polycythemia is clinically defined as elevated red blood cell mass ( hemoglobin > 18.5 g / dl in men , 16.5 g / dl in women or other evidence of increased red cell volume or hemoglobin or hematocrit greater than 99th percentile of method - specific reference range for age , sex , altitude of residence or hemoglobin greater than 17 g / dl in men , 15 g / dl in women if associated with a documented and sustained increase of at least 2 g / dl from an individual 's baseline value that can not be attributed to correction of iron deficiency , or elevated red cell mass greater than 25% above mean normal predicted value 1 . polycythemia can be further classified as primary polycythemia , secondary polycythemia , or polycythemia due to abnormal hypoxia sensing . primary polycythemias are caused by intrinsic defects in the erythroid precursors that result in hyper responsiveness to normal level of serum erythropoietin ( epo ) . secondary polycythemias are driven by the factors ( predominantly epo but also insulin growth factor 1 and cobalt ) extrinsic to the erythroid progenitor cells . generally , in secondary polycythemia , the increased red cell mass represents a physiologic response to tissue hypoxia or abnormally increased level of serum epo 2 . polycythemias due to abnormal hypoxia sensing include chuvash polycythemia , polycythemias associated with von hippel - lindau mutations other than the chuvash polycythemia mutation , and polycythemia due to proline hydroxylase mutation 3 , 4 . acquired conditions that lead to increased epo production , such as chronic hypoxia and a variety of tumors , are the most common causes of secondary polycythemias . these include hemoglobin variants with high affinity for oxygen , congenitally low erythrocyte 2 , 3 biphosphoglycerate levels , and inherited methemoglobinemias . all these conditions are characterized by a left shift in hb dissociation curve which in turn leads to tissue hypoxia and a physiologically appropriate increase in epo levels . congenital cyanotic heart or lung disorders , which lead to tissue hypoxia and increased epo level are also examples of secondary congenital polycythemias but these are characterized by a normal hb dissociation curve . two subjects ( mother and daughter ) , from a family with multiple subjects with polycythemia , were available for evaluation : subject 1(mother ) : she is a 59 year old caucasian woman who presented with lifelong history of polycythemia . her medical history was remarkable for 1 ) multifocal ductal carcinoma in situ ( dcis ) of the breast in 2002 , treated with simple mastectomy and 2 ) colonic diverticula and internal hemorrhoids since 2005 . family history was remarkable for presence of lifelong history of polycythemia in her mother and two daughters . laboratory parameters revealed high hb ( 16 gm% ) , high hematocrit ( 48% ) , normal mcv , normal platelet and white blood cell counts , normal arterial oxygen saturation , and normal liver function tests . subject 2 ( daughter ) : she is a 30 year old caucasian woman who presented for further evaluation of lifelong history of polycythemia . family history was significant for lifelong history of polycythemia in her mother , maternal grandmother and her younger sister . laboratory parameters were remarkable for elevated hemoglobin ( 17.2 gm% ) and hematocrit ( 51.4% ) but normal mcv , normal platelets and white blood cell counts , normal arterial oxygen saturation , and normal liver function tests . both subjects underwent following laboratory tests : serum epo levels were normal in both subjects ( 15 miu / ml and 19 miu / ml respectively , normal range 4.1 - 19.5 miu / ml ) . venous blood gas parameters were obtained which included partial pressure of oxygen ( venous po2 ) , venous ph and venous oxygen saturation . calculation of affinity of hb for oxygen ( p50 ) was done using the mathematical formula as described 5 . routine hb electrophoreses , high performance liquid chromatography ( hplc ) 6 , 7 , isoelectric focusing ( ief ) in polyacrylamide gel , globin chain analysis 8 and peptide mapping 9 were performed . this was followed by beta globin gene sequencing which was performed by abi3730 96-capillary sequencer at the dna sequencing core facility at the university of utah school of medicine ( primers and conditions available upon request ) . the p50 was found to be low at 18 mm hg ( normal range 22.6 to 29.4 ) suggesting increased affinity of hb for oxygen . ief showed a band anodal to hb a and globin chain analysis by hplc revealed an unidentified beta globin variant in both subjects ( figure 1 ) . peptide mapping showed an extra peak at 26.9 min but showed no decrease in any peaks suggesting a mutation somewhere in the core ( figure 2 ) . beta globin gene sequencing revealed a novel mutation ( gtg->ttg ) of codon 109 of exon 3 of beta globin gene . this mutation leads to a previously reported high affinity hb variant known as hb johnstown ( beta109 val->leu ) 10 - 12 ; however , this nucleotide change is novel and previously unreported ; and it leads to a previously described amino acid substitution that was however , caused by a different nucleotide missense mutation . hb johnstown ( beta109 ( g11 ) val->leu ) is a high oxygen affinity hb variant and there are three reports in the literature . it was first reported by jones and colleagues in oregon , in 1990 , in a healthy asymptomatic subject with mild erythrocytosis and left - shifted hemoglobin - o2 dissociation curve10 . as with many other hb variants , hb johnstown is silent on standard hemoglobin electrophoretic analyses , and was identified and isolated by reverse - phase hplc of individual globin chains . structural analysis revealed the substitution beta 109 ( g11 ) val ->leu 10 . in 2000 , the underlying beta globin mutation [ beta - globin codon 109 ( gtg ->ctg ) ] was first reported by ropero and colleagues , in two unrelated families ( total four subjects ) of basque extraction in spain 11 . in one of these families , hb johnstown mutation was present in double heterozygosity with another beta 0 thalassemia mutation ivs-1-nt1 ( g->a ) . in 2004 , feliu - torres and colleagues reported hb johnstown [ beta - globin codon 109 ( gtg ->ctg ) ] in an eight year old girl , who had been referred for evaluation of erythrocytosis , in double heterozygosity with another beta globin mutation [ ivs - i-1(g->a ) ] . however , these reports described a causative g to c mutation that is different that mutation we describe in our subject with hb johnstown , namely g to t , both encoding the same amino acid ; i.e. leucine and present in subjects not known to be of spanish / basque extraction . the sigmoid shape of hb - oxygen dissociation curve is indicative of cooperative interaction between heme and oxygen . oxygen affinity and hb - oxygen dissociation is affected by blood ph , 2 , 3- biphosphoglycerate ( 2 , 3 bpg ) level in the red cell and temperature , and globin structure 13 . affinity of hb with oxygen is expressed as the p50 , which is the partial pressure of oxygen in blood at which 50% of the hb is saturated with oxygen . the venous p50 can be measured directly using a cooximeter which is no longer easily available in routine and even reference laboratories . lichtman and colleagues have reported a mathematical formula which can be used to calculate p50 reliably 5 . calculating p50 using this formula requires the following venous gas parameters : partial pressure of oxygen ( venous po2 ) , venous ph and venous oxygen saturation , and uses anti - log mathematical function that many clinicians find difficult to use for calculation 5 . the p50 of a healthy person with normal hb is 26  1.3 mm hg . the 99% confidence interval for individual observations has been reported to be 22.6 to 29.4 mm hg . an abnormally low p50 reflects an increased affinity of hemoglobin for oxygen and vice versa . elevations and reductions in 2 , 3- bpg level in the erythrocyte will also lead to corresponding changes in p50 values ; however , in only reported subjects this decrease was limited to a p50 value between 20 and 35 mm hg . there should be high suspicion for the presence of a high affinity hb variant if p50 value is < 20 mm hg 5 . during oxygenation and deoxygenation , there is considerable movement along the interface of alpha 1 and beta 2 chains of the hb tetramer . all these substitutions can affect the cooperative nature of oxygen binding with heme , and in turn , can change the affinity of hb for oxygen . the majority of mutations affecting oxygen affinity result in high affinity hb variants which result in leftward shift of the dissociation curve and relative tissue hypoxia 14 . there are 90 high affinity hb variants , listed on the globin server , known to be associated with high affinity for oxygen ( http://globin.bx.psu.edu/hbvar/menu.html accessed on may 04 , 2007 ) 15 . high affinity hb variants release oxygen in the tissue relatively slowly and create relative tissue hypoxia . this leads to increased production of epo from kidneys which results in increased red blood cell mass and polycythemia . at an elevated level of increased red blood cell mass ( which depends upon the oxygen affinity of a given variant ) adequate oxygenation of the tissue is reestablished and epo production plateaus and at this new steady state serum epo is often at normal level . this leads to stabilization of hb level after achieving a certain elevated level of hematocrit . family history of polycythemia in a subject with polycythemia should raise the suspicion for the presence of a high affinity hb variant 14 . a low p50 value ( obtained from venous gas parameters ) is supportive of high oxygen affinity hb variant or decreased 2 , 3 bpg level . in a polycythemic patient , establishing a correct diagnosis of a high affinity hb variant is important as these patients have normal life expectancy and do not require phlebotomy . the therapies used for polycythemia vera such as phlebotomy and chemotherapy should not be used in patients who have polycythemia due to high affinity hb variants . with this report , we are providing an electronic version ( using microsoft excel program ) of the mathematical formula 5 , with which p50 can be calculated in few seconds , provided venous gas parameters are available , without necessity of more sophisticated calculations using antilog parameters . with increased ease and rapidity of calculation using our excel program ( supplementary material ) , we hope that use of p50 will increase , leading to improved detection of hb variants in subjects with familial polycythemia .