The Use of Citrate as an Additive in Blood Collection Tubes

Brian Smith,  Becton Dickinson Director, Medical Affairs – Asia Pacific and Japan shares with us  the first in a series of articles about the tubes we use and see everyday in the pre-analytical area. This newsletter we look at the sodium citrate tube- “blue top”. The use of tri-sodium citrate as an anticoagulant dates back to a time before the First World War when it was developed as an additive for donor blood. The anticoagulant properties of this additive are based on its ability to complex with calcium ions in the blood and thus render them unavailable for participation at various key points of the coagulation pathway. The use of tri-sodium citrate, in conjunction with citric acid for this purpose continues today although the formulation of citrate based additives for donor blood has evolved considerably over the past nine decades. One of the first enhancements was the addition of D-glucose (dextrose) to sustain metabolic activity of the red blood cells and thus prolong the shelf life of the blood. The term ‘ACD’ was coined to describe the ingredients of the additive – ‘acid’, ‘citrate’, ‘dextrose’. Other early improvements to the formulation also included refinement of the citric acid concentration to overcome problems with caramelisation of the glucose during sterilization whilst maximizing the volumetric ratio of blood:additive to minimize dilution. Two basic formulations evolved, namely ACD ‘A’ and ACD ‘B’. Both of these remain in use today although they are used mainly for in vitro diagnostic testing (e.g. human leucocyte antigen testing). Later, changes were made to strengthen the pH buffering capacity of the additive with a move to phosphate buffer systems (hence ‘CPD’ for ‘citrate’, ‘phosphate’, ‘dextrose’). This has benefits for donor blood but offers no advantage over ACD for most in vitro diagnostic testing. Other improvements continue to be seen today – all with the aim of prolonging shelf life in the face of dwindling worldwide supplies of donor blood.

In the clinical laboratory setting, tri-sodium citrate dihydrate is most commonly used as an additive in blood collection tubes required for coagulation testing. Citrate additives are used in two concentrations, namely 0.105 – 0.109 mol/L (3.13 – 3.2%a) and 0.129 mol/L (3.8%a). A volumetric ratio of 9 parts of blood to 1 part of the liquid additive is used – this is critical. An additive concentration between 0.105 and 0.109 mol/L is generally accepted as being superior to the higher (0.129 mol/L) concentration and this is reflected in the current CLSI Approved Standard1. As for donor blood above, the additive acts as an anticoagulant by complexing with calcium. Again, it is often used in combination with citric acid and as such provides weak pH buffering capacity – advantageous with regard to stability of some analytes. This additive is ideally suited to coagulation testing because it provides a means of temporarily halting the coagulation process in the specimen at the point of collection. Once in the laboratory (and following centrifugation to obtain a cell free plasma specimen), the coagulation process can be re-started from the ‘citrate induced hiatus’ at the point of collection with the addition of specific reagents (e.g. tissue thromboplastin in the case of the INR* test or activated partial thromboplastin in the case of the aPTT test) along with additional calciumb. The time required for the re-started coagulation process to proceed to finality (with the formation of an insoluble fibrin clot) is then measured and used to assess the status of a particular coagulation pathway.

* International Normalised Ratio

The addition of aqueous calcium chloride solution (‘reagent calcium’) in the laboratory is a critical step. If there is an excessive amount of citrate in the plasma specimen, some of the reagent calcium will be ‘quenched’ – a potential source of error. The lower concentration of additive (0.105 mol/L – 0.109 mol/L) lessens the likelihood of residual citrate in the plasma specimen. Levels of residual citrate can also be elevated in under-filled specimen tubes and in specimens from polycythaemic patientsc (a significant issue with haematocrits >0.6L/L). Levels of residual citrate will be higher where specimen tubes containing 0.129 mol/L citrate are used in these situations.

This article was adapted from an article in BD Asia Pacific Preanalytical Notes (APPN). We would like to kindly acknowledge BD and the Editorial Boardd for APPN for permission to post this on the APaN website.

a            based on the use of dihydrous tri-sodium citrate (MW 296) in combination, where applicable, with citric acid (MW 192).

b            typically added as 0.025 mol/L CaCl2

c         the amount of liquid additive can be adjusted to compensate for abnormally elevated haematocrit values according to the following formula:

mL of additive per mL of blood = (1 – HCT x 100)/(595 – HCT x 100)

where HCT = haematocrit (L/L)

d APPN Editorial Board:

  • Dr Tan It Koon, Former Head, Clinical Biochemistry Laboratories and IEM Reference Laboratory, Department of Pathology, Singapore General Hospital (SGH), Singapore; Founding and past President of Asian & Pacific Federation of Clinical Biochemistry (APFCB) and Singapore Association of Clinical Biochemistry (SACB); Former Member of IFCC Executive Board and WHO Expert Advisory Panel on Health Laboratory Services.
  • Dr G. Neil Kent, Principal Scientist, Department of Clinical Biochemistry and Clinical Pharmacology and Toxicology, PathWest Laboratory Medicine WA, Perth, Australia.
  • Prof. Hyun-Sook Chi, Department of Laboratory Medicine, University of Ulsan ,College of Medicine and Asan Medical Center, Seoul, Korea.
  • Dr T. F. Ashavaid, Consultant Biochemist and Head, Department of Laboratory Medicine, Joint Director Research, P.D. Hinduja National Hospital and Medical Research Centre, Mahim, Mumbai (Bombay), India.

1  Tubes and Additives for Venous Blood Specimen Collection; Approved Standard – Fifth Edition. CLSI Document H1-A5, 2003

On August 8, 2011, posted in: Uncategorized by

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