1. The laboratory diagnosis and monitoring of anemia
using electronic automated blood cell counters
Dr Eloísa Urrechaga PhD
Consultant Medical Laboratory
Osakidetza. Servicio Vasco de Salud
Spain

2. Conflict of Interest Declaration
I have the following potential conflict(s) of Interest to report
Type of Affiliation / Financial Interest
Name of Commercial Company
Receipt of grants / research supports:
none
Receipt of honoraria or consultation fees:
Participation in a company sponsored speaker’s bureau
-- Clinical utility of erythrocyte and reticulocyte parameters.
Science in the Hematology Laboratory.
Abbott Convention Center Wiesbaden, Germany, 10th October 2012.
-- Potential utility of red blood cell algorithm in microcytic RBC disorders
Sysmex European Hematology Symposium . Lyon, France, 10-12th September 2013.
-- Triaging Microcytic Anemia using Extended RBC Parameters from Automated Hematology Analyzer
Turning Science into caring. 8th Abbott Diagnosis Asia Pacific, China and Japan Scientific Symposium
Bali ,Indonesia, 1-3rd December 2015.
-- New Advanced Clinical Parameters in Anemia Diagnostics
Sysmex European Scientific Symposium. Istanbul , Turkey, 11-12th April , 2016.
-- Beckman Coulter´s Webminar
Tools for assessing iron status. Improving the assessment of iron status and anemia differentiation
with DxH st May 2017.
Stock shareholder
Spouse /Partner no
Other support

3. Learning Objectives
Hematology is the medical specialty devoted to diseases of the blood. In contrast to clinical chemistry, hematology is more focused on counting cells and assessing morphology .
The primary hematology test is the complete blood count (CBC), used for patient screening. Unexpected CBC results typically trigger confirmatory or follow-up testing. Due to technological progress modern analyzers report new parameters that provide further information from the traditional count: extended RBC parameters, markers of hypochromia.
This presentation will briefly review the technical basis of the extended parameters and how the additional information reported can be used to improve the management of anemia.
Understand how extended parameters reflect the patho-phisiology of the different anemias
Understand the complementary information provided by extended RBC parameters to the standard hemogram
Learn to use extended RBC parameters in improving the detection of functional iron deficiency, thalassemia trait, spherocytosis, and latent iron deficiency
Hematology is the medical specialty devoted to diseases of the blood. In contrast to clinical chemistry, hematology is more focused on counting cells and assessing morphology .
The primary hematology test is the complete blood count (CBC), used for patient screening. Unexpected CBC results typically trigger confirmatory or follow-up testing. Due to technological progress modern analyzers report new parameters that provide further information from the traditional count: extended RBC parameters, markers of hypochromia.
This presentation will briefly review the technical basis of the extended parameters and how the additional information reported can be used to improve the management of anemia.
Understand how extended parameters reflect the patho-phisiology of the different anemias
Understand the complementary information provided by extended RBC parameters to the standard hemogram
Learn to use extended RBC parameters in improving the detection of functional iron deficiency, thalassemia trait, spherocytosis, and latent iron deficiency

4. Functional iron deficiency Genetic Differential diagnosis of anemia
Automated red blood cell analyzers provide measurements of MCV, MCH, MCHC, and quantify the heterogeneity in distribution of both mean cell volume (RDW)
Additional technological progress in the last 10–15 years has resulted in automated measurements
of the number of reticulocytes and cellular reticulocyte parameters
Many analyzers are also able to quantify the fraction of erythrocytes with volume or Hb concentration above or below defined thresholds, the RBC subsets
New extended parameters are valuable in diagnosing
Latent Iron deficiency
Functional iron deficiency Genetic
Differential diagnosis of anemia
Acquired
the erythrocyte indices were introduce in the 30s, by Wintrobe; this was a revolution in medicine , we still use MCV and MCV to define anemia, after 80 years
They were further refined with the advent of automated red blood cell analyzers provide measurements of MCV and MCHC,
and quantified the heterogeneity in distribution of both mean cell volume (RDW)
Some of them Hb distribution mean cellular Hb concentration (HDW)
Many analyzers are also able to quantify the fraction of erythrocytes with volume or Hb concentration above or below defined thresholds
Additional technological progress in the last 10–15 years has resulted in automated measurements of the number of reticulocytes and cellular reticulocyte parameters, Hb as I mentioned
Quantification of erythrocyte subsets is valuable in
Functional iron deficiency
Differential diagnosis of microcytic anemia
Hereditary Spherocytosis
I´ll be referring to these diseases and how a modern analyzer reporting extended parameters can aid in the diagnosis of all these common anemias

5. Flow Cytometry High angle Side scatter Hb Absorbance Fluorescence
Low angle
Forward scatter VOLUME
LASER BEAM
Absorbance Fluorescence
RNA content
The first technology used in automatic counters was the Coulter principle, impedance, later flow cytometry was introduced :Technicon was the first manufacturer to use this principle in theH1 and H3 counters in the 80s
For many years was the unique ; after the company was absorbed by Bayer and later by Siemens, now sapphire also uses the same theory
Here is a reticulocyte channel in a modern counter
Red cells are both mature and reticulocyte stained with oxazine are crossing and the laser beam is passing cell-by-cell
two light-scatter measurements: the high-angle light scatter side scatterrelated with the cellular opacity, the Hb the cell contains
and the low-angle light scatter forward related to the size, the volume
reticulocyte are recognised with absorbance or flourescence
This is not exactly the same, the laser is different and also the angles where the light is detected, and dye for reticulocytes is flourescent but , red cells are spherocyted first
reticulocyte channel in a modern Advia
Red cells are stained with oxazine, only recituclocytes; both mature and reticulocyte are crossing and the laser beam is passing and two light-scatter measurements are measure
: the high-angle light scatter (5° to 15°) related with the cellular opacity, the Hb the cell contains
and the low-angle light scatter (2° to 3°) related to the size, the volume
reticulocyte are recognised with absorbance 12

