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 .
 two polycythemic subjects from a family with multiple polycythemic subjects were evaluated . 
 estimation of oxygen affinity of hb from venous blood gas parameters ( p50 ) revealed low p50 suggesting a high affinity hb variant . 
 further work up , which included beta globin gene sequencing , revealed a novel mutation changing a codon to the previously reported high affinity hb - hb johnstown ( beta109 val->leu ) . 
 polycythemic subjects with high affinity hb variant are asymptomatic with normal life expectancy . 
 their differentiation from polycythemia vera ( pv ) is crucial to avoid therapy which is otherwise reserved for pv patients . 
 we provide an electronic version ( in microsoft excel program ) of a previously reported mathematical formula for rapid calculation of p50 from venous blood gases . 
 estimation of p50 is an essential initial step in the evaluation of a subject with personal and family history of polycythemia . 
1. Background
2. Methods
3. Results and Discussion
4. Conclusion
Supplementary Material