to stand at room temperature (18°C–25°C) for 30 min before use. For results, if the control values are outside the stated range, check all the components of the test system to ensure that all are functioning correctly, i.e., assay conditions, reagents, and integrity of the plasma being tested.
Blood was collected in BD vacutainer and gently mixed with anticoagulant immediately after collection. Vacutainer was spun at 1500 rpm for 20 min, and supernatant of platelet-rich plasma (PRP) was aliquoted in separated sterile RIA tubes. The sterile RIA tube containing PRP was spun at 3000 rpm for 20 min. The platelet poor plasma (PPP) was analyzed for doing the coagulation study. In this study, 72 (12%) individuals were smokers, and 66 (11%) individuals were social drinkers out of 600. The platelets count of 600 PPP samples were done on Melet Schloesing (MS-4, France) 3 part hematology analyzer and all had platelet count <10000/mm3. The tests on coagulation study were completed within 4 h of blood collection. All tests were done on Start 4 semiautomated coagulation analyzer (Diagnostica Stago) using manufacturer's protocol.
Prothrombin time test
The assay procedure was performed by placing 100 μl PPP in cuvette preheated to 37°C for 50–60 s and subsequently adding 100 μl of thromboplastin reagent (STA Neoplastin Cl Plus 5) at 37°C. A steel ball (Amelung KC4A) rotates in the sample cuvette under the influence of magnetic field until the formation of fibrin strands around the ball impedes it. This is detected by a change in the magnetic field, and the coagulation time is recorded. Addition of calcified amount of tissue factor (Factor III) to citrated plasma activates Factor VII which induces the formation of a stable plug. The time in seconds from thromboplastin reagent (STA Neoplastin Cl Plus 5) addition to clot formation was defined as the PT.
Activated partial thromboplastin time test
The APTT test was based on the addition of kaolin (pH 7.4), Factor XII activator (CK Prest-5 reagent), and calcium chloride, which induces the formation of a stable plug. The assay procedure is initiated by placing 50 μl of citrated plasma and 50 μl kaolin (CK Prest-5 reagent preheated to 37°C) in a cuvette preheated to 37°C followed by the additional incubation for 180 s at 37°C and then adding 50 μl of calcium chloride that had been preheated to 37°C. On the addition of calcium chloride, the machine started calculating the time and the clotting time. The time in seconds from calcium chloride addition to clot formation was defined as APTT.
The TT is a rapid and simple test for the assessment of fibrin formation. The TT remains normal in deficiencies of Factor XII (fibrin stabilizing factor). The assay procedure was performed by placing 100 μl PPP in cuvette for 50–60 s at 37°C. Subsequently, 100 μl of titrated calcium thrombin (STA Thrombin 2) was added in the sample. A steel ball rotates in the sample cuvette under the influence of the magnetic field until the formation of fibrin strands around the ball impedes it. This is detected by a change in the magnetic field, and the TT is recorded.
Qualitative determination of fibrinogen was based on the addition of a relatively large amount of strong thrombin solution to dilute citrated plasma, ensuring that the clotting time depends only on the fibrinogen contained in the sample. In the assay, 100 μl of diluted plasma (neat plasma is diluted 1:20 by the combination of 50 μl of neat plasma plus 950 μl of Owren's Veronal buffer pH 7.4) was added to the cuvette (37°C) and incubated for 60 s then added 50 μl of fibrinogen reagent (fibriprest-2). On the addition of fibrinogen reagent, the machine calculated the time and the clotting time. The time (seconds) until clot formation was automatically converted into mg/dl.
Statistical analysis was used to determine the mean and standard deviation (SD) of the results. The normal value may vary depending on local conditions. Therefore, it is necessary that each laboratory establish its own normal ranges and acceptable control value for their particular local patient population. In general, values are considered normal if they fall within the range of mean SD (mean ± 2 SD). Thereafter, a range of normal values were calculated for the test.
| Results|| |
The maximum and minimum value of PT was 17.7 and 10.1 s, respectively [Table 1]a. The average mean of PT of 600 individuals was 13 s, and the SD was 1.15 s [Figure 1]a, i.e., 2SD is ± 2.30 s. The range of PT lies between 10.7 and 15.3 s. The value of FNPP for PT was 12.8 s which was slightly lower than the mean value [Table 1]b and [Figure 2]a.