6. RBC extended parameters and Reticulocyte Hb
Hypochromic RBC
Hypo (%)
Siemens Differential diagnosis Anemia
Reticulocyte Hb
CHr (pg)
Siemens
%HPO (%)
Abbott Differential diagnosis Anemia
Mean Reticulocyte Hb
MCHr (pg)
Abbott
Hypo He (%)
Sysmex Functional Iron deficiency
Equivalent Reticulocyte Hb
Ret He (pg)
Sysmex
Low Hb Density
Red Cell Size Factor
Reticulocyte Hb Content
Reticulocyte Hb Expresion
LHD (%)
RSf (fL)
RHCc (pg)
RHE (pg)
Beckman Coulter Latent Iron Deficiency
Beckman Coulter
Horiba ABX
Mindray
17 Table 1 summarizes the parameters now reported by the different analyzers. the biomarkers of hypochromia; is a very active field, This was the last review published on line one month ago, but I discovered his one last week
Each Company applies the technology in a different way in the analyzers, . BC uses impedance Siemens, Abbott, Sysmex flow cytometry aswell
and for this reason these new parameters are exclusive of each manufacturer and they are patented
Flow cytometry provides information about individual cell characteristics.
This is not exactly the same, the laser is different and also the angles where the light is detected, and dye for reticulocytes is flourescent but , red cells are spherocyted first (they look happy aren´t they?)
Modern analyzers report reticulocyte counts but also these parameters related to the characteristics of these cells Hb content, reflect the quality of the erythropoiesis, Reticulocytes are immature cells with a life span of only 1 to 2 days. When released from the bone marrow, measurement of their hemoglobin content or volume give information of the current activity of the bone marrow and the amount of iron immediately available for erythropoiesis.
Red blood cells (RBC) are continuously produced in the bone marrow, In iron deficient erythropoiesis, synthesis of Hb is severely impaired leading to the production of reticulocytes with low Hb concentration
after two days become mature hypochromic cells. Because of the long life span of mature erythrocytes %Hypo values are related to iron status in the last 2-3 months,
Urrechaga E, et al. Biomarkers of hypochromia: the contemporary assessment of Iron status and erythropoiesis. Journal of Biomedicine and Biotechnology 2013http://dx.doi.org/ /2013/603786
Archer NM, Brugnara C . Crit Rev Clin Lab Sci, 2015; 52(5): 256–272

7. the past and the present; this is the histogram of red cells by impedance
MCV is the mean of the volumes of all erythrocytes;
RDW refers to the variety of volumes present in the red cell set so the contribution of marginal-sized subsets to the calculated mean value can be assessed
WE need both measures to describe the whole set; as you can see on these slides; mean value can be simmilar , but the degree of anisocytosis is really different, and it is a relevant information
RDW reflects the variety of volumes
MCH is calculated from red blood cell count and Hb, represents the average in the whole population of red cells
Here are the histogram by optical technology; the advantage is that we get the mean Hb content and the distribution width and the percentage of RBC subsets:
can give complementary information of the contribution of cell with extreme values of Hb (hypochromic and hyperchromic cells) to the mean value
The degree of Polichromasia can be assessed, it can be seen that different subsets coexist reflecting the fluctuations of iron availability to the erythron in the previous weeks,
this is a healthy adult; histograms reflecting both volume and Hb content
what we see in the slide are similar red cells, so narrow histograms, this is are uniform cells

8. Total YLD resulting from all causes of anemia by country.
TotalYLD resulting from all causes of anemia by country.. World Health Organization estimates that one thousand point six 1.6 billion people, corresponding to near twenty five percent 25% of the global population, are affected by anemia.
Iron deficiency anemia, is the most common cause of anemia.
The last systematic review of global anemia burden in the past decade shows that despite efforts to reduce the prevalence of iron deficiency anemia, the number of years lived with disability as a result of this condition has increased.
Nicholas J. Kassebaum et al. Blood 2014;123:
©2014 by American Society of Hematology

9. Iron deficiency Iron is critical for optimal functioning
and survival of cells:
Mitochondrial dysfunction
Deranged enzyme activity
Abnormal transport and structural
proteins
Impaired exercise capacity
Reduced work efficacy
Impaired cognitive performance
Increased morbidity and mortality
3 iron is essential for life is involved in vital redox processes (Fe) and plays a number of key roles in biology the main Hemoglobin transport of O2
and enzymes with Heme as a prosthetic group, Cytochroms are components of the electron-transport chain so energy production
detoxification, antioxidation (xantine oxidase, catalase
Enzymes involved in DNA synthesis, replication, repair and transcription
Due to the easy transition between ferrous/ferric states is indispensable for many redox processes in most cells
Iron is essential to any form of life, primarily to ensure the transport of oxygen or to catalyse reactions
of electron transfer, nitrogen fixation or DNA synthesis,
Many proteins that have crucial roles in cellular physiology require iron to function.
@For research use only. Not for use in diagnostic procedures 9

