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Indian Journal of Transfusion Medicine
  Indian Journal of Transfusion Medicine Indian Journal of Transfusion Medicine

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Leucodepletion

                                       Dr. Mangesh Kulkarni

M.D.

 

                                                                               Dr. Anand Deshpande

M.D.

 

INTRODUCTION
Leucodepletion refers to reduction in leucocyte count to < 5X 106 cells / unit of blood component.  Leucocyte  reduced , leuco-poor and leuco-free are the various terms used but “leucocyte depleted” is the  universally accepted one1.

Most of the complications associated with blood transfusion are due to the presence of leucocytes in blood and blood products. Blood components like packed red cells and platelets are rich in allogenic leucocytes which are responsible for complications like febrile non-hemolytic transfusion reactions(FNHTRs) and platelet refractoriness1.

In the literature the relationship between various  adverse reactions like FNHTRs, platelet refractoriness and presence of leucocytes in blood components is documented. The first paper which showed  role of leucocytes in febrile reactions occurring after platelet transfusion was published by  Brittingham and Chaplin in 1957.Perkins showed  relationshipbetween FNHTRs and leucocyte  contamination of blood components. Opelz and  Terasaki in their “Bombshell” paper published in 1973  claimed improved kidney survival in recepients  who were transfused prior to transplantation, indicating some kind of immunosuppression  after transfusion2.

In 1986 ,in UK first case of bovine spongiform  encephalopathy (BSE) was recognised. But  in 1993,there was an epidemic of BSE with  more than 1000 cases /week. On epidemiologic  investigations, the   relationship between the  agent and some change in the  rendering process for the food supplements for attles was found. Petroleum-based solvents were used for fat extraction in processing  the livestock carcasses. After rise in prices ,this  solvent extraction step was bypassed which  lead to the survival of infectious agent i.e. prion  and its transmission to cattles. In humans ,the same agent caused spongiform encephalopathy labelled as new variant of CJD .In nvCJD, the infectious agent was transmitted with lymphocytes present in blood and blood  products . After this epidemic, UK and other European countries formulated guidelines for universal leucodepletion of blood products and stopped using plasma from donors  who are inhabitants of UK 3,4,5.

On an average, 109 leucocytes are present  in a whole blood unit .The initial standards for leucodepletion for red cell concentrate required  removal of at least 70% of the leucocytes and retention of 70% of the original red cells. Presently blood is considered leucodepleted if number of white cells per unit is < 5 X 106 with retention of at least 80 % of original red cells.6 In case of leucodepleted platelet concentrates prepared from whole blood, standards require the average number of WBC per unit to be less than 8.3 X 105 ,so that a pool of six units  does not contain more than 5 X 106 WBCs per unit 7

Leucodepletion –AABB Standards 8-

  • .  Each transfusion of a leucodepleted  component should contain < 5X 106 leucocytes.
  •  . Leucodepleted RBCs should retain 85 % of original RBCs with leucocyte count reduced to < 5X 106 /unit
  • . Random donor platelets should contain at  least 5.5 X 10 10 platelets in 75 % units with < 8.3 x 105 leucocytes in all components
  • . Apheresis platelets should contain > 3 X 10 11  platelets in 75 % units with < 5 X 106 leucocytes in 100 % components.
  • .The European standards recommend the  residual leucocyte count to be < 1 X 10 6/unit      

METHODS OF LEUCODEPLETION

A)  Centrifugation and removal of buffy coat

Can  be done either manually or using semiautomatic instruments 

(Fig.1).This reduces  eucocytes by 1 log.

 


                                                                             Fig 1:- Instruments for buffy coat removal 

A)   Washing of red cells-can be done manually or semiautomatic equipments are available. Reduction in the shelf life of RBCs to only 24 hrs.and loss of RBCs are major disadvantages.

B)   Freezing and thawing of red cells-Glycerol is used  for freezing but it is a tedious method which requires thawing  and degycerolization  

C)   Filtration- Filtration is the most common technique  used for leucodepletion. The filters used for leucodepletion  were developed in three generations. The first generation filters   remove large clots and particulate debris.The second  generation filters remove microaggregates and leucocytes by 1 log. The most advanced third generation filters remove  leucocytes by 3 log 1.

