malaria is caused by one or a combination of four species of plasmodia and leads to over one million deaths , of which over 75% occur in african children under 5 years infected mainly with plasmodium falciparum ( pf ) . pf and plasmodium vivax ( pv ) are identified as the plasmodium species responsible for causing the most severe form of malaria . in epidemiological studies , intervention studies , and clinical trials , malaria microscopy is routinely relied upon as a primary endpoint measurement of the level of malaria infection . this is expressed as parasite density and is classically defined as the number of asexual forms of parasite relative to a blood volume ( e.g. , microliter ) . in the four basic counting techniques using microscopy , white blood cells ( wbcs ) are relatively used in estimating plasmodium parasitaemia by counting the number of parasites against a predetermined number of wbcs on giemsa stained blood smears . complete blood counts , particularly wbcs count , can be performed with new generation automated haematology analysers   and/or manually using stained microscope smears and the neubauer chamber and counters [ 5 , 6 ] . some health facilities frequently use manual convention methods to determine the complete blood counts of a patient for management as a result of a high cost of purchasing and maintaining fully automated or semiautomated haematology analyzers . another cost burden using an automated system includes the ability to ensure prompt validation , maintenance , and implementation of rigorous quality systems . automated machines may therefore not be the preferred choice to quantify wbcs in resource poor areas . due to the frequent lack of facilities in some malaria endemic countries to quantify wbcs , an assumed wbcs count of 8000/l of blood has been accepted by world health organization as reasonably accurate   to estimate malaria parasite densities . assumed wbcs count of blood may generate systematic errors which could produce incorrect conclusions in patient management or during clinical research that uses malaria parasite counts as an end point [ 3 , 8 ] . medical laboratories need to set relevant reference ranges for wbcs and other common laboratory parameters for clinical management of patients or during malaria clinical research . kintampo health research centre has established reference values for common haematological and biochemical laboratory parameters for its study area covering an area of about 7200  km . the team compared the geometric mean parasite densities ( gmpd ) calculated using absolute wbcs , assumed wbcs , and wbcs reference values of the participants enrolled in the malaria studies in middle ghana to assess the impact of using these as malaria parasite density estimators . the studies used for this data analysis were approved by the ethical review committees of the ghana health service , london school of hygiene and tropical medicine , kintampo health research centre institutional ethics committee , noguchi memorial institute of medical research , and the ghana food and drugs authority . written informed consent was sought from all mothers whose children participated in the studies . the data analysis was carried out on laboratory data from a study area with a profile presented by adu - gyasi et al . , 2012 . briefly , the area is located within the forest - savannah transitional ecological zone in ghana with perennial malaria transmission . pf is the predominating plasmodium species while p. malariae and p. ovale are in the minority . the data was obtained from children under 5 years who were recruited into malarial studies carried out by kintampo health research centre between the periods of october 2008 and march 2011 . in the first study , children were enrolled and followed actively every month for two years . the children were referred to the hospital for care whenever they were unwell to determine the incidence of malaria . the second study which was a clinical trial was to determine the effect of providing micronutrient powder with or without iron on the incidence of malaria among children under three years living in a high malaria - burden area in ghana . in each of the studies , blood samples were collected by finger - prick into 0.5  ml microtainers containing ethylenediaminetetraacetic acid ( k2edta - bd , usa ) from the participants on their scheduled visit days and on any other day participants visited the health facilities with illness . samples were collected and transported according to the standard operating procedures ( sops ) established by the kintampo health research centre . briefly , participant 's finger was rubbed , and the tip was cleaned with disposable alcohol swab and allowed to dry . safety lancet was used to prick and drops of blood were collected into labelled test - tubes to the required volume . collected samples were used for full blood count analysis and for making blood smears . examination of thick and thin smears was carried out as described by adu - gyasi et al . , 2012 . parasite densities for all participants were calculated using assumed wbcs count of 5000/l , 6000/l , 8000/l , and 10000/l of blood . in addition , we used the wbcs