10. Latent iron deficiency
Spectrum of iron deficiency ; this is sequential process, with a progressive reduction of iron stores until depletion, if no detected in time anemia
Iron depletion is the stage at which there is no storage of iron in the body
At this stage there is a change only in plasma ferritin levels and not in hemogram indices.
Next step is  Iron deficient erythropoiesisis
the stage at which the requirement of the erythroid marrow for iron is no longer fully met by the supply, an thus an impairment in Hb synthesis, but do to long life span of erythrocytes Hb and indices may be still normal and only minor changes can be detected in the hemogram
Iron deficiency anemia is the most severe degree of iron deficiency. At this stage, the restriction in hemoglobin production is severe enough to lead to distortion of red cells, with microcytosis and hypochromia
24 Iron metabolism and changes in biomarkers depending on the stage of iron depletion
Normal hemoglobin level does not exclude ID, because individuals with normal body iron stores lose a large amount of body iron during a long period until the impairment of the functional compartment and the erythropoiesis becomes iron deficient
adding information on iron status, extremely useful to the clinicians in the early detection of a negative iron balance and start treatment before IDA is established.
Allowing to make the diagnosis of iron deficiency in an early phase may help to reduce manifestations of iron deficiency in our patients and thus improving public health.
Moreover, less prolonged treatment with supplements and lower consumption of healthcare resources contribute to the efficiency and sustainability of the health services.
@For research use only. Not for use in diagnostic procedures

11. It is well recognized that established iron deficiency anemia has a myriad of physiological and psychological sequelae
The importance of careful assessment, investigation and treatment of patients with ANEMIA is clearly established
Important unanswered questions remain about the pathophysiological consequences of Iron Deficiency
The WHO define ID as a condition in which there are no available iron stores and in which there are signs of a compromised supply of iron to tissues, including the erythron.
Iron stores should be carefully measured in the vulnerable groups outlined and that attempts to replenish iron stores in these individuals: Perinatal, toddlers, school children , adolescents, premenopausal women
Rattehalli D. et al. Iron deficiency without anaemia: do not wait for the Haemoglobin to drop?
Health Policy and Technology 2013; 2: 45-8
Review the available literature examiningth edeleterious effects of iron
A Study of the Effects of Latent Iron Deficiency on Measures of Cognition: A Pilot Randomised Controlled Trial of Iron Supplementation in Young Women
It is well recognized that established anemia has a myriad of physiological and psychological sequels.
Iron stores should be carefully measured in groups at risk and attempt to replenish iron stores in perinatal, toddlers, school children, adolescents and premenopausal women.
iron deficiency without anemia has also Deleterious effects on cognitive development and physical performance
information on iron status, is extremely useful to the clinicians in the early detection of a negative iron balance and start treatment before anemia is established.
Allowing to make the diagnosis of iron deficiency in an early phase may help to reduce manifestations of iron deficiency in our patients and thus improving public health.
Moreover, less prolonged treatment with supplements and lower consumption of healthcare resources contribute to the efficiency and sustainability of the health services

12. Eighty years after its introduction MCV is still
Asberg AE et al. Empty iron stores in children and young adults—the diagnostic accuracy of MCV, MCH, and MCHC. Int Jnl Lab Hem 2014; 36(1):
Eighty years after its introduction MCV is still
used to classify anemia.
When using MCV to classify anemia, we ought not to falsely classify iron-deficient anemic patients as iron replete.
Normal MCV does not exclude iron deficiency
MCV, MCH, and MCHC are only
moderately accurate in diagnosing empty iron stores in children and young adults.
These tests are no more accurate in anemic than in nonanemic patients.
normal values
of MCV, MCH, and MCHC do not exclude empty iron stores in anemic patients.
the role of erythrocyte indices evaluating iron depletion
Eighty years after its introduction, mean cell volume MCV is still used to classify anemia.
Normal MCV does not exclude iron deficiency, we ought not to falsely classify iron-deficient anemic patients as iron replete.
Wintrobe indices are only moderately accurate in diagnosing empty iron stores in children and young adults.
Normal values of MeanCellVolume, MeanCellHeemoglobin do not exclude empty iron stores in anemic patients
These tests are no more accurate in anemic than in nonanemic patients.

13. Beckman-Coulter V C S Red cells Size factor Low Hb Density
Beckman Coulter technology impedance volume, conductivity scatter
This is the main figure cells are classify in the cube
Parameters derived from this technology
RSf averages the volumes of the mature and young red cells
Low Hb density derives from mean cell Hb concentration , sigmoid function and an square root
Good diagnosttic performances to detect iron deficiency

14. Latent iron deficiency
Clinical value of markers of hypochromia in the detection of latent iron deficiency in non-anemic premenopausal women
No ID: Hb > 120 g/L, s-ferritin was within reference interval Latent Iron Defiency: Hb > 120 g/L, s-ferritin < 30 μg/L
No ID
LID P
RBC, 1012/L
4.8 (0.8)
4.37 (0.5)
<0.0001
Hb, g/L
135 (6)
131 (12)
0.006
MCV, fL
92.2 (2.1)
90.2 (3.8)
MCH, pg
32.4 (0.7)
30.6 (2.2)
MCHC, g/L
339 (6)
319 (23)
0.001
RDW, %
Ret He, pg
%Hypo He
@LHD%
Ferritin, µg/L
13.1 (0.6)
33.8 (2.1)
0.3 (0.15)
2.8 (2.0)
86 (3)
14.5 (2.1)
27.8 (2.4)
6.9 (5.5)
5.6 (2.5)
16 (7.6)
AUC
95% CI
Cutoff
Sensitivity %
Specificity %
@LHD %
0.898
5.0%
85.9
84.1
Ret He
0.914
29.9pg
86.8
85.7
%Hypo He
0.934
1.6%
92.1
we studied the Clinical value of markers of hypochromia in the detection of latent iron deficiency in a group at risk , premenopausal women
Two hundred fifty 250 consecutive nonanemic women in fertile age ), whose analyses had been requested by general practicioners, were included.
We found that Ninety-seven (38.8% near thirty nine percent) subjects in this group of apparently healthy women had latent Iron Deficiency.
Two analyzers were used Coulter LH and sysmex five thousand
ROC analysis rendered similar results in the detection of Latent Iron deficiency
The advantage of low hemoglobin density is that there is no need to include reticulocytes to obtain the advanced information
LHD %
97 women (38.8%) in 250 apparently healthy women had latent ID
Urrechaga E et al. Journal Clin Lab Analysis 2016; 30 (5):

