TABLE 2.

Recommendation on essential laboratory data and clinical parameters in the diagnosis of PK deficiency

	Recommendation	Evidence
Clinical presentation	PK deficiency may be suspected in: - patients with variable chronic anemia and/or splenomegaly and/or jaundice, with normal or near-normal red cell morphology. - transfusion dependent cases of unknown etiology - haemolytic patients with unexplained severe neonatal indirect hyperbilirubinemia - presence of high reticulocyte number in splenectomised patients with no diagnosis	Mean: 95% Median:100% (75–100)
Clinical data	-information on clinical history (both recent as well as from infancy, ie neonatal jaundice), family history should always be requested together with samples, as well as the time of last blood transfusion	Mean: 98.6% Median:100% (90–100)
Laboratory data (mandatory in bold)	-complete blood count -RBC morphology -markers of haemolysis (reticulocyte count, LDH, unconjugated bilirubin, haptoglobina,b)	Mean: 97% Median:100% (90–100)
Differential diagnosis	Acquired haemolytic anemia, membranopathies, CDAs, unstable haemoglobins, red cell enzymopathies other than PK deficiency should be excluded (see figure 5)	Mean: 92.1% Median:100% (50–100)
Biochemical testing
Reference test for biochemical assay	RBC PK activity assay by spectrophotometry (Beutler, 84)	Mean: 98.7% Median:100% (80–100)
Storage time of sample	PK enzyme assay may be considered stable at 4°C until up to 21 days after collection.c A maximum of 14 days storage is recommended if PK activity is related to HK activity due to different stability of HK activity	Mean: 95% Median:100% (80–100)
Sample anticoagulant	Citrtate-dextrose solution (ACD); EDTA, citrate phosphate dextrose (CPD), heparin could be considered for the enzyme assay (Beutler, 84): EDTA is the main anticoagulant used in daily practice.	Mean: 100% Median:100%
Sample preparation	Purification on α-cellulose/microcrystalline cellulose column is recommended. Buffy coat removal may be considered as an alternative. PK enzyme activity cannot be performed on whole blood	Mean: 96.7% Median:100% (80–100)
Reticulocytes interference	Reticulocyte number must be taken into account when interpreting results of PK enzyme assay, particularly when of low-normal PK activity levels. Results could be compared with enzyme activities obtained from a control sample with the same degree of reticulocytosis, or by calculating the ratio of PK activity to another cell age dependent enzyme (eg, hexokinase).	Mean: 96.1% Median:100% (70–100)
Interference of donor red blood cells	The enzyme assay should be performed as far as possible after a red cell transfusion. The laboratory should record the time since transfusion. A minimum of 50 days from last transfusion is considered a “safe” period for testing of PK activity, leading to an estimated donor RBC contamination of about 7–14%. Results of enzyme activity need to be interpreted with caution in transfused patients.d	Mean: 96.9% Median:100% (60–100)
Confirmatory tests	In case of decreased PK activity, sequencing of PKLR gene is highly recommended to confirm the diagnosis	Mean: 88.3% Median:100% (10–100)
Molecular testing
Indication	-molecular testing is highly recommended to confirm a suspected case of PK deficiency based on decreased enzyme activity. -molecular testing of PKLR gene by Sanger is suitable for patients with (relatively) decreased PK activity - use of NGS panels is a reliable alternative method for diagnosis of PK deficiency. It is particularly relevant for: - neonates (if family study is not available) - transfusion dependent patients/recently transfused patients - samples with prolonged shipping times	Mean: 91.2% Median:100% (10–100)
PKLR genotype discrepancies	In case of genotype discrepancies (patients with suspected PKD and one or none mutations detected) further investigation are required: -assays for detection of large deletions -re-evaluation of other causes of haemolysis by specific tests or NGS platform In absence of any mutation and decreased PK activity: - NGS tools or, KLF1 gene mutations should be considered	Mean: 92.5% Median:100% (40–100)

The degree of agreement was expressed as percentage of agreement and reported as mean, median and range.

^aDecreased haptoglobin useful only after 6 months of age.

^bOr evaluation of carboxyhaemoglobin evaluation as index of haemolysis.

^cIn the impossibility of shipment at 4°C, the assay must be performed by 3–5 day.

^dIf the transfusion history is not available, a statement that recent transfusion can affect results should be added to the results.