|Figure 1: (a) Frequency histogram represents range of prothrombin time in samples. (b) Frequency histogram represents range of activated partial thromboplastin time in samples. (c) Frequency histogram represents range of thrombin time (thrombin time) in samples. (d) Frequency histogram represents range of fibrinogen (mg/dl) in samples|
Click here to view
|Figure 2: (a) Plot represents comparison between fresh normal pool plasma and individual values of prothrombin time. (b) Plot represents comparison between fresh normal pool plasma and individual values of Activated partial thromboplastin time. (c) Plot represents comparison between fresh normal pool plasma and individual values of thrombin time. (d) Plot represents comparison between fresh normal pool plasma and individual values of fibrinogen (mg/dl)|
Click here to view
Out of 600 samples, the maximum value of APTT was 42.8 s and the minimum value was 23.9 s [Table 1]a. The mean APTT was 34 s, and the SD was 3.6 s [Figure 1]b, i.e., 2 SD is ± 7.2 s; hence, its range lies between 26.8 and 41.2 s. The value for APTT in FNPP was 33.2 s. There is a difference of 0.8 s between mean APTT and FNPP [Table 1]b and [Figure 2]b.
In thrombin, maximum and minimum value was 23.8 and 11.2 s, respectively [Table 1]a. The mean of TT was 17.3 s, and its SD was ± 2.23 s, i.e., 2SD is ± 4.46 s which means its range lies between 12.8 and 21.7 s [Figure 1]c. The value of FNPP for TT was 17 s. The value for TT of FNPP is 0.3 s less than mean normal TT [Table 1]b and [Figure 2]c.
The maximum value of fibrinogen was 399.5 mg/dl (13.3 s), and the minimum value was 215 mg/dl (27.2 s) [Table 1]a. The mean fibrinogen was 298 mg/dl (18.7 s), and its SD was ± 37 mg/dl [Figure 1]d, i.e., 2 SD is ± 74 mg/dl range lies between 224 and 372 mg/dl. The value of fibrinogen in FNPP was 298 mg/dl (19 s) [Table 1]b and [Figure 2]d. There is no difference between mean Fibrinogen and FNPP. Fifty samples at random were revaluated on a similar machine for confirming consistency of results.. The correlation between expected and observed value of coagulation profile of PT, APTT, TT and fibrinogen showed agreement [Figure 3].
|Figure 3: (a) Probability-probability plot symbolizes correlation between expected and observed values of prothrombin time. (b) Probability-probability plot symbolizes correlation between expected and observed values of activated partial thromboplastin time. (c) Probability-probability plot symbolizes correlation between expected and observed values of thrombin time (thrombin time). (d) Probability-probability plot symbolizes correlation between expected and observed values of fibrinogen (mg/dl)|
Click here to view
| Discussion|| |
The mean of PT, APTT, TT, and fibrinogen in the blood samples of volunteers in plains were 13 s, 34 s, 17.3 s, and 298 mg/dl (19 s), respectively. Moreover, the values of FNPP for PT, APTT, TT, and fibrinogen were 12.8 s, 33.2 s, 17 s, and 298 mg/dl (19 s), respectively. These values are in agreement with the normal values (PT = 13 s, APTT = 34 s, TT = 17.3 s, Fib = 223.4-372.6 mg/dl) of the control sample provided by the manufacturer. Ninety-seven percent of PT, 95.2% APTT, 93.3% of TT, and 97.5% fibrinogen lie within range. All the results are normal and lie within range. These results establish reference ranges and normal values of coagulation tests, i.e., PT, APTT, TT, and fibrinogen for further studies.
We observed no effect of age on coagulation assay profile in our study. Overall mean values resembled with various age groups in coagulation cascade [Table 2], [Figure 4] and [Figure 5]. About 11% individuals consume liquor, and 12% were smokers [Table 3]. No significant difference was observed in the data analysis of coagulation cascade for PT, APTT, TT, and fibrinogen for 600 healthy male volunteers with various habit and health parameters [Table 4]. Two percent of the individuals were alcohol consumers and smokers and 11% of individuals were alcohol consumers; but, these habit and health parameters in our study have no effect on their coagulation cascade mean values [Table 4].