15. Reticulocyte Hb can be useful to detect the initial phase of iron deficiency when erythropoiesis become deficient , before anemia is established
Allowing to make the diagnosis of iron deficiency in an early phase
may help to reduce manifestations of iron deficiency in our patients improving public health
Less prolonged treatment with supplements and lower consumption of healthcare resources
contribute to the efficiency and sustainability of the health services
Reticulocyte Hb sysmex and Abbott can detect iron deficient erythropiesis in apprently healthy subjects

16. Anemia of chronic diseases Functional Iron deficiency
The anemia of chronic disease (also called anemia of inflammation) is an acquired disorder of iron metabolism
Cartwright GE. The anemia of chronic disorders.
Semin Hematol. 1966;3:
This condition is associated with infection and inflammation:
Chronic renal failure
Autoimmune disorders
Inflammatory bowel disorders
Heart failure
Chronic obstructive pulmonary disease
Cancer
The underlying etiology : macrophages that normally recycle iron are found to sequester it. Consequently, decreased serum iron is available for erythropoiesis
Functional Iron deficiency : Lack of availability of pool iron storage in relation to the demands, to maintain a erythropoiesis level adapted
I Cavill & IC Macdougall. Blood 1993; 82: 1377
Functional iron deficiency
The anemia of chronic disease (also called anemia of inflammation) is an acquired disorder of iron metabolism, described long ago
Now we now The underlying etiology : macrophages that normally recycle iron are found to sequester it. Consequently, decreased serum iron is available for erythropoiesis.
The main concept is Functional Iron deficiency : Lack of availability of pool iron storage in relation to the demands, to maintain a erythropoiesis level adapted.
This condition is associated with many diseases:
Chronic renal failure
Autoimmune disorders
Inflammatory bowel disorders
Heart failure
Chronic obstructive pulmonary disease
Cancer
Iron-restricted erythropoiesis indicates that the delivery of iron to erythroid
precursors is impaired, no matter how replete the stores.3,4 Stores may be normal
or even increased because of iron sequestration
This concept of iron availability started in when Recombinant human erythropoietin (rHuEpo) has been available for treatment of renal disease anemia since the But soon it became clear the need to supplement iron to manage the anemia; so nephrologist needed reliable laboratory test to diagnosed iron deficiency and to guide the treatment;
They proceed empirically in clinical practice but the advance in basic science finally add the

17. Weiss G. et al N Engl J Med 2005;352:1011-23
                                              
Full Text
                                                                         Mensaje a la biblioteca / Zure liburutegi...                                                                               
                                                                                                                                                                                                          
as you all are guessing hepcidin is an acute phase response Lets move to daily practice
This slide summarizes Mechanisms Underlying Anemia of Chronic Disease and the concept functional iron deficiency.
In A you can see a bacteria
cytokines, interleukin-6 stimulate the hepatic expression of the acute-phase protein hepcidin in B inhibits duodenal absorption of iron
and also in C inhibits iron export from macrophages the mechanism in the same binding to cell-surface ferroportin, triggers its internalization, there is no channel so iron can´t release into circulation
Another acute phase response protein is produced C
ferritin expression is induced for the storage of iron within macrophages.
In summary, these mechanisms lead to a decreased iron in the circulation and thus to a limited availability of iron for erythroid cells.
Erythropoiesis becomes iron deficient. Direct consequence of an imbalance between the iron requirements and the actual supply is a reduction of red cell hemoglobin content, which causes hypochromic reticulocytes and later hypochromic mature red cells.
 ACD is observed in many conditions associated with chronic inflammation. The main concept is functional iron deficiency : iron in the stores but reduced availability of iron so inefficent erythropiesis;
Weiss G. et al N Engl J Med 2005;352:

18. The full reticulocyte screen
The basic standard hemogram and the extended parameters
Reticulocyte Hb, immature fractions , total Hb within the erythrocytes percentages of the subsets and the difference in Hb content in the reticulocytes and mature cells
Also the scattergram and histograms; volume histogram based on sheath flow
The optical histogram corresponding with the blue signal on the Y axis which shows Hb distribution in the whole mature cell population

19. Hyper He
Ret He
Hypo He
In the reticulocyte channel blood cells are stained by a polymethine fluorescent dye, specific for RNA/DNA, and analysed by flow cytometry using a semiconductor laser.
For each individual cell 3 signals are generated
Flourescence =amount of nucleic acids=reticulocytes
Forward scatter, related to size of the cell; side scatter intracellular information
Cell by cell a scattergram is generated 2 signals
fluorescence , indicating mature red cells and reticulocytes.
Forward scatter correlates with erythrocyte and reticulocyte hemoglobin content (RBC He, Ret He).
Ret He is the mean value of the forward light scatter within the reticulocyte population
Are reported by all XE analyzers since 2004
RBC He the amount of Hb within erythocytes

20. NKF-K/DOQI (Kidney / Disease Outcomes Quality Initiative) guidelines
Anemia Evaluation
NKF-K/DOQI (Kidney / Disease Outcomes Quality Initiative) guidelines
Assessment of anemia should involve:
Degree of anemia: Hb
Type of anemia: MCV and MCH
Erythropoiesis: Reticulocytes
Iron stores: Ferritin
Functional iron available for erythropoiesis :
–Transferrin Saturation
– CHr (reticulocyte Hb content) (Siemens)
– Hypo (% hypochromic erytrocytes ) (Siemens)
Inflammation: PCR
Revised European best practice guidelines for the management of anaemia in patients with chronic renal failure.
Nephrol Dial Transplant 2004; 19 (suppl 2): Guideline 1.2
Clinical guidelines defined the targets of therapy
The clinical guidelines for Assessment of anemia in patients receiving therapy recommend the evaluation of anemia should involve different parameters
The most important question is which are the best parameters to assess the iron status
erythrocyte and reticulocyte parameters, reported by the modern analysers are included in the clinical guidelines t control the use of EPO and IV Fe ; cut offs are 28-29pg and 5%
Technicon was the first and for many years the only manufacture to report hypochromic cells