Apheresis with leucodepletion – Apheresis sets used for  most of the cell separators today are equipped with in-built  filters for leucodepletion of the blood products.

Fig. 2: In-laboratory filtration – pre process, in process, post process

The filtration can be carried out at bed side or in laboratorypre  process,in process or post process (Fig. 2). Filtration in  laboratory is superior to bed side filtration as it removes leucocytes before the release of cytokines which are the main  culprit in FNHTRs. Removal of leucocytes within 6-8 hours of collection allows the phagocytosis of bacteria to take place but the cytokines are not released till then.

Advantages of in-laboratory filtration-
1.  Control over the blood temperature
2.  Control over quality assurance
3.  Staff training is easy
4.  Leucocytes are removed before the release of  interleukins

Methods for counting of residual leucocytes 10,11 :
As the number of residual leucocytes in leucodepleted blood components is very low, automated  aematology cell counters  are not accurate in counting them .Some special methods are required for this purpose.

1.   Manual methods- Manual counting method using  Nageotte counting chamber is sensitive and accurate in counting less than 5 leucocytes per microlitre

            Fig. 2: In-laboratory filtration – pre process, in process, post process

2.   Automated methods- Flow cytometry, using propidium  iodine alone or combined with thiazole orange shows  comparable results to Nageotte chamber method.

Approximate number of residual leucocytes in blood components 8

Blood component Average no.of
leucocytes /unit

Fresh whole blood

RBC concentrate

Buffy coat depleted red cells

Red cells leucodepleted by filtration

Washed red cell concentrate

Deglycerolised red cells

Platelet concentrate

Platelet pheresis

Platelet pheresis leucocyte reduced

Platelets leucocyte reduced

Platelets pooled leucocyte reduced

FFP thawed

109

108

108

< 5X106

107

106-107

107

10 6-10 7

<5 X 10 6

<8.3 X 10 5

<5 X10 6

< 0.6 X10 6 -1.5 x 10 7

Quality assurance in leucodepletion :

Quality assurance and standard operating procedure for  leucodepletion has to be formulated .The staff needs to be  properly and adequately trained in methods of leucodepletion.  Every leucodepleted product has to be labelled as –  “leucodepleted product”. Food and Drug administration (FDA)  guidelines recommend testing of 1 % of leucodepleted units  out of which 100 % should contain less than 5 X 106 WBC  per unit 7 . 

INDICATIONS FOR LEUCODEPLETED BLOOD  COMPONENTS1:

  • Febrile non-hemolytic transfusion reactions (FNHTRs) 
  •  Platelet refractoriness 
  •  Prevention of transfusion associated viral infections 
  •  Aplastic anaemia and bone marrow transplantation patients 
  •  Fetal and neonatal transfusions 

I)  Febrile non-hemolytic transfusion reactions (FNHTRs)- 

A)    Associated with red cell transfusion- FNHTRs occur following red cell transfusion with an incidence of 6-8 % and the reason is HLA alloimmunization. One log reduction  of leucocytes by buffy coat removal while preparation of red  cell concentrate is enough for prevention of FNHTRs.

B) Platelet transfusion associated- FNHTRs occur with an incidence of > 30 %.Cytokines released during the stroge of   platelets are responsible for these FNHTRs. So  leucodepletion before release of cytokines  helps in prevention of FNHTRs. 

II)  Platelet refractoriness- is defined as the repeated failure  to obtain satisfactory response to transfusion of platelets.  This refractoriness could be either immune mediated due to  HLA alloimmunization or non-immune mediated due to  splenomegaly or sepsis. At least the immune mediated  platelet refractoriness could be prevented by use of leucodepleted blood products. 

III)  Prevention of transmission associated viral infections-

A) Cytomegalovirus (CMV) infection - Use of blood  from CMV seronegative donors is the standard but costly way of prevention of CMV transmission through transfusion. Leucodepletion of lymphocytes is an effective alternative to screening of donors for CMV seronegativity. 