|Figure 5: (a) Mean dot plot represents relativity of prothrombin time with age in all individuals. (b) Mean dot plot represents relativity of activated partial thromboplastin time with age in all individuals. (c) Mean dot plot represents relativity of thrombin time (thrombin time) with age in all individuals. (d) Mean dot plot represents relativity of fibrinogen (mg/dl) with age in all individuals|
Click here to view
|Table 4: Coagulation profiling in alcohol consumers, nondrinkers, smokers, nonsmokers, and alcohol consumers and smoker|
Click here to view
All results were consistent. Our range of APTT was over 14.4 s from 26.8 s to 41.2 s. The manufacturer datasheet mentions the range of 25–40 s. Our results show 1.8 s longer APTT from its lower limit and 1.2 s longer from its upper limit.
The lower limit for PT was 10.7 s, and the upper limit was 15.3 s. Inclusion of both normal and abnormal controls will enable detection of nonlinearity in the standard curve. A reference standard (calibrator) is used for accuracy. The exact potency is not important although the approximate value should be known to select a preparation at the upper or lower limit of the normal reference range. Normal and abnormal controls are usually obtained from commercial companies.
In normogram, there is no significant relationship between PT, APTT, TT, and fibrinogen (mg/dl) [Table 5].
P value must be less than 0.05 for significant relationship, and it can be seen in comparative study where there is prolonged PT and prolonged APTT.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Alesci S, Borggrefe M, Dempfle CE. Effect of freezing method and storage at -20 degrees C and -70 degrees C on prothrombin time, aPTT and plasma fibrinogen levels. Thromb Res 2009;124:121-6.
Harmening DM, Bethal M. Coagulation Methods in Clinical Hematology and Fundamental of Hemostasis. 5th
ed. Philadelphia. PA. USA: F.A. Davis Co.; 2009. p. 849-81.
Kirkwood TB. Calibration of reference thromboplastins and standardisation of the prothrombin time ratio. Thromb Haemost 1983;49:238-44.
Zucker S, Cathey MH, Sox PJ, Hall EC. Standardization of laboratory tests for controlling anticoagulent therapy. Am J Clin Pathol 1970;53:348-54.
Kazakos GM, Papazoglou LG, Rallis T, Tsimopoulos G, Adamama-Moraitou K, Tea A, et al.
Effects of meloxicam on the haemostatic profile of dogs undergoing orthopaedic surgery. Vet Rec 2005;157:444-6.
Ofosu FA, Liu L, Freedman J. Control mechanisms in thrombin generation. Semin Thromb Hemost 1996;22:303-8.
Phillips DR. Thrombin interaction with human platelets. Potentiation of thrombin-induced aggregation and release by inactivated thrombin. Thromb Diath Haemorrh 1974;32:207-15.
Banfield DK, MacGillivray RT. Partial characterization of vertebrate prothrombin cDNAs: Amplification and sequence analysis of the B chain of thrombin from nine different species. Proc Natl Acad Sci U S A 1992;89:2779-83.
Hantgan RR, Francis CW, Scheraga HA, Marder VJ. Fibrinogen structure and physiology. In: Colman RW, Hirsh J, Marder VJ, Saizman EW, editors. Hemostasis and Thrombosis – Basic Principles and Clinical Practice. Philadelphia: J.B. Lippincott Company; 1987. p. 269-88.
Clauss A. Rapid physiological coagulation method in determination of fibrinogen. Acta Haematol 1957;17:237-46.
Neofotistos D, Oropeza M, Ts'ao CH. Stability of plasma for add-on PT and APTT tests. Am J Clin Pathol 1998;109:758-63.
Hathaway WE, Christian MJ, Clarke SL. Study of the various parameters intervening in the pre analytic variables (revue de la literature). Sang Thromb Vaiss 1998;10:5-18.
Gjonnaess H, Fagerhol MK. Studies on coagulation and fibrinolysis in pregnancy. Acta Obstet Gynecol Scand 1975;54:363-7.
Bain BJ, Imelda B, Mike AL, Lewis SM. Practical Haematology. 11th
ed. E & S Livingstone Edinburgh, Scotland) and J & A Churchill (London, England) Elsevier;2012. p. 404-6.
Hillman L, Lusher CR, Levin Hillman CR, Lusher JM. Determining the sensitivity of coagulation screening reagents: a simplified method. Lab Med 1982;13:162-5.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]