21. Ret He The New Reticulocyte Parameter (RET-Y) of the Sysmex XE 2100
Its Use in the Diagnosis and Monitoring of Posttreatment Sideropenic Anemia
M Buttarello, et al Am J Clin Pathol 2004;121:
Ret Y a measure of reticulocyte size: a sensitive indicator of iron deficiency
TS Kikler et al Clin Lab Haem 2004; 26:
Technicon was the first and for many years the only manufacture to report hypochromic cells
,Symex launched theuri new instrument XE2100
The New Reticulocyte Parameter (RET-Y) of the Sysmex XE 2100
The first repeorts of the cahnges in reticulocyt Hb after therapy
Its Use in the Diagnosis and Monitoring of Posttreatment Sideropenic Anemia : a sensitive indicator of iron deficiency

22. , severe anemia, both histograms are broad and reflect anisocytosis and high hypochomic percentage seen in the scattergram
Patient recovery these shoulders are the past (older cells) and the new after therapy lower lower Hypo
Or high level of polichromasia, means that different subsets coexist reflecting the fluctuations of iron availability to the erythron in the previous weeks, reflects nicely the slide
The instrument assesses erythrocytes on a cell by cell basis so can therefore detect and quantify subpopulations of cells. Percentage hypochromic red cells is the concentration of haemoglobin in individual cells
It is a more sensitive marker because small changes in the number of red cells with inadequate haemoglobin can be measured before there is any change in the mean value, it needs more time to change

24. Thomas W et al. Guideline for the laboratory diagnosis of functional iron deficiency . Br J Hematol 2013; 161:
MCH, MCV 1B
% Hypochromic RBC 1B
Reticulocyte Hb 1B
Protoporphyirin Zn 1B
Bone Marrow 1B
Ferritin 1 A 1 B
sTfR 1A
Sat transferria 1 A 1 B
Erythropoyetin 1 A
Hepcidin UE
These are incorporated to tHe nephrology best practice guidelines, but they are recommended by the british Guideline for the laboratory diagnosis of functional iron deficiency
Can be used in other diseases cancer, reumatoyd arthritis, inflammatory bowel disease
Heart failure
An the good notice is that now reticuloyte Hb and hypochromic cells are reported by many different analyzers
Reticulocytes are immature red blood cells with a life span of only 1 to 2 days. When these are first released from the bone marrow, measurement of their hemoglobin content can provide the amount of iron immediately available for erythropoiesis. A less than normal hemoglobin content in these reticulocytes is an indication of inadequate iron supply relative to demand. The amount of hemoglobin in these reticulocytes also corresponds to the amount of hemoglobin in mature red blood cells. CHr
It is possible then not only to count the reticulocytes but also to assess the quality of the erythropoiesis;
The measurement of CHr is a direct assessment of the incorporation of iron into erythrocyte hemoglobin and thus an estimate of the recent functional availability of iron into the erythron; due to the life span of the reticulocytes CHr is a sensitive indicator of iron deficient erythropoiesis
The percentages of mature erythrocytes
Measurement of %Hypo (defined as the percentage of red blood cells with Hb concentration less than 280 g/L) is a sensitive method for quantifying the hemoglobinization of mature red cells. Because of the long circulating life span of mature erythrocytes %Hypo values are related to iron status in the last 2-3 months, and have been recognised as an indicator of iron deficiency

25. Disorders Characterized by Microcytosis
Figure 1 Disorders Characterized by Microcytosis. Red cells become microcytic because of a lack of hemoglobin. This can result from a lack of iron (deficiency or inflammation), defects in heme synthesis (sideroblastic anemias), or defects in the production of hemoglobin protein (thalassemia).
The microcytic anemias are those characterized by the production of red cells that are smaller than normal. The small size of these cells is due to decreased production of hemoglobin, the predominant constituent of
red cells (Fig. 1). The causes of microcytic anemia are a lack of globin product (thalassemia), restricted iron delivery to the heme group of hemoglobin (anemia of inflammation),
a lack of iron delivery to the heme group (iron-deficiency anemia), and defects in the synthesis of the heme group (sideroblastic anemias).
DeLoughery TG. N Engl J Med 2014;371:

26. Differential diagnosis IDA & Thalassemia trait
Microcytic anemia in the case of thalassemia results from impaired globin chain synthesis and decreased hemoglobin synthesis
Thalassemia syndromes are among the most common genetic disorders worldwide, with 1.7% of the world's population carrying thalassemic genes
Thalassemia is prevalent in some parts of the world: Mediterranean , Middle East, India, Southeast Asia; owing to migration of populations, there is virtually no country in the world in which thalassemia does not affect some percentage of the inhabitants
Microcytic anemia in the case of thalassemia results from impaired globin chain synthesis and decreased hemoglobin (Hb) synthesis
Thalassemia syndromes are among the most common genetic disorders worldwide, Thalassemia syndromes are among the most common genetic disorders worldwide, with 1.7% of the world's population carrying thalassemic genes
Although thalassemia is endemic to the Mediterranean basin and countries of the Far East, owing to migration of populations in recent decades, there is virtually no country in the world in which thalassemia does not affect some percentage of the inhabitants.
As in all chronic diseases, prevention is important in the overall management of the disease.. Appropriate screening, detection of patients, and counsel of couples at risk are the most important procedures for the reduction of morbidity and mortality.

