VALDIDATION OF

MEASUREMENTS FOR
BIOCHEMICAL AND CLINICAL
PARAMETERES
Harry Freitag LM, S.Gz, M.Sc
Division of Molecular Nutrition
Department of Nutrition and Health, FM, UGM
Objectives:
-Be able to formulate potential bias in biochemichal measurements
-Understand validation methods for biochemistry measurements
-Be able to formulate concept of GLOD STANDARD and
RECOMMENDED METHOD
-Be able to formulate potential bias in clinical measurement
-Understand validation methods for clinical measurements
VALIDATION OF BIOCHEMICAL
PARAMETERS
Iron
Definitions of iron deficiency states
1. Iron deficiency (ID) is a reduction in body Fe to the extent that
cellular storage Fe required for metabolic/ physiological functions is
fully exhausted, with or without anaemia.
2. Iron deficiency anaemia (IDA) is defined as ID and a low
haemoglobin (Hb).
3. Iron-deficient erythropoiesis (IDE) is defined as laboratory
evidence of a reduced supply of circulating Fe for erythropoesis,
indicated by either reduced Fe saturation of plasma transferrin or
signs of ID in circulating erythrocytes.
Bone-marrow biopsy
Bone-marrow examination to establish the absence of stainable iron
remains the gold standard for the diagnosis of iron deficiency,
particularly when performed and reviewed under standardized
conditions by experienced investigators.
However, marrow examinations are expensive, uncomfortable, and
require technical expertise, and are not performed routinely in
clinical practice
Haemoglobin
Haemoglobin (Hb) is a widely used screening test for ID, but used
alone has low specificity and sensitivity.
Its sensitivity is low because individuals with baseline Hb values in
the upper range of normal need to lose 20–30 % of their body Fe
before their Hb falls below the cut-off for anaemia
Its specificity is low because there are many causes of anaemia
other than ID.
Mean corpuscular volume and
reticulocyte Hb content
Measured on widely-available automated haematology analysers, the mean
corpuscular volume (MCV) is a reliable, but relatively late indicator of ID, and its
differential diagnosis includes thalassemia.
The reticulocyte Hb content (CHr) has been proposed as a sensitive indicator
that falls within days f the onset of IDE
However, false normal values can occur when the MCV is increased or in
thalassemia; its wide use is limited as it can only be measured on one model of
analyzer.
For both MCV and CHr, low specificity limits their clinical utility
Erythrocyte zinc protoporphyrin
Erythrocyte zinc protoporphyrin (ZnPP) increases in IDE because
zinc replaces the missing Fe during formation o the
protoporphyrin ring
The ratio of ZnPP/haem can be measured directly on a drop of
blood using a portable hematofluorometer.
In adults, ZnPP has a high sensitivity in diagnosing iron deficiency
In infants and children,ZnPP may also be a sensitive test for
detecting iron deficiency
Erythrocyte zinc protoporphyrin
However, the specificity of ZnPP in identifying iron deficiency
may be limited, because ZnPP can beincreased by lead
poisoning, anaemia of chronic disease,chronic infections
and inflammation, haemolytic anaemias,or
haemoglobinopathies
The effect of malaria on ZnPP in children is equivocal
Transferrin saturation
Transferrin saturation is a widely used screening test for ID,
calculated as the ratio of plasma Fe to total Fe-binding
capacity.
Although relatively inexpensive, its use is limited by diurnal
variation in serum Fe and the many clinical disorders that
influence transferrin levels
Serum ferritin
Serum ferritin (SF) may be the most useful laboratory measure of Fe
status; a low value is diagnostic of IDA in a patient with anaemia
In healthy individuals, SF is directly proportional to Fe stores: 1mg/l
SF corresponds to 8–10 mg body Fe or 120mg storage Fe/kg body
weight
It is widely available, well-standardized, and has repeatedly been
demonstrated to be superior to other measurements for identifying
IDA.
Serum transferrin receptor
The serum transferrin receptor (TfR) is a transmembrane
glycoprotein that transfers circulating Fe into developing red
cells; <80 % of TfR in the body are found on erythroid
precursors.