reference values established for children aged less than one year ( 10000/l of blood ) and also for children less than five years ( 9200/l ) . data obtained from the database of the malarial studies was checked for completeness and consistency and all queries were resolved . analyses were done using stata ( version 12 ; stata corp . , tx , usa ) and graphpad prism version 5.0 ( graphpad software , inc . ) particularly for geometric mean and intervals . of the 39851 results received over the study period , data of 4944 participant results were consistent and complete for the purpose of our analysis . of the total , 50.6% ( 2498/4944 ) were males and 49.4% ( 2443/4944 ) were females . the mean ( sd ) wbcs and gmpd of the 4944 positive samples were 10500/l ( 4.1 ) and 10644/l ( 95% ci 9986/l to 11347/l ) of blood , respectively . there was no significant difference between the mean ( sd ) wbcs 10600/l ( 4.0 ) for males and 10500/l ( 4.1 ) for females ( p = 0.255 ) . neither was there a significant difference between the gmpd 11058/l ( 95% ci 10114/l to 12091/l ) of blood for males ( table 1 ) and 10217/l ( 95% ci 9324/l to 11197/l ) of blood for females ( table 2 ) . with an assumed wbcs count of 8000/l , a gmpd of 8679/l ( 95% ci , 8140/l to 9253/l ) of blood was estimated ( table 3 ) . in using the wbcs reference values established among children in the study area , ( 10400/l ) , a gmpd of 11282/l ( 95% ci , 10581/l to 12029/l ) of blood and a wbcs count of 9200/l produced a gmpd of 9981/l ( 95% ci , 9361/l to 10641/l ) of blood . the difference in the gmpd calculated using absolute wbcs compared to densities estimated with assumed wbcs was significantly lower for 8000/l . however , gmpd for assumed wbcs count of 10.0  10/l , wbcs reference values of 10400/l , and 9200/l estimated a geometric mean parasite density of 10848/l ( 95% ci , 10175/l to 11567/l ) , 11282/l ( 95% ci , 10581/l to 12029/l ) , and 9981/l ( 95% ci , 9361/l to 10641/l ) of blood that was not significantly different from estimates obtained with the absolute wbcs ( table 3 ) . the interval of gmpd obtained when an assumed wbcs of 8000/l , 6000/l and 5000/l were used and that from absolute wbcs count did not overlap . however , the intervals of gmpd from absolute wbcs count and that obtained with assumed wbcs counts of 9200/l , 10000/l and 10400/l overlapped showing no significant difference in the gmpd ( table 3 ) . a correlation analysis of the malaria parasite densities with the wbcs reference value and assumed wbcs compared to the absolute wbcs showed a significant level of correlation ( p < 0.0001 ) with all the assumed wbcs count ( table 4 ) . this has become dominated by the convenient but inaccurate assumption of a constant wbcs count of 8000/l of peripheral blood [ 3 , 15 ] due to lack of capacity to measure patients absolute wbcs . the mean wbcs count from the participants with malaria infection compares with the wbcs reference values , 10400/l for children less than 1 year and 9200/l for children up to 5 years . the parasite densities from the wbcs reference values agree with the density obtained from using participant 's absolute wbcs . it is agreed that the best solution in estimating parasite densities would be to use the corresponding absolute wbcs count for each age group . though there is a significant level of correlation , estimating the parasite density of plasmodium species with the assumed wbcs count of 8000/l of blood , compared to the absolute and reference value wbcs count , would mean underestimating significantly the parasite density of plasmodium species infections for patients in the study area . therefore , establishing regional based reference wbcs as suggested   to estimate parasite densities in malarial infections will be appropriate . this was evident in the fact that the geometric mean parasite density , 11282/l ( 95% ci , 10581/l to 12029/l ) and 9981/l ( 95% ci , 9361/l to 10641/l ) of blood , estimated by using the established wbcs standard values of 10,400/l and 9200/l of blood for children less than 1 year and up to 5 years old , respectively , overlaps with the geometric mean parasite density obtained by using the mean absolute wbcs count , 10644/l ( 95% ci 9986/l to 11347/l ) of blood , of the participants . results obtained when an assumed wbcs count of 10000/l of blood was used to estimate the parasite density   were also consistent with that from the absolute wbcs count of participants . the parasitaemia obtained by the use of the other assumed wbcs count was significantly lower ( table 3 ) . since lack of resources in some settings in africa makes it difficult to estimate malaria parasite density based on actual wbcs count of patients , where available , the study recommends the use of an established wbcs reference value for a known population and where the reference value could be implied . the reference value established in kintampo has been predicted as a malaria density estimator when compared with the absolute wbcs of participants . in environments where reference values have not been established , the study affirms the use of an assumed wbcs count of 10000/l of blood to estimate malaria parasite density as documented .