27. accumulation of excess unmatched globin leads to the premature destruction of erythroid precursors in marrow
The erythopoiesis in thalassemia is ineffective but proceeding at a high rate
Iron absorption is increased in conditions of ineffective erythropoiesis. The poor elimination of reactive oxygen species, lso hamper erythropoiesis and the oxidant injury may cause hemolysis
apoptosis of the maturing nucleated erythroid cells so limited differentiation plays a role in the development of ineffective erythropoiesis. This would further exacerbate anemia and increase iron absorption
This is reflect in erythrocytosis but the red cells are microcytic, Hb synthesis is impaired
In iron deficiency states,
RBCs are continuously produced in the bone marrow as the iron stores progressively decrease. As a result, they tend to be more and more microcytic. Because of their long life span of approximately 4 months, several cohorts of normocytic and increasingly microcytic RBCs coexist in the peripheral blood, leading to anisocytosis.
On the contrary, the underlying pathogenetic anomaly in β thalassemia has no fluctuations, and as a result, the bone marrow produces a constantly uniform population of microcytic erithrocytes.
Rund & Rachmilewitz Beta-thalassemia. New England Journal of Medicine 2005: 353(11):
Copyright © 2005 Massachusetts Medical Society. All rights reserved.

28. On the contrary iron deficiency proceeds continuously from replete iron stores to IDA
In iron deficiency states,
RBCs are continuously produced in the bone marrow as the iron stores progressively decrease. As a result, they tend to be more and more microcytic. Because of their long life span of approximately 4 months, several cohorts of normocytic and increasingly microcytic RBCs coexist in the peripheral blood, leading to anisocytosis and polichromasia

29. Siemens & Abbott Sapphire + Alinity
Volume/Hb Concentration cytogram Mie Map
Volume/Hemoglobin Concentration (V/HC) cytogram Mie Map
Hemoglobin concentration is plotted along the x axis and cell volume is plotted along the y axis. Only red blood cells appear on this cytogr
each mature RBC is represented by a red dot ALL RBC are represented, classified according morpholgy , Hb concnetration
The two vertical linesin the graph represent the CHC cut-off values for calculating hypo- and hyperchromic RBC proportions (28 and 41 g/dL, respectively). And cell size
Likewise, the horizontal lines are at 60 and 120 fL, the values for defining micro- and macrocytic RBC.

30. If you are lucky enough to work with a sapphire, this morphology are pretty nicely catched by the analyzer, not only by the standard parameters, also the extended
A carrier has much more microcytes than a iron deficient at the same Hb level
Iron deficient tends to be more hypochomic; anisocyosis and polichromasia is reflected by this image , an open fan , while the signal in a carrier is so narrow reflect that all the cell are similar, of course in the micro area
 (poster Mie Map by Abbott  CELL-DYN Sapphire : the search for the hidden trait)

31. RBC /L Hb 120 g/L MCV 68.1 fL MCH 24.5 pg MCHC 331 g/L RDW % RBC /L Hb 95 g/L MCV 73.7 fL MCH 22.5 pg MCHC 309 g/L RDW 16.5 %
5.34
124
74.3
23.2
312
16.4
5.03
118
75.1
22.7
301
17.8
IDA and thalassemia have different etiologies, which renders different profiles in the hemogram data; nevertheless, the results can be very similar and based on the individual parameters of the hemogram is difficult to recognise a carrier in daily practice.
For a certain degree of anemia MCV and MCH tend to be lower in β thalassemia than in IDA and RDW higher in IDA, RBC higher in a carrier

32. Erythrocyte Indices differential diagnosis of microcytic anemia
Indices Year
IDA
β Thalassemia
Mentzer = MCV / RBC
> 13
< 13
Srivastava = MCH / RBC
England & Fraser = MCV - RBC - 5*Hb
> 3.8
>0
< 3.8
<0
Ricerca = RDW / RBC
> 4.4
< 4.4
Green & King = MCV2 * RDW / 100* Hb
> 65
< 65
MH ratio ( Technicon) = %micro / %hypo
Sirdah = MCV - RBC - 3*Hb
MH ratio (Siemens)
<1
>27
<3.4
>1
<27
>3.4
Ehsani = MCV - (10* RBC)
> 15
< 15
ThalIndex=(0.615*MCV)+(0.518*MCH)
+(0.446*RDW)
MH ratio (Abbott)
>59
< 6.4
<59
> 6.4
IDA and thalassemia have different ethiologies, which renders typical profiles in the hemogram data, erythrocytosis and microcytosis in carriers, anisocytosis in iron deficiency; nevertheless, the results can be very similar and based on the individual parameters of the hemogram is difficult to recognise a carrier in daily practice.
 A number of indices have been proposed to differentiate IDA from heterozygous -thalassemia The usefulness of the indices is to detect patients with a high probability to be thalassemia carriers, so the best index must have as high a sensitivity as possible to detect almost all thalassemia patients. “Suspicious” samples can be selected for HbA2 quantification and molecular analysis to confirm the presumptive diagnosis of the disease
formulas that incorporate MCV, MCH, RBC count, RDW and Hb in various combinations to enhance the differences of the individual parameters in both diseases. The fact that new indices have been published in recent years demonstrates the interest in the matter.
The erythopoiesis in thalassemia is ineffective but proceeding at a high rate
This is reflect in erythrocytosis but the red cells are microcytic, Hb synthesis is impaired
For a certain degree of anemia MCV and MCH tend to be lower in β thalassemia than in IDA
Another significant difference between these two types of anemia is the level of anisocytosis . In iron deficiency states,
RBCs are continuously produced in the bone marrow as the iron stores progressively decrease. As a result, they tend to be more and more microcytic. Because of their long life span of approximately 4 months, several cohorts of normocytic and increasingly microcytic RBCs coexist in the peripheral blood, leading to anisocytosis.
On the contrary, the underlying pathogenetic anomaly in β thalassemia has no fluctuations, and as a result, the bone marrow produces a constantly uniform population of microcytic erithrocytes.
 The fact that new indices have been published in recent years demonstrates the interest in the matter;
patients with thalassemia carriers and IDA.
The measurement of microcytic and hypochromic red cells shows different results in
also you can see indices based on erythrocyte subsets
volume
, as a result of the chronic increase of erythropoiesis. β thalassemia patients have erythrocytosis but due to impaired globin synthesis Microcytes are produced with very small
Iron deficient erythropoiesis is characterized by the production of RBC with a decrease Hb concentration, high number of hypochromic cells, the number of microcyte increases with the degree of anemia but microcytes of β thalassemia are generally smaller, with more preserved Hb concentration