 B) New variant Crutzfeldt-Jakob disease (nvCJD) – is a rare form of spongioform encephalopathy first described in 1996 with total 170 cases till December 2009 in UK ,of whom 167 had died 5. Also six cases in France and one each in Canada,Ireland, Italy and USA.The infectious agent is transmitted through lymphocytes present in blood. To prevent transmission of nvCJD ,UK and other European countries follow policy of use of universal leucodepleted blood products 3,4,5. 

C) Other Viral infections - Leucodepletion is not useful in prevention of transmission of other viruses like HIV 1& 2, HBV, HCV, HTLV, Parvovirus B19. 

IV) Leucodepleted blood products are indicated in patients with aplastic anaemia and patients planned for bone marrow transplantation. 

V)   Blood components used for foetal and neonatal transfusions should be leucodepleted. 

LEUCODEPLETION IS NOT USEFUL IN - 

A. Fresh frozen plasma (FFP) and cryoprecipitate transfusion - as these blood components are prepared with  least cellular contamination and hence donot require  leucodepletion. 

B. Prevention of transfusion associated graft versus host disease (TAGVHD) - Gamma irradiation is the method of choice for prevention of TAGVHD. 

C. Transfusion associated acute lung injury (TRALI) - is due to the leucocyte antibodies present in donor plasma  and leucodepletion is not useful in prevention of TRALI. 

CURRENT SCENARIO WORLDWIDE - 

Most of the European countries and UK , Canada follow 100%  “Universal leucodepletion “for all blood products 3,4,5. In USA most of the blood components are leucodepleted.In developing  countries like India,cost is the major hurdle for use of universal leucodepletion of blood products, but in multiply transfused cases the cost of leucodepletion is worth. In last 10 years , worldwide increased use of leucodepleted blood and blood  products has played a major role in the management of  transfusion reactions and HLA- sensitisation. 

References: 

  1. Guidelines on the clinical use of leucocyte-depleted blood componenets.British committee of standards in Haematology  (BCSH),Blood Transfusion Task Force, 27Nov 1997. 
  2. Opelz G,Sengar D.P.S.,Terazaki P.I. The effect of a blood transfusion on subsequent kidney transplantation. Transplantation Proceedings, 1973, 5, 253-259. 
  3. Dr.Bruce Evatt,Dr.paul Giangrande.Blood transfusion and the risk of new variant Creutzfeldt-Jakob disease (nvCJD). World Federation of Hemophilia Task force on TSEs-bulletin 1,19 th sept,2000. 
  4. Fredrick A, Murphy.Mad cow disease, The BSE epidemic in Great Britain. www.accessexcellence.org 
  5. Andrews N J. Incidence of variant of Creutzfeldt-jakob disease anddeaths in the UK January 1994-December 2009. Statistics unit, Centre for infections, health protection agency Updated 22nd June2010. 
  6. Novotny VMJ,van Doorn R, Witvliet MD et al.Occurence of allogenic HlA and non HLA antibodies after transfusion  of prestorage filtered platelets and red blood cells: A aprospective study. Blood1995;85: 1736-41. 
  7. Silva MA. Ed. Standards for blood banks and transfusion services. 23 rd ed.Bethesda MD.Am.Assocn of Blood Banks :2005 
  8. Mark Brecher.Editor.Technical Manual.AABB.15 the edition. Mayland,USA.2005,492-3. 
  9. Transfusion Medicine Technical Manual, Second Edition  2003,DGHS, Ministry of Health and Family Welfare, Govt.of India. 
  10. Muller TH,Doscher A,Schunter F,Scott CS.Maual and automated methods for determination of leucocyte counts in extreme low levels: comparative evaluation of the Nageotte chamber and the Abott Cell Dyn 3500 analyser. Transfus Sci.1997;18: 505-15. 
  11. Dzik WH,Ragosta A,Cusak WF.Flow-cytometric method or counting very low numbers of leucocytes in platelet  products.Vox Sang 1990;59:153-9.

 

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Dr. Mangesh Kulkarni
MD
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