A circulating, soluble form of TfR consists of the extracellular
domain of the receptor. The total mass of cellular TfR and,
therefore, of serum TfR depends both on the number of
erythroid precursors in the bone marrow and on the number
of TfRs per cell, a function of the iron status of the cell
Serum transferrin receptor
However, normal expansion of the erythroid mass during growth, as
well as diseases common in developing countries, including
thalassemia, megaloblastic anaemia due to folate deficiency, or
hemolysis due to malaria, may increase erythropoiesis and TfR
independent of iron status
Thus, the diagnostic value of TfR for IDA is uncertain in children from
regions where inflammatory conditions, infection and malaria are
endemic.
I want to develop new methods to
measure iron status, How should I validate
the new methods??
Background: Spot ferritin assay on dried serum spot (DSS) samples
provides reliable and accurate assessment. Standard DSS
preparations, however, involve precise serum aliquots and require
some skill and training of field personnel.
Objective: We evaluated the validity of the spot ferritin assay on DSS
samples prepared by simplified approaches and standard technique
in Guatemala City.
Design: Venous blood (5 mL) was obtained from 104 subjects aged
2415y(xSD) and transferred into non heparin-containing (2plain and 2 self-
sealing) capillary blood collection tubes.
Three DSS samples were prepared: A (standard, 20L serum), B (blot,30 –
35mm serum column), and C (dispenser, 20L serum pushed directly from
self-sealing capillary tubes with a dispenser).
Spots were air ]dried and placed in hermetic plastic bags with a desiccant.
Two weeks later, entire spots for DSS A and C samples and a circle in the
center for DSS B samples were analyzed
Relation between the difference in serum ferritin values determined with the use of
the dried serum spot (DSS) ferritin method and the traditional ferritin method,
against the average of values obtained with the 2 methods
Correlations between serum ferritin values measured with the
traditional method and ferritin values measured with dried serum spot
(DSS) ferritin methods.
VALIDATION OF CLINICAL PARAMETERS
BACKGROUND
A simple field test for vitamin A deficiency and xerophthalmia is
urgently needed.
Biochemical parameters of vitamin A status, theoretically the most
sensitive criteria, are impractical in most field and clinic situations,
while a history of nightblindness is considered too subjective to be
ofvalue.
Objectively testing scotopic vision in preschool-age children is
impractical for most field and clinic work
BACKGROUND
Bitot’s spots with conjunctival xerosis (X1B) are the most prevalent,
accepted clinical criterion.
Nightblindness (XN) is generally considered the earliest, mildest
expression of clinical xerophthalmia.
But rigorous, objective assessment of scotopic vision is difficult if not
impossible in young children, especially under field conditions, while
asking a parent or guardian whether a child is nightblind is
considered too subjective to be of value.
METHODS
One of the project’s major activities is a 2-year, prospective,
longitudinal field study of 5000 rural, preschool-age children
residing in Purwakarta Regency, West Java, a 2-hr drive from
the major cities of Jakarta, to the north, and Bandung, to
the southeast.
METHODS
The children were examined by the ophthalmologist and pediatrician
and their height and weight measured.
During the first clinical round blood samples were obtained from
every child with evidence of active xerophthalmia, normal age/sex/
locale matched controls (the next appropriate child from the same,
or occasionally an adjacent RT), and a 5% subsample of all study
children.
METHODS
Presence or absence ofa history of nightblindness was determined
for every child by asking the accompanying guardian or relative
whether the child had “buta ayam” or “kotokeun” (“chicken
blindness” in Indonesian and Sundanese, respectively), local terms
for nightblindness.
If the guardian was unsure of its meaning (a rare occurrence) he or
she were asked whether the child had particular difficulty locating
food or toys after dusk or in a poorly lighted room.
Conclusion
The present study suggests that a properly elicited history of
nightblindness can be as valid evidence of vitamin A
deficiency as the presence of X1B.
Mean serum vitamin A levels among children with isolated
XN, isolated X1B and coexistent XN/X1B were cmically and
statistically significantly greater than among their age/sex/
neighborhood matched controls