33. Abbott CELDYN Sapphire
There was an inverse relationship between the percentages of the microcytes (MCV < 60 fL) and the hypochromic cells (MCHC 280 g/L) in IDA and Thalassemia
Technicon H*1
d ’Onofrio G et al . Automated measurement of red blood cell microcytosis and hypochromia in iron deficiency and beta-thalassemia trait. Arch Pathol Lab Med. 1992; 116:
Siemens Advia 2120
Urrechaga E. Discriminant value of % Microcytic / % Hypochromic ratio in the differential diagnosis of microcytic anemia. Clin Chem Lab Med 2008;46(12):
Abbott CELDYN Sapphire
E Urrechaga, JJML Hoffmann, S Izquierdo, JF Escanero. Differential Diagnosis of Microcytic Anemia: the Role of Microcytic and Hypochromic Erythrocytes. Int Jnl Lab Hematol 2015; 37:
Onofrio et al used H*1. They found that there was an inverse relationship between the percentages of the microcytes (MCV < 60 fl) and the hypochromic cells (MCHC 280 g/L) in IDA and TT. Percentile of microcytes (mean 33.1%) was higher than the hypochromic cells (mean 13.9%) in TT. On the contrary, in IDA, the percentage of hypochromic cells (mean 34.6%) was higher than the percentage of microcytes (mean 12.8%)
Years latter working with next generation of analyzer we found the same trends

34. Johannes J.M.L. Hoffmann *, Eloisa Urrechaga, Urko Aguirre Discriminant indices for distinguishing thalassemia and iron deficiency in patients with microcytic anemia: a meta-analysis Clinical Chemistry & Laboratory Medicine 2015; 53(12):
The fact that new indices have been published in recent years demonstrates the interest in the matter; so in this point Dr Hoffmann, responsible in abbott for medical and scientific affairs in Europe, decided that this was the time to analyse all indices , so he proposed to do a metaanalysis
The primary outcome in the analyses was the performance of different markers in the differential diagnosis of microcytic negative (TN), false positive (FP) and false negative (FN)
values. These values were retrieved from the publications found and entered into a database. In studies with sensitivities and specificities of 100%
Pooled positive likelihood ratio (PLR) and negative likelihood ratio (NLR) with their 95% confidence intervals (95% CI) were calculated using random effects models
[25] . Summarized sensitivities and specificities were also computed to assess the clinical effectiveness, even though both screening parameters are not considered appropriate
for meta-analyses [26] .
As an accuracy measure we calculated the diagnostic odds ratio (DOR), an indicator of test accuracy that comprises a combination of sensitivity and specificity and is
independent of disease prevalence, making it very appropriate for comparing different studies [27, 28] . The higher the DOR value, the better discriminatory test performance
is present.
Summary receiver operating characteristic (SROC) curves were used to summarize overall test performance and to calculate the area under the SROC curve (AUC), which is quite robust to heterogeneity

35. We identified 147 reports in which at least one discriminant index had been evaluated. Twelve of these discriminant indices had been evaluated by five or more studies
( Tables 1 and 2 ). In total, 99 such studies comprised 135,409 patient results, ranging from 3091 for the M/H ratio to 22,022 for the England and Fraser index ( Table 2 ).
Some 30 other discriminant formulas had been investigated in < 5 studies and therefore were excluded from our current meta-analysis.
Out of these 99 studies, 36 (36%) were from Europe, 24 (24%) from the Mediterranean region, 20 (20%) from Southeast Asia, 14 (14%) were from North America and
the few remaining studies were from Latin America and Australia. Forty-one studies (41%) investigated adults, 11 (11%) included only children, 13 (13%) were focused on
mixed populations of adults and children and 35 (35%) did not provide the patients ’ age.
With regard to the hematology analyzers used, 32 (32%) conducted the analyses with Coulter, 20 (20%) with Bayer and 18 (18%) with Sysmex; the remaining 30% were performed with other analyzers
or the analyzer type was not specified.
Our results: MHratio `performed the best; may be is more clear to look at the summary RO C plots; see the highest point for MH ratio
So you can use it with confidence to detect carriers, but only in adults; this has never been applied in children

36. Hemolytic Anemias
Hemolysis is the premature destruction of erythrocytes
A hemolytic anemia will develop if bone marrow activity cannot compensate for the erythrocyte loss
hemolysis is the premature destruction of erythrocytes. A hemolytic anemia will develop if bone marrow activity cannot compensate for the erythrocyte loss. The severity of the anemia depends on whether the onset of hemolysis is gradual or abrupt and on the extent of erythrocyte destruction. Mild hemolysis can be asymptomatic while the anemia in severe hemolysis can be life threatening and cause angina and cardiopuThere are multiple causes for hemolytic anemia, and the clinical presentation can differ depending on the etiology. An array of laboratory tests are available for detecting hemolysis, and specialized tests may be indicated to diagnose the cause for hemolysis . There are differences in the management of various types of hemolytic anemias (see Treatment).lmonary decompensation.

