At a Glance

Pyruvate kinase deficiency (PKD) is an autosomally recessive inherited disease that produces ineffective conversion of phosphoenolpyruvate in the Embden-Meyerhof Pathway, resulting in decreased production of ATP. It is the second most frequent enzymatic defect after G-6-P-D deficiency.

Since red blood cells (RBCs) are incapable of producing ATP through oxidative mechanisms, the presence of PKD limits the availability of ATP to circulating red blood cells. A family history of congenital hemolytic anemia or anemia of childhood is typically present. However, mild forms of the disease can delay diagnosis to late childhood or early adulthood. The signs and symptoms may include otherwise unexplained anemia, icterus, jaundice, gallstones, and fatigue. Also, patients frequently present with an enlarged spleen. The decrease in ATP concentration results in increased fragility of red blood cells, producing weakened red blood cell membranes and hemolysis. Occasional PKD also occurs in patients with hematopoeitc neoplasias as a secondary condition.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

A standard complete blood count (CBC) will show varying degrees of anemia, ranging from 5 to 12 g/dL hemoglobin. Reticulocytosis is proportional to the severity of anemia but will show a 50% increase if the patient has been splenectomized. In most cases, the morphological RBC characteristics are unremarkable and are normocytic/normochromic. With hemolysis or splenectomy, reticulocytes, polychromatophilia, macrocytosis, and spicular RBCs (echinocytes) may be apparent. Heinz bodies and spherocytes are absent.

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Osmotic fragility testing may serve as a useful screen for PKD. Unlike hereditary spherocytosis, PKD autohemolysis does not correct with the addition of glucose. It may be normalized after the addition of ATP (being aware of circumstances that will cause false-negative results, such as prior transfusions). A second helpful test is the fluorescent screening test for PKD. Because PKD patients have an impaired ability to convert NADPH to NADP+ and NADPH is a fluorescent compound, fluorescence should fail to disappear in PKD patients after incubation of 45-60 minutes. Again, false-negative results can occur with prior transfusion, and there is little relationship of this result to the severity of hemolysis. Some patients with high reticulocyte counts may also have normal fluorescent screening tests.

RBC enzyme activity may also be performed. This type of testing is more frequently available in reference laboratories. Quantitative RBC enzyme assays give definitive confirmation of the results of screening tests and allow detection of heterozygotes for possible genetic counseling. Most techniques use spectrophotometry measuring the oxidative results of the NADPH to NADP+ conversion. Specimens are stable for several days at 4°C if collected in EDTA, heparin, or acid-citrate-dextrose (ACD) tubes, allowing transport to an appropriate reference laboratory. (Table 1)

Table 1
Osmotic Fragility Fluorescent Screening Test RBC PK Enzymatic Activity
Positive, corrects with ATP but not sucrose Positive fluorescence after 60 minute incubation Negative or reduced (heterozygotes)

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

The presentation of PKD is quite variable, depending on several factors. Homozygosity is the most likely form of presentation. Heterozygotes are usually asymptomatic. The prevalence rate of a heterozygous carrier of one PKD gene is estimated at 1% in an American population, reflecting its presentation in widely distributed ethnic groups from northern Europe to Japan. One of the more notable populations with a predilection for PKD is the Amish population in the United States. Screening of the four most common gene mutations demonstrates an estimated prevalence of 51 cases per million persons in the Caucasian population. Interestingly, this is 50 times higher than the number of individuals diagnosed with PKD, suggesting the disorder is frequently under-diagnosed.

Other factors that might affect the laboratory results include prior transfusion, which dilutes the natural proportion of defective RBCs. Also, some of the atypical findings on CBC (e.g., echinocytes, macrocytosis, polchromatophilia) may not be apparent in nonsplenectomized patients. After splenectomy, CBC findings may be more apparent. Elevated reticulocytes in a given sample may also affect the fluorescent test, since reticulocytes still retain the ability to convert NADPH to NADP+. Enzymatic analysis is impaired if not collected in appropriate tubes or if delayed in arriving at the reference laboratory.

What Lab Results Are Absolutely Confirmatory?

The definitive confirmation of the results of screening tests is the quantitative RBC enzyme assay. The blood should be collected in EDTA, heparin, or ACD, and the enzyme is stable for several days at 4°C under these conditions. The blood should not be allowed to freeze, because washed blood cells are used in enzyme assays and the enzymes are usually less active in hemolyzed specimens.

The definitive test at the molecular level is analysis of the DNA sequence showing evidence of an enzymatic defect. The variability of the disease presentation probably reflects the variability genetically. PKD exists as four isoenzymes. Two isoenzymes are encoded by a genetic locus on badn 15q22; two others are encoded on band 1q21. The two 15q22 isoenzymes are found in striated muscles, brain, leukocytes, platelets, and other tissues. The two 1q21 isoenzymes are found in liver, normoblasts, reticulocytes, and erythrocytes. Varying degrees of compensation can occur in the liver with 1q21 mutations. Actually, enzymatic defects that have been observed range from decreased substrate affinity to thermal instability. Mutations that affect enzyme kinetics and thermostability produce more severe PKD.

What Confirmatory Tests Should I Request for My Clinical Dx? In addition, what follow-up tests might be useful?

Molecular analysis, including genetic counseling, may be helpful in PKD observed in a primary case, in siblings, and in parents anticipating more children. The more common mutations are noted in two chromosomal locations (1q21 and 15q22). More than 180 different mutations have been identified as causes of hemolysis. Also, patients having 1q21-associated PKD may demonstrate deficiencies in the liver, contributing to the increased bilirubin frequently observed.

The variability in disease expression may be partially attributed to the compensatory expression of the tissue-distributed isoenzyme. Morbidity in the newborn is usually the result of severe PKD, expressed as anemia and hyperbilirubinemia or both. Death in utero or shortly after birth from nonimmune hydrops fetalis should cause review of PKD. Blood or exchange transfusion can be a consideration in severe cases of anemia. Iron overload is another serious complication of PKD and should be monitored for patients known to have this disease.

What Factors, if Any, Might Affect the Confirmatory Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

A younger patient presenting with hemolytic anemia and normal blood cell morphology should first be evaluated for possible noncongenital causes. Such causes are evaluated with a direct antiglobulin test (Coomb’s test) to rule out an autoimmune hemolytic process. A sucrose test also rules out paroxymal nocturnal hemglobinuria. If the anemia is thought to be a hereditary process, clinical evaluation may require distinguishing this disease from hereditary spherocytosis or hemoglobinopathies. Both hereditary spherocytosis and PKD produce autohemolysis on osmotic fragility testing. PKD can be distinguished from hereditary spherocytosis, because PKD does not correct with the addition of glucose. If a hemoglobinopathy is suspected, then hemoglobin electrophoresis and propanol stability testing (to identify unstable hemoglobins) may be performed.

The primary challenge to diagnosing PKD is the variability in the disease severity. The severity can range from severe neonatal anemia to a young adult with mild chronic anemia. There is considerable variation in the primary CBC markers for PKD. The CBC may range from normocytic/normochromic presentation to increased reticulocytes, polychromatophilia, macrocytosis, and the presence of echinocytes. Additionally, intervening therapies, such as splenectomy and transfusion, can produce dilutional normal results. Conversely, splenectomy may actually enhance morphological RBC changes associated with PKD. Ultimately, the confirmatory tests are actual enzymatic testing of patient RBCs and DNA mutational analysis.