37. Hemólisis is the premature destruction of erythrocytes
Hemólisis is the premature destruction of erythrocytes. A hemolytic anemia will develop if bone marrow activity cannot compensate for the erythrocyte loss. Pathophysiology
Hemolysis can be due to hereditary and acquired disorders. The etiology of premature erythrocyte destruction is diverse and can be due to conditions such as intrinsic membrane defects, abnormal hemoglobins, erythrocyte enzymatic defects, immune destruction of erythrocytes, mechanical injury, and hypersplenism
Autoimmune hemolytic anemia and hereditary spherocytosis are examples of extravascular hemolysis because the red blood cells are destroyed in the spleen and other reticuloendothelial tissues. Intravascular hemolysis occurs in hemolytic anemia due to the following:
Prosthetic cardiac valves
Glucose-6-phosphate dehydrogenase (G6PD) deficiency
Thrombotic thrombocytopenic purpura
Disseminated intravascular coagulation
Transfusion of ABO incompatible blood
Paroxysmal nocturnal hemoglobinuria (PNH)

38. Hereditary Spherocytosis
The red cell surface, a net of proteins keep the shape
Spectrin ankyrin
Hereditary spherocytosis (HS) is the most common cause of genetic based chronic hemolysis in north Europe and America, affects 1 in 2000
but is likely even higher as many mild cases of HS go undetected
Is characterized by a loss of surface area leading to the release of microparticles.called spherocytes
The key defect in HS is structural abnormalities of the proteins that form the RBC cytoskeleton, mainly a- and b-spectrin, ankyrin, band 3
Due to these abnormalities, the equilibrium surface/volume is disrupted, and the cell tends to be spheric, more stable due to lower volume
; the same Hb packed in a lower volume means hyperchromia
Spectrin is the major component of the protein network covering the erythrocyte surface
A link with ankyrin couples the actin /spectrin complex to the membrane
Provides mechanical support to the membrane bilayer, allows survival of cells in circulation

39. All the reference tests suffer from specific limitations
All the reference tests suffer from specific limitations. The aim of this study was to develop easy to use diagnostic tool for screening of hereditary spherocytosis based on routinely acquired haematological parameters
Other technologies Impedance employs the sphered volume, sysmex percentage of microcytes, percentage of hypochromic cells, reticulocyte counts, but reticulocyte must be requested
With Sapphire you can see in the CHC histogram and mie map a dual peak; cells with higher HCH are spherocytes

40. AUC 0.972 Cut off 4.9 % Sens 96.4 % Spec 99.1 %
Int J Lab Hematol 2015; 37:
AUC
Cut off 4.9 %
Sens %
Spec %
Hyperchromic RBC in various patients groups. Median values (black diamonds) and central 95% percentiles are shown. Abbreviations: AIHA: autoimmune haemolytic anaemia; Enz def: RBC enzyme deficiency; Hb pathy: haemoglobinopathy; HE: hereditary elliptocytosis; HS: hereditary spherocytosis.
As seen in the previous slides the percentage of hyperchomic cells is higher in hereditary spherocytosis than seen in other hemolysis, immune or enzyme defects
Roc analysis rendered the best AUC AUC Cut off 4.9 % Sens 96.4 % Spec 99.1%better than MCHC or bilirrubin
HS is a very heterogeneous
disorder, both clinically and at the molecular level As a consequence of the variable expression, HS is
not always easy to diagnose, in particular in mild cases
cytometric analysis of eosin-50-maleimide (EMA) binding to
erythrocytes is accepted as one of the most sensitive and specific HS tests available Although the
test is relatively simple to perform, it requires the availability of a flow cytometer and some expertise in
defining the optimal cut-off level
The flow cytometric eosin-50-maleimide test (EMA) is highly sensitive
and specific for HS as a confirmatory method. The aims of this
study were to assess the utility of hyperchromic erythrocytes in HS
screening and to evaluate the EMA test performed on CELL-DYN
Sapphire analyser compared with the reference method.
ilable for non-commercial use only. Please note that permission may be required for re-use of images in which the copyright is owned by a third party.
Multidimensional optical scatter of sphered erythrocytes can identify and enumerate hyperchromic erythrocytes, which might be used for hereditary spherocytosis (HS) screening.
The flow cytometric eosin-5′-maleimide test (EMA) is highly sensitive and specific for HS as a confirmatory method. The aims of this study were to assess the utility of hyperchromic erythrocytes in HS screening and to evaluate the EMA test performed on CELL-DYN Sapphire analyser compared with the reference method.

41. In summary…… Extended RBC parameters
Expand information at a cellular level
Correlate with the pathophysiology of disease
Operational Efficiency
Rapid and Automated
Financial Justification
Improve the clinical relevant information Quality of erythropoiesis
Aids clinicians in
Assessing true status of iron
Detect Functional Iron Deficiency = patients who can benefit from therapy
Differential diagnosis genetic or acquired
Microcytic Anemia
Hemolytic anemia
Fluorescent Flow Cytometry Expand information at a cellular level
Measures Hemoglobin content on red cells
Monitors changes in hemoglobin incorporation into erythron
Provides information of the quality of erythropoiesis
Operational Efficiency
Rapid and Automated
Rapid Information to Clinicians iron status
Faster Response to Changes from Therapy
Financial Justification
Aids clinicians in
Assessing true status of iron
Detect Functional Iron Deficiency patients who can benefit from therapy
Differential diagnosis of acquire anemia vs genetic anemia,